SYNTÉZů, CHARAKTERIZACE A BIOLOGICKÁ ůktivitů KOMPLEX

Transkript

1 UNIVERZITů PůLůCKÉHO V OLOMOUCI Přírodovědecká fakulta Katedra anorganické chemie SYNTÉZů, CHARAKTERIZACE A BIOLOGICKÁ ůktivitů KOMPLEX P ECHODNÝCH KOV S R ZNÝMI N-DONOROVÝMI LIGůNDY Habilitační práce Mgr. Pavel Štarha, Ph.D. Olomouc, 2015

2 Poděkování Velmi rád bych zde upřímně poděkoval spolupracovníkům, kolegům a v mnoha případech i kamarádům z Katedry anorganické chemie, Katedry buněčné biologie a genetiky, Katedry biofyziky a Regionálního centra pokročilých technologií a materiálů (RCPTM) Přírodovědecké fakulty Univerzity Palackého v Olomouci za vynikající a podnětnou spolupráci, podporu a kvalitní pracovní zázemí, kterých se mi dostávalo po celou dobu mého působení na olomoucké Alma Mater. Speciální poděkování patří prof. RNDr. Zdeňku Trávníčkovi, Ph.D., jehož odborné a věcné vedení, stejně jako příležitost podílet se na výzkumu moderních a zajímavých tematik na půdě špičkově vybaveného pracoviště, byly, jsou a zcela nepochybně také budou pro moji profesní kariéru naprosto zásadní. Dále chci poděkovat prof. RNDr. Zdeňku Dvořákovi, DrSc. et Ph.D., prof. RNDr. Viktoru Brabcovi, DrSc. a členům jejich výzkumných týmů z Katedry buněčné biologie a genetiky resp. Katedry biofyziky, Přírodovědecké fakulty Univerzity Palackého v Olomouci, za detailní studia biologických vlastností níže komentovaných sloučenin, jejichž výsledky zásadním způsobem přispěly, a doufám, že i v budoucnu budou přispívat, do mnou studované problematiky. Díky patří i generálnímu řediteli RCPTM prof. RNDr. Radku Zbořilovi, Ph.D. za umožnění využití mnohých analytických technik z přístrojového parku centra pro potřeby mé výzkumné činnosti. Velké díky tímto posílám také do Department of Chemistry, University of Warwick, Coventry, UK, kde mi Prof. Peter J. Sadler FRS FRSE dal jedinečnou příležitost pracovat po dobu šesti měsíců ve špičkovém výzkumném týmu na jedné z nejlépe hodnocených britských univerzit a načerpat tak nové a neocenitelné vědomosti a dovednosti, ze kterých budu nepochybně čerpat při své nastávající vědecko-výzkumné činnosti. Děkuji rodičům za trpělivou podporu nejen v dobách studií na Přírodovědecké fakultě Univerzity Palackého v Olomouci, ale i na počátku mého profesního působení tamtéž. V neposlední řadě chci poděkovat své rodině, manželce Ivaně a dcerám Aničce a Emičce, za jejich lásku, trpělivost a nikdy nekončící přísun životní energie. 2

3 Obsah 1. Úvod Chemoterapeutika na bázi komplexů přechodných kovů Protinádorová chemoterapeutika na bázi platiny Historie a současnost Mechanismus účinku Chemoterapeutika jiných přechodných kovů Platnaté komplexy s různými N-donorovými ligandy Platnaté komplexy s deriváty 7-azaindolu Cis-dichloroplatnaté komplexy s deriváty 7-azaindolu In vitro protinádorová aktivita a molekulární farmakologie Protinádorová aktivita in vivo Karboxylato komplexy s deriváty 7-azaindolu Oxalatoplatnaté komplexy s deriváty 7-azaindolu Fototoxické deriváty karboplatiny s deriváty 7-azaindolu Selektivní malonato a dekanoato komplexy Možnosti cíleného transportu komplexů na bázi platiny Platnaté komplexy s deriváty N6-benzyladeninu Protinádorová aktivita in vitro Protinádorová aktivita in vivo Komplexy jiných přechodných kovů s různými N-donorovými ligandy Protizánětlivá aktivita Au(I) komplexů s deriváty N6-benzyladeninu Protinádorově účinné Au(I) komplexy s deriváty 7-azaindolu Závěr Literatura Seznam příloh Přílohy

4 1. Úvod Léčiva na bázi koordinačních sloučenin platiny počítají svoji historii od 60. let minulého století, kdy Barnett Rosenberg, profesor chemie a biofyziky na Michigan State University, objevil protinádorové vlastnosti cis-diammin-dichloroplatnatého komplexu (cis-[pt(nh 3 ) 2 Cl 2 ]), známého od té doby pod triviálním a posléze i obchodním názvem cisplatina [1 4]. Tato látka byla v roce 1λ78 schválena jako chemoterapeutikum protinádorové terapie a dodnes je vůdčím léčivem při léčbě různých typů rakoviny. Klinický úspěch cisplatiny byl základem rozsáhlého výzkumu mnoha vědeckých týmů, jehož výsledkem bylo a, s ohledem na nezastupitelnost metaloterapeutik na bázi platiny v onkologické praxi, stále je mnoho nových komplexů platiny, u kterých byly studovány jejich biologické vlastnosti. Některé z těchto látek pak, obdobně jako cisplatina, dosáhly klinického využití. Tyto jsou známé pod názvy karboplatina, oxaliplatina, nedaplatina, lobaplatina a heptaplatina [1 3,5]. Je obecně známým faktem, že chemoterapie léčivy na bázi platiny je spojená s negativními vedlejšími účinky, jako jsou nefrotoxicita, neurotoxicita, ototoxicita, myelosuprese nebo resistence některých typů nádorů, a to ať už vrozená nebo získaná. Přirozenou snahou bioanorganických chemiků je vývoj takových látek, které při srovnatelné nebo dokonce vyšší protinádorové aktivitě zmíněné vedlejší účinky nevykazují. Tohoto lze dosáhnout různými způsoby - vhodnou modifikací klinicky užívaných léčiv na bázi platiny užitím jiných ligandů, aktivací (např. fotoaktivace) původně neaktivních látek ve fyziologickém prostředí, zaměřením biologického účinku na jiný cíl než je jaderná DNA v případě konvenčních platnatých metaloterapeutik (např. mitochondrie a redoxní mechanismus účinku) nebo využitím principů cíleného transportu u klinicky používaných nebo nově připravených komplexů platiny (např. nanočástice oxidů železa nebo liposomy) [6 13]. Nezbytné je v této souvislosti zmínit také studium (zatím bez praktického využití v onkologické praxi) biologicky aktivních koordinačních sloučenin jiných (někdy též neplatinových) přechodných kovů, z nichž se jako medicinálně nejperspektivnější jeví komplexy ruthenia a v poslední době též osmia a iridia [11,14 20]. Některé z těchto přístupů moderní bioanorganické chemie, resp. výzkumu a vývoje nových medicinálně perspektivních koordinačních sloučenin přechodných kovů, budou v předložené habilitační práci komentovány jako výsledky vědecko-výzkumné činnosti, na které se autor po ukončení doktorského studia v listopadu β010 podílel v rámci svého působení na Katedře anorganické chemie a Regionálního centra pokročilých technologií a materiálů. Za reprezentativní příklad 4

5 jistě mohou posloužit cis-dichloroplatnaté komplexy s halogen-deriváty 7-azaindolu, které vykazují několikanásobně vyšší in vitro cytotoxickou aktivitu na různých typech lidských nádorových buněčných linií (např. karcinom vaječníku, prsní karcinom nebo osteosarkom s hodnotami IC 50 = 1,8, β,0 resp. β,5 M) ve srovnání s cisplatinou (IC 50 = 12,0, 19,6 resp. γ4,β M vůči zmíněným lidským nádorovým buněčným liniím) a jejichž in vivo protinádorová aktivita, která je srovnatelná s cisplatinou, vyvolává ve výrazně menší míře negativní vedlejší účinky na testovaná zvířata; IC 50 = letální koncentrace pro 50% testovaných buněk [21]. Předložená habilitační práce je rozdělena do dvou základních částí. V kapitole 2 jsou popsány literární poznatky o protinádorových léčivech na bázi platiny a jiných přechodných kovů se zaměřením na terapeutika na bázi platiny, jejichž deriváty byly v dosavadní vědeckovýzkumné kariéře autora jeho hlavním předmětem studia. Podkladem pro následující kapitoly 3 a 4 předložené habilitační práce je soubor jedenácti publikací, na kterých se uchazeč autorsky spolupodílel a jež byly publikované v renomovaných a v oblasti bioanorganické chemie respektovaných časopisech, jakými jsou např. Journal of Medicinal Chemistry, PLoS ONE nebo Journal of Inorganic Biochemistry. Část předložených výsledků je předmětem patentové ochrany celkem tří národních patentů a jedné národní a dvou mezinárodních přihlášek vynálezu. Závěrem lze uvést, že cíle vědecko-výzkumné činnosti uchazeče byly po ukončení doktorského studia následujícíμ 1/ design, příprava a studium fyzikálně-chemických vlastností koordinačních sloučenin platiny a jiných biologicky perspektivních přechodných kovů; 2/ studium biologické (především protinádorové) aktivity připravených sloučenin na úrovni in vitro i in vivo, se zaměřením na schopnost látek překonávat získanou rezistenci vůči klinicky používaným chemoterapeutikům a na selektivní účinek těchto látek vůči nádorovým buňkám vedoucí ke snížení negativních vedlejších účinků; 3/ studium cíleného transportu připravených biologicky aktivních koordinačních sloučenin metodou funkcionalizace magnetických nanočástic na bázi oxidů železa. 5

6 2. Chemoterapeutika na bázi komplex p echodných kov Bioanorganická chemie koordinačních sloučenin přechodných a vnitřně přechodných prvků je nesmírně bohatým a pestrým odvětvím chemie, které pro smysluplné naplnění svých základních cílů nutně musí spolupracovat a překrývat se s mnoha jinými chemickými (např. analytická chemie, biochemie) i nechemickými (např. biofyzika, toxikologie, farmakologie) vědními obory [2,14,19]. Její praktický význam je, s ohledem na klinické využití mnoha koordinačních sloučenin (např. protinádorová léčiva na bázi platiny, MRI kontrastní látky na bázi gadolinia), obrovský a i do dalších let tento vědní obor skýtá mnohé perspektivy, což lze dedukovat z neutuchajícího pronikání koordinačních sloučenin do klinických experimentů, v jehož různých fázích se nyní nachází několik desítek komplexů různých d- a f- prvků. Nelze však nesouhlasit s obecně přijímaným tvrzením, že jestli je některá skupina látek vůdčí mezi biologicky aktivními sloučeninami na bázi komplexů přechodných kovů, jsou to, díky více než 50% podílu na celosvětové protinádorové chemoterapii, komplexy platiny. Obrázek 1. Strukturní vzorce klinicky používaných metaloterapeutik na bázi platiny 2.1. Protinádorová chemoterapeutika na bázi platiny Původním protinádorovým léčivem na bázi koordinačních sloučenin platiny je cisplatina (cis-diammin-dichloroplatnatý komplex), kterou do celosvětové klinické praxe následovaly karboplatina (diammin-cyklobutan-1,1`-dikarboxylatoplatnatý komplex [22]) a oxaliplatina (1R,2R-diaminocyklohexan-oxalatoplatnatý komplex [23,24]), na lokálních asijských trzích je dále používána nedaplatina (diammin-glykolatoplatnatý komplex [25]), lobaplatina (1,2-bis(aminomethyl)cyklobutan-laktatoplatnatý komplex [26]) a heptaplatina ((4R,5R)-4,5-bis(aminomethyl)-2-isopropyl-1,3-dioxolan-malonatoplatnatý komplex [27]) 6

7 (obrázek 1) [1 3,14]. V různých fázích klinických studií se v současné době nachází několik dalších komplexů platiny, ať už v oxidačním stupni +II nebo +IV. Některé z těchto látek budou blíže okomentovány níže Historie a současnost Základy bioanorganické a medicinální chemie protinádorově aktivních koordinačních sloučenin platiny byly položeny na počátku 60. let β0. století americkým univerzitním profesorem Barnettem Rosenbergem, který jako první popsal cytotoxické účinky cis-[pt(nh 3 ) 2 Cl 2 ] (cisplatina, obrázek 1) [4]. Zajímavostí jistě je, že původní výzkum Prof. Rosenberga nebyl orientován na biologicky aktivní komplexy platiny či jiných přechodných kovů, ale na vliv elektromagnetického (jak elektrické, tak i magnetické složky) záření na buněčné dělení bakterií. Experimenty výzkumného týmu v testovací komoře obsahující platinové elektrody sice vedly k výrazným morfologickým změnám studovaných E. coli, ale podrobnější studium tohoto procesu prokázalo, že za to nenese přímou odpovědnost procházející elektrický proud, ale sloučeniny, které se uvolňují ze zmíněných platinových elektrod ponořených do média. Detailní analýza prokázala, že se do roztoku uvolňuje více platinu obsahujících sloučenin, které byly posléze nasyntetizovány a samostatně testovány. Proběhnuvší biologické experimenty prokázaly jako látku odpovědnou za potlačení buněčného dělení právě zmíněnou cisplatinu (nejednalo se o původní látku, byla již více než sto let známa jak tzv. Peyronova sůl [28]; pro přesnost a úplnost - cisplatina byla jedna ze dvou aktivních látek, avšak druhá z nich, cis-[pt(nh 3 ) 2 Cl 4 ], klinické využití nenachází). Tento veskrze průlomový objev odstartoval rapidní výzkum protinádorové aktivity cisplatiny a jejího mechanismu účinku, kdy byly v nebývale krátké době léčeni první pacienti (1971) a již v roce 1978 byla cisplatina přijata Food and Drug Administration (FDA) ke klinickému využití v onkologické praxi [1 3]. Je třeba už na tomto místě zmínit, že již ve zmíněných 70. letech minulého století bylo patrné, že vynikající terapeutické schopnosti cisplatiny, která byla účinná a od počátku používaná na více typech nádorů, jsou spojeny s celou řadou negativních vedlejších účinků, jako je nefrotoxicita (toxický efekt pro ledviny), neurotoxicita (porušení normální funkce nervového systému), ototoxicita (negativní vliv na fyziologii a funkci vnitřního ucha) nebo myelosuprese (potlačení krvetvorby v kostní dřeni). V současné literatuře se lze dokonce setkat s pochybnostmi, jestli by, navzdory své aktuální vůdčí pozici mezi protinádorovými chemoterapeutiky, byla cisplatina schválena ke komerčnímu využití, pokud by klinické testování a schvalovací proces probíhal o několik desetiletí později. Neméně závažnými 7

8 problémy spojenými s terapií cisplatinou a jinými léčivy na bázi platiny jsou nutnost intravenózní aplikace a také rezistence (rozlišujeme vrozenou a získanou, viz níže [3,29]). Zmíněné jevy, k nimž je třeba připočítat psychické problémy onkologických pacientů, které jsou jimi vyvolány, nutně vedly k derivatizaci cisplatiny a intenzivní syntéze a studiu biologických vlastností mnoha set nových komplexů platiny (následně také komplexů jiných, neplatinových přechodných kovů). Pouze zlomek z těchto sloučenin dosáhl fáze klinického testování a pouze méně než deset látek bylo do dnešní doby přijato jako protinádorové léčivo do klinické praxe (viz výše; obrázek 1) [3]. Cisplatina je účinná především proti rakovině vaječníků a varlat, úspěšně používána je také k léčbě nádorů hlavy, krku, močového měchýře, melanomu nebo osteosarkomu. Obecně platí, že stejné nádory, jaké jsou léčeny cisplatinou, lze léčit i užitím karboplatiny (schválená celosvětově) a nedaplatiny (schválená v Japonsku). Výhodou těchto derivátů cisplatiny, které opravňují jejich klinické využití, jsou výrazně nižší nefrotoxicita, ototoxicita a neurotoxicita ve srovnání s cisplatinou. Nicméně též platí, že tyto látky jsou méně protinádorově aktivní než cisplatina. Obdobný farmakologický profil zmíněných léčiv pak souvisí s tzv. křížovou rezistencí karboplatiny, resp. nedaplatiny s cisplatinou. Z medicinálního a také, v jistém smyslu, z molekulárně-farmakologického pohledu se od výše zmíněných léčiv liší oxaliplatina [23,24]. Tato látka byla vyvinuta již v roce uvedení cisplatiny do klinické praxe (1978), pro celosvětové klinické využití byla schválena až téměř po více než dvaceti letech (2002). Oxaliplatina je také látkou, která přinesla svěží vítr do v té době již trochu unavené problematiky léčiv na bázi platiny (toho času pouze cisplatiny a karboplatiny) - je to komplex s jiným typem nosného N-donorového ligandu (obrázek 1). Jednoznačnou výhodou oxaliplatiny oproti výše uvedeným látkám je použití (v kombinované terapii s 5-fluorouracilem a leukovirinem) vůči jiným typům nádorů, především pak vůči cisplatinou a karboplatinou neléčitelné rakovině tlustého střeva. Aplikace oxaliplatiny navíc není spojena s většinou známých negativních vedlejších účinků (myelosuprese, nefrotoxicita, ototoxicita), klinicky aplikovatelná dávka oxaliplatiny je omezena jí vyvolávanou neurotoxicitou. Na základě řečeného lze předpokládat, a ve skutečnosti to také platí, že oxaliplatina nevykazuje křížovou rezistenci s cisplatinou a je účinná vůči některým nádorům se získanou resistencí vůči cisplatině [30]. Obdobně se můžeme vyjádřit i k dalším léčivům na bázi platiny, které stejně jako oxaliplatina, obsahují N-donorové ligandy odlišné od NH 3 obsaženého v cisplatině, karboplatině a nedaplatině. Jsou jimi na lokálních asijských trzích používané lobaplatina (Čína; léčba chronické myelogenní leukémie, nemalobuněčného 8

9 karcinomu plic nebo neoperabilních karcinomů prsu) a heptaplatina (Jižní Korea; léčba rakoviny žaludku) (obrázek 1). Lze konstatovat, že hlavním cílem bioanorganických chemiků již není vývoj co nejaktivnějších nebo ve srovnání s cisplatinou méně toxických sloučenin, ale správné pochopení mechanismů rezistence nádorů vůči platinovým metaloterapeutikům a z toho vyplývající cílená syntéza látek, které překonávají schopnost nádorových buněk snížit transport léčiva z extracelulárního prostoru do prostoru intracelulárního, cytoplasmatickou detoxifikaci, schopnost buněk opravovat DNA adukty s léčivy na bázi platiny a toleranci nádorových buněk k poškození cílové molekuly jaderné DNA [7,31,32]. Příklady takových látek jsou známy jak z onkologické praxe (oxaliplatina, lobaplatina, heptaplatina), tak i mezi klinicky testovanými agens (satraplatina, bis-acetato-ammin-dichloro-cyklohexylaminplatičitý komplex; pikoplatina, cis-ammin-dichloro-2-methylpyridinplatnatý komplex [33,34]; obrázek β). Zajímavou možností je i zlepšení transportu (např. liposomální formulace známých léčiv na bázi platiny, příp. navýšení lipofility léčiva, z něhož plyne vyšší transport do buňky (lipoplatina, aroplatina [12,35])), nebo aktivní ovlivnění hladiny fyziologických látek zapojených do mechanismu rezistence nádorových buněk vůči účinkům léčiv na bázi platiny. Toto je z praxe známo např. pro glutathion (vytváří adukty s léčivy na bázi platiny v rámci intracelulární inaktivace a tím zvyšuje jejich transport z nádorové buňky) a L-buthioninsulfoximin (L-BSO; inhibitor enzymu -glutamylcystein syntetázy, který je zapojen do fyziologické syntézy glutathionu), kdy lze preaplikací nebo kombinovanou terapií účinně navýšit aktivitu léčiv i na původně necitlivých nádorových buňkách [36]. Obrázek 2. Strukturní vzorce klinicky testovaných satraplatiny a pikoplatiny Mechanismus účinku Cisplatina je chemicky jednoduchý platnatý komplex obsahující dva N-donorové NH 3 ligandy (tzv. carrier ligands), které zůstávají ve fyziologickém prostředí koordinovány na centrální atom, a dva chloridové ionty (tzv. leaving groups), které jsou ve fyziologickém prostředí nahrazeny ligandy jinými (viz níže) [1 3]. Z chemického úhlu pohledu je cisplatina 9

10 poměrně nereaktivní látka, která navíc, vzhledem k její nízké rozpustnosti ve vodě, musí být pacientům aplikována intravenózně. Základním dějem po aplikaci léčiva je jeho transport do cílové buňky, resp. buněčná akumulace (obrázek γ). Mechanismy transportu léčiv na bázi platiny sice stále nejsou zcela jednoznačně objasněny, je však známo, že je mnoho vlivů na transport do buňky (např. koncentrace Na + /K + iontů, ph), a především, že kromě pasivní difúze existuje minimálně jeden aktivní transport přes plazmatickou membránu z extracelulárního prostoru směrem do buňky, a to přes měďnaté transportéry CTR1 [37]. V buňce, kde je koncentrace chloridových iontů nižší (~10 mm) ve srovnání s extracelulárním prostředím (~100 mm), jsou chloridové ionty cisplatiny nahrazovány molekulami vody (obrázek γ) [38 40]. Z mechanistického hlediska chápeme tento proces jako nezbytnou aktivaci léčiva, jelikož jsou to právě kladně nabité mono- nebo diaqua komplexy (příp. produkty jejich protolytických reakcí), které se mohou vázat na cílovou molekulu jaderné DNA. Vysoce labilní Pt O vazba (platina voda) je v jádře nahrazena stabilnější vazbou Pt N (platina nukleobáze), kterou aktivované léčivo vytváří odštěpením zmíněných aqua ligandů a následnou koordinací na dusíkové atomy nukleobází, především pak guaninu, v menší míře adeninu (obrázek 3). Ve většině případů se jedná o bifunkcionální adukty na jednom vlákně DNA (tzv. intrastrand adukty), které podle toho, na jakou nukleobázi sahají, rozlišujeme na adukty mezi dvěma sousedními guaniny (1,2-d(GpG); 60 65% aduktů), mezi sousedním guaninem a adeninem (1,2-d(ApG); 20 β5%) nebo mezi dvěma guaniny, mezi nimiž je ještě jedna nukleobáze (1,3-d(GpXpG); 2%; X = adenin, cytosin nebo uracil). Dále byly detekovány monofunkcionální adukty na guanin (2%) a také adukty mezi dvěma guaniny obou vláken DNA (tzv. interstrand adukty; 2%). Takto vzniklé adukty tvořené cisplatinou a DNA mění sekundární strukturu DNA a vedou k řadě procesů, jako je rozpoznání a oprava poškození DNA, zastavení buněčného cyklu nebo apoptóza [41]. Obrázek 3. Schematické znázornění buněčné akumulace a následných vnitrobuněčných procesů cisplatiny (nahoře) a typy Pt DNA aduktů tvořených cisplatinou (dole). Převzato z [40]. 10

11 Z terapeutického pohledu toto způsobuje inhibici DNA a RNA syntézy, jinými slovy je potlačeno buněčné dělení, což vede nádorovou buňku k buněčné smrti, z makroskopického pohledu tedy k léčbě nádorového onemocnění. Také ostatní léčiva na bázi platiny musí nezbytně podstoupit aktivaci nahrazením leaving group (což jsou u všech klinických analogů cisplatiny bidentátně koordinované karboxylátové dianionty; obrázek 1). Ačkoli tento hydrolytický proces téměř neprobíhá ve vodném prostředí, v prostředí fyziologickém jsou zmíněné karboxylato ligandy nahrazeny, podobně jako u cisplatiny, dvěma molekulami vody, přičemž do tohoto děje aktivně vstupují HCO 3 a H 2 PO 4 ionty nebo jiné fyziologicky přítomné látky (např. guanosin-5'-monofosfát, GMP) [42 44]. Aktivované diaqua komplexy opět atakují molekulu jaderné DNA, jak je popsáno výše pro cisplatinu. Je určitě vhodné zde zmínit, že v případě karboplatiny a nedaplatiny jsou aktivované částice chemickými analogy aktivovaných částic cisplatiny o složení cis-[pt(nh 3 ) 2 (H 2 O) 2 ] 2+, což je vysvětlením obdobného terapeutického profilu a vzájemné křížové rezistence těchto tří metaloterapeutik. Naproti tomu aktivace oxaliplatiny, lobaplatiny a heptaplatiny poskytuje jiné typy částic, jelikož tato léčiva obsahují ve svých molekulách jiné typy N-donorových ligandů (obrázek 1). Toto způsobuje rozsáhlejší změny na cílovém místě v souvislosti s přítomností objemného N-donorového ligandu, jako jsou třeba sterické efekty ve velkém žlábku DNA, které mohou vést k účinnější inhibici DNA syntézy. Zmíněné bylo prokázáno v případě oxaliplatiny, jejíž schopnost tvořit kovalentní adukty s jadernou DNA je sice nižší ve srovnání s cisplatinou, i tak je ale oxaliplatina více cytotoxická než cisplatina, protože stejný počet DNA aduktů oxaliplatiny efektivněji inhibuje buněčné dělení ve srovnání s cisplatinou [45,46]. Asi nejzávažnějším problémem chemoterapie léčivy na bázi platiny, a to jak z pohledu klinické aplikace, tak i optikou bioanorganických chemiků zabývajících se vývojem nových látek tohoto typu, je rezistence. Jak již bylo uvedeno výše, rozlišujeme rezistenci vrozenou (chápejme ji tak, že některé typy nádorů, jako jsou např. karcinomy prostaty, tlustého střeva a konečníku nebo prsu, odolávají biologickému účinku cisplatiny a karboplatiny; jinak řečeno, těmito léčivy mohou být léčeny pouze některé typy nádorů) a získanou (některé typy původně léčitelných nádorů, známo je to např. pro nádory vaječníků léčené cisplatinou a karboplatinou, jsou vůči opakované chemoterapii těmito léčivy na bázi platiny necitlivé, jinak řečeno recidiva některých nádorů vyžaduje použití jiného, často méně účinného chemoterapeutika) [3,29]. V zásadě platí, že rezistentní nádorové buňky vykazují jiné mechanismy akumulace léčiva, rozpoznání a opravy vzniklých Pt DNA aduktů a apoptózy [47,48]. Jinak řečeno, za rezistencí stojí dva základní mechanismy - omezení množství léčiva 11

12 atakujícího cílovou molekulu DNA a omezení schopnosti vzniklých Pt DNA aduktů vyvolat buněčnou smrt. Bylo experimentálně prokázáno, že rezistentní buňky přijímají méně léčiva, než jejich analoga před vypěstováním rezistence [47]. Jak tomuto mechanismu rezistence předejít ukazuje výše zmíněná satraplatina. Tento platičitý komplex přechází ve fyziologickém prostředí ve vícero látek, z nichž jedna je cis-ammindichloro-cyklohexylaminplatnatý komplex, který, ačkoli je chemicky nepříliš odlišný od cisplatiny, výrazně lépe proniká do nádorových buněk [49]. Jiný přístup k navýšení transportu léčiv na bázi platiny jsou výše zmíněné liposomální formulace s konvenčními (lipoplatina [12]; obrázek 4) nebo novými lipofilními (ProLindac [50]) komplexy nebo funkcionalizované nanočástice [6 8,51 54]. Pro názornost uveďme, že enkapsulace léčiva (cisplatiny v případě lipoplatiny) do liposomové formulace vede k výrazně vyšší účinnosti cisplatiny, čehož přímým Obrázek 4. Schematické znázornění lipoplatiny, liposomální formulace nesoucí jako léčivo cisplatinu (vlevo) a porovnání její aktivity ( ) s cisplatinou ( ), směsí cisplatiny a liposomů ( ) a samotnými liposomy (čárkovaná čára). Převzato z regulon.com a z [55]. důsledkem je nižší toxicita vůči nenádorovým buňkám, a tedy i nižší negativní vedlejší účinky [55]. V případě výše zmíněného ProLindac, derivátu oxaliplatiny s odstupujícím ligandem na bázi hydroxypropylmethakrylamidu, je z ekvitoxické dávky do cílové nádorové buňky účinně dopraveno více než desetkrát vyšší množství platiny ve srovnání s oxaliplatinou [56]. Již výše v práci byl zmíněn glutathion (GSH) jako jedna ze síru obsahujících biomolekul (můžeme zmínit ještě metalothioneiny [57]), které jsou přítomné v intracelulárním prostoru, kde se díky vysoké afinitě síry k platině ochotně koordinují na zmíněný kov, a tím usnadňují jeho transport ven z buňky (eflux) skrze GS-X pumpy. Roli glutathionu při rezistenci nádorových buněk potvrzují i četné publikace, kde je popsána vyšší hladina GSH u rezistentních buněk ve srovnání s buňkami původními [36]. Tuto schopnost nádorových buněk chránit se cytotoxickému účinku léčiv na bázi platiny překonává, pravděpodobně díky sterickému bránění koordinace síru obsahujících biomolekul, pikoplatina (obrázek β). Tato látka odvozená od cisplatiny náhradou jedné NH 3 molekuly 2-methylpyridinem (2-pikolin; na tomto místě je vhodné upozornit na chemickou podobnost zmíněného β-methylpyridinu s níže diskutovaným 7-azaindolem a jeho deriváty, jejichž 12

13 koordinační sloučeniny byly hlavním předmětem studia uchazeče) vede ke schopnosti této látky působit na nádorové buňky necitlivé vůči účinku jak cisplatiny, tak i oxaliplatiny. Nádorová buňka má dále celou řadu možností, jak se vyrovnat s tím, že se léčivo na bázi platiny dostalo do buňky a v buňce do jádra, kde se kovalentně navázalo na molekulu DNA. Pro příklad možností oprav Pt DNA aduktů lze uvést MMR (mismatch repair), což je opravný systém, v rámci kterého jeho proteiny rozpoznávají poškození DNA a stimulují buňku k opravným procesům [58]. Narušením této dráhy lze naopak navyšovat citlivost léčených nádorů vůči účinku aplikovaných léčiv. Nelze v této souvislosti opomenout ani fakt, že i přes velmi dobře známé negativní vedlejší účinky léčiv na bázi platiny se je stále nedaří nahradit méně toxickými léčivy, ať už na bázi neplatinových přechodných kovů nebo látkami organickými. Jednou z alternativ, jak omezit toxicitu léčiv na bázi platiny, je kombinovaná terapie, jak ji známe u oxaliplatiny podávané od počátku jejího klinického využití s jiným protinádorovým chemoterapeutikem 5-fluoruracilem. Obdobně je v klinické praxi karboplatina podávaná při léčbě nemalobuněčného karcinomu plic s paklitaxelem a bevacizumabem [59]. Celá problematika rezistence nádorových buněk vůči cytotoxickému ataku chemoterapeutik na bázi komplexů platiny je sice stále více a více prozkoumanou a objasněnou oblastí, nicméně celková vzájemná provázanost jednotlivých faktorů (viz obrázek 5), které do ní přispívají, je i nadále z velké části zahalena rouškou tajemství, a to Obrázek 5. Schematické znázornění drah zapojených do zprostředkování biologického účinku vyvolaného léčivy na bázi platiny (vlevo) a mechanismy inhibující cytotoxický účinek (apoptózu) léčiv na bázi platiny v rezistentních liniích (vpravo). Převzato z [48]. 13

14 především z toho úhlu pohledu, který se týká cíleného designu a následného biologického účinku nových léčiv. Dosavadní poznatky poukazují na to, že cílení na jeden libovolný faktor buněčné rezistence není cestou, protože léčená buňka dokáže ztrátu/omezení jednoho mechanismu rezistence efektivně nahradit mechanismy jinými Chemoterapeutika jiných p echodných kov Mnoho koordinačních sloučenin přechodných (Ti, V, Cr, Mn, Fe, Co, Cu, Zn, Y, Zr, Mo, Tc, Ru, Pd, Ag, La, Ta, Os, Pt, Au) a vnitřně přechodných (Ce, Sm, Gd, Ho, Lu, Ac) prvků nachází v současné době praktické medicinální využití nebo k němu směřuje v různých fázích klinických experimentů [2,14,19]. Z uvedených souvisí s prací autora protinádorově aktivní komplexy platiny (viz výše) a vybraných neplatinových prvků VIII.B. skupiny periodického systému, dále pak protizánětlivé komplexy zlata v oxidačním stupni +I. Ačkoli klinický úspěch chemoterapeutik na bázi platiny je i z dnešního pohledu neoddiskutovatelný, již v době uvedení těchto léčiv na trh bylo patrné, že je nutné vyvinout takové látky, které rozšíří spektrum léčitelných tumorů, budou méně toxické (omezení negativních vedlejších účinků metaloterapeutik na bázi platiny používaných v onkologické praxi) a schopné překonávat rezistenci vůči klinicky používaným chemoterapeutikům. Tyto požadavky vedly bioanorganické chemiky nejen k syntéze nových, vhodně strukturně modifikovaných komplexů platiny (viz výše), ale také k vývoji a studiu biologických vlastností u koordinačních sloučenin jiných přechodných prvků. Mezi těmito se jako velmi vhodní kandidáti pro klinické využití jeví komplexy ruthenia [14,60 62]. Historii těchto látek počítáme již od počátku 80. let minulého století, kdy byla publikována vysoká aktivita jednoduchého Ru(III) komplexu, fac-[rucl 3 (NH 3 ) 3 ], vůči EMT-6 sarkomu u myší [60,63]. Fáze klinických testů dosáhly až rozpustnější iontové komplexy ruthenia KP1019/NKP-1339 nebo NAMI-A (obrázek 6) [64]. Z výhod těchto látek uveďme pro příklad schopnost KP1019/NKP-1339 snížit objem kolonorektálního karcinomu myší o 95 %, aniž by byla u testovaných zvířat pozorována jakákoli mortalita, a pouze zanedbatelný úbytek hmotnosti (6%), komplex NAMI-A je zajímavý svojí výraznou antimetastatickou aktivitou [65]. Nicméně je nutné zmínit, že mechanismus účinku těchto látek není stále zcela objasněný. Předpokládá se in vivo redukce z Ru(III) na Ru(II), ovlivnění redoxní rovnováhy léčených nádorových buněk a interakce komplexů s molekulou jaderné DNA [66]. Významnou skupinou látek, které navazují na výše uvedené Ru(III) komplexy, jsou organokovové koordinační sloučeniny s arenem navázaným na centrální atom [67,68], které se od výše uvedených komplexů mechanisticky liší a jejichž biologická aktivita (schopnost překonávat 14

15 rezistenci nádorů vůči cisplatině, selektivní účinek) je velice perspektivní pro následné klinické využití [14,69]. V posledních letech je stále větší pozornost věnována komplexům osmia, především pak organokovovým sloučeninám se složením odvozeným od výše uvedených komplexů ruthenia. Obecně lze konstatovat, že komplexy osmia jsou kineticky inertnější ve srovnání s analogickými komplexy ruthenia, mechanisticky jsou si však obě skupiny podobné [např. 69,70]. Zajímavý je jistě i fakt, že izostrukturní komplexy osmia v mnoha případech převyšují analogické komplexy ruthenia, jak lze demonstrovat na komplexech [Os(µ 6 -p-cym)(impy)cl]pf 6, [Os(µ 6 -p-cym)(impy)i]pf 6, Obrázek 6. Strukturní vzorec komplexních aniontů sloučenin NAMI-A (vlevo) a KP1019/NKP-1339 (vpravo) [Ru(µ 6 -p-cym)(impy)cl]pf 6 a [Os(µ 6 -p-cym)(impy)i]pf 6, jejichž in vitro cytotoxicita (IC 50, tj. letální koncentrace pro 50% testovaných buněk) je rovná γ,0, 1,β, 16,β a γ,0 µm [69]; p-cym = p-cymen, Impy = N1,N1-dimethyl-N4-[(2-pyrid yl)methylen]anilin. Zmiňme zde také podobné organokovové komplexy iridia, které jsou v posledních letech v literatuře zmiňovány jako látky výrazně protinádorově aktivní a vysoce klinicky perspektivní [např. 18,71]. Také tyto komplexy mají, obdobně jako předešlé komplexy ruthenia a osmia, na centrální atom koordinovaný aromatický kruh, kterým je v tomto případě různě derivatizovaný cyklopentadien (např. 1,β,γ,4,5-pentamethylcyklopentadien nebo 1,2,3,4-tetramethyl-5-bifenyl-cyklopentadien (Cp biph )). Také tyto komplexy svojí cytotoxicitou překonávají klinicky používanou cisplatinu, současně je jejich účinek selektivní vůči nádorovým buňkám a jsou schopné působit na nádorové buňky rezistentní vůči cisplatině. Příkladem uveďme alespoň komplexy [(η 5 -Cp biph )Ir(phen)Cl]PF 6 a [(η 5 -Cp biph )Ir(bpy)Cl]PF 6 a jejich vysokou in vitro cytotoxicitu (IC 50 = 0,72, resp. 0,57 µm); phen = 1,10-fenanthrolin; bpy = 2,2`-bipyridin [72]. Historii protizánětlivě aktivních koordinačních sloučenin zlata počítáme od β0. let 20. století, kdy byla do léčby revmatoidní artritidy, jednoho ze zánětlivých onemocnění, přijata zlatná thiolátová léčiva. Příkladem takových léčiv je i dodnes používaný orálně podávaný auranofin (triethylfosfin-(2,3,4,6-tetra-o-acetyl-1-thio-β-d-glukopyranosato- S)zlatný komplex (obrázek 7), který je klinicky používaný od roku 1λ85 [73]. Mezi další klinicky používané protizánětlivé Au(I) komplexy patří vedle zmíněného auranofinu ještě myocrisin (aurothiomalát sodný), sanochrysin (aurothiosulfát sodný), solganol (aurothioglukosa) nebo allochrysin (aurothiopropanolsulfonát sodný) [2]. Auranofin je 15

16 lipofilnější než ostatní zmíněné komplexy. Je podáván orálně a v menší míře vyvolává negativní vedlejší účinky spojené s aplikací Au(I) komplexů do organismu, především pak poškození ledvin. Samotný mechanismus účinku auranofinu není zcela objasněn, je ale prokázáno, že ještě před vstupem do buňky dochází k odštěpení S-donorového ligandu, resp. k jeho nahrazení jiným síru obsahujícím ligandem Obrázek 7. Strukturní vzorec auranofinu (např. glutathionem) [74]. Postupně je ve vnitrobuněčném prostoru nahrazen i druhý původní ligand auranofinu (triethylfosfan) a vzniká konjugát Au(I) komplexu a proteinu (albuminu). Takto se dostává do cílového místa, kde je pravděpodobně transformován na [Au(CN) 2 ], který působí proti zánětu [75]. 16

17 Výsledková část habilitační práce 3. Platnaté komplexy s r znými N-donorovými ligandy Vývoj a studium protinádorových vlastností nových komplexů platiny byl v prvních dekádách po objevu cisplatiny orientován do dvou základních směrů - oba berou jako základní molekulu právě zmíněnou cisplatinu, jež je derivatizována buď záměnou odstupujících chloridových iontů nebo N-donorových ligandů výchozí cisplatiny. Z výše uvedeného textu je patrné, že oba přístupy byly úspěšné z pohledu uvedení léčiv do klinické praxe. Zopakujme zde ještě jednou, že to bylo právě zavedení jiných N-donorových ligandů do struktury protinádorově aktivních komplexů platiny, které vlilo novou energii do vývoje metaloterapeutik na bázi platiny, a to tím, že bylo prokázáno (oxaliplatina) a následně potvrzeno (lobaplatina, heptaplatina, nebo klinicky studovaná pikoplatina), že právě typ N-donorového ligandu má vliv na typ léčitelných a léčených nádorů. Současný přístup hledání nových potenciálních léčiv na bázi platiny lze pak shrnout tak, že je snahou výzkumných skupin připravit komplexy platiny s novými nosnými N-donorovými ligandy (např. cis-dichloroplatnaté komplexy, představující přímé deriváty cisplatiny), které prokáží dostatečný, pokud možno vyšší ve srovnání s cisplatinou, biologický účinek, který pak je následně modifikován, ať už derivatizací samotného N-donorového ligandu nebo zavedením jiných odstupujících skupin místo zmíněných chloridových iontů. Neopomenutelným přístupem pak je také studium biologických vlastností nově vyvinutých komplexů platiny v různých nadmolekulárních formulacích (liposomy, funkcionalizované nanočástice atd.). Heterocyklické sloučeniny, které byly použity jako N-donorové ligandy různých typů platnatých komplexů, jejichž studiem se uchazeč zabýval nebo se na něm podílel, jsou v zásadě dvojího typu - jedná se o deriváty 7-azaindolu a N6-benzyladeninu. Obě uvedené skupiny organických látek vykazují určité společné vlastnosti. Jsou odvozené od biomolekul (indolu a adeninu), jsou to polydentátní ligandy koordinačních sloučenin a, v neposlední řadě, mnohé organické látky obsahující uvedené základní motivy vykazují výrazné biologické účinky včetně účinků protinádorových Platnaté komplexy s deriváty 7-azaindolu Přehled o rozsahu chemie koordinačních sloučenin platiny se 7-azaindolem (obrázek 8) a jeho deriváty si můžeme udělat z relevantních chemických databází. Krystalografická 17

18 strukturní databáze (CSD, [76]) čítá ke dni celkem 53 molekulových struktur, ve kterých je na centrální atom platiny koordinován přes atom/atomy dusíku 7-azaindol [např. 77] nebo jeho derivát [např ]. Pokud by poznatky z krystalografické databáze měly být zobecněny, jedná se ve většině případů o jednojaderné organokovové komplexy s bidentátně koordinovaným N-donorovým ligandem tvořeným dvěma 7-azaindolovými motivy spojenými přes oba N1 atomy organickým linkerem (ten se v některých případech sám koordinuje přes atom uhlíku) [79,80]. Z těch několika málo odlišných struktur jmenujme alespoň látky popsané v níže uvedených pracích, na kterých se uchazeč podílel (obsahují monodentátní ligandy na bázi 7-azaindolu), nebo zajímavé struktury komplexů o složení [NBu 4 ][Pt(C 6 F 5 ) 2 (aza)(aza )] (obsahuje jednu molekulu 7-azaindolu koordinovanou typicky přes atom N7 a jeden deprotonizovaný 7-azaindol koordinovaný přes atom N1) a [NBu 4 ] 2 [{Pt(C 6 F 5 ) 2 } 2 (µ-oh)(µ-aza )] (s deprotonizovaným 7-azaindolem koordinovaným na dvě kovová centra přes atomy N1 a N7) [77], příp. komplexy se zajímavými bidentátními deriváty 7-azaindolu (např. 1-(2- pyridyl)-7-azaindolem), které se na centrální atom koordinují přes N7 atom 7-azaindolového kruhu a pyridylový dusík, což dohromady dává stabilní, šestičetný, centrální atom obsahující cyklus [78]. V databázi SciFinder bylo nalezeno celkem 1γ6 látek obsahujících 7-azaindolový motiv a platinu, 111 z nich představuje koordinační sloučeniny s Pt N vazbou mezi centrálním atomem a některým z dusíků 7-azaindolového cyklu. U většiny z těchto látek nacházíme atom platiny koordinovaný na dusíkový atom N7, pět látek obsahuje můstkující ligand na bázi deprotonizovaného 7-azaindolu [77,81,82], ve struktuře tří komplexů se 7-azaindolový kruh koordinuje na centrální atom výhradně přes N1 dusík pyrolového kruhu [77]. Obrázek 8. Strukturní vzorec 7-azaindolu zobrazený se schématem číslování jeho uhlíkových a dusíkových atomů. Následující text bude zaměřen na diskuzi výsledků dosažených v rámci studia platnatých komplexů s N-donorovými deriváty na bázi 7-azaindolu publikovaných v pracích, na kterých se uchazeč experimentálně a autorsky podílel. Je vhodné už na tomto místě zmínit fakt, že vysoká medicinální perspektiva níže diskutovaných cis-dichloroplatnaých a cis-dijodoplatnaých komplexů s deriváty 7-azaindolu byla zohledněna podáním jedné mezinárodní (EP ) a tří národních přihlášek vynálezu, na jejichž základě byly prozatím uděleny národní patenty CZ (Dichlorido komplexy platiny s halogenderiváty 7-azaindolu, způsob jejich přípravy a použití těchto komplexů jako léčiv v protinádorové 18

19 terapii. Původciμ Z. Trávníček, P. Štarha, I. Popa. Majitelμ Univerzita Palackého v Olomouci. Datum uděleníμ ) a CZ (Použití dichlorido komplexů platiny s halogenderiváty 7-azaindolu pro přípravu léčiv pro léčbu nádorových onemocnění. Původciμ Z. Trávníček, P. Štarha, Z. Dvořák. Majitelμ Univerzita Palackého v Olomouci. Datum uděleníμ ) Cis-dichloroplatnaté komplexy s deriváty 7-azaindolu Syntéza platnatých dichloro komplexů s deriváty 7-azaindolu je zajímavá pro svoji jednoduchost, rychlost a možnost poskytnout vysoce biologicky aktivní látky jednokrokovou syntézou, což neplatí pro cisplatinu, od které lze zmíněné komplexy formálně odvozovat. Pokud se ve stručnosti podíváme na průmyslovou syntézu cisplatiny, tak jejím základním krokem je výroba tetrajodoplatnatanu z výchozího tetrachloroplatnatanu, následuje interakce s amoniakem resp. vhodnou amonnou solí za vzniku cis-diammin-dijodoplatnatého komplexu, který podléhá další reakci se stříbrnou solí (např. AgNO 3 ), čímž vzniká komplexní cis-diammin-diaquaplatnatý kationt, který je následně převeden na cisplatinu reakcí s nadbytkem alkalického chloridu. Tuto obecně platnou (např. i pro karboxylato komplexy, viz níže pro oxaliplatinu) a široce používanou metodu přípravy různých typů platnatých komplexů známe z literatury jako tzv. Dharovu metodu [83]. V práci Štarha et al., Polyhedron, 2012 (příloha 1) je popsána syntéza dichloroplatnatého komplexu se 7-azaindolem, která pak byla základem pro syntézu série analogů s četnými halogenderiváty 7-azaindolu (Štarha et al., J. Inorg. Biochem., 2012 (příloha β), Muchová et al., J. Biol. Inorg. Chem., 2013 (příloha γ), Štarha et al., PLoS ONE, 2014 (příloha 4)). Nespornou výhodou pro nově vznikající sérii dichloro komplexů bylo to, že k syntéze cis-dichloroplatnatého komplexu se 7-azaindolem v chemické a izomerické čistotě odpovídající minimálním požadavkům na biologické testování ( λ5%) nebylo potřeba sáhnout k výše rozepsané Dharově metodě - tyto látky byly připraveny přímou jednokrokovou syntézou z K 2 [PtCl 4 ] (viz obrázek 9). Výhody jsou nasnadě - méně reakčních kroků zkracuje reakční čas a zvyšuje výtěžek, navíc tím, že odpadá nutnost použití Ag(I) soli, je současně Obrázek 9. Signály N1 H vodíkových atomů cis- a trans-izomeru komplexu [PtCl 2 (3Claza) 2 ] prokazující λ5% izomerickou čistotu studovaného cis-izomeru. 19

20 vyřešen problém kontaminace produktu stříbrem, jak je známo z průmyslové výroby cisplatiny In vitro protinádorová aktivita a molekulární farmakologie Zmíněný komplex cis-[ptcl 2 (aza) 2 ], popsaný v práci Štarha et al., Polyhedron, 2012 (příloha 1), nevykazoval žádnou in vitro cytotoxickou aktivitu (IC 50 > 1,0 µm) na lidském prsním karcinomu MCF7 a osteosarkomu HOS, toto lze ovšem připočíst omezené rozpustnosti studovaného komplexu v médiu biologického experimentu. Koncept studia platnatých komplexů s N-donorovými ligandy na bázi 7-azaindolu nicméně od počátku nestál pouze na samotném 7-azaindolu, ale především na jeho derivátech. Z více důvodů (komerční dostupnost, různé typy substituentů, různé substituované polohy, možnost dalších chemických derivatizací) se jako velmi vhodné jevily halogenderiváty 7-azaindolu. Celkem bylo pro syntézu cis-dichloroplatnatých komplexů použito osm takových derivátů, konkrétně se jedná o monosubstituované deriváty γ-chlor-7-azaindol (3Claza), 3-brom-7-azaindol (3Braza), 3-jod-7-azaindol (3Iaza), 4-chlor-7-azaindol (4Claza), 4-brom-7- azaindol (4Braza) a 5-brom-7-azaindol (5Braza), a disubstituované deriváty γ-chlor-5-brom- 7-azaindol (3Cl5Braza) a 3-jod-5-brom-7-azaindol (3I5Braza). Detailně prostudované, tzn. in vitro cytotoxicita vůči panelu lidských nádorových buněčných linií, studium molekulárně-farmakologických vlastností a dokonce také in vivo studium protinádorových vlastností na myším modelu leukémie, jsou prozatím komplexy s 3Claza, 3Iaza a 5Braza, což je detailně popsáno a diskutováno v pracích Štarha et al., J. Inorg. Biochem., 2012 (příloha 2), Muchová et al., J. Biol. Inorg. Chem., 2013 (příloha γ) a Štarha et al., PLoS ONE, 2014 (příloha 4). U komplexů s ostatními jmenovanými ligandy je známa jejich in vitro cytotoxicita na sérii vybraných lidských nádorových buněčných linií (viz níže). V práci Štarha et al., J. Inorg. Biochem., 2012 (příloha β) je popsána jednokroková syntéza tří cis-dichloroplatnatých komplexů s 3Claza, 3Iaza nebo 5Braza, jejíž výchozí platnatou solí je výše zmíněný tetrachloroplatnatan draselný. Produkty byly detailně fyzikálně-chemicky charakterizované např. multinukleární NMR spektroskopií, hmotnostní spektrometrií a, v případě komplexu cis-[ptcl 2 (3Claza) 2 ], také monokrystalovou rentgenovou strukturní analýzou (viz příloha β, obr. 2 na str. 60). Komplexy byly testovány in vitro vůči třem vybraným lidským nádorovým buněčným liniím, konkrétně vůči MCF7, HOS a karcinomu prostaty LNCaP. Získané výsledky ukázaly, že nové látky jsou výrazně (ve většině případů statisticky významně na p < 0,05 hladině významnosti) aktivnější ve srovnání s cisplatinou (viz příloha β, tabulka 2 a obr. 3 na str. 61). Nejaktivnějším byl 20

21 komplex s 5Braza, který byl na zmíněných lidských nádorových buněčných liniích přibližně λ,8, 1γ,7 resp. β,5 aktivnější ve srovnání s cisplatinou, což jsou rozhodně výsledky velice perspektivní a předurčující danou látku pro studium dalších aspektů protinádorové aktivity. Již v práci Štarha et al., J. Inorg. Biochem., 2012 (příloha β) byla studována schopnost výše uvedených komplexů a pro srovnání také cisplatiny vytvářet adukty s fragmentem DNA a schopnost těchto vzniklých aduktů inhibovat RNA syntézu T7 RNA polymerázou. Na základě získaných výsledků (viz příloha β, obr. 4 na str. 61) lze konstatovat, že studované cis-dichloroplatnaté komplexy se váží na DNA podobným způsobem jako cisplatina a efektivně inhibují syntézu RNA výše zmíněnou RNA polymerázou, což je mechanismus známý z literatury jako jeden z těch, které přivádí buňku k její smrti, příp. apoptóze [84]. Hlubší molekulárně farmakologické poznatky o cis-dichloroplatnatých komplexech s deriváty 7-azaindolu (byly publikovány pouze výsledky s komplexy obsahující 3Claza a 3Iaza, výsledky získané na komplexu s 5Braza byly v některých experimentech ovlivněny jeho nižší rozpustností ve srovnání se zbylými dvěma analogy), resp. o faktorech souvisejících s jejich výraznou in vitro cytotoxicitou, jsou rozepsány a diskutovány v práci Muchová et al., J. Biol. Inorg. Chem., 2013 (příloha γ). Studie probíhaly na lidské nádorové linii karcinomu vaječníku A2780, která v oblasti protinádorově aktivních komplexů platiny platí za modelovou linii standardně používanou nejen pro stanovení cytotoxicity, ale i jako model pro studium resistence (u linie Aβ780 lze opakovaným přidáváním daného léčiva (např. cisplatiny) vypěstovat rezistenci) či mnohých mechanistických dějů. Studium prokázalo, že nahrazení obou NH 3 molekul ve struktuře cisplatiny halogenderiváty 7-azaindolu výrazně ovlivnilo základní mechanistický krok, kterým je akumulace působícího terapeutika cílovou buňkou. Konkrétně bylo prokázáno, že obsah platiny v Aβ780 buňkách atakovaných komplexy se 7-azaindoly je po 24 h přibližně 45 vyšší, než je tomu u buněk, které interagovaly s cisplatinou. S tímto úzce souvisí i platinace DNA, vypovídající jednak o množství platiny uvnitř nádorové buňky, resp. jejího jádra, druhak o schopnosti léčiva interagovat s cílovou molekulou jaderné DNA. I zde byl po β4 h odhalen nezanedbatelný, zhruba čtrnáctinásobný rozdíl mezi novými komplexy a cisplatinou (viz příloha γ, tabulka 1 na str. 585). Protože nelze pro všechny komplexy platiny automaticky předpokládat molekulu jaderné DNA jako jediný možný cíl, je vhodné přesvědčit se o jejich schopnosti vázat se na DNA experimentálně. V diskutované práci (Muchová et al., J. Biol. Inorg. Chem., 2013) je tedy, kromě výše uvedeného pokusu popisujícího míru platinace jaderné DNA, použit ještě model interakce studovaných látek s DNA v bezbuněčném prostředí. Z výsledků experimentů plyne, že se studované komplexy efektivně váží na molekulu DNA a že ca λ0 % molekul 21

22 komplexů se 7-azaindoly je po β4 h vázáno na DNA bifunkcionálně. Bylo kvantifikováno množství interstrand aduktů (viz příloha γ, obr. 4 na str. 585), které pro nové komplexy činilo ca 5%, pro cisplatinu to bylo procent šest. V souladu s předpoklady pak zbytek připadá na intrastrand adukty. Je známo, že cisplatina (stejně jako mnohé další komplexy platiny) vyvolává apoptózu a v menší míře také nekrózu a že tyto látky zastavují buněčný cyklus v S a/nebo Gβ fázi. Pro komplexy se 7-azaindoly bylo zjištěno, že ve studovaných ovariálních buňkách ve srovnání s cisplatinou výrazně více indukují apoptózu, která navíc u komplexů se 7-azaindoly významně převyšuje nekrózu (viz příloha γ, obr. β na str. 58γ). Analýza průtokovou cytometrií prokázala, že se velká část buněk atakovaných komplexy se 7-azaindoly, na rozdíl od cisplatiny, nachází v sub-g1 fázi buněčného cyklu, zato poměr v S a Gβ fázích je opačný, tedy v těchto fázích buněčného cyklu nacházíme především buňky, k nimž byla jako testovaná látka přidána cisplatina (viz příloha γ, obr. γ na str. 58γ). Zajímavý je také nárůst procentového zastoupení buněk léčených komplexy se 7-azaindoly v subg1 fázi při zvýšení koncentrace studovaných látek z 3,0 na 5,0 µm, a to na úkor buněk v G0/G1 fázi. Z uvedeného je patrný odlišný terapeutický mechanismus účinku nových komplexů s halogenderiváty 7-azaindolu od cisplatiny. I po zmíněném navýšení koncentrace testovaných látek se 7-azaindoly zůstává poměr buněk v G2 fázi nižší ve srovnání s cisplatinou léčenými buňkami a dokonce i s kontrolní neléčenou skupinou buněk. Toto je zajímavé s ohledem na výsledný cytotoxický účinek studovaných látek, který je výrazně vyšší ve srovnání s cisplatinou. Lze z toho usuzovat, že změny vyvolané vytvořením Pt DNA aduktů s komplexy se 7-azaidoly mohou být pro buňku daleko závažnější, hůře opravitelné a efektivněji vedoucí buňku k její smrti (apoptóze). I z tohoto důvodu byly na studovaných látkách, a pro srovnání i na cisplatině, provedeny experimenty na uvedenými komplexy platiny modifikovaném puc1λ plasmidu, které poskytly informace o efektivitě oprav poškození DNA studovanými komplexy platiny. Jak je patrné z dosažených výsledků (viz příloha γ, obr. 6 na str. 587), množství produktů oprav poškozené DNA, přeneseně tedy schopnost buňky působit mechanismy nukleotidové excisní reparace (NER) vůči terapeutikem vyvolanému poškození DNA, je pro nové platnaté komplexy s halogenderiváty 7-azindolu nižší ve srovnání s cisplatinou. Uvedené lze chápat jako naprosto zásadní poznatek, který je jednou z hlavních příčin vyššího cytotoxického účinku komplexů se 7-azaindoly ve srovnání s konvenční cisplatinou a může souviset i se schopností testovaných látek působit na nádorové buňky rezistentní vůči cisplatině. Výše je uvedeno, že buňky ovariálního karcinomu jsou vhodným a často používaným modelem pro studium schopnosti testovaných látek překonávat získanou rezistenci tohoto typu nádoru vůči 22

23 farmakologickému účinku cisplatiny. Výsledkem tohoto experimentu je porovnání hodnot IC 50 získaných na cisplatina-senzitivní (A2780) a cisplatina-rezistentní (Aβ780R) buněčné linii lidského karcinomu vaječníku (obrázek 10). Poměr IC 50 (A2780R) ku IC 50 (Aβ780), známý z literatury jako tzv. rezistenční faktor (RF; též, faktor rezistence nebo rezistenční index) je pak pro testované komplexy po 72 h roven 0,38 pro cis-[ptcl 2 (3Claza) 2 ] a 0,44 pro cis-[ptcl 2 (3Iaza) 2 ], což jsou hodnoty zásadně Obrázek 10. Graf zobrazující výsledky in vitro cytotoxicity cis-[ptcl 2 (3Claza) 2 ], cis-[ptcl 2 (3Iaza) 2 ] a cisplatiny vůči cisplatina-senzitivní (Aβ780) a cisplatina-rezistentní (Aβ780R) buněčné linii lidského karcinomu vaječníku. odlišné od cisplatiny s RF = 3,9. Z celkového pohledu tedy lze konstatovat, že studované cis-dichloroplatnaté komplexy s halogenderiváty 7-azaindolu vykazují výrazné farmakologické výhody ve srovnání s konvenčním protinádorovým chemoterapeutikem cisplatinou, které jsou důležitým faktorem, na jehož základě mohly být tyto látky přijaty do návazných biologických studií zahrnujících také in vivo experimenty na myších. V přímé návaznosti na výše diskutované práce Štarha et al., J. Inorg. Biochem., 2012 (příloha β) a Muchová et al., J. Biol. Inorg. Chem., 2013 (příloha γ) byla v práci Štarha et al., PLoS ONE, 2014 (příloha 4) u výše zmíněných cis-dichloroplatnatých komplexů s halogenderiváty 7-azaindolu 3Claza, 3Iaza a 5Braza popsána jejich in vitro cytotoxicita na širokém panelu lidských nádorových buněčných linií a především in vivo protinádorová aktivita na myších (viz níže). In vitro cytotoxicita byla studována na lidských nádorových buněčných liniích A2780, A2780R, karcinomu plic A549, karcinomu děložního čípku HeLa a maligního melanomu G-γ61 (viz příloha 4, tabulka 1 a obr. 4 na str. 7), čímž doplnily MCF7, HOS a LNCaP (Štarha et al., J. Inorg. Biochem., 2012, příloha β) na celkový počet osmi nádorových linií. Stejně jako v práci Štarha et al., J. Inorg. Biochem., 2012 (příloha β) byl i v této práci tím nejaktivnějším komplexem ten, který obsahuje 5-brom-7-azaindol. Tato látka byla na uvedených liniích použitých v diskutované práci Štarha et al., PLoS ONE, 2014 (příloha 4) přibližně 5.γ (A54λ), β.γ (HeLa), 5.7 (G-γ61), 6.7 (Aβ780) a 1β.λ (Aβ780R) cytotoxičtější (statisticky významně na hladině významnosti p < 0,05), než použitý standard cisplatina. Také v této práci byl z poměru aktivit na Aβ780R a Aβ780 vypočítán rezistenční faktor, který je pro studované komplexy přibližně β nižší ve srovnání s cisplatinou, což lze v rámci diskutované práce dát do přímé souvislosti s neochotou nových 23

24 platnatých komplexů interagovat se síru obsahujícími biomolekulami, pro které je z literatury znám jejich přímý vliv na rezistenci nádorových buněk vůči cisplatině a jiným protinádorově aktivním platnatým komplexům. Za zmínku jistě stojí i další cis-dichloroplatnaté komplexy s odlišně substituovanými halogenderiváty 7-azaindolu, které byly v návaznosti na výše uvedené práce připraveny, charakterizovány a testovány na jejich in vitro cytotoxickou aktivitu vůči panelu lidských nádorových buněčných linií (manuskript zaslaný do MedChemComm). V konkrétním přiblížení se jedná o komplexy s 4Claza, 3Braza, 4Braza, 3Cl5Braza a 3I5Braza. Složení bylo prokázáno kombinací hmotnostní spektrometrie (byly detekovány molekulové píky studovaných látek; obrázek 11) a multinukleární NMR spektroskopie, která jednoznačně potvrdila přítomnost organických ligandů ve struktuře studovaných koordinačních sloučenin (v 1 H, 13 C a 15 N NMR spektrech byly detekovány všechny signály příslušných derivátů 7-azaindolu) a jejich koordinaci na centrální atom přes atom dusíku N7, což lze konstatovat na základě porovnání koordinačních posunů (Δδ, ppm) vypočtených jako rozdíl chemických posunů příslušných signálů ve spektrech komplexu a volného ligandu (Δδ = δ komplex δ ligand ). Pro atom N1 je Δδ = 2.5 γ.6 ppm, zatímco pro N7 je hodnota koordinačního posunu ( 101.0) ppm. In vitro cytotoxicita byla studována vůči celkem osmi lidským nádorovým buněčným liniím (jmenovitě Aβ780, Aβ780R, HOS, MCF7, Gγ61, A549, HeLa a LNCaP) a linii zdravých lidských fibroblastů MRC5 (tabulka 1). Z výsledků testování in vitro cytotoxicity shrnutých v tabulce 1 je patrné, že všechny komplexy s monosubstituovanými deriváty 7-azaindolu (A C) byly statisticky významně (p < 0,05) aktivnější než cisplatina vůči HOS, MCF7, Aβ780, Aβ780R, A54λ a HeLa. Poněkud odlišně se chovaly komplexy D a E s disubstituovanými deriváty 7-azaindolu, jejichž aktivita se vzájemně řádově liší a vyšší in vitro cytotoxicitu tak ve srovnání s cisplatinou vykazuje pouze komplex D, a to na liniích HOS, Aβ780 a Aβ780R. Uvedené statisticky významné rozdíly ve srovnání s konvenčním chemoterapeutikem Obrázek 11. ESI hmotnostní spektrum studovaného komplexu cis-[ptcl 2 (3Braza) 2 ] s označeným (*) píkem {[PtCl 2 (3Braza) 2 ] H} a s vloženým porovnáním jeho experimentálně zjištěného a teoretického izotopového rozložení 24

25 cisplatinou poukazují na schopnost studovaných komplexů překonávat vrozenou rezistenci některých typů nádorů vůči biologickému účinku zmíněného léčiva. Aplikovaný model testování schopnosti studovaných látek překonávat získanou rezistenci buněk karcinomu vaječníku prokázal, že všechny nové komplexy se 7-azaindoly tuto schopnost mají (viz hodnoty RF v tabulce 1). Studium vlivu testovaných látek na zdravé lidské fibroblasty prokázalo, že s výjimkou komplexu D je vliv všech nových platnatých komplexů s deriváty 7-azaindolu na nenádorové buňky výrazný a ve srovnání s cisplatinou vyšší (viz hodnoty SF v tabulce 1). Jedním dechem je však nutné dodat, že toto nemusí korelovat s celkovým vlivem testovaných látek na organismus a vyvolávání negativních vedlejších účinků, jak je popsáno v práci Štarha et al., PLoS ONE, 2014 (příloha 4). Co se týká porovnání in vitro cytotoxicity těchto pěti cis-dichloroplatnatých komplexů s výše uvedenými komplexy s jinými deriváty 7-azaindolu (3Claza, 3Iaza a 5Braza), lze konstatovat, že s výjimkou HeLa buněčné linie je nejaktivnější látkou cis-[ptcl 2 (5Braza) 2 ]. Tabulka 1. Výsledky testování in vitro cytotoxicity cis-dichloroplatnatých komplexů s 4-chlor-7-azaindolem (4Claza; komplex A), 3-brom-7-azaindolem (3Braza; B), 4-brom-7-azaindolem (4Braza; C), 3-chlor-5-brom-7- azaindolem (3Cl5Braza; D) a 3-jod-5-brom-7-azaindolem (3I5Braza; E) a cisplatiny vůči lidským nádorovým buněčným liniím prsního karcinomu MCF7, osteosarkomu HOS, cisplatina-senzitivního karcinomu vaječníku A2780, cisplatina-rezistentního karcinomu vaječníku Aβ780R, karcinomu plic A54λ, karcinomu děložního čípku HeLa, maligního melanomu Gγ61 a karcinomu prostaty LNCaP, a buněčné linii lidského zdravého fibroblastu MRC5. Buňky interagovaly s testovanými látkami β4 h. Data jsou uvedena jako IC 50 ± SD ( M). Linie / faktor A B C D E Cisplatina MCF7 γ.5±1.0* 5.γ±0.8* β.7±1.β* >10.0 a >50.0 a 18.1±5.1 HOS 7.5±β.4* 5.γ±β.1* 4.5±β.7* 4.γ±0.5* 46.7±4.0 β5.4±8.5 A2780 γ.8±0.β* 4.1±0.4* γ.8±0.5* 4.8±1.0* γγ.4±γ.γ β1.8±γ.λ A2780R γ.6±0.7* γ.6±0.7* γ.5±1.1* γ.8±1.0* β4.7±1.0 γβ.0±λ.6 A ±4.β* 1λ.0±5.6* 11.1±0.γ* >10.0 a >50.0 a >50.0 a HeLa 11.λ±1.β* 17.1±0.8* λ.β±β.0* >10.0 a >50.0 a γλ.λ±4.6 G361 γ.β±0.5 β.λ±0.4 β.7±0.4 γ.0±0.5 >50.0 a 5.8±β.4 LNCaP γ.λ±0.1 4.λ± ±0.6 >10.0 a >50.0 a γ.8±1.5 MRC5 γ.5±0.γ 4.0±0.γ γ.6±0.γ 17.0±4.8 >20.0 a >50.0 a RF b SF c >0.60 >2.29 * značí statisticky významný rozdíl hodnot od standardu cisplatiny na p < 0,05 hladině významnosti (ANOVA) a ) IC 50 nebylo dosaženo do koncentrace dané rozpustností příslušného komplexu v testovacím médiu b ) RF = rezistenční faktor vypočítaný jako IC 50 (A2780R)/IC 50 (A2780) c ) SF = faktor selektivity vypočítaný jako IC 50 (MRC5)/IC 50 (A2780) 25

26 Obrázek 12. ESI hmotnostní spektra směsi studovaného komplexu cis-[ptcl 2 (3Braza) 2 ] s glutathionem zaznamenaná ihned (0 h), 1 nebo β4 h po smíchání ve směsi voda/methanol (1:1, v/v) s šedým polem vyznačujícím oblast, ve které byl po β4 h detekován adukt o složení {[PtCl(3Braza) 2 (GSH)] 2H} a s vloženým porovnáním jeho experimentálně zjištěného a teoretického izotopového rozložení Obrázek H NMR spektra komplexu cis-[ptcl 2 (4Braza) 2 ] v DMF-d 7 a ve směsi DMF-d 7 /H 2 O (4:1, v/v) bez nebo s glutathionem (GSH) měřená po dobu dvou dnů v intervalu 24 h. = N1 H, cis-[ptcl 2 (4Braza) 2 ]; * = N1 H, trans-[ptcl 2 (4Braza) 2 ]; = N1 H, hydrolytický produkt; = N1 H, cis-[ptcl(4braza) 2 (GSH)]; ^ = C6 H, cis-[ptcl 2 (4Braza) 2 ]; # = C6 H, trans-[ptcl 2 (4Braza) 2 ]; = N H glycinu a cysteinu obsažených v GSH Stejně jako v případě předchozí série cis-dichloroplatnatých komplexů s deriváty 7-azaindolu byla i v případě těchto látek, konkrétně u reprezentativního komplexu cis-[ptcl 2 (3Braza) 2 ], studována optikou ESI hmotnostní spektrometrie schopnost těchto látek interagovat se, z mechanistického pohledu aktivity protinádorově aktivních komplexu platiny, stěžejní síru obsahující biomolekulou glutathionem (GSH). Na rozdíl od experimentů popsaných v práci Štarha et al., PLoS ONE, 2014 (příloha 4) byly v tomto případě interakční experimenty provedeny na delší časové škále (β4 h). Po uplynutí zmíněné doby byl v ESI 26

27 hmotovém spektru detekován pík odpovídající aduktu {[PtCl(3Braza) 2 (GSH)] 2H} (obrázek 12). Z tohoto lze usuzovat, že u těchto komplexů je jeden chloridový aniont nahrazován molekulou glutathionu. 1 H NMR spektroskopie byla užita ke studiu výše zmíněné interakce s glutathionem a hydrolytické stability ve směsném rozpouštědle DMF-d 7 /H 2 O (4:1, v/v), tentokráte u komplexu cis-[ptcl 2 (4Braza) 2 ]. Spektra byla zaznamenávána v intervalu β4 h po dobu pěti dnů. Také tyto NMR experimenty jednoznačně prokázaly schopnost těchto komplexů interagovat s glutathionem a také podléhat hydrolytickým dějům za vzniku nových částic předpokládaného složení cis-[pt(4braza) 2 Cl(H 2 O)] +, cis-[pt(h 2 O) 2 (4Braza) 2 ] 2+, případně produktům jejich protolytických dějů (obrázek 13). U komplexů (aplikovány v koncentraci β0 µm) byla dále na purifikovaném proteasomu získaném z lidských nádorových buněk karcinomu vaječníku Aβ780 studována jejich schopnost inhibovat aktivitu β0s proteasomu. Bylo zjištěno, že žádný z komplexů neinhiboval katalytickou aktivitu zmíněného proteasomu Protinádorová aktivita in vivo V práci Štarha et al., PLoS ONE, 2014 (příloha 4) jsou, kromě výše zmíněného, popsány výsledky in vivo protinádorové aktivity u cis-dichloroplatnatých komplexů s 3Claza, 3Iaza a 5Braza na myším modelu lymfocytární leukémie L1210 (samcům myší byla injekcí aplikována suspenze β 10 6 buněk myší leukémie L1β10; po desetidenní indukční době byly u myší makroskopicky patrné sekundární tumory; následujících sedm dnů byla zvířata léčena intraperitoneálně injekčně podávanými komplexy v dávce γ mg/kg). U testovaných myší byla denně zaznamenávána jejich hmotnost a zdravotní stav jako takový. Zvířata byla utracena, pokud hmotnost zvířete klesla pod 80% - rakovinné tkáně byly následně odebrány k histologickému a histochemickému studiu (např. imunohistochemická detekce transkripčního faktoru p5γ, kaspáz 3 a 8, a zánětlivého cytokinu TNF-α). Za základní výsledek považujeme stanovení průměrné doby přežití vztažené vůči neléčené skupině myší (kontrola), což je v práci popsáno jako %T/C vypočtené jako podíl průměrné doby přežití léčené a neléčené skupiny myší (viz příloha 4, tabulka β na str. 8). Je patrné, že terapeutický účinek nových platnatých komplexů se 7-azaindoly (%T/C = 97,1 100,0) neznamenal pro léčená zvířata žádnou výraznou změnu v porovnání s neléčenou skupinou, což ale nelze brát jako negativní výsledek, přihlédneme-li k hodnotě %T/C = λγ,γ, která je výsledkem pro skupinu zvířat léčených cisplatinou. Už jen z tohoto důvodu nelze brát hodnotu %T/C jako jediný výsledek proběhnuvšího in vivo testování, ale je nutné pohlédnout na výsledky komplexněji se zvážením více experimentálních parametrů. Takovými parametry, 27

28 stanovenými post mortem, byly hmotnost zvířat, hmotnost nádorové tkáně a její procentuální poměr k celkové tělesné hmotnosti (viz příloha 4, tabulka β na str. 8). Hodnoty těchto parametrů v zásadě korelovaly s výše uvedenými hodnotami %T/C, jelikož pro cisplatinu byl sice pozorován nejvýraznější úbytek hmotnosti nádorové tkáně, který byl ovšem doprovázen také výrazným snížením celkové hmotnosti testovaných zvířat, což v důsledku vedlo k vysoké mortalitě zvířat této skupiny a tedy k nízké hodnotě %T/C. Na druhou stranu vliv platnatých komplexů se 7-azaindoly na hmotnost nádorové tkáně nebyl tak výrazný, tyto látky však v daleko menší míře působily na celkovou hmotnost testovaných zvířat. S tímto souvisí také hodnocení celkového zdravotního stavu testovaných zvířat v průběhu experimentu, který u nových komplexů se 7-azaindolovými deriváty nevykazoval žádné rozdíly oproti normálnímu chování, zato zvířata léčená cisplatinou vykazovala, kromě zmíněného úbytku na hmotnosti, ještě další negativní příznaky spojené s aplikací metaloterapeutik na bázi platiny, jako je únava, nechutenství nebo nenormální projevy chování. Vše zmíněné přispělo ke konečnému pozitivnímu hodnocení cis-dichloroplatnatých komplexů s halogenderiváty 7-azaindolu ve srovnání s cisplatinou, kdy nižší protinádorovou aktivitu na úrovni in vivo převyšovala absence negativních vedlejších účinků spojená s aplikací zmíněných nových komplexů do těla léčených myší. V lyzátech nádorových tkání byly stanoveny vybrané proteiny, jmenovitě kaspáza 8 (Casp-8), kaspáza 3 (Casp-3) a p53. Je známo (viz výše), že vznik aduktů terapeutických komplexů na bázi platiny a DNA (Štarha et al., J. Inorg. Biochem., 2012 (příloha β) a Muchová et al., J. Biol. Inorg. Chem., 2013 (příloha γ) v případě zde studovaných komplexů s deriváty 7-azaindolu) vede k výrazným změnám ve struktuře DNA, které v konečném důsledku vedou k pozastavení buněčného cyklu, resp. k apoptóze. Poškození DNA bývají, mimo jiných mechanismů, rozpoznávány také transkripčním faktorem p53, který v takových případech spouští transkripci genu pβ1 a tím děje zastavující buněčný cyklus, což může být v případě přetrvávajícího poškození DNA následováno ději apoptotickými. Logicky s uvedeným tvrzením pak zní konstatování, že většina nádorových buněk má zmutovaný gen pro p5γ, a naopak že u efektivně léčených buněk jsou hodnoty p5γ vyšší. Na procesu apoptózy se pak aktivně podílí další z proteinů stanovovaných v diskutované práci Štarha et al., PLoS ONE, 2014 (příloha 4), kterými byly kaspázy γ a 8 (v obecném přiblížení řečeno, vyšší hodnoty těchto proteinů jsou důsledkem efektivního cytotoxického účinku testovaných látek). Z výsledků experimentů popsaných v diskutované práci vyplynuly rozdíly mezi testovanými komplexy cis-[ptcl 2 (3Claza) 2 ] a cis-[ptcl 2 (3Iaza) 2 ] ve srovnání s komplexem cis-[ptcl 2 (5Braza) 2 ] (viz příloha 4, obr. 7 na str. 10). V případě kaspázy γ byl 28

29 komplex obsahující ve své struktuře 5-brom-7-azaindol tím, který jako jediný výrazně navyšoval hladinu tohoto proteinu (hladinu kaspázy γ nepatrně navyšovaly ještě komplex cis-[ptcl 2 (3Iaza) 2 ] a cisplatina). Obdobně také v případě kaspázy 8 a p53 byla pouze u komplexu s 5Braza (a cisplatiny) zjištěna vyšší hladina zmíněných proteinů. Společně s výše uvedeným byl stanoven také mitotický index (MI; viz příloha 4, tabulka γ na str. λ), jehož nízké hodnoty indikují cytotoxický účinek studovaných komplexů na nádorovou tkáň (regresi tumoru) - také z hodnot MI je patrný rozdíl mezi cis-[ptcl 2 (5Braza) 2 ] a jeho dvěma analogy s odlišně substituovanými deriváty 7-azaindolu. Dalším z hodnotících parametrů, který v sobě zahrnuje většinu z výše uvedených výsledků, je tzv. index reaktivity. Je dán semikvantitativním vyhodnocením výsledků provedených histologických a histochemických metod (viz příloha 4, tabulka γ na str. λ) a lze jej brát jako parametr vypovídající o míře reakce nádorové tkáně na chemoterapeutický účinek aplikovaných látek nebo o stupni regrese tumoru. V případě cisplatiny je její nízký index reaktivity (1,γ5) dán jejím výrazným cytotoxickým účinkem (viz úbytek nádorové tkáně), který je spojený s nízkou mitotickou aktivitou a naopak výraznou nekrózou a krvácením. Z pohledu nepříliš výrazné in vivo protinádorové aktivity (ve smyslu redukce hmotnosti nádorové tkáně a průměrné doby přežití) byla poněkud překvapivá hodnota indexu reaktivity (1,71) získána u komplexu cis-[ptcl 2 (5Braza) 2 ]. Tato sice nekoreluje, jako v případě výše zmíněné cisplatiny, s úbytkem hmotnosti nádorové tkáně, i tak je ale nutné ji brát jako hodnotu perspektivní, protože nám indikuje regresi tumoru nezávisle od jejího pomalejšího nástupu u zmíněného komplexu ve srovnání s cisplatinou. Hodnoty indexu reaktivity pro další dva testované komplexy s deriváty 7-azaindolu jsou výrazně vyšší než u zmíněného komplexu s 5Braza. Z mnoha zásadních poznatků práce Štarha et al., PLoS ONE, 2014 (příloha 4) lze rozhodně vyzdvihnout fakt, že komplex cis-[ptcl 2 (5Braza) 2 ] obstál ve studiu protinádorové aktivity na úrovni in vivo provedeném na myším modelu leukémie L1β10, což jej favorizuje do dalších stádií základního farmakologického testování (metabolismus látky a lékové interakce na úrovni enzymů P450, farmakokinetika atd.) s výhledem na následný preklinický výzkum této látky Karboxylato komplexy s deriváty 7-azaindolu Je zajímavým faktem, že většina komplexů platiny, které v návaznosti na cisplatinu vstoupily do fáze klinických testů, obsahuje ve své molekule jeden bidentátní nebo dva monodentátní karboxylato ligandy (např. karboplatina, oxaliplatina, heptaplatina atd.; viz obrázek 1) [85]. Různorodost těchto O-donorových ligandů a možnost jejich derivatizace 29

30 umožňují bioanorganickým chemikům vhodně modifikovat farmakologicky důležité vlastnosti výsledných komplexů platiny (např. hydrolytická stabilita nebo lipofilita). Je také nutné zdůraznit, že karboxylato ligandy obsažené v klinicky testovaných nebo používaných léčivech na bázi platiny představují vhodné a mnoha studiemi prověřené odstupující skupiny. Z toho důvodu byly některé z nich vybrány pro přípravu nových komplexů platiny s deriváty 7-azaindolu, jinak řečeno k modifikaci chemických a farmakologických vlastností zjištěných u základní série cis-dichloroplatnatých komplexů s uvedenými N-donorovými ligandy Oxalatoplatnaté komplexy s deriváty 7-azaindolu Studium cis-dichloroplatnatých komplexů se 7-azaindolem a jeho halogenderiváty provázelo v pracích Štarha et al., Polyhedron, 2012 (příloha 1) a Štarha et al., J. Inorg. Biochem., 2012 (příloha β) také studium oxalatoplatnatých komplexů s analogickými N-donorovými ligandy. Syntéza platnatých oxalato komplexů s deriváty 7-azaindolu je, stejně jako v případě výše diskutovaných dichloro komplexů, atraktivní svojí jednoduchostí a rychlostí - i tyto látky totiž lze získat v jednom reakčním kroku. Uvedené je zajímavé, přihlédneme-li k výrobě oxaliplatiny, od které opět zde diskutované oxalatoplatnaté komplexy s deriváty 7-azaindolu formálně odvozujeme. Její výroba probíhá podle výše zmíněné Dharovy metody [83], čili reakcí 1R,2R-diaminocyklohexan-dichloroplatnatého (nebo jiného dihalogeno) komplexu s alespoň dvěma molárními ekvivalenty stříbrné solí (např. AgNO 3 ) za vzniku komplexního diaquaplatnatého kationtu. Přidáním kyseliny šťavelové nebo její alkalické soli pak dostáváme výslednou oxaliplatinu. Příprava oxalatoplatnatého komplexu se 7-azaindolem je popsána v práci Štarha et al., Polyhedron 2012 (příloha 1). Komplex byl připraven v jednom reakčním kroku syntézou vycházející z K 2 [Pt(ox) 2 ] βh 2 O (tento je komerčně dostupný nebo jednoduše připravitelný reakcí K 2 [PtCl 4 ] s nadbytkem alkalického šťavelanu, např. K 2 (ox) H 2 O [86]) interagujícím se stechiometrickým množstvím 7-azaindolu (aza) za vzniku požadovaného produktu, [Pt(ox)(aza) 2 ]. Stojí za zmínku, že jednokroková syntéza oxalatoplatnatých komplexu vycházející z alkalického bis(oxalato)platnatanu byla v literatuře poprvé popsána autorem této práce [87] a je jedinou známou alternativou výše popsané Dharovy metody přípravy oxalatoplatnatých komplexů. Výhody jsou opět zřejmé, můžeme zde zopakovat výše uvedené pro dichloro komplexy - jeden reakční krok znamená kratší reakční čas a vyšší výtěžek, není také nutné řešit kontaminaci produktu stříbrem, jak je známo z průmyslové výroby zde zmíněné oxaliplatiny [např. 88]. Ale zpět ke zmíněnému komplexu [Pt(ox)(aza) 2 ], jenž byl, stejně jako jeho dichloro analog, prokázán jako in vitro cytotoxicky neaktivní do koncentrace dané jeho rozpustností 30

31 v testovacím médiu (IC 50 > 0,1 µm vůči MCF7 a HOS), jak je popsané v práci Štarha et al., Polyhedron, 2012 (příloha 1, str. 407). V navazující práci Štarha et al., J. Inorg. Biochem., 2012 (příloha β) jsou, vedle výše diskutovaných cis-dichloroplatnatých komplexů, popsány také tři komplexy oxalatoplatnaté s 3Claza, 3Iaza a 5Braza. Také tyto komplexy byly připraveny zmíněnou jednokrokovou syntézou vycházející z bis(oxalato)platnatanu draselného. U těchto komplexů byla studována jejich in vitro cytotoxicita vůči lidským nádorovým buněčným liniím MCF7, HOS a LNCaP, avšak závěr z těchto biologických experimentů je obdobný jako pro [Pt(ox)(aza) 2 ], tedy že jsou necytotoxické do koncentrace dané jejich rozpustností v použitém médiu (viz příloha β, tabulka β na str. 61). Na tato zjištění bylo navázáno prací P. Štarha et al., Molecules, 2014, (příloha 5), která si kladla za cíl studium vlivu zavedení odlišně substituovaných derivátů 7-azaindolu do molekuly oxalatoplatnatého komplexu na in vitro protinádorovou aktivitu. Do série ke čtyřem výše uvedeným komplexům s aza, 3Claza, 3Iaza a 5Braza tak přibyl také komplex s bromem v poloze 3 7-azaindolového kruhu (3Braza) a navíc komplexy se substituenty v poloze 4 (4Claza a 4Braza). Všechny tři nově připravené komplexy byly testovány na jejich in vitro cytotoxicitu vůči lidským nádorovým buněčným liniím HOS a MCF7. Stejně jako v předchozích případech nebyla ani u komplexů [Pt(ox)(4Claza) 2 ] a [Pt(ox)(4Braza) 2 ] zjištěna žádná aktivita až do koncentrace dané jejich rozpustností (IC 50 > 1,0 M). Ovšem v případě komplexu [Pt(ox)(3Braza) 2 ], který byl v testovacím médiu velmi dobře rozpustný (>50,0 M), byla na zmíněných dvou liniích zjištěna zajímavá in vitro cytotoxicita srovnatelná s cisplatinou a vyšší než u oxaliplatiny (viz příloha 5, tabulka 1 na str a obr. 3 na str ). Na základě těchto výsledků byl tento komplex testován na šesti dalších lidských nádorových buněčných liniích, kterými byly Aβ780, Aβ780R, Gγ61, HeLa, A54λ a LNCaP. Z výsledků (viz příloha 5, tabulka 1 na str a obr. 3 na str ) plyne, že [Pt(ox)(3Braza) 2 ] je na liniích Aβ780 a HeLa více in vitro cytotoxický než cisplatina a na liniích Aβ780, HeLa a Gγ61 více než oxaliplatina. Na liniích Aβ780R, A54λ a LNCaP nebyla zjištěna žádná aktivita až do limitní testované koncentrace (IC 50 > 50,0 M). U in vitro protinádorově aktivního komplexu [Pt(ox)(3Braza) 2 ] bylo studováno ( 1 H a 195 Pt NMR, ESI hmotnostní spektrometrie) jeho chování ve vodu obsahujících rozpouštědlech (směs DMF-d 7 /H 2 O, 9:1 v/v; směs voda/methanol, 1:1 v/v) a schopnost interagovat s vybranými biomolekulami (cystein, GSH, GMP). Komplex byl ve směsi DMF-d 7 /H 2 O stabilní a nepodléhal hydrolýze - NMR spektra neobsahovala ani po pěti dnech žádné nové signály (viz příloha 5, obr. 1 na str. 108γ6). V souladu s tímto zjištěním není překvapivé, že ani hmotnostní spektra nevykazovala v čase žádné změny spojitelné 31

32 se vznikem nových platinu obsahujících částic (viz příloha 5, obr. β na str. 108γ7). Co se týká schopnosti studovaného komplexu interagovat s uvedenými biomolekulami, která byla studována ESI hmotnostní spektrometrií, tak ani zde nebyla nedetekována žádná nově vzniklá platinu obsahující částice, která by byla prokazatelným produktem interakce některé z biomolekul se studovaným komplexem. Jedinou výjimkou je pík velmi nízké intenzity odpovídající cystein (cys) obsahující částici {[Pt(cys)(ox)(3Braza) 2 ]+H} +, obsahující pravděpodobně monodentátně koordinovaný oxalátový aniont jako důsledek rozštěpení jedné Pt O vazby a otevření pětičetného PtO 2 C 2 kruhu (viz příloha 5, obr. β na str. 108γ7) Fototoxické deriváty karboplatiny s deriváty 7-azaindolu Další ze studovaných typů platnatých komplexů, Pt(cbdc)(naza) 2 ] (cbdc = cyklobutan-1,1-dikarboxylátový aniont; naza = 3Claza, 3Braza, 3Iaza, 4Claza, 4Braza a 5Braza), lze odvodit od karboplatiny nahrazením obou NH 3 molekul dvěma deriváty 7-azaindolu (obrázek 14). Tyto komplexy byly připraveny podle výše zmíněné Dharovy metody, s klíčovým meziproduktem o složení cis- PtI 2 (naza) 2 ]. Práce popisující zmíněné komplexy a jejich in vitro cytotoxicitu a fototoxicitu byla akceptována k publikaci v časopise PLoS One (manuskript je k habilitační práci přiložen jako příloha 6). Obrázek 14. Strukturní vzorec studovaných derivátů karboplatiny Zde si dovolím malou odbočku, jelikož i u zmíněných dijodo komplexů byla studována jejich in vitro cytotoxicita vůči lidským nádorovým buněčným liniím Aβ780, A2780R, MCF7, HOS, A549, G361, HeLa a karcinomu prostaty 22Rv1 (tabulka 2). Poněkud Tabulka 2: In vitro cytotoxicita cis-[pti 2 (naza) 2 ] komplexů vůči lidským nádorovým buněčným liniím karcinomu vaječníku Aβ780, cisplatin-resistentního karcinomu vaječníku Aβ780R, prsního karcinomu MCF7, osteosarkomu HOS, karcinomu plic A54λ, maligního melanomu Gγ61, karcinomu děložního čípku HeLa a karcinomu prostaty 22Rv1 a jejich srovnání s cisplatinou. Výsledky jsou dány jako hodnoty IC 50 ( M). 2Me4Claza = 2-methyl-4-chloro-7-azaindol A2780 A2780R MCF7 HOS A549 G361 HeLa 22Rv1 cis-[pti 2 (aza) 2 ] γ,5±0,7* γ,γ±0,γ* 1,7±0,8* 0,8±0,4* 1β,γ±1,1 β,λ±0,6* 7,0±0,6* 4,6±1,β* cis-[pti 2 (3Claza) 2 ] 4,1±0,8* γ,γ±0,5* 1,5±0,4* 1,γ±0,8* 6,4±1,4 β,γ±1,0* 4,8±0,4* 4,8±β,4* cis-[pti 2 (3Iaza) 2 ] β,8±0,γ* γ,β±0,β* 1,λ±0,5* β,β±1,2* 8,8±β,β γ,1±0,β* 4,7±0,8* 4,β±1,β* cis-[pti 2 (3Braza) 2 ] β,γ±1,1* β,6±0,8* 1,8±0,γ* β,8±1,0* λ,8±1,γ 1,6±0,7* 6,β±0,7* 4,5±1,6* cis-[pti 2 (4Claza) 2 ] γ,7±1,1* γ,4±0,β* 1,5±0,5* 0,5±0,β* 4,7±0,γ γ,β±0,β* γ,8±0,γ* 4,β±0,1* cis-[pti 2 (4Braza) 2 ] γ,7±1,1* 3,3±0,4* 1,0±0,4* 0,4±0,1* 4,γ±0,λ γ,β±0,β* γ,8±0,β* γ,8±0,1* cis-[pti 2 (5Braza) 2 ] γ,4±0,β* γ,5±0,5* 1,6±0,8* 1,4±1,1* 7,γ±1,6 γ,4±0,γ* 5,4±1,β* 5,1±0,8* cis-[pti 2 (2Me4Claza) 2 ] 1,7±1,1* 1,0±0,4* β,1±1,0* 0,7±0,β* γ,5±0,β 1,7±1,γ* γ,8±0,1* γ,5±0,β* Cisplatina β1,6±0,6 β0,β±4,7 17,λ±γ,5 18,λ±1,7 >50,0 5,γ±0,7 γ0,4±11,0 β6,λ±γ,5 32

33 překvapivě, vzhledem k faktu, že dijodoplatnaté komplexy byly už v prvních letech po objevu cisplatiny postulovány jako její necytotoxické a tedy farmakologicky neperspektivní analogy [89] a že není v literatuře popsáno příliš dijodoplatnatých komplexů vykazujících výraznou in vitro cytotoxicitu [např ], byla u dijodo komplexů se 7-azaindoly zjištěna výrazná in vitro cytotoxicita, která je mnohonásobně a statisticky významně (p < 0,05) vyšší ve srovnání s cisplatinou a ve většině případů i s dichloro analogy uvedených dijodo komplexů. Farmakologická perspektiva se odrazila v podání přihlášky vynálezu, jež chrání využití těchto látek jako léčiv v protinádorové terapii (PV β , 04/2014). Bylo také zjištěno, že dijodo komplexy pravděpodobně působí jiným mechanismem, než jaký je prokázán pro jejich dichloro analogy (viz výše), protože v experimentu na Aβ780 buňkách má cis-[pti 2 (4Braza) 2 ] odlišnou (vyšší) IC 50 (tj. nižší cytotoxicitu) než směs cis-[ptcl 2 (4Braza) 2 ] a dvou ekvivalentů KI. Navíc byla aktivita zmíněného dijodo komplexu modifikována (navýšena) přídavkem 5,0 µm L-BSO, a to konkrétně z hodnoty IC 50 = γ,7 µm na IC 50 = 1,0 µm. Na základě tohoto experimentu samozřejmě nelze rozhodnout, zdali je to dáno neochotou studovaných dijodoplatnatých komplexů podléhat inaktivačním reakcím s glutathionem (jehož fyziologickou syntézu L-BSO inhibuje), nebo významným ovlivněním redoxních dějů v buňce (glutathion je znám svojí schopností regulovat vnitrobuněčnou hladinu volných radikálů zasahujících do redoxních dějů) [11,95,96]. Toto vyžaduje další studium, jehož výsledky budou součástí připravované publikace. Vrátíme-li se ke zmíněným derivátům karboplatiny, lze konstatovat, že z výsledků in vitro cytotoxicity je patrná poměrně výrazná aktivita na A2780 ovariálním karcinomu (až 4,6 vyšší než u cisplatiny), nižší cytotoxicita pak byla zjištěna v případě lidských Tabulka 3. Výsledky testování in vitro cytotoxicity derivátů karboplatiny s deriváty 7-azaindolu a standardů karboplatiny a cisplatiny vůči lidským nádorovým buněčným liniím cisplatina-senzitivním karcinomu vaječníku A2780 a karcinomů prostaty LNCaP a PC-3. Buňky interagovaly s testovanými látkami 24 h. Data jsou uvedena jako hodnoty IC 50 ± SD (μm). Komplex A2780 LNCaP PC-3 [Pt(cbdc)(3Claza) 2 ] 11,8±6,2* 30,8±3,6 36,3±2,3 [Pt(cbdc)(3Iaza) 2 ] 10,3±4,3* >20,0 a 18,5±0,9 [Pt(cbdc)(3Braza) 2 ] 14,4±6,0 >50,0 a 42,3±0,8 [Pt(cbdc)(4Claza) 2 ] 5,3±0,9* 18,7±5,1 17,6±8,8 [Pt(cbdc)(4Braza) 2 ] 5,1±0,9* 23,5±3,8 26,6±4,1 [Pt(cbdc)(5Braza) 2 ] 4,7±1,9* 22,1±1,4 29,6±9,4 Karboplatina >50,0 a >50,0 a >50,0 a Cisplatina 21,8±3,9 >50,0 a >50,0 a * značí statisticky významný rozdíl hodnot od standardu cisplatiny na p < 0,05 hladině významnosti a) IC 50 nebylo dosaženo do uvedené koncentrace 33

34 nádorových buněčných linií karcinomu prostaty LNCaP a PC-3 (tabulka 3). U komplexu s 3Braza bylo dále experimentálně stanoveno, že po přidání 5,0 µm L-BSO statisticky významně (p < 0,05) vzrostla (ca 4,4 ) cytotoxicita na IC 50 = γ,γ±0,γ µm. S ohledem na fakt, že pro karboplatinu, od které jsou zde studované komplexy odvozeny, je známo, že její in vitro cytotoxicita může být efektivně navýšena aktivací UVA zářením [97], rozhodli jsme se i pro její, zde popsané deriváty se 7-azaindoly tuto problematiku prostudovat. Studium bylo zaměřeno na vliv UVA záření na složení reprezentativního komplexu a na změny biologických vlastností spojených s ozářením komplexů. Jak je patrné z obrázku 15, viabilita buněk byla výrazně nižší (p 0,05; ANOVA), pokud byly testované buňky Aβ780 a LNCaP po akumulaci komplexu ozářeny UVA zářením (20 min, 365 nm). V souladu s výše uvedeným vlivem UVA záření na viabilitu nádorových buněk se studované komplexy ve výrazně vyšší míře vázaly na CT DNA, jinými slovy, míra platinace DNA po UVA ozáření byla ve srovnání s neozářenými vzorky ca β γ vyšší. Obrázek 15. In vitro viabilita lidských nádorových buněk cisplatina-senzitivního karcinomu vaječníku A2780 a karcinomu prostaty LNCaP pro komplexy [Pt(cbdc)(3Iaza) 2 ] (A), [Pt(cbdc)(3Braza) 2 ] (B), [Pt(cbdc)(4Claza) 2 ] (C) a [Pt(cbdc)(4Braza) 2 ] (D) aplikovaných v 10 µm koncentraci ve tmě a po ozáření UVA zářením. * značí statisticky významný rozdíl hodnot na ozářených a neozářených buňkách (p < 0,05) Vzhledem k tomu, že biologické experimenty poskytly odlišné výsledky pro UVA ozářené a neozářené komplexy, bylo nutné pokusit se zjistit, jaký vliv má zmíněné záření na složení komplexů. 1 H NMR studiem bylo zjištěno, že komplexy (bez UVA ozáření) jsou v DMF-d 7 stabilní po dobu minimálně 14 dní, během kterých nebyly v protonových spektrech detekovány žádné změny. U reprezentativního komplexu [Pt(cbdc)(4Braza) 2 ] byla ve směsném rozpouštědle DMF-d 7 /H 2 O (1:1, v/v) po 24 h (bez UVA ozáření) detekována nová sada signálů (např. N1 H signál při 11,94 ppm, obrázek 16) odpovídající monofunkcionální částici o složení [Pt(4Braza) 2 (cbdc )(H 2 O)] (cbdc = monodentátně koordinovaný cbdc) [42 44]. Ve spektrech komplexů (rozpuštěných také ve směsi DMF-d 7 /H 2 O, 1:1, v/v) po působení UVA záření (20 min; max = 365 nm, 4,3 mw cm 2 ) byly 34

35 detekovány tři N1 H signály 4Braza (1γ,0β, 1β,γ5 a 11,8β ppm) ve srovnání s jedním signálem ve spektru původního komplexu (13,02 ppm) (obrázek 16). Po β4 h stání při laboratorní teplotě a laboratorním osvětlení nebyly detekovány žádné změny ve zmíněném protonovém NMR spektru. Obrázek H NMR spektra (N1 H oblast ligandu 4Braza) zaznamenaná v čase (A a C - čerstvé roztoky; B a D - po β4 h stání za laboratorní teploty a laboratorního osvětlení) pro vzorky před (A a B) a po (C a D) UVA iradiaci (β0 min, max = 365 nm) pro komplex [Pt(cbdc)(4Braza) 2 ] ve směsi DMF-d 7 /H 2 O (1:1, v/v). Obrázek H NMR spektra (N1 H oblast ligandu 4Braza) zaznamenaná v čase (po 24 (A) nebo 96 h (B a C) stání za laboratorní teploty a laboratorního osvětlení) pro vzorky po UVA iradiaci (β0 min, max = 365 nm) pro komplex [Pt(cbdc)(4Braza) 2 ] ve směsi DMF-d 7 /H 2 O (1:1, v/v) (A a B) nebo pro tento komplex s přídavkem roztoku volného 4Braza (C) Následné 1 H NMR experimenty (tzv. spikování volným 4Braza) prokázaly, že jeden (11,8β ppm) ze zmíněných N1 H signálů náleží volnému 4Braza uvolněnému z původního komplexu (viz obrázek 17). Dále získané výsledky poukazovaly na to, že druhý nový N1 H signál (1β,γ5 ppm) odpovídá molekule 4Braza obsažené v nově vzniklé platinu obsahující částici o pravděpodobném složení cis-[pt(h 2 O) 2 (cbdc )(4Braza)]. U této látky se pak cbdc ligand vyskytuje s největší pravděpodobností jako monodentátní (značeno jako cbdc ), na což poukazuje nový kvintet (β,08 ppm) charakteristický pro CH 2 CH 2 CH 2 vodíky tohoto ligandu (1,89 ppm v původním komplexu). Fakt, že dochází k rozštěpení jedné z Pt O vazeb a tím k otevření šestičlenného PtO 2 C 3 cyklu dokládá i zjištění, že signál detekovaný při 12,35 ppm přechází v čase v signály dva (1β,γ5 a 1β,18 ppm), což je pravděpodobně spojeno s izomerací uvedené částice. Uvolňování 4Braza ze struktury původního komplexu prokázalo také ESI-MS studium, protože ve spektru UVA ozářeného vzorku byla několikanásobně vyšší 35

36 intenzita píku částic {4Braza+H} + a {[Pt(cbdc)(4Braza)] H}, než ve spektru vzorku před ozářením. Analogické 1 H NMR a ESI-MS experimenty byly provedeny také pro směs studovaného komplexu s GSH nebo GMP v DMF-d 7 /H 2 O (1:1, v/v). V případě GSH nebyla uvedenými technikami prokázána žádná interakce. V případě směsi s GMP byly v protonovém NMR spektru po ozáření rozpoznány signály uvolněného 4Braza a navíc byl u původního C8 H signálu GMP (8,37 ppm) detekován signál nový při 8,γ1 ppm, což indikuje interakci studovaného komplexu s GMP. V hmotových spektrech pak byly nalezeny píky {[Pt(cbdc)(4Braza)(GMP)] Na+2H} +, {[Pt(cbdc)(4Braza)(GMP)]+H} + a {[Pt(cbdc)(4Braza)(GMP)] 2Na+H} při 920,2, 942,3 a 896,3 m/z, což domněnku o interakci komplexu s GMP potvrdilo. Transkripční mapování Pt DNA aduktů, provedené pro vybrané komplexy [Pt(cbdc)(3Iaza) 2 ] a [Pt(cbdc)(4Braza) 2 ] metodou studia in vitro RNA syntézy T7 RNA polymerázou na psp73kb/hpai DNA fragmentu, prokázalo, že místa inhibice jsou pro tyto komplexy jak s UV ozářením tak i bez něj podobné cisplatině (tedy především GG nebo AG místa; obrázek 18). Nicméně, výrazný je rozdíl v efektivitě inhibice mezi ozářeným a neozářeným komplexem, konkrétně UVA ozářené komplexy inhibovaly, při stejné míře platinace DNA, RNA syntézu výrazně efektivněji než komplexy neozářené. Toto souvisí se známým rozdílem mezi schopností mono- a bifunkcionálních Pt DNA aduktů inhibovat RNA syntézu [98 100]. Jinak řečeno, komplexy po ozáření UVA zářením se na DNA vážou jiným způsobem (bifunkcionálně) než komplexy neozářené (monofunkcionálně). Výsledky experimentů provedených na stejných komplexech a lineární psp73kb/ecori DNA, které byly zaměřené na kvantifikování interstrand a intrastrand aduktů komplexů s DNA, pak prokázaly, že UVA ozářené komplexy vytvářely oba typy aduktů, zatímco neozářené komplexy vytvářely pouze intrastrand adukty. Obrázek 18. Inhibice RNA syntézy T7 RNA polymerázou na psp73kb/hpai fragmentu modifikovaném komplexem [Pt(cbdc)(4Braza) 2 ] Toto velmi zajímavé zjištění týkající se odlišné povahy (mono- vs. bifunkcionální) vzniklých Pt DNA aduktů v závislosti na tom, jestli byl platnatý komplex ozářen UVA zářením nebo ne, bylo podpořeno výsledky fluorescenčních experimentů provedených na 36

37 DNA s ethidium bromidem (EtBr). Je známo, že vznik bifunkcionálních aduktů (např. u cisplatiny) efektivně brání interkalaci EtBr do DNA, což je pozorováno jako pokles fluorescence [ ]. Uvedené ale neplatí pro adukty monofunkcionální (např. chloro-diethylentriaminplatnatý komplex, dienplatin), které způsobují pouze nepatrný pokles intenzity fluorescence. Z výsledků získaných pro výše uvedené komplexy plyne stejný závěr, jako je uvedený pro modelové sloučeniny cisplatinu a dienplatinu. Jinými slovy, modifikace DNA UVA ozářenými komplexy vedla k výraznému poklesu fluorescence podobnému jako u cisplatiny, zatímco modifikace DNA neozářenými komplexy způsobila ne tak výrazný pokles intenzity pozorované fluorescence (obrázek 19). Souhrnně řečeno, tato práce prokázala deriváty karboplatiny se 7-azaindoly jako in vitro cytotoxické látky, jejichž aktivita může být různými mechanismy (L-BSO, fotoaktivace) dále navyšována. Rozdíl mezi aktivními částicemi vzniklými z původních komplexů za normálních podmínek a po ozáření UVA zářením byl ve výborné shodě prokázán chemickými i biologickými metodami. Velmi zajímavé je také zjištění, že studované komplexy uvolňují pod vlivem UVA záření N-donorový ligand. Toto by mohlo být velice zajímavé z biologického a farmakologického pohledu, protože pokud by takový uvolňující se N-donorový ligand byl sám o sobě určitým způsobem protinádorově aktivní, synergicky by tím přispíval k aktivitě platinu obsahující částice s perspektivou dosažení vyššího biologického efektu. Obrázek 19. A. Vznik interstrand (ICL) aduktů pro UVA ozářený (linie 1 5) a neozářený (linie 6 10) komplex [Pt(cbdc)(4Braza) 2 ]. B. Závislost EtBr fluorescence na r b pro DNA modifikovanou UVA ozářeným ( ) a neozářeným ( ) komplexem [Pt(cbdc)(4Braza) 2 ]. Data jsou uvedena jako průměr ± SD. Data pro cisplatinu (čárkovaně) a dienplatinu (tečkovaně) jsou pro srovnání vložena také Selektivní malonato a dekanoato komplexy U aktuálně studovaných malonato (mal) a dekanoato (dec) komplexů platiny obecného složení [Pt(mal)(naza) 2 ] a cis-[pt(dec) 2 (naza) 2 ] (obrázek 20) byla prozatím testována jejich in vitro cytotoxicita vůči cisplatin senzitivní (Aβ780) a rezistentní (Aβ780R) lidské nádorové linii karcinomu vaječníku, linii normálního lidského fibroblastu (MRC5) a primární kultuře lidských hepatocytů (Hep). Mezi těmito komplexy výrazně vynikly látky [Pt(mal)(3Iaza) 2 ] 37

38 Obrázek 20. Strukturní vzorce studovaných malonato (nahoře) a dekanoato (dole) komplexů s deriváty 7-azaindolu. (IC 50 = 26,6±8,λ µm vůči A2780 a 28,λ±6,7 µm vůči A2780R) a cis-[pt(dec) 2 (3Braza) 2 ] (14,5±0,6 µm vůči Aβ780 a 14,5±γ,8 µm vůči A2780R), nikoli uvedenými hodnotami in vitro cytotoxicity (tabulka 4), ale především svojí selektivitou, tedy schopností působit na nádorové buňky a současně nevykazovat žádný biologický účinek na buňkách zdravých. V konkrétním přiblížení byly tyto komplexy na nenádorových buněčných liniích neaktivní až do nejvyšší testované koncentrace (vůči MRC5: IC 50 > 50,0 µm pro [Pt(mal)(3Iaza) 2 ] a IC 50 > 25,0 µm pro cis-[pt(dec) 2 (3Braza) 2 ]; vůči Hep: IC 50 > 250,0 µm pro [Pt(mal)(3Iaza) 2 ] i cis-[pt(dec) 2 (3Braza) 2 ]). Tabulka 4. Výsledky testování in vitro cytotoxicity malonato a dekanoato komplexů s deriváty 7-azaindolu a cisplatiny vůči lidským nádorovým buněčným liniím Aβ780 a Aβ780R, nenádorové linii MRC5 a primární kultuře lidských hepatocytů (Hep). Buňky interagovaly s testovanými látkami β4 h. Data jsou uvedena jako hodnoty IC 50 ± SD ( M). Komplex A2780 A2780R MRC5 Hep [Pt(mal)(3Braza) 2 ] >50,0 a β7,γ±8,λ >50,0 a 32,8 [Pt(mal)(3Iaza) 2 ] β6,6±8,λ β8,λ±6,7 >50,0 a >250,0 a [Pt(mal)(4Braza) 2 ] β4,4±1,β ββ,4±1,8 β1,λ±γ,1 nt [Pt(dec)(3Braza) 2 ] 14,5±0,6* 14,5±γ,8 >25,0 a >250,0 a [Pt(dec)(3Iaza) 2 ] 1γ,0±1,1* 15,8±4,β >25,0 a nt [Pt(dec)(4Claza) 2 ] >50,0 a β4,6±1,β >50,0 a 105,1 [Pt(dec)(4Braza) 2 ] 14,γ±γ,7* 1λ,5±β,1 β6,β±4,8 nt Cisplatina β6,γ±γ,6 >50,0 a >50,0 a >250,0 a * značí statisticky významný rozdíl hodnot od standardu cisplatiny na p < 0,05 hladině významnosti nt, nebylo testováno a) IC 50 nebylo dosaženo do uvedené koncentrace Možnosti cíleného transportu komplexů na bázi platiny Jak je uvedeno výše v práci, jednou z možností, jak navýšit aktivitu metaloterapeutik a/nebo snížit jejich negativní vedlejší účinky, je jejich cílený transport za využití různých typů nanočástic jako transportních systémů. Jednou z nejperspektivnějších oblastí je pak studium funkcionalizovaných magnetických nanočástic na bázi oxidů železa pro magnetický transport 38

39 léčiv. Do této oblasti zapadá i práce Štarha et al., Molecules, 2014, 1622 (příloha 7), ve které je popsána příprava a charakterizace maghemitových core-shell nanočástic obalených 4-aminobenzoovou kyselinou, která váže biologicky aktivní částici, cis-[pt(h 2 O) 2 (naza)] 2+. Studované nanočástice byly připravené srážecí metodou ze směsi FeCl 3 6H 2 O, FeCl 2 4H 2 O a zmíněné organické karboxylové kyseliny a následným přídavkem vodného roztoku NH 3. Syntéza nebyla provedena v inertní atmosféře, ale v atmosféře oxidační (vzduch), která zajistila oxidaci vznikajícího Fe 3 O 4 (magnetit) na -Fe 2 O 3 (maghemit). Vzniklé nanočástice následně interagovaly s aktivovaným platnatým komplexem (vznikl aktivací stříbrnou solí z původních cis-dichloroplatnatých komplexů s deriváty 7-azaindolu 3Claza a 5Braza) za vzniku výsledné funkcionalizované nanočástice (viz příloha 7, obr. 1, str. 1624). Přítomnost obalující organické kyseliny i funkcionalizujícího komplexu byla potvrzena FTIR spektroskopií a TG/DTA termickou analýzou (viz příloha 7, obr. 2 na str. 1625). Platina pak byla jednoznačně detekovaná v EDS spektrech výsledných systémů. U vzniklých nanokompozitů předpokládáme vazbu Pt N mezi funkcionalizujícím komplexem a obalující karboxylovou kyselinou, což bylo nepřímo potvrzeno detailním FTIR studiem. Připravené nanočástice mají sférický tvar s průměrnou velikostí 1γ,0±β,1 nm, jak je patrné z TEM snímků (viz příloha 7, obr. 3 na str. 1627). Makroskopicky pozorovatelné superparamagnetické vlastnosti (viz příloha 7, obr. 1 na str. 1624) byly u studovaných nanočástic experimentálně prokázány detailním studiem Mössbauerovou spektroskopií (viz příloha 7, tabulka 1 a obr. 4 na str. 1628). V diskutované práci nejsou popsány biologické vlastnosti (např. stabilita v roztoku, uvolňování léčiva v různých podmínkách, toxicita a cytotoxicita výchozích i výsledných funkcionalizovaných systémů atd.) uvedených nanosystémů. Výzkum uvedeného nyní probíhá a jeho výsledky budou popsány a diskutovány v navazující publikaci. Na práci Štarha et al., Molecules, 2014, 1622 (příloha 7) bylo navázáno studiem odvozených typů nanočástic, jejichž základem je kompozit zlata navázaného na maghemitovém jádře, u kterých je na zmíněné zlato navázána síru obsahující karboxylová kyselina (např. kyselina lipoová nebo merkaptopropionová), která je pak dále funkcionalizovaná (Štarha et al., Int. J. Mol. Sci., 2015 (příloha 8)). V případě naší aktuální práce byly nanočástice s kyselinou lipoovou funcionalizovány aktivovanou cisplatinou (příprava a charakterizace analogických systémů s platnatými komplexy s deriváty 7-azaindolu v současné době probíhá) (obrázek β1). Výhody kompozitu zlato/maghemit oproti výše zmíněným maghemitovým nanočásticím jsou takové, že kombinují vlastnosti obou komponent, tady maghemitu (superparamagnetismus, potenciál při MRI zobrazování) 39

40 a zlata (chemická odolnost, fototermální efekt) [104]. Zlato navíc chrání maghemitové jádro před chemickou a enzymatickou degradací ve fyziologickém prostředí a je vhodné pro chemickou derivatizaci (např. zmíněnými síru obsahujícími molekulami) [105,106]. Z literatury jsou známé podobné systémy zlatem obalených nanočástic na bázi oxidu železa a jejich rozmanité využití [např ]. Avšak je prozatím známa pouze jedna práce, která popisuje tyto nanočástice funkcionalizované modifikovanou cisplatinou nebo jinou biologicky aktivní sloučeninou na bázi platiny [112]. Naše práce přináší ve srovnání se zmíněnou publikací jednodušeji připravitelné systémy, které pak účinněji vážou aktivní komplex platiny (cisplatinu). Studované nanočástice jsou připraveny následujícím postupem. Ze směsi Fe(III) a Fe(II) solí (molární poměr βμ1) jsou srážecí metodou připraveny magnetitové (Fe 3 O 4 ) nanočástice [113], které jsou následně oxidovány zředěnou kyselinou dusičnou na nanočástice maghemitové ( -Fe 2 O 3 ), symbolizované dále jako mag [114]. Následné navázání zlata na maghemit (mag/au nanočástice) je provedeno postupným přidáváním 1% HAuCl 4 [112]. Dalším přídavkem je pak na zlato navázána kyselina lipoová (mag/au HLA) [115]. mag/au HLA nanočástice jsou v případě připravované práce funkcionalizovány aktivovanou cisplatinou, cis-[pt(nh 3 ) 2 (H 2 O) 2 ] 2+ (CDDP*), čímž je získán finální produkt mag/au LA CDDP*. Obrázek 21. Předpokládané složení studovaných mag/au LA CDDP* nanočástic (vlevo), část XPS jejich spektra (vpravo nahoře) a jejich TEM snímek (vpravo dole) Připravené mag/au LA CDDP* nanočástice (pro srovnání a jednoznačnější interpretaci také syntetické meziprodukty) byly charakterizovány rozličnými technikami. Z HRTEM a TEM snímků je patrné, že se jedná o kulovité nanočástice dobré velikostní distribuce, jejichž průměrná velikost je 12,2±1,9 nm (viz příloha 8, obr. 2 na str. 2037). Přítomnost platiny (přeneseně aktivované cisplatiny) byla prokázána výsledky XPS a EDS spektroskopie a ICP-MS. V XPS spektru mag/au LA CDDP* nanočástic (viz příloha 8, obr. 4 a 5 na str resp. 2040) byly, kromě Fe2p, O1s, C1s, Au4d, Au4f, Au5p a S2p píků detekovaných také v XPS spektru meziproduktu mag/au HLA, nalezeny fotoelektronové píky jednoznačně přiřaditelné aktivované cisplatině (N1s, Pt4d a Pt4f). Stejné výsledky poskytla také zmíněná EDS spektroskopie, kterou byla také detekována platina. Velmi 40

41 důležitým poznatkem přispěla do aktuálního studia TG/DTA termická analýza, z jejichž výsledků lze usuzovat, že aktivovaná cisplatina se na mag/au HLA nanočástice váže jednou Pt O vazbou. Obsah Pt v mag/au LA CDDP* nanočásticích byl studován metodou ICP-MS. Molární poměr PtμAu je 1:2,6 a platina představuje 4,0% celkové hmotnosti studovaných nanočástic. Studium stability mag/au LA CDDP* nanočástic prokázalo, že výrazně závisí na ph. V kyselém prostředí je uvolňování aktivované cisplatiny zanedbatelné. V neutrálním prostředí dosahuje hodnot dostačujících pro následné testování in vitro cytotoxicity nebo jiných biologických vlastností. Nejnižší stabilitu, tedy nejvyšší obsah uvolňované platinu obsahující částice, prokázaly studované nanočástice v zásaditém prostředí. Ačkoli dosavadní výsledky představují prozatím pouze chemickou část studované problematiky, jeví se diskutované nanosystémy jako slibné i pro následné biologické studium (in vitro a in vivo protinádorová aktivita, toxicita, MRI zobrazování atd.). Je zde také rozsáhlý prostor modifikace studovaných nanočástic ve smyslu jiné síru obsahující látky, jiné platinu obsahující funkcionalizující látky (např. zmíněné platnaté komplexy se 7-azaindoly) nebo třeba navázání specifických látek pro selektivní rozpoznání cílových nádorových buněk. Uvedená témata budou předmětem dalšího studia Platnaté komplexy s deriváty N6-benzyladeninu N6-benzyladenin (6-benzylaminopurin) a jeho deriváty jsou biologicky aktivní sloučeniny, které v závislosti na míře substituce purinového kruhu vykazují buď cytokininovou aktivitu (cytokininy jsou jednou ze základních skupin rostlinných hormonů) nebo aktivitu protinádorovou vycházející ze schopnosti derivátů vhodně substituovaných na purinovém kruhu inhibovat cyklin-dependentní kinázy (CDK) živočišných (včetně lidských) buněk. CDK jsou proteiny přirozeně regulující buněčný cyklus, jejichž inhibicí je tento děj pozastaven [ ]. Nejznámějším CDK inhibitorem na bázi derivátů N6-benzyladeninu je 2-(1-ethyl-2-hydroxyethylamino)-N6-benzyl-9-isopropyladenin (roskovitin), který je pod jménem Seliciclib klinicky testován na pacientech trpících nemalobuněčným karcinomem plic [117,119]. Optikou koordinačního chemika jsou deriváty N6-benzyladeninu, tedy dusíkaté heterocyklické sloučeniny, vhodnými vícedentátními N-donorovými ligandy pro syntézu komplexů přechodných kovů. V literatuře jsou popsány komplexy Fe(II/III) [120], Co(II) [121], Ni(II) [122], Cu(II) [123], Zn(II) [124], Ru(III) [125], Pd(II) [126], Au(I) (viz práce Trávníček et al., J. Med. Chem., 2012, příloha 10), Au(III) [127], Pt(IV) [128] a v neposlední řadě také Pt(II) dichloro komplexy [ ]. 41

42 Protinádorová aktivita in vitro V rámci kontinuity výzkumu biologicky aktivních koordinačních sloučenin platiny na Katedře anorganické chemie Přírodovědecké fakulty Univerzity Palackého v Olomouci se v návaznosti na výše zmíněné dichloroplatnaté komplexy s deriváty N6-benzyladeninu uchazeč věnoval studiu biologicky aktivních komplexů platiny - tentokrát se jednalo o oxalato komplexy - v rámci své disertační práce [87,135,136], kde jsou také detailně okomentovány způsoby jejich přípravy a jejich protinádorová aktivita, která byla v mnoha případech výrazně vyšší ve srovnání s cisplatinou. Příprava oxalatoplatnatých komplexů s deriváty N6-benzyladeninu a jejich využití v protinádorové terapii je předmětem patentové ochrany národního patentu CZγ0β6βγ (udělen βγ. 6. β011) a mezinárodní přihlášky vynálezu WOβ010/1β1575 (publikováno β β010). Jediná práce, která prozatím navázala na postgraduální studium oxalatoplatnatých komplexů s deriváty N6-benzyladeninu, popisuje u série čtyř oxalato komplexů s roskovitinem a jeho benzyl substituovanými deriváty jejich schopnost ovlivňovat lidský jaterní mikrosomální cytochrom P450 [137]. Studium aktivity devíti CYP enzymů zmíněného mikrosomálního systému (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4), prokázalo, že studované oxalato komplexy výrazně ovlivňují CYP3A4, a to na různých koncentračních úrovních. Toto pravděpodobně souvisí se schopností tohoto enzymu interagovat s objemnými molekulami. Vzhledem k tomu, že CYPγA4 je enzymem metabolizujícím většinu léčiv, je nutné uvedené výsledky brát jako nevýhodu ve smyslu jejich možného dalšího farmakologického využití. Oxalatoplatnaté komplexy jsou popsány také v práci Štarha et al., Molecules, 2014, Tentokráte ovšem byly jako N-donorové ligandy použity jiné deriváty N6-benzyladeninu, a to benzyl substituované deriváty N6-benzyladenosinu (obrázek ββ). Komplexy byly připraveny výše popsanou jednokrokovou syntézou vycházející z bis(oxalato)platnatanu draselného Obrázek 22. Strukturní vzorec studovaných oxalatoplatnatých komplexů s deriváty N6-benzyladenosinu. a charakterizovány širokou škálou fyzikálně-chemických technik včetně ESI hmotnostní spektrometrie a multinukleární ( 1 H, 13 C, 15 N, 195 Pt) NMR spektroskopie (obrázek βγ), které prokázaly složení a čistotu komplexů, včetně koordinace použitých derivátů 42

43 Obrázek 23. Výsledky 1 H 15 N gs-hmbc experimentů pro N6-(2-chlorobenzyl)adenosin (2ClL; vlevo) a komplex (vpravo) obsahující 2ClL jako N7-koordinovaný ligand, prokazující zmíněný typ koordinace přes atom N7 adeninového kruhu. N6-benzyladenosinu přes dusíkový atom N7. Komplexy byly testovány na in vitro cytotoxicitu vůči dvěma lidským nádorovým buněčným liniím (HOS a MCF7), avšak nebyl u nich zjištěn žádný biologický efekt až do koncentrace dané jejich rozpustností v testovacím médiu. Příčinou cytotoxické inaktivity byla nízká (ca 50 nižší ve srovnání s cisplatinou) buněčná akumulace studovaných oxalatoplatnatých komplexů zjištěná na linii MCF7. S uvedeným souvisí také fakt, že, jak plyne z výsledků ESI-MS a 1 H NMR, komplexy nepodléhají hydrolýze. Analogické N-donorové ligandy, tedy deriváty N6-benzyladenosinu substituované v tomto případě nejen na benzenovém kruhu, ale v některých případech i na purinovém skeletu, byly použity také v práci Štarha et al., Molecules, 2013 popisující a diskutující přípravu, charakterizaci a in vitro cytotoxicitu trans-dichloroplatnatých komplexů (syntéza cis-izomerů nevedla k izolování produktů o akceptovatelné izomerické čistotě) (obrázek 24). Cíle práce lze ztotožnit s těmi uvedenými výše pro práci Štarha et al., Molecules, 2014, 3832, nicméně ani v tomto případě nebyla u testovaných komplexů zjištěna žádná in vitro cytotoxicita vůči HOS a MCF7 Obrázek 24. Strukturní vzorec derivátů N6-benzyladenosinu použitých jako ligandy pro studované trans-dichloroplatnaté komplexy. 43

44 Obrázek 25. ESI hmotnostní spektra studovaných komplexů 12 (a) a 7 (b) v methanolu (nahoře) a jejich směsí s L-methioninem měřené ihned (uprostřed) a 12 h (dole) po přípravě studované směsi. lidským nádorovým buněčným liniím, a to až do maximální testované koncentrace (IC 50 > 50,0 µm). Jedním z možných mechanismů odpovídajícím za inaktivitu v uvedeném biologickém experimentu je interakce s jednou z hlavních fyziologických síru obsahujících biomolekul L-methioninem, jak bylo v práci prokázáno ESI-MS studiem (viz obrázek 25). 44

45 Jiný typ Pt(II) komplexů s deriváty N6-benzyladeninu, odvozený od karboplatiny nahrazením obou NH 3 molekul v její struktuře zmíněnými různě substituovanými deriváty N6-benzyladeninu (viz příloha 9, obr. 1 na str. 653), je popsán v práci Dvořák et al., Toxicol. in Vitro, 2011 (příloha 9), kde je detailně rozebrána in vitro cytotoxická aktivita sedmi Pt(II) komplexů vůči celkem sedmi lidským nádorovým buněčným liniím, včetně jejího srovnání s klinicky užívanými metaloterapeutiky cisplatinou, karboplatinou a oxaliplatinou. Výrazná aktivita byla pozorována na liniích karcinomu vaječníku Aβ780, karcinomu vaječníku rezistentního vůči cisplatině Aβ780R, prsního karcinomu MCF7 a maligního melanomu Gγ61, na kterých byla pro nejaktivnější komplex [Pt(cbdc)(4CF 3 L) 2 ] (4CF 3 L = 2-chloro-N6-(4-trifluormethylbenzyl)-9-isopropyladeninu) určena aktivita odpovídající hodnotám IC 50 rovnajícím se 6,4±0,1, 5,6±1,7, 1λ,0±6,6, resp. 11,γ±β,8 µm (viz příloha 9, obr. 2 4 na str. 654 a 655). Zmíněné látky účinně překonávaly získanou rezistenci nádorových buněk vůči cisplatině (studováno na modelu karcinomu vaječníku A2780) - jejich rezistenční faktor se rovná 0,λ 1,4 (pro cisplatinu je to β,6). Nebyl také pozorován žádný vliv studovaných Pt(II) komplexů na zdravé lidské hepatocyty. Molekulární farmakologií cis-dichloroplatnatých komplexů s deriváty N6-benzladeninu ze skupiny CDK inhibitorů a jejich syntetických prekurzorů (viz výše) se zabývají práce [134,138]. Ve druhé zmíněné, na které se uchazeč autorsky spolupodílel, je popsán a s cisplatinou srovnán mechanismus účinku potenciálního protinádorového léčiva o složení cis-[ptcl 2 (boh) 2 ], kde boh symbolizuje CDK inhibitor ze skupiny derivátů N6-benzyladeninu bohemin, 2-(3-hydroxypropylamino)-N6-benzyl-9-isopropyladenin [129]. Bylo zjištěno, že se komplex efektivně váže na CT-DNA (t 50% = 640±27 min), avšak ve významně menší míře v porovnání s cisplatinou (t 50% = ca 120 min). Kvantifikace monofunkcionálních aduktů s DNA (experiment s [ 14 C]thiomočovinou, inkubace 48 h) prokázala, že v případě studovaného komplexu je takových aduktů 10% (2% pro cisplatinu), jinak řečeno, λ0% ze vzniklých aduktů komplexu s boheminem je po 48 h vázáno bifunkcionálně. Studovaný komplex také účinně rozvolňoval supercoiled DNA, podobný efekt byl pozorován na oligodeoxyribonukleotidovém duplexu, se kterým, s ohledem na složení použitého oligodeoxyribonukleotidu, komplex s boheminem vytváří 1,2-GG intrastrand adukt. Vliv studovaného komplexu na DNA byl zkoumán také chemickou metodou založenou na schopnosti některých agens (KMnO 4, diethylpyrokarbonát) reagovat pouze s nukleobázemi jednovláknové nebo rozvolněné dvouvláknové DNA, nikoli s intaktní dvouvláknovou DNA. Také v tomto případě byl cis-dichloroplatnatý komplex s boheminem tím, který více rozvolňuje DNA, ve srovnání s cisplatinou. Z dosažených výsledků je patrné, 45

46 že komplex s boheminem vykazuje poněkud odlišnou aktivitu, z mechanistického úhlu pohledu, ve srovnání s cisplatinou. Proto byl studován, a s cisplatinou porovnán, vliv interakce komplexu s boheminem s DNA na rozpoznání vzniklých aduktů proteiny HMG (high-mobility group) domény. Je zajímavé, že ačkoli je vazba HMG proteinů na DNA poškozenou cisplatinou nebo jinými cytotoxickými léčivy na bázi platiny důležitým dějem zodpovídajícím za vlastní cytotoxický účinek, nebyla, na rozdíl od cisplatiny, pro komplex s boheminem provedeným experimentem detekována. Na druhou stranu to byl komplex s boheminem, který účinněji inhiboval polymerizaci DNA a transkripci DNA RNA polymerázou II ve srovnání s cisplatinou. U komplexu s boheminem byla dále zjištěna, v porovnání s cisplatinou, vyšší míra syntézy spojené s opravami DNA, jinými slovy účinnější oprava Pt DNA aduktů vytvořených mezi komplexem s boheminem, než u aduktů s cisplatinou. UV-Vis elektronová spektroskopie pak byla použitá jako nástroj ke studiu interakce obou výše uvedených cis-dichloroplatnatých komplexů s glutathionem. Bylo zjištěno, že komplex s boheminem interaguje s uvedenou biomolekulou výrazně méně ve srovnání s cisplatinou. Závěrem lze k této práci říci, že komplex s CDK inhibitorem ze skupiny vícesubstituovaných derivátů N6-benzyladeninu boheminem a cisplatina mají některé mechanistické procesy společné, bez výrazných rozdílů (např. vazebná místa na DNA, rozvolnění DNA atd.), jiné procesy jsou odlišné, ať už v prospěch jednoho nebo druhého protinádorově aktivního platnatého komplexu. Potvrdit nebo vyvrátit farmakologický potenciál studované látky tedy musí další studie Protinádorová aktivita in vivo Perspektivní hodnoty in vitro cytotoxické aktivity výše zmíněných oxalatoplatnatých komplexů, které ve své struktuře obsahovaly deriváty N6-(substituovaný benzyl)-9- isopropyladeninu různě substituované na atomu Cβ purinového skeletu, a to buď chlorem (tj. syntetické prekurzory roskovitinových derivátů [87]; např. zde studovaný komplex [Pt(ox)(2,4diOMeL) 2 ], kde 2,4diOMeL = 2-chlor-N6-(2,4-dimethoxybenzyl)-9- isopropyladeninu) nebo 2-aminobutan-1-olem (vlastní deriváty roskovitinu [135]; např. zde studovaný komplex [Pt(ox)(4OMeros) 2 ], kde 4OMeros je derivát roskovitinu 2-(1-ethyl-2- hydroxyethylamino)-n6-(4-methoxybenzyl)-9-isopropyladenin), byly základem pro vstup těchto látek do fáze in vivo testování, kdy byl pro studium vybrán model lymfocytární leukémie L1β10 na myších (připravovaná práce; metodika je popsána v práci Štarha et al., PLoS ONE, 2014, příloha 4). 46

47 Z výsledků plyne, že se in vivo biologický účinek obou látek výrazně liší a je přesně opačný, než tomu bylo ve fázi in vitro experimentů (pro příklad zde uveďme hodnoty IC 50, na modelové lidské nádorové buněčné linii karcinomu vaječníku Aβ780, která je pro [Pt(ox)(2,4diOMeL) 2 ] rovna 4,0±1,0 µm a pro [Pt(ox)(4OMeros) 2 ] je to 14,4±1,4 µm [135,136]). Za hlavní výsledek in vivo experimentu lze pokládat průměrnou délku prodloužení života ve srovnání s kontrolní (neléčenou) skupinou a cisplatinou. Cisplatina vyvolávala u testovaných zvířat výrazné nežádoucí účinky, které se následně projevovaly na celkovém zdravotním stavu, což vedlo, vedle snižování objemu nádorových tkání, k celkovému úbytku hmotnosti, apatii, nechutenství a nakonec i systémovému selhání. Obdobné účinky, ale v menší míře, vyvolával komplex [Pt(ox)(2,4diOMeL) 2 ], nejméně toxických projevů bylo pozorováno u [Pt(ox)(4OMeros) 2 ]. Uvedené se u cisplatiny a komplexu [Pt(ox)(2,4diOMeL) 2 ] projevilo ve snížení průměrné doby přežití testovaných myší ve srovnání s neléčenou kontrolou. Naopak aplikace komplexu [Pt(ox)(4OMeros) 2 ] výrazně, ve srovnání s cisplatinou dokonce statisticky významně (na hladině významnosti p < 0,05; ANOVA), prodloužila průměrnou délku života myší (obrázek 26). Perspektivu do dalšího studia vnáší i výsledky histologického hodnocení prokazující toxické účinky (např. cirhóza jater, poškození střevního endotelu, nefrotoxicita), které sice byly detekovány po aplikaci všech testovaných sloučenin, avšak s výrazným rozdílem mezi zvířaty léčenými cisplatinou (irreparabilní poškození tkání) a komplexy s deriváty N6-benzyladeninu (reverzibilní poškození tkání). Obrázek 26. Výsledky testování in vivo protinádorové aktivity vybraných oxalatoplatnatých komplexů s deriváty N6-benzyladeninu. 2,4diOMeL = 2-chlor-N6-(2,4- dimethoxybenzyl)-9-isopropyladenin, 4OMeros = 2-(1-ethyl-2- hydroxyethylamino)-n6-(4-methoxybenzyl)-9-isopropyladenin. 47

48 4. Komplexy jiných p echodných kov s r znými N-donorovými ligandy Již na předešlých stranách této práce je uvedeno, že moderní bioanorganická chemie biologicky aktivních komplexů přechodných kovů má čím dál širší záběr, alespoň pokud se na problematiku díváme optikou typů přechodných kovů, jež jsou v posledních letech klinicky nebo preklinicky studovány [2,9 11,13 16,18 20]. Je sice nutné jedním dechem dodat, že to jsou stále komplexy platiny, které v těchto počtech nad ostatní přechodné kovy vyčnívají, nicméně z pohledu klinického využití si s nimi v mnohém nezadají zlatné komplexy používané jako jedna z možností v terapii zánětlivých onemocnění, jako je např. revmatoidní artritida. Také naše pracoviště v poslední době pravidelně přispívá do problematiky protizánětlivě aktivních komplexů zlata pracemi popisujícími látky na bázi komplexů zlata s různými N-donorovými ligandy, které v četných případech překonávají protizánětlivé účinky různých konvenčních terapeutik, ať už na bázi zlata nebo jiného typu Protizánětlivá aktivita ůu(i) komplex s deriváty N6-benzyladeninu V práci Trávníček et al., J. Med. Chem., 2012 (příloha 10) se zabýváme studiem protizánětlivé aktivity zlatných komplexů s benzyl substituovanými deriváty N6-benzyladeninu (obrázek β7), a to na úrovni in vitro i in vivo. U látek byla studována (a s klinicky užívaným protizánětlivým léčivem na bázi zlata auranofinem porovnávána) jejich schopnost regulovat hladinu pro- a protizánětlivých cytokinů spojených s projevy zánětlivých onemocnění, jako je TNF-α (faktor nádorové nekrózy α), IL-1 (interleukin-1beta), HMGB1 (high-mobility group protein B1), IL-10 (interleukin-10) a IL-1RA (receptorový antagonista interleukinu-1), a to na modelu lipopolysacharidem (LPS) stimulovaných makrofágů buněčné linie lidské akutní monocytické leukémie (THP-1). In vivo experimenty byly založeny na studiu změn objemu otoku tlapek u potkanů po aplikaci látky vyvolávající zánět (karagenanu). Obrázek 27. Obecný strukturní vzorec studovaných komplexů zlata s deriváty N6-benzyladeninu. Složení celkem osmi komplexů obecného složení [Au(nL)(PPh 3 )] (viz příloha 10, obr. 1 na str. 4569) obsahujících 2F, 3F, 2Cl, 3Cl, 4Cl, 3Me, 4Me nebo 3OMe deriváty N6-beznyladeninu (nl) bylo prokázáno ESI+ hmotnostní spektrometrií, kterou byly detekovány molekulové píky [Au(nL)(PPh 3 )+H] +, a 1 H a 13 C NMR spektroskopií, kterou byly 48

49 detekovány všechny vodíkové a uhlíkové atomy připravených látek, s výjimkou N9 H vodíkových atomů použitých derivátů N6-benzyladeninu, které se ve struktuře studovaných komplexů nachází deprotonizované (během přípravy v bazickém prostředí odštěpují zmíněný N9 H vodík). Zmíněný Nλ atom pak byl na základě zjištěných hodnot koordinačních posunů atomů purinového skeletu prokázán jak atom, kterým se tyto ligandy koordinují na centrální atom. ESI-MS byla také použita jako nástroj ke studiu interakce studovaných komplexů se síru obsahujícími biomolekulami cysteinem a glutathionem. Z výsledků plyne, že uvedené biomolekuly (především cystein) s komplexy interagují ve smyslu nahrazení N-donorových ligandů na bázi N6-benzyladeninu. Ještě před vlastním studiem protizánětlivých vlastností byla u komplexů studována a prokázána (LD 50 ca β M) in vitro cytotoxicita vůči THP-1 buňkám (viz příloha 10, obr. 2 na str. 4570). Určitě stojí za zmínku, že při nižších koncentracích studované komplexy nejen že THP-1 buňky neinhibovaly, ale dokonce jejich buněčné dělení stimulovaly. Na modelu prozánětlivého cytokinu TNF-α bylo zjištěno, že po 2 a 4 h studované komplexy různou měrou, avšak více ve srovnání s auranofinem, snižují genovou transkripci projevující se snížením hladiny mrna spojené s TNF-α (viz příloha 10, obr. 3 na str. 4571). Tyto komplexy také ca β,5 snižují sekreci TNF-α. Obdobné výsledky byly získány také na dalším použitém prozánětlivém cytokinu, kterým tentokráte je IL-1 (viz příloha 10, obr. 4 na str. 4571). Fyziologickým antagonistou zmíněného cytokinu IL-1 je protizánětlivý cytokin IL-1RA, přičemž důležitým parametrem při hodnocení protizánětlivých vlastností studovaných látek je jejich vzájemný poměr. Pro tento cytokin platí, že ačkoli studované zlatné komplexy s deriváty N6-benzyladeninu snižovaly jeho transkripci, nevykazovaly žádný výrazný efekt na sekreci IL-1RA (viz příloha 10, obr. 5 na str. 4572). Pro mnoho zánětlivých onemocnění včetně revmatoidní artritidy platí, že jsou doprovázeny zvýšenou produkcí prozánětlivého IL-1 a/nebo naopak sníženou produkcí protizánětlivého IL-1RA, což způsobuje, v konečném důsledku stejně se projevující, absolutní nebo relativní zvýšení hladiny IL-1. Z tohoto důvodu byl pro studované látky stanoven poměr zmíněných cytokinů, tedy IL-1 /IL-1RA (viz příloha 10, obr. 6 na str. 4572). Z výsledků vyplynulo, že dva ze tří studovaných komplexů (komplexy s 2F a 2Cl deriváty N6-benzyladeninu) mají výrazně nižší hodnotu poměru IL-1 /IL-1RA ve srovnání se třetím studovaným komplexem (komplex s 3Me derivátem N6-benzyladeninu), auranofinem i prednisonem (kortikosteroid pro léčbu zánětlivých onemocnění) a naopak, že se tato hodnota neliší od kontrolní skupiny buněk, u kterých nebyla lipopolysacharidem vyvolána zánětlivá reakce. V případě prozánětlivého HMGB1 studované zlatné komplexy během prvních čtyř hodin neovlivňovaly jeho transkripci 49

50 odlišně od auranofinu, avšak po osmi hodinách už mezi nimi lze pozorovat významný rozdíl (viz příloha 10, obr. 7 na str. 4573). Kromě výše zmíněného IL-1RA byl studován také jiný protizánětlivý cytokin, a to IL-10, pro který bylo zjištěno, že jeho transkripci studované látky ovlivňují pouze nepatrně a víceméně srovnatelně s auranofinem a prednisonem (viz příloha 10, obr. 8 na str. 4573). Na úrovni in vitro byl tedy jednoznačně prokázán vliv studovaných zlatných komplexů s deriváty N6-benzyladeninu na transkripci a expresi pro- a protizánětlivých cytokinů, což lze chápat jako výborný základ pro navazující studium protizánětlivé aktivity in vivo. Tyto experimenty byly provedeny tak, že testovaným zvířatům byl podán karagenan, kterým byl vyvolán zánět projevující se jako otok jejich tlapky. Následně byla zvířata léčena jak studovanými komplexy s N6-benzyladeniny, tak také auranofinem a indomethacinem (standardy), které byly intraperitoneálně aplikovány v dávce 10 mg/kg (5 mg/kg pro indomethacin). Změny objemu otoku tlapek testovaných potkanů byly měřeny pletysmometricky po dobu 6 h po podání karagenanu. Jako nejaktivnější ze studie vyšel komplex [Au(2FL)(PPh 3 )], který významně (více než auranofin a indomethacin) snižoval objem otoku potkaních tlapek (viz příloha 10, obr. 9 na str. 4573). Další dva studované komplexy [Au(2ClL)(PPh 3 )] a [Au(3MeL)(PPh 3 )] objem otoku tlapek nesnižovaly. Pro komplex [Au(3MeL)(PPh 3 )] platí, že byl obdobně aktivní jako indomethacin a aktivnější než auranofin, zatímco pro nejméně aktivní [Au(2ClL)(PPh 3 )] lze konstatovat, že jeho aktivita byla podobná té zjištěné pro auranofin. Pro lepší pochopení mechanismů in vivo protizánětlivé aktivity a rozdílů mezi jednotlivými komplexy a použitými standardy byla provedena cytologická a histochemická analýza zvířecích tkání izolovaných po ukončení pletysmometrických pokusů. Vedle klasického hodnocení histologických změn prokazujících zánětlivé procesy v hodnocených tkáních (viz příloha 10, obr. 10 na str. 4574) byla provedena také imunohistochemická analýza kaspázy 3, TNF-α, IL-6 a E selektinu (CD62E) (viz příloha 10, tabulka 1 na str. 4574). Semikvantitativním hodnocením byla zjištěna následující míra poškození tkáníμ [Au(3MeL)(PPh 3 )] > [Au(2ClL)(PPh 3 )] > [Au(2FL)(PPh 3 )] = auranofin. I z těchto výsledků tedy plyne schopnost studovaných zlatných komplexů snižovat projevy zánětlivých procesů. Celkově vzato, z výsledků prezentovaných v práci Trávníček et al., J. Med. Chem., 2012 (příloha 10) plyne, že studované komplexy jsou srovnatelně nebo více in vitro a in vivo protizánětlivě aktivní ve srovnání s konvenčním protizánětlivým léčivem na bázi zlata auranofinem. 50

51 Na výsledky publikované v práci Trávníček et al., J. Med. Chem., 2012 (příloha 10) pak bylo navázáno dalšími pracemi našeho výzkumného týmu, které se zabývají zlatnými komplexy a jejich biologickou (nejen protizánětlivou, ale také protinádorovou) aktivitou [127,139,140]. Jednou z prací, která na uvedenou studii navazuje, je Hošek et al., PLoS ONE, 2013 (příloha 11), ve které je popsána analogická studie podobných komplexů zlata, které se od výše diskutovaných liší pouze tím, že obsahují deriváty N6-benzyladeninu substituované nejen na benzenovém kruhu, ale také na atomu Cβ purinového skeletu (obrázek β8). Tři reprezentativní komplexy, konkrétně to jsou Obrázek 28. Obecný strukturní vzorec studovaných komplexů zlata s deriváty 2-chlor- N6-benzyladeninu. [Au(nL)(PPh 3 )], kde nl symbolizuje deprotonizovaný β-chlor-n6-benzyladenine (L) nebo jeho 2-methoxy (2OMeL) a 4-methyl (4MeL) deriváty, byly podrobeny studiu jejich in vitro a in vivo protizánětlivé aktivity. Také v případě těchto sloučenin byla pozorována in vitro cytotoxicita na THP-1 buňkách (LD 50 ~ 1.5 µm) a při nižších koncentracích naopak navýšení metabolické aktivity nad úroveň kontrolních buněk (viz příloha 11, obr. 2 na str. 4). Na prozánětlivých cytokinech TNF-α a IL-1 bylo zjištěno navýšení jejich exprese závislé na koncentraci komplexů (viz příloha 11, obr. 3 na str. 5 a obr. 4 na str. 6), které navíc bylo výraznější ve srovnání s auranofinem. Ve srovnání s předchozí prací Trávníček et al., J. Med. Chem., 2012 (příloha 10) byl nově studován také vliv komplexů zlata na metaloproteinázy (MMP), který byl výrazný a ve srovnání s auranofinem vyšší (viz příloha 11, obr. 5 na str. 7). S ohledem na poměrně vysokou in vitro protizánětlivou aktivitu bylo poněkud překvapivé zjištění, že na úrovni in vivo studované komplexy žádnou protizánětlivou aktivitu nevykazují. Po jejich aplikaci nebyl pozorován žádný pozitivní vliv na velikost vnějším zásahem vyvolaného edému na tlapkách testovaných potkanů. Histologické studium edematické tkáně ukázalo obdobné projevy zánětlivých reakcí u vzorků všech komplexů (viz příloha 11, obr. 7 na str. 9). Dále byly imunohistochemicky stanoveny kaspáza 3, TNF-α, IL-6 a E selektin, ovšem toto studium bylo ztíženo faktem, že všechny izolované vzorky si byly vzájemně velice podobné bez výrazných rozdílů. Lze ovšem konstatovat, že histologické a histochemické změny korelovaly s výsledky pletysmometrických měření. Stejně jako u předchozích komplexů (viz výše) byla i u zde diskutovaných komplexů prokázána (ESI-MS studium) jejich schopnost interagovat s jednou ze základních síru obsahujících biomolekul 51

52 cysteinem, který ve struktuře původního komplexu nahrazuje příslušný derivát β-chlor-n6- benzyladeninu (viz příloha 11, obr. 8 na str. 10). Celkově vzato, výsledky práce Hošek et al., PLoS ONE, 2013 (příloha 11) získané pro studované komplexy zlata s deriváty 2-chlor-N6-benzyladeninu na úrovni in vitro jsou v kontrastu s výsledky in vivo experimentů a následných histologických a imunohistochemických experimentů. Vysvětlení tohoto rozdílu vyžaduje další studium, možným vysvětlením je nižší stabilita studovaných komplexů ve fyziologických podmínkách, než je tomu v případě komplexů s odlišně substituovanými deriváty N6-benzyladeninu (Trávníček et al., J. Med. Chem., 2012, příloha 10), u kterých výsledky in vitro, in vivo a ex vivo experimentů vzájemně korelovaly Protinádorově účinné ůu(i) komplexy s deriváty 7-azaindolu U zlatných komplexů není studována pouze jejich protizánětlivá aktivita, jak je popsáno výše pro Au(I) komplexy s deriváty N6-benzyladeninu, ale i jiné typy biologických aktivit. Jednou z takových, kde Au(I) komplexy často vykazují farmakologicky perspektivní hodnoty, je aktivita protinádorová [141,142]. S cílem připravit takové Au(I) komplexy, které by mohly být vysoce protinádorově aktivní, byly nasyntetizovány komplexy obecného složení [Au(PPh 3 )(naza)], které obsahují deriváty 7-azaindolu (obrázek β9). Z koordinačně-chemického pohledu je jistě zajímavé, že se deriváty 7-azaindolu koordinují na centrální atom přes dusíkový atom N1 deprotonizovaný přídavkem báze (u výše zmíněných komplexů platiny tomu bylo přes atom dusíku N7), jak bylo prokázáno detailním NMR studiem. Látky byly testovány vůči cisplatina-senzitivnímu karcinomu vaječníku (A2780), cisplatina-rezistentnímu karcinomu vaječníku (Aβ780R) a buněčné linii zdravého lidského fibroblastu (MRC5) (tabulka 5). Z výsledků je patrné, že studované Au(I) komplexy jsou aktivnější na obou nádorových liniích (u většiny látek to lze interpretovat jako schopnost překonávat rezistenci nádorových buněk vůči účinku cisplatiny), ale i na linii nenádorové. Toto se logicky projevuje do nepříliš vysokého a farmakologicky neperspektivního indexu selektivity většiny studovaných látek. Výjimkami jsou komplexy s 3Iaza, 5Braza a 2Me4Claza, jejichž index selektivity je vyšší než 8, čímž tyto látky předurčuje pro další biologická studia (více nádorových buněčných linií, lidské hepatocyty, mechanistická studia, příp. in vivo experimenty). Obrázek 29. Obecný strukturní vzorec studovaných komplexů zlata s deriváty 7-azaindolu. 52

53 Tabulka 5. Výsledky testování in vitro cytotoxicity trifenylfosfan-zlatných komplexů s deriváty 7-azaindolu a cisplatiny vůči lidským nádorovým buněčným liniím cisplatina-senzitivního karcinomu vaječníku A2780, cisplatina-rezistentního karcinomu vaječníku A2780R a buněčné linii lidského zdravého fibroblastu MRC5. Buňky interagovaly s testovanými látkami 24 h. Data jsou uvedena jako IC 50 ± SD (μm). Komplex A2780 A2780R MRC5 RF SF [Au(PPh 3 )(aza)] 3,8±1,1* 4,4±0,8 7,7±1,7 1,16 2,03 [Au(PPh 3 )(3Claza)] 22,4±5,7 21,7±0,8 31,4±4,9 0,97 1,40 [Au(PPh 3 )(3Braza)] 23,γ±γ,4 21,γ±0,8 27,γ±1,7 0,91 1,17 [Au(PPh 3 )(3Iaza)] 3,5±0,4* 11,0±4,7 29,β±5,9 3,14 8,34 [Au(PPh 3 )(5Braza)] 3,1±0,5* 14,4±γ,3 26,0±4,2 4,65 8,39 [Au(PPh 3 )(3Cl5Braza)] 22,λ±β,3 13,8±β,1 26,5±γ,7 0,60 1,16 [Au(PPh 3 )(3I5Braza)] 20,β±β,5 22,1±0,3 27,γ±β,6 1,09 1,35 [Au(PPh 3 )(2Me4Claza)] 2,8±0,7* 8,λ±γ,8 β4,6±0,4 3,18 8,78 Cisplatina 20,γ±β,3 >50,0 a >50,0 a * značí statisticky významný rozdíl hodnot od standardu cisplatiny na p < 0,05 hladině významnosti a) IC 50 nebylo dosaženo do uvedené koncentrace 53

54 5. Závěr V předložené habilitační práci jsou po teoretickém úvodu do uchazečem studované problematiky biologicky aktivních komplexů přechodných kovů předloženy a diskutovány výsledky uchazečovy vědecko-výzkumné činnosti (celkem je okomentováno jedenáct příloh a několik dalších prací, ať už publikovaných nebo v různých fázích přípravy), kterou se od ukončení doktorského studia na podzim roku β010 zabýval na půdě Katedry anorganické chemie a Regionálního centra pokročilých technologií a materiálů, Přírodovědecké fakulty Univerzity Palackého v Olomouci. Tuto činnost lze rozdělit na dva vzájemně nepoměrné tematické celky, kterými jsou protinádorově aktivní komplexy platiny s různými N-donorovými ligandy (deriváty 7-azaindolu, deriváty N6-benzyladeninu) a protizánětlivě aktivní komplexy zlata, které ve své struktuře obsahují stejné typy N-donorových ligandů. V rámci obou zmíněných tematických celků se podařilo připravit látky, které svými biologickými účinky mnohonásobně převyšují konvenční chemoterapeutika na bázi platiny (cisplatina), resp. zlata (auranofin). Za všechny zde bude zmíněn alespoň cis-[pti 2 (4Braza) 2 ] komplex obsahující 4-brom-7-azaindol, který vykazuje téměř padesátkrát vyšší in vitro cytotoxicitu vůči nádorovým buňkám lidského osteosarkomu (HOS), komplex [Pt(ox)(4OMeros) 2 ] s 4-methoxy derivátem roskovitinu, který v in vivo experimentech probíhajících na myších s implementovanou lymfocytární leukémií efektivně prodlužoval (na rozdíl od cisplatiny) průměrnou délku života testovaných zvířat a jehož aplikace navíc nevyvolávala negativní vedlejší účinky spojené s terapií léčivy na bázi platiny, nebo protizánětlivě aktivní komplex [Au(2FL)(PPh 3 )] s N6-(2-fluorbenzyl)adeninem, který během in vivo experimentů snižoval objem vnějším zásahem vyvolaného zánětlivého otoku potkaních tlapek, což nedokázala ani klinicky používaná léčiva auranofin (metaloterapeutikum na bázi zlata) a indomethacin (kortikosteroid). U většiny připravených farmakologicky perspektivních látek byly v rámci širšího autorského kolektivu (kolegové z Katedry buněčné biologie a genetiky a z Katedry biofyziky resp. Biofyzikálního ústavu AV ČR) na různých úrovních studovány aspekty jejich mechanismu účinku. Rozhodně lze konstatovat, že se v souladu s cíli uchazečovy vědecko-výzkumné práce vytyčenými v Úvodu předložené habilitační práce podařilo připravit biologicky perspektivní koordinační sloučeniny vybraných přechodných kovů, které se ukázaly býti vysoce biologicky (především protinádorově) aktivními, a které, v případě protinádorově aktivních komplexů platiny, účinně překonávaly získanou rezistenci nádorových buněk vůči klinicky 54

55 používanému chemoterapeutiku cisplatině a působily selektivně na nádorové buňky. Mnohé z těchto látek mají potenciál pro to být dále studovány směrem k budoucím preklinickým a klinickým studiím. Obdobně byly připraveny a fyzikálně-chemicky prostudovány magnetické nanočástice na bázi oxidů železa funkcionalizované biologicky aktivními komplexy platiny, které plní požadavky pro magnetický cílený transport zmíněných terapeutik. Uvedená tématika, tedy vývoj nových biologicky aktivních koordinačních sloučenin přechodných kovů, i v současnosti je, a s výhledem do blízké budoucnosti také bude, hlavním vědecko-výzkumným zaměřením uchazeče. Z hlavních směrů výzkumu, ať už probíhajících nebo plánovaných, lze uvést např. studium karboxylato komplexů platiny s biologicky perspektivními N-donorovými ligandy pro účinnější akumulaci cílovou nádorovou buňkou, studium fotoaktivovatelných komplexů platiny s protinádorově aktivním N-donorovým ligandem odstupujícím po aplikaci vhodného záření a následně synergicky biologicky působícím pro účinnější chemoterapii bez negativních vedlejších účinků, organokovové komplexy biologicky perspektivních neplatinových přechodných kovů (např. Ru, Ir, Os) s vybranými N-donorovými ligandy vykazující jiný typ protinádorové aktivity ve srovnání s komplexy na bázi platiny nebo již výše v práci zmíněné studium funkcionalizovaných nanočástic na bázi oxidů železa pro magneticky asistovaný cílený transport léčiv potenciálně spojený také s diagnostickou funkcí. 55

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66 [10] Trávníček, Z.; Štarha, P.; Vančo, J.; Šilha, T.; Hošek, J.; Suchý, P.; Pražanová, G. Anti-inflammatory active gold(i) complexes involving 6-substituted-purine derivatives. J. Med. Chem. 55 (2012) [11] Hošek, J.; Vančo, J.; Štarha, P.; Paráková, L.; Trávníček, Z. Effect of 2-chlorosubstitution of adenine moiety in mixed-ligand gold(i) triphenylphosphine complexes on anti-inflammatory activity: the discrepancy between the in vivo and in vitro models. PLoS ONE 8 (2013) e

67 PŘÍLOHA 1 Štarha, P.; Marek, J.; Trávníček, Z. Cisplatin and oxaliplatin derivatives involving 7-azaindole: Structural characterisations Polyhedron 33 (2012)

68 Polyhedron 33 (2012) Contents lists available at SciVerse ScienceDirect Polyhedron journal homepage: Cisplatin and oxaliplatin derivatives involving 7-azaindole: Structural characterisations Pavel Štarha a, Jaromír Marek b, Zdeněk Trávníček a, a Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, CZ Olomouc, Czech Republic b Department of Inorganic Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, CZ Olomouc, Czech Republic article info abstract Article history: Received 31 October 2011 Accepted 30 November 2011 Available online 13 December 2011 Keywords: Platinum(II) complexes 7-Azaindole X-ray structure Multinuclear NMR In vitro cytotoxicity Platinum(II) dichlorido and oxalato (ox) complexes of the composition cis-[ptcl 2 (Haza) 2 ](I) and [Pt(ox) (Haza) 2 ]H 2 O(IIH 2 O) were prepared by a one-step syntheses from K 2 [PtCl 4 ] and K 2 [Pt(ox) 2 ]2H 2 O, respectively (Haza symbolises 7-azaindole). The complexes were characterised by a set of spectroscopic methods (IR, Raman and multinuclear and 2D NMR), as well as by mass spectrometry, elemental (C, H, and N) and thermal (TG/DTA) analyses. Single-crystal X-ray analysis was performed for cis-[ptcl 2 (Haza) 2 ]DMF (IDMF) and [Pt(ox)(Haza) 2 ] (II), obtained by recrystallization of the complexes I and IIH 2 O from N,N 0 -dimethylformamide (DMF). Both complexes, of a square-planar geometry, involve a tetracoordinated central Pt(II) atom with two monodentate N7-coordinated 7-azaindole molecules in cis-positions, and with the coordination sphere completed by two chloride anions (I; PtN 2 Cl 2 donor set) or one bidentatecoordinated oxalate dianion (II; PtN 2 O 2 donor set). The complexes did not show any in vitro cytotoxic effect, as evaluated by an MTT assay against breast adenocarcinoma and osteosarcoma human cancer cell lines up to a concentration of 1.0 lm (for I) and 0.1 lm (for IIH 2 O), given by their limited solubility in the medium used. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Up until now, several platinum(ii) complexes involving 7-azaindole (Haza) have been reported [1 7]; those whose structure contains derivatives of Haza are described in the literature [8 18]. From a coordination chemistry point of view, Haza may serve as a monodentate or bidentate N-donor ligand, which can be coordinated to the metal centre(s) through its N1 and/or N7 atoms. As for the platinum(ii) complexes, the following coordination modes of the 7-azaindole moiety to the Pt(II) atom were determined by crystallographic studies. [NBu 4 ][Pt(C 6 F 5 ) 2 (Haza)(aza)]Haza involves the Haza molecule coordinated through the N7 atom, however its deprotonated form (aza) is coordinated through the N1 atom [3]. The same work also reported the X-ray structures of [NBu 4 ] 2 [{Pt(C 6 F 5 ) 2 } 2 (l-oh)(l-aza)] (aza coordinates through N1 and N7), [Pt(aza)(dmba)(PPh 3 )] and [Pt(aza)(dmba)(dmso)] (both with aza coordinated through N1); dmba stands for N,C-bidentate coordinated N,N 0 -dimethylbenzylamine. A deprotonated form of 7-azaindole coordinates through N1 and N7 to Pt(II) and Ag(I) in the structure of [NBu 4 ][{(C 6 F 5 ) 3 Pt}(l-aza){Ag(Me 2 CNH)}] (Me 2 CNH = acetone imine) [5]. 1-Diphenylphosphino-7-azaindole (dppaza) is bound to the Pt(II) atom through N7 in [PtCl 2 (dppaza)] Corresponding author. Tel.: ; fax: address: zdenek.travnicek@upol.cz (Z. Trávníček). [8]. Zhao et al. prepared platinum complexes involving 1-(2 0 -pyridyl)-7-azaindole (paza), which is bidentate-coordinated through the N7 atom of the 7-azaindole ring and the nitrogen atom of pyridyl in the case of [Pt(paza)(Me) 2 ], while in the structure of an organoplatinum tetrameric macrocycle, [Pt(paza)(Me)] 4, it is also bound through the C2 atom of the 7-azaindole moiety [11]. A group of numerous complexes with two 7-azaindole molecules linked together by various linkers bound to the N1 atoms and coordinated to platinum through N7, such as [Pt(Ph) 2 (1,3-bam)], have been reported in the literature [12] [bam = N,N-bis(7-azaindolyl)methane, Ph = phenyl]. Similar compounds have also been described in the literature, as follows: [PtCl(1,3-bab)] and [PtCl(Brbab)] with 1,3-bis(N7-azaindolyl)benzene (1,3-bab) and 1-bromo-3,5-bis(N7-azaindolyl)benzene (Brbab), coordinated through the mentioned N7 atoms as well as through one carbon atom of the aromatic ring [13]; [Pt(1,2-bab)Me 2 ], [Pt(1,2- bab)ph(sme 2 )][BAr 0 4], [Pt(1,2-bab)(Bz)(SMe 2 )][BAr 0 4], [Pt(1,2- bab)(bz)(mecn)][bar 0 4] were reported in [15], where Bz = benzyl and Ar 0 = 3,5-bis(trifluoromethyl)phenyl; Zhao et al. prepared [Pt(L)(CHMePh)(MeCN)][BAr 0 4] (L stands for 1,2-bab and bam) and [Pt(1,2-bab)(l 3 -CHMePh)][BAr 0 4] [16]. A different coordination mode of the 7-azaindole moiety was described for the complexes with bam and its isomer linked through the N1 and N7 atoms (L 0 ), which are involved in [Pt(L 0 )Me 2 ] and [Pt(L 0 )Ph(SMe 2 )][BAr 0 4], where the azaindole-based ligands are coordinated via the /$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi: /j.poly

69 P. Štarha et al. / Polyhedron 33 (2012) nitrogen atoms not substituted by a CH 2 linker [14]. A different linker, namely Me 3 Si-substituted methylene, connects both 7-azaindole moieties within the bam derivative (Me 3 Si-bam) structure of [Pt(Me 3 Si-bam)Me 2 ] and [PtI 2 (Me 3 Si-bam)][{PtIMe 3 } 4 ] [17]. As it is known from the literature, as well as from the recently reported results of our research team, platinum(ii) complexes involving an organic compound, which is physiologically potent itself, as a ligand coordinated to the metal centre show significant biological effects, such as in vitro cytotoxicity. With respect to the fact that the derivatives of 7-azaindole are known to be physiologically active substances, which show various types of activities in dependence on their structure [19 25], we decided to prepare simple platinum(ii) dichlorido and oxalato complexes with these substances (concretely halogeno derivatives of 7-azaindole) and to study their in vitro cytotoxicity. Preliminary results regarding one of the mentioned complexes showed that its in vitro cytotoxicity was evaluated as several times higher than that of cisplatin (unpublished data). The herein described cis-[ptcl 2 (Haza) 2 ](I) and [Pt(ox)(Haza) 2 ](IIH 2 O) were prepared and characterised as model compounds with respect to syntheses optimizations and physicochemical properties. It has to be stated that the title complexes were, together with cis-[pti 2 (Haza) 2 ], reported by Harrison et al. [1] and their in vivo toxicity and antitumor activity against Yoshida Sarcoma, Osteosarcoma, ADJ/PC6A tumour and P388 Lymphocytic leukaemia were studied on rats. Interestingly, neither [1], nor any of the articles cited therein, nor any other literature source have described the synthesis and characterisation of these compounds as yet. 2. Experimental 2.1. Starting materials K 2 [PtCl 4 ], K 2 (ox)h 2 O, 7-azaindole and solvents were purchased from Sigma Aldrich Co., Acros Organics Co. and Lachema Co. and they were used as received. K 2 [Pt(ox) 2 2H 2 O was prepared according to the literature [26] Synthesis of cis-[ptcl 2 (Haza) 2 ](I) and [Pt(ox)(Haza) 2 ]H 2 O (IIH 2 O) 7-Azaindole (1.0 mmol) and 0.5 mmol of K 2 [PtCl 4 ] (for I) or K 2 [Pt(ox) 2 ]2H 2 O (for IIH 2 O) were separately dissolved in a minimum volume of hot ethanol and hot distilled water, respectively (Scheme 1). The solutions were mixed together and the reaction mixture was stirred at 50 C for 2 days. The obtained products were filtered off and washed with distilled water and ethanol, and then dried at a temperature of 40 C. The results of the elemental analysis, mass spectrometry and IR, Raman and NMR spectroscopy are given in Supplementary data Physical methods Elemental analyses were carried out on a Flash 2000 CHNS Elemental Analyzer (Thermo Scientific). Infrared spectra were recorded on a Nexus 670 FT-IR (Thermo Nicolet) in the cm 1 (KBr pellets) and cm 1 (Nujol technique) regions. Raman spectra were recorded using an NXR FT-Raman Module (Thermo Nicolet) between 150 and 3750 cm 1. The reported IR and Raman signal intensities have been defined as w = weak, m = middle, s = strong and vs = very strong. Mass spectra of acetonitrile solutions of the complexes were obtained by LCQ Fleet ion trap mass spectrometry using the ESI technique in both the positive and negative modes (Thermo Scientific). All the observed isotopic distribution representations were compared with the theoretical ones (QualBrowser software, version 2.0.7, Thermo Fischer Scientific). Simultaneous TG/DTA analyses were performed using an Exstar TG/DTA 6200 thermal analyzer (Seiko Instruments Inc.); 100 ml min 1 dynamic air atmosphere, C (2.5 C min 1 ). 1 H, 13 C and 195 Pt NMR spectra and two dimensional correlation experiments ( 1 H 1 H gs-cosy, 1 H 13 C gs-hmqc, 1 H 13 C gs-hmbc) of the DMF-d 7 solutions were measured at 300 K on a Varian 400 device. 1 H and 13 C spectra were adjusted against the signals of tetramethylsilane (Me 4 Si). 195 Pt spectra were calibrated against K 2 [PtCl 6 ]ind 2 O, found at 0 ppm. 1 H 15 N gs-hmbc experiments were obtained at natural abundance and calibrated against the residual signals of N,N 0 -dimethylformamide (DMF) adjusted to 8.03 ppm ( 1 H) and ppm ( 15 N). The splitting of proton resonances in the reported 1 H spectra is defined as s = singlet, d = doublet, t = triplet, br = broad band, dd = doublet of doublets, m = multiplet Single crystal X-ray analysis In efforts to obtain crystals suitable for a single-crystal X-ray analysis, the compounds cis-[ptcl 2 (Haza) 2 ] (I) and [Pt(ox)(Haza) 2 ]H 2 O(IIH 2 O) were recrystallized from DMF, which led to the formation of cis-[ptcl 2 (Haza) 2 ]DMF (IDMF) and [Pt(ox)(Haza) 2 ] (II). The X-ray data of IDMF and II were collected on an Xcalibur 2 diffractometer (Oxford Diffraction Ltd.) with a Sapphire2 CCD detector and with MoKa radiation (Monochromator Enhance, Oxford Diffraction Ltd.). Data collection and reduction were performed using the CrysAlis software [27]. The same software was used for data correction for absorption effects by the empirical absorption correction using spherical harmonics, implemented in the SCALE3 ABSPACK scaling algorithm. The structure was solved by direct methods using SHELXS-97 and refined on F 2 using the full-matrix least-squares procedure (SHELXL-97) [28]. Non-hydrogen atoms were refined anisotropically and hydrogen atoms were located in difference maps, fixed in their theoretical geometrical positions with distances of N H = 0.88 Å, C H = 0.95 Å and Scheme 1. Schematic pathway for the preparation of cis-[ptcl 2 (Haza) 2 ](I) and [Pt(ox)(Haza) 2 ]H 2 O(IIH 2 O) given together with the atom numbering and colour labelling of ring A (Haza moiety involving C and N atoms), ring B (Haza moiety involving C 0 and N 0 atoms) and ring C (atoms of the PtX 2 N 2 coordination environment, where X symbolises Cl for I and O for IIH 2 O). (Colour online.)

70 406 P. Štarha et al. / Polyhedron 33 (2012) C methyl H = 0.98 Å, and refined isotropically by using the riding model with U iso (H) = 1.2U eq (C, N) [1.5U eq (C) for methyl atoms]. The crystal data and structure refinements are given in Table 1. Molecular graphics as well as additional structural calculations were drawn and interpreted using DIAMOND [29] and Mercury [30] In vitro cytotoxicity In vitro cytotoxic activity was determined by an MTT assay [MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in human breast adenocarcinoma (MCF7; ECACC No ) and human osteosarcoma (HOS; ECACC No ) cancer cell lines purchased from the European Collection of Cell Cultures (ECACC). For more details see Supplementary data. 3. Results and discussion 3.1. General and spectroscopic properties We prepared I and IIH 2 O using the simple one-step syntheses starting from K 2 [PtCl 4 ] and K 2 [Pt(ox) 2 ]2H 2 O, respectively, which reacted with two molar equivalents of 7-azaindole (see Scheme 1). The contents of C, H and N were proven by elemental analysis, showing the composition and purity of the prepared complexes. The results of thermal analysis proved complex I to be non-solvated, while IIH 2 O was found to be monohydrated (Dm between 26 and 132 C: calc. 3.3% for H 2 O; found 3.7%); the TG and DTA curves of IIH 2 O are given in Supplementary data (Fig. S1). ESI- mass spectrometry of I (see Fig. S2 in Supplementary data) detected the molecular peak of [PtCl 2 (Haza)(aza)], its [{PtCl 2 (aza) 2 }+Na] adduct with the sodium cation, as well as Table 1 Crystal data and structure refinements for cis-[ptcl 2 (Haza) 2 ]DMF (IDMF) and [Pt(ox)(Haza) 2 ](II). IDMF Empirical formula C 14 H 12 Cl 2 N 4 PtC 3 H 7 NO C 16 H 12 N 4 O 4 Pt Formula weight T (K) 120(2) 120(2) Wavelength (Å) Crystal system orthorhombic monoclinic Space group P P2 1 /c a (Å) (12) (3) b (Å) (16) (6) c (Å) (3) (4) a ( ) b ( ) (4) c ( ) V (Å 3 ) (5) (10) Z 4 4 D calc (g cm 3 ) Absorption coefficient (mm 1 ) Crystal size (mm) F (000) h Range for data collection ( ) h h Index ranges (h, k, l) 10 6 h h k k l l 6 13 Reflections collected/unique (R int ) 18048/3409 (0.0213) 9856/2778 (0.0368) Data/restraints/parameters 3409/0/ /0/226 Goodness-of-fit (GOF) on F Final R indices [I >2r(I)] R 1 = , wr 2 = R 1 = , wr 2 = R indices (all data) R 1 = , wr 2 = R 1 = , wr 2 = Largest peak and hole (e Å 3 ) 0.746, , II peaks whose m/z values and isotopic distribution corresponded to the [PtCl(aza) 2 ] and [PtCl 2 (aza)] fragments (see Supplementary data). Similarly, the mass spectrum of IIH 2 O contains the molecular peaks of the [Pt(ox)(Haza)(aza)] and [{Pt(ox)(aza) 2 }+Na] adducts (Fig. S2). Fragments of the compositions [Pt(ox)(aza)] and [aza] were also observed in the mass spectrum of the studied oxalato complex. Further, mass spectrometry performed in the positive mode (ESI+) detected the [{PtCl 2 (Haza) 2 }+K] + adduct of complex I with the potassium cation at m/z and fragments whose m/z values of and correspond to the composition of [{Pt(Haza)(aza)}] + and [Haza+H] +, respectively. The spectrum also contains the peaks of [{PtCl 2 (Haza) 2 } 2 +K] +, [{PtCl 2 (Haza) 2 } 2 +Na] + and [PtCl(Haza) 3 ] +, which are formed by the recombinations of the molecules of the studied complex with other ones or with the free Haza molecule. The ESI+ mass spectrum of IIH 2 O contains the [{Pt(ox)(Haza) 2 }+Na] + adduct at m/z, its fragments of [{Pt(Haza)(aza)}] +, [{Pt(ox)(Haza)}+Na] + and [Haza+H] +, and [{Pt(ox)(Haza) 2 } 2 +K] +, [{Pt(ox)(Haza) 2 } 2 +Na] + and [{Pt(ox)(Haza) 2 } 2 +H] + adducts. The Raman and IR spectra of I and IIH 2 O(Supplementary data) contain all the vibrations of 7-azaindole [31], whose maxima positions were slightly shifted and their intensities changed as well, due to the coordination of Haza to the Pt(II) atom. However, several facts should be discussed in more details: (1) the Raman vibrations of free Haza at 1586 and 1603 cm 1 (IR: 1584 and 1600 cm 1 ) were found as a single vibration in this region for I and IIH 2 O; (2) the band in the IR spectrum of free Haza detected at 1499 cm 1 was split in the spectra of both complexes; (3) the Raman vibration of Haza at 1044 cm 1 was not detected in the spectra of the complexes; (4) the Raman vibration at 1338 cm 1 (IR: 1343 cm 1 ) remained unchanged in the case of IIH 2 O, but it split into two peaks in the spectra of I; (5) the Raman spectrum of I contains, contrary to Haza, a vibration of a medium intensity at 338 cm 1 assignable to the Pt Cl bond, and another additional band was observed at 1090 cm 1 ; (6) additional bands at 1128, 484 and 467 cm 1 were detected in the IR spectrum of I as compared to free Haza; (7) the maximum of Haza of the strong intensity at 765 cm 1 was not found in the IR spectra of the complexes; (8) as for the difference between the spectra of free Haza and IIH 2 O, several more bands were detected in the Raman (values in the text) and IR (values in parentheses) spectra of this complex, those at 933, 1383 (1393), 1654 (1654), (1671) and 1689 (1705) cm 1 belong to the vibrations of the oxalate dianion, and the vibration with a maximum at 483 (484) cm 1 can be assigned to a five-membered ring (PtO 2 C 2 ) deformation [32]. All the signals in the 1 H, 13 C and 1 H 15 N gs-hmbc spectra of 7- azaindole were found in the corresponding spectra of both complexes (Table 2). The 1 H 15 N gs-hmbc experiments proved the coordination via the N7 atom of Haza, since the coordination shifts (calculated as Dd = d complex d ligand ) of this atom equal ppm for I and ppm for IIH 2 O, while the Dd values of the second nitrogen atom involved in the structure of Haza (N1) are 2.34 ppm (I) and 1.72 ppm (IIH 2 O). The coordination of Haza to the metal centre through N7 causes an electron density redistribution, which leads to the upfield shift of the C7a atom, which neighbours the coordination site. Other carbon atoms, including the C6 atom situated, as well as the above mentioned C7a, next to N7, are shifted downfield. Surprisingly, the Dd values of the carbons neighbouring the coordination site of the Haza moiety are not the highest ones (Table 2): Dd (C6) in the case of both studied complexes is exceeded by Dd (C3a), and Dd (C4) and Dd (C7a) are exceeded by all the carbon atoms of I and IIH 2 O, except for the C5 atom of I. The 13 C NMR spectrum of IIH 2 O involves one more signal (166.1 ppm) as compared with that of free Haza and I, which belongs to the carbon atoms (C11, C12) of the oxalate

71 P. Štarha et al. / Polyhedron 33 (2012) Table 2 The results of NMR spectroscopy given as 1 H, 13 C and 15 N NMR chemical shifts d/coordination shifts (Dd = d complex d ligand ) [ppm] obtained and calculated for the prepared complexes cis-[ptcl 2 (Haza) 2 ](I) and [Pt(ox)(Haza) 2 ]H 2 O(IIH 2 O). 1 H NMR 13 C NMR 15 N NMR N1H C2H C4H C5H C6H C2 C3 C3a C4 C5 C6 C7a N1 N7 I 13.13/ 1.43 IIH 2 O 13.07/ / / / / / / / / / / / / / / / / / / / / / / / / / / Fig. 1. The molecular structure of cis-[ptcl 2 (Haza) 2 ]DMF (IDMF) with the nonhydrogen atoms depicted as thermal ellipsoids at the 50% probability level and given with the atom numbering scheme (except for C3A 0 and C7A 0 ). Fig. 3. Part of the crystal structure of cis-[ptcl 2 (Haza) 2 ]DMF (IDMF) showing selected non-covalent interactions of the N HO, N HC, C HC and C HCl types depicted as dashed green (hydrogen bonds) and orange lines; hydrogen atoms not involved in the depicted interactions are omitted for clarity. (Colour online.) d = ppm for I, and ppm for IIH 2 O. It corresponds with our previously obtained 195 Pt NMR spectroscopy results of cisdichlorido [33] and oxalato [26,34] complexes involving monodentate-coordinated N-donor N6-benzyladenine-based ligands, whose signals were found at ca and 1685 ppm, respectively. Fig. 2. The molecular structure of [Pt(ox)(Haza) 2 ](II) with the non-hydrogen atoms depicted as thermal ellipsoids at the 50% probability level and given with the atom numbering scheme (except for C3A 0 and C7A 0 ). dianion, bidentately-coordinated to the metal centre. The highest changes of the chemical shift in the 1 H NMR spectra were found for the N1H and C6H protons, which in the case of N1H is probably given by the steric hindrance caused by the coordination of the Haza molecules to the central Pt(II) atom and by the involvement of the N1H proton in the non-covalent bond system. The coordination shift of the C6H proton relates to the coordination through the neighbouring N7 atom. The 195 Pt NMR spectra showed one signal, however, it was detected at very different positions: 3.2. Single crystal X-ray analysis of cis-[ptcl 2 (Haza) 2 ]DMF (IDMF) and [Pt(ox)(Haza) 2 ](II) The complexes show a slightly distorted square-planar geometry in the vicinity of the tetracoordinated central Pt(II) atom. The dichlorido complex I (Fig. 1) adopts a PtCl 2 N 2 donor set with the chlorine atoms mutually arranged in cis positions, while a PtO 2 N 2 coordination environment of II (Fig. 2) involves a bidentate-coordinated oxalate anion with two Haza ligands in a cis fashion. The bond lengths and angles formed by the atoms of both coordination environments can be found in Table 3. The Pt N bond lengths of both structures are somehow shorter as compared with the average value of this bond determined for platinum(ii) dichlorido [2.013(25) Å] and oxalato [1.997(14) Å] complexes with two N-donor imine ligands deposited in Crystallographic Structural

72 408 P. Štarha et al. / Polyhedron 33 (2012) In vitro cytotoxicity As was already mentioned above, Harrison et al. [1] reported I and II as substances showing no in vivo toxic and antitumor effect against Yoshida Sarcoma, Osteosarcoma, ADJ/PC6A tumour and P388 Lymphocytic leukaemia on rats. On the other hand, these complexes were prepared as model compounds of platinum(ii) dichlorido and oxalato complexes involving various derivatives of 7-azaindole whose in vitro cytotoxic effects were found to be significantly higher as compared with the platinum-based anticancer drug cisplatin (unpublished results). Because of these facts, we decided to evaluate the in vitro cytotoxicity of I and IIH 2 O, however, it could not be exactly determined owing to the limited solubility of the complexes in the used medium, and thus the obtained IC 50 values can be expressed only as >1.0 lm for I and >0.1 lm for IIH 2 O against both cell lines (IC 50 of cisplatin, formerly determined by the same method, equals 19.6 lm against MCF7 and 34.2 lm against HOS; [26]). Fig. 4. Part of the crystal structure of [Pt(ox)(Haza) 2 ](II) showing selected noncovalent interactions of the N HO, C HO, C HC and CC types depicted as dashed green (hydrogen bonds) and orange lines; hydrogen atoms not involved in the depicted interactions are omitted for clarity. (Colour online.) Table 3 Selected bond lengths (Å) and angles ( ) of cis-[ptcl 2 (Haza) 2 ]DMF (IDMF) and [Pt(ox)(Haza) 2 ](II). Bond lengths IDMF II Bond angles IDMF Pt N (2) 2.001(4) N7 Pt N (9) 87.8(2) Pt N (2) 2.009(4) N7 Pt X (7) 92.12(14) Pt Cl (7) N7 Pt X (7) (14) Pt Cl (7) N7 0 Pt X (7) (14) Pt O (3) N7 0 Pt X (7) 97.4(2) Pt O (3) X1 Pt X (3) 82.68(13) X = Cl for IDMF and O for II. Database (CSD, ver , August 2011 update [35]). Similarly, the Pt Cl [2.296(13) Å] and Pt O [2.009(18) Å] bond lengths of the previously mentioned reported structures found in CSD can be compared with the parameters determined for our complexes, and it can be said that the values correlate well, except for the Pt O2 bond which is significantly longer. The differences of the angles around the central atom are caused by the presence of the bidentate oxalate anion in the structure of II, resulting in significantly different values of analogous X1 Pt X2 angles (X = Cl for IDMF and O for II), as well as in different values for the other angles given in Table 2. The planes (see Scheme 1) fitted through atoms of both Haza moieties (ring A involves C and N atoms, ring B involves C 0 and N 0 atoms) and atoms of the PtX 2 N 2 coordination environment (ring C; X symbolises Cl for IDMF and O for II) form the following dihedral angles: \AB = 82.23(7) (IDMF) and 86.60(12) (II), \AC = 66.81(5) (IDMF) and 68.49(10) (II), \BC = 88.82(4) (IDMF) and 89.41(7) (II). The crystal structures of IDMF (see Fig. 3) and II (see Fig. 4) contain N HO hydrogen bonds with NO distances and N HO angles ranging from 2.803(3) to 3.067(6) Å, and 163.9(2) to 174.4(3), respectively, which fall to the interval of values typical for hydrogen bonds of medium strength (DA Å and \NHO ; see Ref. [36]). The other non-covalent contacts detected in the crystal structures are of the types N HC, C HC and C HCl (for IDMF; Fig. 3), and C HC, C HO and CC (for II; Fig. 4), whose parameters are summarised in Table S1 in Supplementary data. II 4. Conclusions The cis-[ptcl 2 (Haza) 2 ](I) and [Pt(ox)(Haza) 2 ]H 2 O(IIH 2 O) complexes with 7-azaindole were synthesised and thoroughly characterised. The crystallographic study of the title complexes showed a slightly distorted square-planar geometry with two monodentate 7-azaindole molecules coordinated through the N7 atoms to the metal centre. The in vitro cytotoxicity of both I and IIH 2 O was studied, but the complexes were inactive up to a concentration of 1.0 lm, and 0.1 lm, respectively, given by their limited solubility in the medium used. For all that, we believe in the biological potential of such types of complexes, which motivates us for future work. Now we are studying structural analogues of the herein presented compounds, involving halogeno-derivatives of 7-azaindole. Based on the preliminary results we can confirm the generally known fact that only an insignificant structural modification of the molecule may significantly improve its biological interest because the changes lead to the preparation of biologically attractive platinum(ii) compounds with in vitro cytotoxicity several times better than that of cisplatin. After the completion of these results, they will become the subject of a future publication. Acknowledgements The authors gratefully thank the Grant Agency of the Czech Republic (P207/11/0841), the Operational Program Research and Development for Innovations European Regional Development Fund (CZ.1.05/2.1.00/ ), the Operational Program Education for Competitiveness European Social Fund (CZ.1.07/2.3.00/ ) of the Ministry of Education, Youth and Sports of the Czech Republic, and Palacký University (PrF_2011_014) for financial support. The authors also thank Dr. Radim Vrzal for carrying out the cytotoxicity tests. Appendix A. Supplementary data CCDC and contain the supplementary crystallographic data for cis-[ptcl 2 (Haza) 2 ]DMF (IDMF) and [Pt(ox)(Haza) 2 ](II). These data can be obtained free of charge via or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) ; or deposit@ccdc.cam.ac.uk. Supplementary data associated with this article can be found, in the online version, at doi: /j.poly

73 P. Štarha et al. / Polyhedron 33 (2012) References [1] R.C. Harrison, C.A. McAuliffe, M.E. Friedman, Inorg. Chim. Acta 92 (1984) 43. [2] E.J. New, C. Roche, R. Madawala, J.Z. Zhang, T.W. Hambley, J. Inorg. Biochem. 103 (2009) [3] J. Ruiz, V. Rodríguez, C. de Haro, A. Espinosa, J. Pérez, C. Janiak, Dalton Trans. 39 (2010) [4] J.M. Casas, B.E. Diosdado, J. Forniés, A. Martín, A.J. Rueda, A.G. Orpen, Inorg. Chem. 47 (2008) [5] J.M. Casas, J. Forniés, A. Martín, A.J. Rueda, Organometallics 21 (2002) [6] S.W. Lai, M.C.W. Chan, K.K. Cheung, S.M. Peng, C.M. Che, Organometallics 18 (1999) [7] J.A. Bailey, V.M. Miskowski, H.B. Gray, Inorg. Chem. 32 (1993) 369. [8] H.L. Milton, M.V. Wheatley, A.M.Z. Slawin, J.D. Woollins, Inorg. Chem. Commun. 7 (2004) [9] A.D. Burrows, M.F. Mahon, M. Varrone, Dalton Trans. (2003) [10] Z.M. Hudson, S.B. Zhao, R.Y. Wang, S. Wang, Chem. Eur. J. 15 (2009) [11] S.B. Zhao, R.Y. Wang, S. Wang, J. Am. Chem. Soc. 129 (2007) [12] S.B. Zhao, G.H. Liu, D. Song, S. Wang, Dalton Trans. (2008) [13] D. Song, Q.G. Wu, A. Hook, I. Kozin, S.N. Wang, Organometallics 20 (2001) [14] D. Song, S. Wang, Organometallics 22 (2003) [15] S.B. Zhao, D.T. Song, W.L. Jia, S.N. Wang, Organometallics 24 (2005) [16] S.B. Zhao, G. Wu, S. Wang, Organometallics 25 (2006) [17] S.B. Zhao, R.Y. Wang, S. Wang, Organometallics 28 (2009) [18] S.B. Zhao, Q. Cui, S. Wang, Organometallics 29 (2010) 998. [19] P.J. Cox, T.N. Majid, S. Amendola, S.D. Deprets, C. Edlin, J.Y.Q. Lai, A.D. Morley, US Patent , [20] M. Klein, US Patent , [21] H.J. Dyke, S. Price, K. Williams, US Patent , [22] G.N. Anilkumar, S.B. Rosenblum, S. Venkatraman, F.G. Njoroge, J.A. Kozlowski, US Patent , [23] D. Simard, Y. Leblanc, C. Berthelette, M.H. Zaghdane, C. Molinaro, Z. Wang, M. Gallant, S. Lau, T. Thao, M. Hamel, R. Stocco, N. Sawyer, S. Sillaots, F. Gervais, R. Houle, J.F. Lévesque, Bioorg. Med. Chem. Lett. 20 (2010) [24] S. Hong, S. Lee, B. Kim, H. Lee, S.-S. Hong, S. Hong, Bioorg. Med. Chem. Lett. 21 (2011) 841. [25] C. Marminon, A. Pierré, B. Pfeiffer, V. Pérez, S. Léonce, P. Renard, M. Prudhomme, Bioorg. Med. Chem. 11 (2003) 679. [26] P. Štarha, Z. Trávníček, I. Popa, J. Inorg. Biochem. 104 (2010) 639. [27] CrysAlis CCD and CrysAlis RED, Version , Oxford Diffraction Ltd., England, [28] G.M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr. 64 (2008) 112. [29] K. Brandenburg, DIAMOND, Release 3.1f, Crystal Impact GbR, Bonn, Germany, [30] C.F. Macrae, I.J. Bruno, J.A. Chisholm, P.R. Edgington, P. McCabe, E. Pidcock, L. Rodriguez-Monge, R. Taylor, J. van de Streek, P.A. Wood, J. Appl. Crystallogr. 41 (2008) 466. [31] B. Karthikeyan, Spectrochim. Acta, Part A 64 (2006) [32] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, fifth ed., Wiley-Interscience, New York, [33] L. Szüčová, Z. Trávníček, I. Popa, J. Marek, Polyhedron 27 (2008) [34] Z. Trávníček, P. Štarha, I. Popa, R. Vrzal, Z. Dvořák, Eur. J. Med. Chem. 45 (2010) [35] F.A. Allen, Acta Crystallogr., Sect. B: Struct. Sci. 58 (2002) 380. [36] G. Gilli, P. Gilli, The Nature of the Hydrogen Bond: Outline of a Comprehensive Hydrogen Bond Theory, Oxford, New York, 2009.

74 PŘÍLOHA 2 Štarha, P.; Trávníček, Z.; Popa, A.; Popa, I.; Muchová, T.; Brabec, V. How to modify 7-azaindole to form cytotoxic Pt(II) complexes: Highly in vitro anticancer effective cisplatin derivatives involving halogeno-substituted 7-azaindole J. Inorg. Biochem. 115 (2012) 57 63

75 Journal of Inorganic Biochemistry 115 (2012) Contents lists available at SciVerse ScienceDirect Journal of Inorganic Biochemistry journal homepage: How to modify 7-azaindole to form cytotoxic Pt(II) complexes: Highly in vitro anticancer effective cisplatin derivatives involving halogeno-substituted 7-azaindole Pavel Štarha a, Zdeněk Trávníček a,, Alexandr Popa a, Igor Popa a, Tereza Muchová b,c, Viktor Brabec c a Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, CZ Olomouc, Czech Republic b Department of Biophysics, Faculty of Sciences, Palacký University, 17. listopadu 12, CZ Olomouc, Czech Republic c Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolská 135, CZ Brno, Czech Republic article info abstract Article history: Received 13 April 2012 Accepted 17 May 2012 Available online 24 May 2012 Keywords: Platinum(II) complexes 7-azaindole derivatives X-ray structure Multinuclear NMR In vitro cytotoxicity DNA binding The platinum(ii) dichlorido and oxalato complexes of the general formula cis-[ptcl 2 (nhaza) 2 ] (1 3) [Pt(ox)(nHaza) 2 ](4 6) involving 7-azaindole halogeno-derivatives (nhaza) were prepared and thoroughly characterized. A single-crystal X-ray analysis of cis-[ptcl 2 (3ClHaza) 2 ] DMF (1 DMF; 3ClHaza symbolizes 3-chloro-7-azaindole) revealed a distorted square-planar arrangement with both the 3ClHaza molecules coordinated through their N7 atoms in a cis fashion. In vitro cytotoxicity of the complexes was evaluated by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay against the HOS (osteosarcoma), MCF7 (breast adenocarcinoma) and LNCaP (prostate adenocarcinoma) human cancer cell lines. The dichlorido complexes 1 3 (IC 50 =3.8, 3.9, and 2.5 μm, respectively) showed significantly higher in vitro anticancer effect against HOS as compared with cisplatin, whoseic 50 =37.7 μm. The biological effect of cisplatin against MCF7 (IC 50 =24.5 μm) and LNCaP (IC 50 =3.8μM) was also exceeded by 1 3 (except for 2 against LNCaP), but the difference can be classified as significant only in the case of 1 (IC 50 =3.4 μm) and 3 (IC 50 =2.0 μm) against MCF7. The molecular pharmacological studies (RNA synthesis by T7 RNA polymerase in vitro) provedthat1 3 bind to DNA in a similar manner as cisplatin, since the RNA synthesis products of 1 3 and cisplatin showed a similar sequence profile of major bands Elsevier Inc. All rights reserved. 1. Introduction The 7-azaindole (Haza) derivatives have been utilized and patented as biologically effective organic compounds with various types of activities, such as anti-proliferative [1], protein-kinase inhibition [2] and treatment of kinase-induced diseases [3], anticancer and anti-inflammatory [4], andantiviral[5]. Other works such as those of Simard et al. [6], Marminon et al. [7] and Hong et al. [8] are also noteworthy, since they describe Haza derivatives of various structural types showing high biological activity, namely anti-proliferative activity [6,7] or CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) receptor antagonistic activity [8]. With respect to these facts it is quite surprising that only three works have dealt with platinum(ii) complexes involving 7-azaindolebased ligands and described a biological study of such compounds. The cis-[ptcl 2 (Haza)(NH 3 )] and trans-[ptcl 2 (Haza)(NH 3 )] complexes Corresponding author. Tel.: ; fax: address: zdenek.travnicek@upol.cz (Z. Trávníček). were studied for their cytotoxic activity in vitro against A2780 human ovarian carcinoma cells and they showed lower activity as compared with cisplatin (see Section 3.2) [9]. J. Ruiz et al.[10] prepared several mono- and dinuclear complexes of platinum with 7-azaindole, such as [Pt(dmba)(aza)(dmso)] showing submicromolar cytotoxic activity against A2780, its cisplatin-resistant analog A2780R and human breast cancer cells T47D; dmba = N,N -dimethylbenzylamine, aza = a deprotonated form of 7-azaindole. The complexes cis-[ptcl 2 (Haza) 2 ], [Pt(ox)(Haza) 2 ]andcis-[pti 2 (Haza) 2 ] were studied in vivo for their antitumor activity against Yoshida sarcoma, osteosarcoma, ADJ/PC6A tumor and P388 lymphocytic leukemia on rats, but surprisingly no activity was observed [11]. One more conclusion of the literature research regarding 7-azaindole derivatives (nhaza) has to be mentioned 3-chloro-7- azaindole (3ClHaza), 3-iodo-7-azaindole (3IHaza) and 5-bromo-7- azaindole (5BrHaza) have not been reported as ligands of transition metal complexes to date. In other words, these heterocyclic compounds can be taken into account as novel ligands from the coordination chemistry point of view. One of our latest works reported synthesis, characterization and cytotoxicity in vitro, determined by an MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide] assay against breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines, of /$ see front matter 2012 Elsevier Inc. All rights reserved. doi: /j.jinorgbio

76 58 P. Štarha et al. / Journal of Inorganic Biochemistry 115 (2012) two of the above-mentioned substances [12]. The cis-[ptcl 2 (Haza) 2 ] (IC 50 >1.0 μm against both cell lines) and [Pt(ox)(Haza) 2 ](IC 50 >0.1 μm against both cell lines) complexes showed very limited solubility in the medium used (0.1% DMF in water; DMF = N,N -dimethylformamide), which disallowed exact evaluation of their cytotoxicity in vitro. Thus, one of the topics of the present work was how to modify the Haza molecule to increase both solubility and cytotoxicity of the final platinum(ii) complexes. One of the important early phases of the mechanism by which platinum compounds exert their anticancer activity is the formation of adducts on nuclear DNA by these agents [13 15]. Hence, data on the DNA binding mode of platinum complexes are of interest. In the present study, we have also applied a methodology based on transcription mapping of DNA adducts in a cell-free medium previously developed for cisplatin and its analogs to investigate the reaction of 1 3 with natural DNA. 2. Materials and methods 2.1. Preparation of the platinum(ii) dichlorido (1 3) and oxalato (4 6) complexes K 2 [PtCl 4 ] (0.5 mmol) was dissolved in a minimum of distilled water and poured into the ethanolic solution of 3ClHaza, 3IHaza, and 5BrHaza (1.0 mmol), respectively. The mixture was stirred at 50 C for two days and then the product was filtered off and washed by distilled water (5 ml) and ethanol (5 ml). The obtained complexes cis-[ptcl 2 (3ClHaza) 2 ](1), cis-[ptcl 2 (3IHaza) 2 ] 0.75EtOH (2) andcis-[ptcl 2 (5BrHaza) 2 ] 0.5EtOH (3) weredriedatthetemperature of 40 C (Fig. 1). K 2 [Pt(ox) 2 ] 2H 2 O (0.5 mmol) dissolved in a minimum volume of distilled water was added to 1.0 mmol of 3ClHaza, 3IHaza or 5BrHaza dissolved in a minimum volume of ethanol. The obtained products of the composition [Pt(ox)(3ClHaza) 2 ] 0.2EtOH (4), [Pt(ox)(3IHaza) 2 ] 0.75H 2 O (5), [Pt(ox)(5BrHaza) 2 ] 0.75H 2 O(6) werefiltered off after two days of stirring at 50 C, washed by 5 ml of distilled water and 5 ml of ethanol, and dried at the temperature of 40 C (Fig. 1). 1: Anal. calc. for C 14 H 10 N 4 Cl 4 Pt: C, 29.44%; H, 1.76%; N, 9.81%. Found: C, 29.02%; H, 1.65%; N, 9.33%. 1 H NMR (DMF-d 7,SiMe 4,ppm):δ (br, N1H, 1H), 9.02 (d, 5.6, C6H, 1H), 8.07 (d, 6.5, C4H, 1H), 8.05 (s,c2h,1h),7.29(m,c5h,1h). 13 C NMR (DMF-d 7, SiMe 4, ppm): δ (C6), (C7a), (C4), (C2), (C3a), (C5), (C3). 15 N NMR (DMF-d 7, ppm): δ (N1), (N7). 195 Pt NMR (DMF-d 7,K 2 PtCl 6, ppm): δ : Anal. calc.forc 14 H 10 N 4 Cl 2 I 2 Pt 0.75EtOH: C, 23.61%; H, 1.85%; N, 7.10%. Found: C, 23.32%; H, 1.68%; N, 6.73%. 1 H NMR (DMF-d 7, SiMe 4,ppm):δ (br, N1H, 1H), 8.98 (dd, 5.7, 1.1, C6H, 1H), 8.09 (d, 2.6, C2H, 1H), 7.82 (d, 7.6, C4H, 1H), 7.28 (m, C5H, 1H). 13 CNMR(DMF-d 7,SiMe 4, ppm): δ (C7a), (C6), (C2), (C4), (C3a), (C5), (C3). 15 NNMR(DMF-d 7, ppm): δ (N1), (N7). 195 Pt NMR (DMF-d 7,K 2 PtCl 6, ppm): δ : Anal. calc.forc 14 H 10 N 4 Cl 2 Br 2 Pt 0.5EtOH: C, 26.37%; H, 1.91%; N, 8.20%. Found: C, 25.93%; H, 1.74%; N, 8.27%. 1 HNMR(DMF-d 7, SiMe 4, ppm): δ (br, N1H, 1H), 9.25 (d, 1.7, C6H, 1H), 8.31 (d, 1.6, C4H, 1H), 7.91 (t, 3.0, C2H, 1H), 6.65 (m, C3H, 1H). 13 C NMR (DMF-d 7, SiMe 4, ppm): δ (C7a), (C6), (C4), (C2), (C3a), (C5), (C3). 15 N NMR (DMF-d 7, ppm): δ (N1), (N7). 195 Pt NMR (DMF-d 7,K 2 PtCl 6, ppm): δ : Anal. calc.forc 16 H 10 N 4 Cl 2 O 4 Pt 0.2EtOH: C, 32.97%; H, 1.89%; N, 9.38%. Found: C, 33.25%; H, 1.78%; N, 9.35%. 1 HNMR(DMF-d 7, SiMe 4, ppm): δ (br, N1H, 1H), 8.82 (dd, 5.8, 1.0, C6H, 1H), 8.22 (dd, 7.9, 1.0, C4H, 1H), 8.09 (s, C2H, 1H), 7.32 (m, C5H, 1H). 13 C NMR (DMF-d 7,SiMe 4, ppm):δ (C11, C12), (C6), (C7a), (C4), (C2), (C3a), (C5), (C3). 15 N NMR (DMF-d 7,ppm):δ (N1), (N7). 195 Pt NMR (DMF-d 7,K 2 PtCl 6, ppm): δ : Anal.calc.forC 16 H 10 N 4 I 2 O 4 Pt 0.75H 2 O: C, 24.49%; H, 1.48%; N, 7.14%. Found: C, 24.18%; H, 1.20%; N, 6.65%. 1 H NMR (DMF-d 7,SiMe 4, ppm): δ (br, N1H, 1H), 8.79 (dd, 5.9, 1.1, C6H, 1H), 8.11 (s, C2H, 1H), 7.96 (dd, 8.0, 1.2, C4H, 1H), 7.30 (m, C5H, 1H). 13 CNMR(DMF-d 7, SiMe 4, ppm): δ (C11, C12), (C7a), (C6), (C2), (C4), (C3a), (C5), (C3). 15 NNMR(DMF-d 7,ppm):δ (N1), (N7). 195 Pt NMR (DMF-d 7,K 2 PtCl 6, ppm): δ : Anal. calc. for C 16 H 10 N 4 Br 2 O 4 Pt 0.75H 2 O: C, 27.82%; H, 1.68%; N, 8.11%. Found: C, 27.86%; H, 1.39%; N, 7.61%. 1 H NMR (DMF-d 7, SiMe 4, ppm): δ (br, N1H, 1H), 9.16 (d, 1.9, C6H, 1H), 8.47 (d, 1.9, C4H, 1H), 7.96 (d, 3.5, C2H, 1H), 6.74 (d, 3.4, C3H, 1H). 13 C NMR (DMF-d 7, SiMe 4,ppm):δ (C11, C12), (C7a), (C6), (C4), (C2), (C3a), (C5), (C3). 15 N NMR (DMF-d 7,ppm):δ (N1), (N7). 195 Pt NMR (DMF-d 7,K 2 PtCl 6,ppm):δ The results of thermogravimetric (TG) and differential thermal (DTA) analyses, electrospray ionization (ESI) mass spectrometry, and IR and Raman spectroscopy are given in the Supplementary data Materials and physical measurements Fig. 1. Synthesis and schematic representation of the cis-[ptcl 2 (3ClHaza) 2 ](1), cis- [PtCl 2 (3IHaza) 2 ] 0.75EtOH (2), cis-[ptcl 2 (5BrHaza) 2 ](3), [Pt(ox)(3ClHaza) 2 ] 0.2EtOH (4), [Pt(ox)(3IHaza) 2 ] 0.75H 2 O(5) and [Pt(ox)(5BrHaza) 2 ] 0.75H 2 O(6) complexes given together with the 7-azaindole derivatives involved in their structures. K 2 [PtCl 4 ], K 2 (ox) H 2 O, 3-chloro-7-azaindole (3ClHaza), 3-iodo-7- azaindole (3IHaza) and 5-bromo-7-azaindole (5BrHaza) and solvents were purchased from Sigma-Aldrich Co., Acros Organics Co., Lachema Co. and Fluka Co. and they were used as received. K 2 [Pt(ox) 2 ] 2H 2 O was prepared according to the literature [16]. Elemental analyses were carried out on a Flash 2000 CHNS Elemental Analyzer (Thermo Scientific). Infrared spectra were recorded on a Nexus 670 FT-IR (Thermo Nicolet) in the cm 1 (ATR technique) and cm 1 (Nujol technique) regions. Raman spectra of 1, 2, 4 and 6 (the complexes 3 and 5 burnt under laser beam) were recorded using an

77 P. Štarha et al. / Journal of Inorganic Biochemistry 115 (2012) NXR FT-Raman Module (Thermo Nicolet) between 150 and 3750 cm 1. Mass spectra of the acetonitrile solutions of complexes were obtained by an LCQ Fleet ion trap mass spectrometer using the ESI-technique (Thermo Scientific). All the observed isotopic distribution representations were compared with the theoretical ones (QualBrowser software, version 2.0.7, Thermo Fischer Scientific). Simultaneous TG/DTA analyses were performed using an Exstar TG/DTA 6200 thermal analyzer (Seiko Instruments Inc.); ceramic crucible, 100 ml min 1 dynamic air atmosphere, C temperature range and temperature gradient of 2.5 C min 1. 1 H, 13 Cand 195 Pt NMR spectra and two dimensional correlation experiments ( 1 H 1 Hgs- COSY, 1 H 13 C gs-hmqc, 1 H 13 C gs-hmbc; 1 H 15 N gs-hmbc; gs = gradient selected, COSY = correlation spectroscopy, HMQC = heteronuclear multiple quantum coherence, HMBC = heteronuclear multiple bond coherence) of the DMF-d 7 solutions were measured at 300 K on a Varian 400 device at MHz ( 1 H), MHz ( 13 C), MHz ( 195 Pt) and MHz ( 15 N). 1 Hand 13 Cspectra were adjusted against the signals of tetramethylsilane (Me 4 Si). 195 Pt spectra were calibrated against potassium hexachloroplatinate (K 2 PtCl 6 )in D 2 Ofoundat0ppm. 1 H 15 N gs-hmbc experiments were obtained at natural abundance and calibrated against the residual signals of DMF adjusted to 8.03 ppm ( 1 H) and ppm ( 15 N). The splitting of proton resonances in the reported 1 Hspectraisdefined as s = singlet, d = doublet, t = triplet, br = broad band, dd = doublet of doublets, m = multiplet. The single crystal X-ray data of the selected crystal of cis-[ptcl 2 (3- ClHaza) 2 ] DMF (1 DMF), obtained by a slow evaporation of a DMF solution, was obtained using an Xcalibur 2 diffractometer (Oxford Diffraction Ltd.) with a Sapphire2 CCD detector, and with Mo Kα (Monochromator Enhance, Oxford Diffraction Ltd.). Data collection and reduction were performed using the CrysAlis software [17]. Thesame software was used for data correction for an absorption effect by the empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. The structure was solved by direct methods using SHELXS-97 and refined on F 2 using the full-matrix least-squares procedure (SHELXL-97) [18]. Non-hydrogen atoms were refined anisotropically and hydrogen atoms were located in a difference map and refined by using the riding model with C\H=0.95 and 0.98 Å, and N\H=0.88Å, with U iso (H)=1.2 U eq (CH, NH) and 1.5 U eq (CH 3 ). The crystal data and structure refinements are given in Table S1. The molecular graphics as well as additional structural calculations were drawn and interpreted using DIAMOND [19] and Mercury [20]. Plasmid psp73kb (2455 bp) was isolated according to standard procedures. Restriction endonucleases NdeI andhpai were obtained from New England Biolabs (Beverly, MA). T7 RNA polymerase and RNasin ribonuclease inhibitor were purchased from Promega (Mannheim, Germany). Ribonucleotide triphosphates were from Roche Diagnostics, GmbH (Mannheim, Germany). [α- 32 P]rCTP was from MP Biomedicals, LLC (Irvine, CA) In vitro cytotoxic activity testing In vitro cytotoxic activity was determined by an MTT assay in human breast adenocarcinoma (MCF7; ECACC no ), human osteosarcoma (HOS; ECACC no ) and prostate adenocarcinoma (LNCaP; ECACC no ) cancer cell lines purchased from European Collection of Cell Cultures (ECACC). The cells were cultured according to the ECACC instructions and they were maintained at 37 C and 5% CO 2 in a humidified incubator. Human cancer cells were treated with 1 6 and cisplatin (applied up to 50 μm) for 24 h, using multi-well culture plates of 96 wells. In parallel, the cells were treated with vehicle (DMF; 0.1%, v/v) and Triton X-100 (1%, v/v) to assess the minimal (i.e. positive control) and maximal (i.e. negative control) cell damage, respectively. The MTT assay was measured spectrophotometrically at 540 nm (TECAN, Schoeller Instruments LLC) Statistical evaluation The data were expressed as the percentage of viability, when 100% and 0% represent the treatments with DMF and Triton X-100, respectively. The cytotoxicity data from the cancer cell lines were acquired from three independent experiments (conducted in triplicate) using cells from different passages. The IC 50 values were calculated from viability curves. The results are presented as arithmetic mean±sd. The significance of the differences between the results was assessed by ANOVA analysis with pb0.05 considered to be significant (QC Expert 3.2, Statistical software, TriloByte Ltd.) DNA transcription by RNA polymerase in vitro A double-stranded DNA template was prepared by digesting the psp73kb plasmid (2455 bp) with NdeIandHpaI restriction endonucleases. The resulting two fragments, 212- and 2243-bp long, were separated on a 1% agarose gel in the buffer containing 40 mm Tris acetate (ph 8), 1 mm EDTA and 0.5 mg ml 1 ethidium bromide. The 212-bp fragment was isolated from the gel, purified using Wizard_SV and PCR Clean-Up System and incubated with the complexes 1, 2, 3 or cisplatin in NaClO 4 (10 mm) for 24 h at 37 C in the dark. At the end of the incubation, the samples were precipitated by ethanol and dissolved in the TE buffer (10 mm Tris Cl, ph 7.4, 1 mm EDTA). The level of platination (r b, the number of molecules of the platinum complex bound per nucleotide residue) in aliquots of these samples was checked by flameless atomic absorption spectrometry. In this way, the analyses of DNA transcription were performed in the absence of unbound (free) platinum complexes. Transcription of the 212-bp (NdeI/HpaI) restriction fragment with DNA-dependent T7 RNA polymerase and electrophoretic analysis of transcripts were performed according to the protocols recommended by Promega (Promega Protocols and Applications, (1989/90)) and previously described in detail [21]. 3. Results and discussion 3.1. Preparation and structural characterization The pale yellow platinum(ii) complexes of the composition cis- [PtCl 2 (nhaza) 2 ] xsolv (1 3; xsolv stands for 0.75EtOH for 2 and 0.5EtOH for 3) were prepared from K 2 PtCl 4 in the mixture of water and ethanol at 50 C with a yield of 70 80% (Fig. 1). The oxalato complexes, [Pt(ox)(nHaza) 2 ] xsolv (4 6; xsolv = 0.2EtOH for 4 and 0.75H 2 Ofor5 and 6), were isolated as the pale yellow (4, 6) orpale gray (5) powders with yields of ~90% (Fig. 1). The empirical formulas, including the water (5 and 6) and ethanol (2, 3 and 4)molecules of crystallizations, resulted from the elemental (C, H, N) and thermal (TG/DTA) analyses (see Supplementary data, Fig. S1). Negativemode electrospray mass spectrometry detected molecular peaks of all the studied complexes at m/z =570 (1), 752 (2), 658 (3), 586 (4), 770 (5) and676(6) (see Supplementary data, Fig. S2). All the mass spectra of Pt(II)-dichlorido complexes (1 3) also contain a peak whose mass corresponds to the fragment of the composition [PtCl 2 (naza)]. Analogical fragments, [Pt(ox)(naza)], were also detected in the spectra of the oxalato complexes (4 6). All the 1 Hand 13 CsignalsoffreenHaza molecules were detected in the NMR spectra of 1 6 and their positions were, due to their coordination to the PtCl 2 (for 1 3) or Pt(ox) (for 4 6) motif, somehow shifted as compared with the free nhaza molecules (Table 1). The 13 C NMR signals of 4 6 observed at ppm are assignable to the carbon atoms (C11, 12) of the oxalate dianion. The 1 H 15 N gs-hmbc experiments of the complexes showed both the nitrogen atoms of the 7- azaindole moiety, however, they were found to be significantly differently shifted (Table 1), which proved the N7-coordination mode of nhaza molecules to the Pt(II) ion, as it was crystallographically determined for 1 DMF (see below). The 195 Pt chemical shifts of Pt(II)-

78 60 P. Štarha et al. / Journal of Inorganic Biochemistry 115 (2012) Table 1 The 1 H, 13 C and 15 N NMR coordination shifts (Δδ=δ complex δ ligand ; ppm) of the 7-azaindole moiety atoms and 195 Pt chemical shifts (δ; ppm) of H NMR 13 C NMR 15 N NMR 195 Pt N1H C2H C3H C4H C5H C6H C2 C3 C3a C4 C5 C6 C7a N1 N dichlorido (1 3) (~ 2120 ppm) and oxalato (4 6) (~ 1780 ppm) complexes differ (see also Table 1) similarly to the platinum(ii) dichlorido and oxalato complexes with unsubstituted 7-azaindole [12]. Although the studied complexes involve differently halogenosubstituted 7-azaindole derivatives, their IR and Raman spectra are very similar and involve the bands (recently reported for 7-azaindole [22]) detected at and cm 1, which are assignable to the C\N, and C\C stretching vibrations, respectively, supporting the presence of the nhaza molecules within the structures of the complexes. The IR and Raman spectra of the studied dichlorido complexes (1 3) showed the bands of the ν(pt\cl) vibration at ca. 340 cm 1, while the bands assignable to ν(pt\n) were observed at ca. 510 cm 1 [23]. The spectra of the oxalato complexes (4 6) contain the peaks of the oxalate dianion vibrations, such as ν as (C_O) at cm 1 and ν s (C\O) at cm 1. The bands of a PtO 2 C 2 ring deformation appeared in the IR and Raman spectra of 4 6 at ca. 450 cm 1 (see Supplementary data for more detailed information about IR and Raman spectra). A single crystal X-ray analysis of 1 DMF proved the tetracoordinated Pt(II) atom within a square-planar PtCl 2 N 2 donor set formed by two chlorine ions and by two N7 nitrogen atoms of 3ClHaza molecules, through which they are coordinated to the metal center, while the second nitrogen of the 7-azaindole moiety, i.e. N1, binds a hydrogen atom (Fig. 2). The distortion of the square-planar geometry of 1 DMF can be clearly demonstrated by the values of the N7\Pt1\Cl1 and N7A\Pt1\Cl2 bond angles, which were found to be (10) and (10), respectively. The angle formed by the planes fitted through the non-hydrogen atoms of both 3ClHaza molecules equals 88.41(8). These planes form the angles of 80.22(6) and 82.18(6) with the plane created through the atoms of the PtCl 2 N 2 chromophore. The crystal structure of 1 DMF is stabilized by N\H O hydrogen bonds and other non-covalent contacts of the C\H Cl, C\H C, N\H C, C Cl and C C types (see Supplementary data, Fig. S3 and Table S2). evaluation (pb0.05) showed complex 3 as significantly more active than 1 and 2 against all the MCF7, HOS and LNCaP cells, and complex 1 was found to be significantly more effective against MCF7 as compared with complex 2. It has to be stated that only the dichlorido complexes 1 3 were evaluated as in vitro cytotoxic against the MCF7 and HOS cancer cells. The oxalato analogs of 1 3, i.e. 4 6, were found to be inactive up to the concentration of 10.0 (4), 25.0 (5) and 0.5 (6) μm, primarily because of their limited solubility in the used water/dmf mixture. However, the oxalate dianion was recently predicted [24] and several times reported (e.g. lit. [25 27]) in a biological perspective as suitable leaving group of cytotoxic platinum(ii) complexes and the substances involving this group were found to be more effective against cancer cells as compared with compounds involving different leaving groups including chlorine ions. With respect to this fact, it is quite a surprise that the oxalato complexes 4 6 were found to be at least three-times (for 4) and even ten-times (for 6) less effective as compared with their highly in vitro cytotoxic dichlorido analogs 1, and 3, respectively In vitro cytotoxic activity The complexes 1 6 and clinically used platinum-based drug cisplatin (applied within the concentration range of μm, unless their solubility is lower) were studied by an MTT assay for their in vitro cytotoxicity against the MCF7 breast adenocarcinoma, HOS osteosarcoma and LNCaP prostate adenocarcinoma human cancer cell lines. The obtained IC 50 values (μm) of 1 3 and cisplatin are given in Table 2 and graphically depicted in Fig. 3. The figure does not contain the results of the remaining substances (4 6), which were found to be cytotoxic inactive in the studied concentration ranges given by their solubility in the used water/dmf mixture, thus their cytotoxicity can be evaluated as >10.0 (for 4), >25.0 (for 5) and >0.5 (for 6) μm asitisgivenin Table 2. The prepared complexes 1 3 (except for 2 against MCF7) are significantly more cytotoxic in vitro (ANOVA, pb0.05) against MCF7 and HOS as compared with cisplatin is (see Fig. 3), with the IC 50 values (Table 2) at least 2.5-times lower than cisplatin. In the case of the LNCaP cancer cell line, none of the complexes showed significantly higher biological effect as compared to cisplatin. Analogical statistical Fig. 2. Themolecularstructureofcis-[PtCl 2 (3ClHaza) 2 ] DMF (1 DMF) with the nonhydrogen atoms depicted as thermal ellipsoids at the 50% probability level and given with the atom numbering scheme. The DMF molecule of crystallization is omitted for clarity. Pt1\N7=2.020(3) Å, Pt1\N7A=2.012(3) Å, Pt1\Cl1=2.2946(10) Å, Pt1\Cl2=2.2982(10) Å, N7\Pt1\N7A=90.10(12), N7\Pt1\Cl2=88.91(9), N7A\Pt1\Cl1=87.76(9) and Cl1\Pt1\Cl2=93.35(4).

79 P. Štarha et al. / Journal of Inorganic Biochemistry 115 (2012) Table 2 The results of the in vitro cytotoxic activity testing of 1 6 and cisplatin against human breast adenocarcinoma (MCF7), osteosarcoma (HOS) and prostate adenocarcinoma (LNCaP) cell lines: cells were treated with tested compounds for 24 h; measurements were performed in triplicate, and cytotoxicity experiment was repeated in three different cell passages; data are expressed as IC 50 ±SD (μm). Complex MCF7 HOS LNCaP 1 3.4±0.3* 3.8±0.1* 3.3± ± ±0.2* 3.8± ±0.2* 2.5±0.1* 1.5±0.4 4 >10.0 a >10.0 a nt 5 >25.0 a >25.0 a >25.0 a 6 >0.5 a >0.5 a nt Cisplatin 19.6± ± ±1.5 nt, not tested. Asterisks (*) indicate significantly different values (pb0.05) between 1 6 and cisplatin. a IC 50 values were not reached due to the limited solubility and they can be expressed as >0.5, >10.0 and >25.0 μm. In the case of 2 and 5, which both involve the 3IHaza carrier ligands, the oxalato complex is several times less soluble in the used water/ DMF mixture than the dichlorido one. A comparison with the above-mentioned platinum(ii) complexes involving 7-azaindole as N-donor ligand, whose biological activity was determined (see Section 1), is not relevant, because they were tested against different cells and they also differ in composition and the type of ligands involved in their structures. Concretely, the dichlorido complex cis-[ptcl 2 (Haza)(NH 3 )] (IC 50 =3.6 μm), which is structurally related to 1 3, and its trans isomer (IC 50 =6.0 μm) showed 2.6- and 4.3-times lower biological effect against A2780, respectively, than cisplatin (IC 50 =1.4 μm) [9]. The cytotoxicity in vitro of structurally different [Pt(dmba)(aza)(dmso)] complex, which involves only one 7-azaindole molecule within its structure, was found to be submicromolar against A2780, A2780cis and T47D with IC 50 values equalled 0.34 μm, 0.45 μm, and 0.53 μm, respectively, which is 2.6-times (A2780), 24.4-times (A2780cis) and even times (T47D) more active than cisplatin [10]. Finally, the most active complex 3 involves 5-bromo-7-azaindole within its structure, which allows us to carry out another conclusion regarding the in vitro cytotoxicity of the prepared compounds. It seems that the substitution of the C5 position of the 7-azaindole moiety is more convenient for the resulting biological, particularly in vitro antitumour, activity, but this assumption has to be proved by testing Fig. 4. Inhibition of RNA synthesis by T7 RNA polymerase on the NdeI/HpaI fragment of psp73kb plasmid modified by complexes 1, 2, 3 or cisplatin. (A) Autoradiogram of 6% polyacrylamide/8 M urea sequencing gel. Lanes: control nonmodified template; cispt, 1, 2, 3 the template modified by cisplatin, complex 1, 2, and 3 at r b =0.01, respectively; A, U, G, C chain terminated marker RNAs. (B) Schematic diagram showing the portion of the nucleotide sequence of the template of the NdeI/HpaI fragment used to monitor inhibition of RNA synthesis by Pt II complexes. The arrow indicates the start of the T7 RNA polymerase. (o), major stop signals (from A). The numbers correspond to the nucleotide numbering in the sequence map of psp73kb plasmid. of the complexes with identically substituted 7-azaindole ring in its C3 and C5 positions Transcription mapping of DNA adducts Fig. 3. The results of the in vitro cytotoxicity testing obtained by an MTT assay against human MCF7 breast adenocarcinoma, HOS osteosarcoma and LNCaP prostate adenocarcinoma cell lines for complexes 1 3 (the results determined for 4 6 are not depicted, because their IC 50 values were not reached due to the limited solubility). The cells were exposed to the compounds for 24 h. Measurements were performed in triplicate and each cytotoxicity experiment was repeated three times. The given IC 50 ±S.D. (μm) values represent an arithmetic mean. The asterisk (*) denotes significant difference (pb0.05) between the studied complexes and cisplatin. The mechanism of action of clinically used anticancer platinum drugs, such as cisplatin and its analogs, involves coordination to purine DNA bases. The resulting DNA damages are then encountered and processed by specific cellular proteins, events that determine the ultimate outcome of DNA damage [15,28]. Among these proteins, RNA polymerase has become a major focus of study because of its various roles in processing damaged DNA. Several types of DNA lesions, including cisplatin cross-links, inhibit transcription by blocking RNA polymerase [29,30]. Arrested RNA polymerase not only functions as a damage recognition factor, eliciting transcription-coupled repair, but also triggers programmed cell death, or apoptosis [31]. In order to determine binding of 1, 2 and 3 to natural DNA, sequence specificity of this binding and the effects on RNA synthesis,

80 62 P. Štarha et al. / Journal of Inorganic Biochemistry 115 (2012) we also employed in the present work a method which consists in RNA synthesis by T7 RNA polymerase in vitro. This method was used in the same way as in several previous studies of the sequence specificity of various DNA-damaging agents including platinum drugs [21,32 34]. T7 RNA polymerase was chosen to initiate these investigations because it is well characterized, its promoter is clearly defined, and the purified enzyme is commercially available. RNA synthesis by various RNA polymerases including T7 RNA polymerase on DNA templates containing several types of bifunctional adducts of platinum complexes can be prematurely terminated at the level or in the proximity of adducts [21,32]. Importantly, monofunctional DNA adducts of several platinum complexes including cisplatin are unable to terminate RNA synthesis [21,35]. Cutting of psp73kb DNA [12] by NdeI and HpaI restriction endonucleases yielded a 212-bp fragment (a substantial part of its nucleotide sequence is shown in Fig. 4B). This fragment contained T7 RNA polymerase promoter in the upper strand close to its 3 -end (Fig. 4B). The experiments were carried out using this linear DNA fragment, randomly modified by cisplatin, 1, 2, or3 at r b =0.01, for RNA synthesis by T7 RNA polymerase (Fig. 4A, lanes cispt, 1, 2, and 3, respectively). RNA synthesis on the template modified by the platinum complexes yielded fragments of defined sizes, which indicates that RNA synthesis on these templates was prematurely terminated. These results suggest that new Pt complexes 1, 2 and 3 were able to bind DNA forming adducts capable to stall RNA polymerase. The sequence analysis revealed that the major bands resulting from termination of RNA synthesis by the adducts of all platinum(ii) dichlorido complexes involving 7-azaindole halogeno-derivatives were similar to those produced by cisplatin, i.e. they appeared mainly at GG and AG sites. This indicates that 1, 2, and3 bind to DNA preferentially at the sites similar to preferential DNA binding sites of cisplatin and that they also form similar types of DNA adducts with a similar frequency as cisplatin. These initial data might be also interpreted to mean that the enhanced activity of the analogs of cisplatin 1, 2,or3 is associated with some features of the damaged DNA and/or its cellular processing not perceptible by the method used in the present work or to an event that is not related to DNA binding. There are many biochemical factors affecting activity of platinum compounds in tumor cells [15,28,36,37]. Further studies are, therefore, warranted to reveal a relative contribution of all potential factors contributing to the enhanced potency of analogs of cisplatin containing 7-azaindole substituted by halogeno-substituents. 4. Conclusions The synthesized platinum(ii) dichlorido and oxalato complexes of the composition cis-[ptcl 2 (3ClHaza) 2 ](1), cis-[ptcl 2 (3IHaza) 2 ] 0.75EtOH (2), cis-[ptcl 2 (5BrHaza) 2 ] 0.5EtOH (3), [Pt(ox)(3ClHaza) 2 ] 0.2EtOH (4), [Pt(ox)(3IHaza) 2 ] 0.75H 2 O(5) and [Pt(ox)(5BrHaza) 2 ] 0.75H 2 O (6) involving 7-azaindole-based N-donor ligands (nhaza) were thoroughly characterized (e.g. multinuclear and 2D NMR spectroscopy, ESI mass spectrometry, X-ray analysis). Their geometry was determined as the distorted square-planar with the appropriate nhaza molecules coordinated through their N7 atoms, as proved by a singlecrystal X-ray analysis of the 1 DMF representative. The complexes were screened for their in vitro cytotoxicity against the MCF7, HOS and LNCaP human cancer cell lines. The complexes 1 3 showed significantly higher biological effect (pb0.05) against MCF7 and HOS as compared with commercially used platinum-based drug cisplatin (except for 2 against MCF7). The IC 50 values of the most active complex3 equalled 2.0 μm (MCF7) and 2.5 μm (HOS), which is about 10- times, and 14-times, respectively, higher cytotoxic activity than cisplatin. The mechanism of action of 1 3 was studied by means of transcription inhibition by DNA adducts of 1 3, which is in the case of cisplatin considered to be one of the major routes by which this anticancer drug kills cancer cells [31]. Thus, the results of the present work also suggest that active platinum complexes 1 3 bind to major pharmacological target of platinum antitumor drugs, DNA, and that the resulting DNA adducts effectively inhibit DNAdependent-RNA-synthesis. As one of the key factors that is important for platinum drug-mediated cytotoxicity is the arrest of RNA synthesis by Pt-DNA adducts [38] then the latter observation may also imply that inhibition of transcription by DNA adducts of 1 3 might significantly contribute to their cytotoxic effects. The reported complexes with high cytotoxicity in vitro (i.e. 1 3) involve halogeno-substituted derivatives of 7-azaindole within their structures in contrast to previously reported platinum(ii) dichlorido complex with unsubstituted 7-azaindole, cis-[ptcl 2 (Haza) 2 ], which was found to be both in vitro [12] and in vivo [11] cytotoxic inactive. The slight modification of 7-azaindole molecule by the halogeno substituents improved the solubility and bio-availability of the platinum(ii) complexes with these ligands involved in their structure. With respect to this fact, our future work dealing with the platinum complexes involving 7-azaindole derivatives will be focused on the complexes with a 7-azaindole moiety substituted by more halogeno-substituents or by substituent/s of different types, which is believed to lead to even more biologically effective platinum(ii) complexes. Acknowledgments The authors (PS, AP, IP, ZT) gratefully thank the Czech Science Foundation (GAČR P207/11/0841), Operational Program Research and Development for Innovations European Regional Development Fund (CZ.1.05/2.1.00/ ), Operational Program Education for Competitiveness European Social Fund (CZ.1.07/2.3.00/ ) of the Ministry of Education, Youth and Sports of the Czech Republic and by Palacký University in Olomouc (PrF_2012_009). The research of TM and VB was supported by the Czech Science Foundation (GAČR P301/10/0598) and by Palacký University in Olomouc (PrF_2012_026). Appendix A. Supplementary data CCDC contains the supplementary crystallographic data for cis-[ptcl 2 (3ClHaza) 2 ] DMF (1 DMF). Data can be obtained free of charge via or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) ; or deposit@ccdc.cam.ac.uk. The results of thermal analysis, ESI-mass spectrometry and IR and Raman spectroscopy, Figure S1 (TG/DTA thermal analysis of 2 and 5), Figure S2 (ESI mass spectrum of 4), Figure S3 (a part of the crystal structure of 1 DMF showing the packing of the molecules within the unit cell and non-covalent contacts), Table S1 (crystal data and structure refinement for 1 DMF) and Table S2 (interatomic parameters of the non-covalent contacts of 1 DMF) are given in Supplementary data. Appendix A. Supplementary data Supplementary data to this article can be found online at dx.doi.org/ /j.jinorgbio References [1] L.D. Arnold, X. Chen, H. Dong, A. Garton, M.J. Mulvihill, C.P.S. Smith, G.H. Thomas, T.M. Krulle, J. 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82 PŘÍLOHA 3 Muchová, T.; Prachařová, J.; Štarha, P.; Olivová, R.; Vrána, O.; Benešová, B.; Kašpárková, J.; Trávníček, Z.; Brabec, V. Insight into the toxic effects of cis-dichloridoplatinum(ii) complexes containing 7- azaindole halogeno derivatives in tumor cells J. Biol. Inorg. Chem. 18 (2013)

83 J Biol Inorg Chem (2013) 18: DOI /s ORIGINAL PAPER Insight into the toxic effects of cis-dichloridoplatinum(ii) complexes containing 7-azaindole halogeno derivatives in tumor cells Tereza Muchova Jitka Pracharova Pavel Starha Radana Olivova Oldrich Vrana Barbora Benesova Jana Kasparkova Zdenek Travnicek Viktor Brabec Received: 5 March 2013 / Accepted: 27 April 2013 / Published online: 15 May 2013 Ó SBIC 2013 Abstract The cisplatin analogues cis-[ptcl 2 (3ClHaza) 2 ] (1) and cis-[ptcl 2 (3IHaza) 2 ] (2) (3ClHaza and 3IHaza are 3-chloro-7-azaindole and 3-iodo-7-azaindole, respectively) are quite toxic to ovarian tumor cells, with moderately better IC 50 values than for cisplatin in the cisplatin-sensitive cell line A2780. We investigated potential factors which might be involved in the mechanism underlying the cytotoxic effects of 1 and 2 and compared these factors with those involved in the mechanism underlying the effects of conventional cisplatin. Our data indicate that the higher cytotoxicity of 1 and 2 originates mainly from their efficient cellular accumulation, different effects at the level of cell cycle regulation, and reduced propensity for DNA adduct repair. Studies of their reactivity toward cellular components reveal efficient binding to DNA, which is typically required for an active platinum drug. Further results suggest that 1 and 2 are Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. T. Muchova J. Pracharova R. Olivova B. Benesova J. Kasparkova Department of Biophysics, Faculty of Sciences, Palacky University, 17. listopadu 12, Olomouc, Czech Republic P. Starha Z. Travnicek Department of Inorganic Chemistry, Faculty of Science, Regional Centre of Advanced Technologies and Materials, Palacky University, 17. listopadu 12, Olomouc, Czech Republic O. Vrana V. Brabec (&) Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, Brno, Czech Republic brabec@ibp.cz capable of circumventing resistance to cisplatin induced by alterations in cellular accumulation and DNA repair. Hence, the latter two factors appear to be responsible for differences in the toxicity of 1 or 2, and cisplatin in tumor cells. The results of this work reinforce the idea that direct analogues of conventional cisplatin-containing halogenosubstituted 7-azaindoles offer much promise for the design of novel therapeutic agents. Keywords Platinum drugs Cytotoxicity Cellular uptake Cell cycle DNA damage DNA repair Abbreviations 3ClHaza 3-Chloro-7-azaindole 3IHaza 3-Iodo-7-azaindole CT Calf thymus DMF N,N 0 -Dimethylformamide EtBr Ethidium bromide FAAS Flameless atomic absorption spectrometry GSH Glutathione IC 50 Compound concentration that produces 50 % cell growth inhibition ICP-MS Inductively coupled plasma mass spectroscopy MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide SD Standard deviation Introduction The design of new potential platinum drugs to overcome tumor resistance to conventional cisplatin is an active area of bioinorganic chemistry and molecular pharmacology. This is because clinical use of platinum-based drugs is 123

84 580 J Biol Inorg Chem (2013) 18: limited by side effects and poor activity in certain types of cancer resulting from acquired or intrinsic resistance [1, 2]. These limitations have given impetus to the design and synthesis of new platinum-based chemotherapeutics with improved pharmacological properties. Hence, the search continues for platinum compounds with novel preclinical properties, such as activity in cisplatin-resistant cells or a pattern of cytotoxicity significantly different from that of cisplatin and its clinically used analogues against a panel of cell lines of various origins [3]. One approach that we have used to circumvent the shortcomings of conventional classic bifunctional platinum-based drugs, such as cisplatin, carboplatin, and oxaliplatin, is to prepare and characterize dichloridoplatinum(ii) and oxalatoplatinum(ii) complexes of the general formulas cis-[ptcl 2 (nhaza) 2 ] (Fig. 1) and [Pt(ox)(nHaza) 2 ](H 2 ox is oxalic acid) involving 7-azaindole halogeno derivatives (nhaza) [4]. These new complexes obey the originally devised structure activity relationships for antitumor candidates in the platinum family: such compounds should be neutral Pt II species with two am(m)ine ligands or one bidentate chelating diamine, and should have leaving ligands that can be replaced during aquation reactions [5]. The dichloridoplatinum(ii) complexes, cis-[ptcl 2 (nhaza) 2 ] [nhaza is 3-chloro-7-azaindole (3ClHaza) for complex 1, 3-iodo-7-azaindole (3IHaza) for complex 2 or 5-bromo-7- azaindole], previously demonstrated [4] promising in vitro cytotoxicity against the HOS (osteosarcoma), MCF7 (breast adenocarcinoma), and LNCaP (prostate adenocarcinoma) human cancer cell lines, i.e., against the tumor cell lines inherently resistant to cisplatin. Also importantly, it was the first time that 7-azaindole halogeno derivatives had been used as ligands of transition metal complexes. Preliminary results have also suggested that the cis-dichloridoplatinum(ii) complexes containing 7-azaindole halogeno derivatives effectively bind, in a cell-free medium, to DNA, which is considered a major pharmacological target of antitumor platinum-based drugs [6, 7], and that some features of the DNA binding mode of Fig. 1 The Pt II complexes used in this work these new Pt II complexes are similar to those of cisplatin [4]. The mode of action of cisplatin and its direct analogues is a multistep process which includes (1) cell accumulation, (2) drug activation or inactivation by sulfur-containing compounds, (3) DNA binding, and (4) cellular responses to the DNA damage, including DNA adduct repair, perturbed cell cycle, and the inhibition of apoptosis or necrosis [2, 8]. The cellular uptake, DNA binding in cells, reactivity with glutathione (GSH), DNA repair synthesis, perturbation of the cell cycle, and inhibition of apoptosis and necrosis were investigated to provide insight into the potency of cis-dichloridoplatinum(ii) complexes containing 7-azaindole halogeno derivatives. Complexes 1 and 2 were selected for this study. Materials and methods Starting materials and reagents Cisplatin, N,N 0 -dimethylformamide (DMF), propidium iodide, and glutathione (GSH) were obtained from Sigma- Aldrich (Prague, Czech Republic). Compounds 1 and 2 were synthesized and characterized as described previously [4]. Stock solutions of 1, 2, and cisplatin were prepared at a concentration of M in DMF, stored at 4 C in the dark, and diluted with water to the appropriate concentration just before use. The final concentration of DMF was less than 0.5 %. Stock solutions of platinum complexes for the cytotoxicity and cellular uptake studies were also prepared in DMF and were used immediately after dissolution. The concentrations of platinum in the stock solutions and after dilution with water were determined by flameless atomic absorption spectrometry (FAAS). Calf thymus (CT) DNA (42 % G? C, mean molecular weight approximately ) was also prepared and characterized as described previously [9, 10]. Plasmid puc19 (2,686 bp) was isolated according to standard procedures. Restriction endonucleases, the Klenow fragment from DNA polymerase I, and plasmid pbr322 (4,361 bp) were purchased from New England Biolabs. Ethidium bromide (EtBr) was from Merck (Darmstadt, Germany). Agarose was from FMC BioProducts (Rockland, ME, USA). Proteinase K and ATP were from Boehringer (Mannheim, Germany). 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Calbiochem (Darmstadt, Germany). Radioactive products were obtained from MP Biomedicals (Irvine, CA, USA). RPMI 1640 medium, fetal bovine serum, and trypsin/edta were from PAA (Pasching, Austria). Gentamicin was from Serva (Heidelberg, Germany). The cell-free extract was prepared from the repairproficient HeLa S3 cell line as reported previously [11]. 123

85 J Biol Inorg Chem (2013) 18: In vitro growth inhibition assay Human ovarian carcinoma cisplatin-sensitive A2780 cells and cisplatin-resistant A2780cisR cells (cisplatin-resistant variant of A2780 cells) were kindly supplied by B. Keppler, University of Vienna (Austria). The A2780cisR cells were grown in RPMI 1640 medium supplemented with gentamicin (50 lg ml -1 ) and heat-inactivated fetal bovine serum (10 %). The acquired resistance of A2780cisR cells was maintained by supplementing the medium with cisplatin (1 lm) every second passage. The cells were cultured in a humidified incubator at 37 C ina5%co 2 atmosphere and subcultured two or three times a week with an appropriate plating density. Stock solutions were freshly prepared in DMF immediately prior to testing. The cells were seeded in 96-well tissue culture plates at a density of 10 4 cells per well in 100 ll of medium. After overnight incubation (16 h), the cells were treated with the compounds at final concentrations in the range of lm in a final volume of 200 ll per well. The final DMF concentration in all wells was 0.1 %, which was shown not to affect cell growth. The cell lines were incubated for 24 or 72 h with the platinum compounds, and cell death was evaluated using a system based on the tetrazolium compound MTT in the same way as described previously [12, 13]. Briefly, 10 ll of a freshly diluted MTT solution (2.5 mg ml -1 ) was added to each well, and the plate was incubated at 37 C in a humidified 5 % CO 2 atmosphere for 4 h. At the end of the incubation period, the medium was removed, and the formazan product was dissolved in 100 ll of dimethyl sulfoxide. Cell viability was evaluated by measurement of the absorbance at 570 nm, using a Tecan Infinite M200 absorbance reader (Schoeller). Compound concentrations that produce 50 % cell growth inhibition (IC 50 ) were calculated from curves constructed by plotting cell survival (%) versus drug concentration (lm). All experiments were done in triplicate. The reading values were converted to the percentage of the control (percentage cell survival). Cytotoxic effects were expressed as IC 50. Detection of apoptosis and necrosis The cell death detection ELISA plus kit (Roche Molecular Biochemicals, Mannheim, Germany) was used as an indicator of apoptosis and necrosis [14]. In this assay, internucleosomal DNA fragmentation was quantitatively assayed by antibody-mediated capture and detection of cytoplasmic mononucleosome- and oligonucleosomeassociated histone DNA complexes. Briefly, after centrifugation (200g), 20 ll of the supernatant was used in the ELISA for detection of necrosis. A2780 cells were resuspended in 200 ll of the lysis buffer supplied by the manufacturer and incubated for 30 min at room temperature. After the nuclei (200g, 10 min) had been pelleted, 20 ll of the supernatant (cytoplasmic fraction) was used in the ELISA for detection of apoptosis following the manufacturer s standard protocol. Following incubation with peroxidase substrate for 20 min, the absorbance was determined at nm (reference wavelength) with a microplate reader (Tecan Sunrise absorbance reader, Schoeller). Signals from wells containing the substrate only were subtracted as background. Other parts of this assay and data analysis were performed according to the manufacturer s instructions. Cell cycle analysis After 24 h, floating cells (A2780) were collected, and attached cells were harvested by trypsinization (trypsin/ EDTA in phosphate-buffered saline). Total cells (floating and attached) were washed twice in phosphate-buffered saline (4 C), fixed in 70 % ethanol, and stored at -20 C. Cell pellets were subsequently rinsed with phosphate-buffered saline and sediment was stained with a solution of propidium iodide (50 lg ml -1, Sigma) supplemented with RNase A (10 lg ml -1, Qiagen) for 30 min at 25 C in the dark. DNA content was measured using flow cytometry (Cell Lab Quanta SC MPL, Beckman Coulter). The percentages of cells in the individual cell cycle phases were analyzed using Multicycle AV for Windows (Verity Software House). Cellular platinum complex accumulation Cellular accumulation of 1, 2, and cisplatin was measured in A2780 cells. The cells were seeded in 100-mm tissue culture dishes (30,000 cells per square centimeter). After overnight incubation, the cells were treated with the Pt II complex (10 lm) for 5 or 24 h [the concentration was verified by the measurement of platinum in the growing medium by FAAS or inductively coupled plasma mass spectroscopy (ICP-MS)]. The attached cells were washed twice with phosphate-buffered saline (4 C), and the pellet was stored at-80 C. The pellets were digested by a highpressure microwave digestion system (MARS5, CEM) with HCl to give a fully homogenized solution, and final platinum content was determined by FAAS. The cellular platinum uptake values were corrected for adsorption effects [15]. For other details, see Results. All experiments were performed in triplicate. DNA platination in cells exposed to platinum complexes A2780 cells grown to near confluence were exposed to 10 lm complex 1, 2, or cisplatin for 5 or 24 h. After the 123

86 582 J Biol Inorg Chem (2013) 18: incubation, the cells were trypsinized and washed twice in ice-cold phosphate-buffered saline. Cells were then lysed in DNAzol (MRC) supplemented with RNase A (100 lg ml -1 ). The genomic DNA was precipitated from the lysate with ethanol, dried, and resuspended in water. The DNA content in each sample was determined by UV spectrophotometry. To avoid the effect of high DNA concentration on ICP-MS detection of platinum in the samples, the DNA samples were digested in the presence of hydrochloric acid (11 M) using a high-pressure microwave mineralization system (MARS5, CEM). Experiments were performed in triplicate, and the values are the mean±the standard deviation (SD). Interstrand DNA cross-linking in a cell-free medium Complex 1, complex 2, and cisplatin were incubated for 24 h with 0.5 lg of a linear 2,686-bp fragment of puc19 plasmid linearized by EcoRI. The linear fragment was first 3 0 -end-labeled by means of the Klenow fragment of DNA polymerase I in the presence of [a- 32 P]dATP. The platinated samples were analyzed for DNA interstrand crosslinks by previously published procedures [16, 17]. The number of interstrand cross-links was analyzed by electrophoresis under denaturing conditions on alkaline agarose gel (1 %). After the electrophoresis had been completed, the intensities of the bands corresponding to single strands of DNA and interstrand cross-linked duplex were quantified. The frequency of interstrand cross-links was calculated as ICL/Pt (%)=XL/5372r b (the DNA fragment contained 5,372 nucleotide residues), where ICL/Pt (%) is the number of interstrand cross-links per adduct multiplied by 100, and XL is the number of interstrand cross-links per molecule of the linearized DNA duplex, and was calculated assuming a Poisson distribution of the interstrand crosslinks as XL=-ln A, where A is the fraction of molecules running as a band corresponding to the non-cross-linked DNA. Characterization of DNA adducts by thiourea Incorporation of [ 14 C]thiourea into DNA under controlled conditions ( M, 10 min, 25 C [18]) was used to quantitate platinum DNA monofunctional adducts of 1, 2, or cisplatin. The measurements were performed in 10 mm NaClO 4 at 25 C. The molar ratio of free Pt II complex to nucleotide phosphates at the onset of incubation with CT DNA, r i, was 0.05 [the DNA concentration was 0.24 mg ml -1 (0.75 mm related to the phosphorus content), and the concentration of Pt II complexes was M]. Samples (120 ll) were withdrawn after 24 h, and each sample was divided into two aliquots (60 ll). In one aliquot, conversion of the monofunctional adducts to bifunctional cross-links was blocked by addition of NaCl (the final concentration was 0.15 M) and quick cooling to -20 C. The unbound Pt II complex was removed from these samples by centrifugation through a Sephadex G50 coarse column, and the molar ratio of covalently bound molecules of Pt II complexes per nucleotide residue, r b, was determined by FAAS. In the other aliquot, the conversion of the adducts was blocked by addition of [ 14 C]thiourea (the final concentration was M, and the specific activity was 2.5 mci mmol -1 ) and NaCl (the final concentration was 0.15 M) so that the final volume of these samples was 1.0 ml. The samples were further incubated at 25 C for 10 min and subsequently were layered on Millipore filters (the diameter of pores was 0.1 lm); the unreacted thiourea and complexes formed between unbound Pt II complexes and thiourea were removed by washing the filters with 15 ml of 5 % (v/v) trichloroacetic acid.the filters were dried under an infrared lamp and transferred to glass tubes, to which 5 ml of toluene scintillator was added. The radioactivity was measured with a TriCarb 2800 TR liquid scintillation analyzer (PerkinElmer) (2 9 2 min). The content of free coordination sites in DNA adducts of Pt II complexes not involved in the binding to DNA was determined as the amount (%) of radioactive thiourea bound to platinated DNA; the concentration of thiourea corresponding to twofold concentration of Pt II complexes (having two potential DNA binding sites) bound to DNA in each sample determined by FAAS (r b ) was taken as 100 %. It was also verified that, at M thiourea, complete saturation of monofunctional or bifunctional adducts was obtained with no apparent reversal of platination [19, 20]. Reactions of Pt II complexes with glutathione Reactions of 1, 2, and cisplatin were investigated by monitoring the UV absorbance at 260 nm of solutions containing the platinum complex and GSH as described in previous work [21, 22] with minor modifications. The Pt II complexes (30 lm) were mixed with GSH (15 mm) at 37 C in 4.6 mm NaCl plus 100 mm NaClO 4, ph 6.0 and 50 % DMF. Reactions were initiated by mixing the Pt II complex with the buffer, followed by immediate addition of GSH. The experiments were performed in triplicate. The absorbance at 260 nm reflects the presence of platinum sulfur and disulfide bonds. The kinetic data were fitted by nonlinear regression (GraphPad Prism) to one-phase or two-phase exponential association. The decision that a fit to two-phase exponential association was more appropriate for each dependence was made by comparing the fits of two equations by using an F test (GrahPad Prism). 123

87 J Biol Inorg Chem (2013) 18: DNA repair synthesis by human cell extract Repair DNA synthesis of cell-free extracts was assayed using puc19 plasmid. Each reaction mixture of 50 ll contained unmodified pbr322 (500 ng) and unmodified or platinated puc19 (500 ng), ATP (2 mm), KCl (30 mm), creatine phosphokinase (rabbit muscle) (0.05 mg ml -1 ), dgtp (20 mm), dctp (20 mm), dttp (20 mm), datp (8 mm), [a- 32 P]dATP (74 kbq) in a buffer composed of N-(2-hydroxyethyl)piperazine-N-ethanesulfonic acid KOH (40 mm, ph 7.5), MgCl 2 (5 mm), dithiothreitol (0.5 mm), creatine phosphate (22 mm), bovine serum albumin (1.4 mg ml -1 ), and cell-free extract (150 lg). Reaction mixtures were incubated for 3 h at 30 C and the reactions were terminated by adding EDTA to a final concentration of 20 mm, sodium dodecyl sulfate to 0.6 %, and proteinase K to 250 lg ml -1 and then incubating the mixture for 30 min at 45 C. The products were extracted with 1 vol of 1:1 phenol chloroform. The DNA was precipitated from the aqueous layer by the addition of 0.02 vol NaCl (5 M), glycogen (5 mg), and 2.5 vol ethanol. After 20 min of incubation on dry ice and centrifugation at 12,000g for 30 min at 4 C, the pellet was washed with 0.5 ml of 70 % ethanol and dried in a vacuum centrifuge. DNA was finally linearized by SspI before electrophoresis on a 1 % agarose gel. Gels were stained with EtBr for photodocumentation, and the radioactivity associated with the bands was quantitated using a FUJIFILM BAS 2500 bioimaging analyzer with AIDA Image Analyzer (raytest Isotopenmessgeräte, Straubenhardt, Germany). Experiments were performed in duplicate. Other physical methods Absorption spectra were measured with a Beckman 7400 DU spectrophotometer equipped with a thermoelectrically controlled cell holder. The FAAS measurements were conducted with a Varian AA240Z Zeeman atomic absorption spectrometer equipped with a GTA 120 graphite tube atomizer. The analysis with the aid of ICP-MS was performed using an Agilent 7500 instrument (Agilent, Japan). The gels were visualized with a FUJIFILM BAS 2500 bioimaging analyzer, and the radioactivity associated with the bands was quantified with AIDA Image Analyzer. Statistical evaluation of the untreated control cells and drug-treated cells was performed using Student s t test. If not stated otherwise, a probability of 0.05 or less was deemed statistically significant. ovarian carcinoma cell line, which is commonly used to test the cytotoxic activity of cisplatin analogues. The tumor cell line was incubated for 24 or 72 h with the platinum compounds and cell survival in cultures treated with the platinum compounds was evaluated as described in Materials and methods. The IC 50 values (mean±sd of three independent experiments) obtained for 1, 2, and cisplatin after 24 h were 3.1 ± 0.3, 4.2 ± 0.9, and 14.9±1.1 lm, respectively; the IC 50 values obtained after 72 h were 1.6±0.3, 1.6±0.3, and 3.8±0.5 lm, respectively. These results show that both complex 1 and complex 2 were markedly more cytotoxic than cisplatin in cisplatin-sensitive A2780 cells after 24 h treatment, whereas the longer treatment (72 h) resulted in only moderately higher cytotoxicity of complexes 1 and 2. Cell death detection To analyze the characteristics of the cell death induced by complexes 1 and 2 and to identify whether cell death is related to apoptotic or necrotic processes, the level of apoptosis and necrosis induced by these drugs and cisplatin at a concentration of 3 lm over 24 h was quantified by a specific ELISA kit. This analysis allows the appearance and relative amounts of cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) to be measured after the induction of apoptosis or when these fragments are released from necrotic cells. Figure 2a shows DNA fragmentation induced by 1, 2, and cisplatin in A2780 cells as a result of apoptotic processes. These results demonstrate that treatment with these antitumor agents led to apoptotic events in the A2780 cell line. Importantly, complexes 1 and 2 induced a significantly higher level of DNA fragmentation due to apoptosis in comparison with cisplatin in A2780 cells. Results Cytotoxicity The cytotoxic activity of complexes 1 and 2 was determined against the A2780 (cisplatin-sensitive) human Fig. 2 Effects of 1, 2, and cisplatin on activation of the apoptotic pathway (a) and necrosis (b) in A2780 cells determined by DNAfragmentation ELISAs. Cells were treated for 24 h with 1, 2, or cisplatin at a concentration of 3 lm. The results are expressed as the mean of two independent experiments with duplicate runs 123

88 584 J Biol Inorg Chem (2013) 18: Fig. 3 Effects of 1, 2, and cisplatin on cell cycle distribution. Untreated (control) A2780 cells or A2780 cells treated at a concentration of 3 lm (a) or 5 lm (b) for 24 h were harvested, fixed, stained with propidium iodide, and assessed for cell cycle distribution by fluorescence-activated cell sorting. The estimated percentages of A2780 cells in different phases of the cell cycle are indicated. The results are expressed as the mean ± the standard deviation (SD) of three independent experiments with duplicate runs. An asterisk denotes a significant difference (p\ 0.05) from the untreated control, and a hash denotes a significant difference (p\0.05) between 1 or 2 and cisplatin A similar procedure was used to detect the extent of necrosis induced by 1, 2, or cisplatin. Importantly, the level of necrosis was significantly lower compared with that of apoptosis triggered by 1, 2, and cisplatin (Fig. 2b). Cell cycle analysis The status of the cell cycle for cells treated with complexes 1 and 2 and for comparative purposes also with cisplatin was analyzed. The analysis of cell cycle perturbation was performed using A2780 cells exposed to 1, 2, or cisplatin at concentrations of 3 or 5 lm for 24 h (Fig. 3). An evaluation of the effects on A2780 cells produced by complexes 1 and 2 in comparison with untreated control A2780 cells showed several significant differences in cell cycle modulation and these became more pronounced with increased concentration of the drug. Exposure of A2780 cells to 1, 2, or cisplatin caused the appearance of a population in the sub-g 1 region of the profile, where apoptotic and necrotic cells are found. The appearance of a sub-g 1 peak is consistent with the onset of internucleosomal DNA cleavage in late apoptosis [23]. Importantly, the efficiency of the compounds tested to cause the appearance of a population in the sub-g 1 region of the profile was different: the sub-g 1 population for cells treated with complexes 1 and 2 was significantly greater than that for cells treated with cisplatin (Fig. 3). This result is consistent with the morphological changes of A2780 cells observed by light microscopy (Figs. S1, S2). Treatment of these cells with complexes 1 or 2 for 24 h resulted in a high number of detached (apoptotic/ necrotic) cells, whereas most of the cells treated with cisplatin under the same conditions were still growing and dividing. Also, importantly, treatment with complex 1 or complex 2 affected the G 0 /G 1 populations much less than treatment with cisplatin, which markedly decreased the G 0 / G 1 populations. Similarly, treatment with complexes 1 and 2 somewhat decreased the peaks corresponding to the S phase in contrast to the peaks corresponding to this phase in case of treatment with cisplatin, which were only affected slightly (Fig. 3). In addition, cisplatin caused an accumulation of cells in the G 2 phase in contrast to treatment with complexes 1 and 2, which decreased the G 2 populations considerably less (see Fig. 3). Thus, different effects were observed for the cis-dichloridoplatinum(ii) complexes containing 7-azaindole halogeno derivatives and cisplatin. The fact that complexes 1 and 2 and cisplatin had different effects on cell cycle progression suggests that substitution of the NH 3 groups in cisplatin by 3ClHaza or 3IHaza changed the mechanism of action of the parent platinum drug. Cellular accumulation The factor that is usually thought to contribute to drug cytotoxicity is cellular accumulation. To examine the accumulation of complexes 1 and 2, the cellular levels of these compounds were measured after 5 and 24 h exposure of the A2780 cells to the drugs at a concentration of 10 lm. The accumulation of complexes 1 and 2 in A2780 cells was, after 5 and 24 h exposure, approximately 12-fold to 15-fold and 45-fold to 46-fold greater, respectively, than that of cisplatin (Table 1). DNA-bound platinum in cells exposed to the platinum complexes Platinum levels on nuclear DNA were determined after the exposure of A2780 cells to 10 lm 1, 2, or cisplatin for 5 or 24 h. The levels of platinum on DNA were determined by 123

89 J Biol Inorg Chem (2013) 18: Table 1 Accumulation of platinum complexes in A2780 cells and platinum content of DNA isolated from A2780 cells exposed to complex 1, complex 2, or cisplatin ICP-MS (Table 1). The platinum content of DNA for the A2780 cells treated with 1 or 2 for 5 or 24 h was approximately 4.5-fold to 4.7-fold or 13.8-fold to 14.8-fold greater, respectively, than that for the cells treated with cisplatin. DNA binding in cell-free media Complex 1 Complex 2 Cisplatin Accumulation 0.705± ± ±0.002 after 5 h a Accumulation 3.732± ± ±0.007 after 24 h a Pt content of 0.72± ± ±0.04 DNA after 5 h b Pt content of DNA after 24 h b 4.70± ± ±0.08 For the structures of the complexes, see Fig. 1. Cells were exposed to 1, 2, or cisplatin (10 lm) for 5 or 24 h. The results are expressed as the mean ± the standard deviation of three independent experiments. a The accumulation of Pt complexes is in nanomoles of Pt per cells. b The Pt content of DNA is in picmoles of Pt complex per microgram of DNA. The platinum complexes were incubated with CT DNA at various r i values (r i is defined as the molar ratio of free platinum complex to nucleotide phosphate) in 10 mm Na- ClO 4 at 37 C in the dark, aliquots withdrawn after 24 h were quickly cooled in an ice bath, and then the free (unbound) platinum compound was removed by gel filtration chromatography using Sephadex G50 columns. The content of platinum and the concentration of DNA in these DNA samples was determined by FAAS and absorption spectrophotometry. Hence, it was possible to prepare samples of DNA modified by these Pt II complexes at a preselected value of r b (the number of molecules of the platinum complex bound per nucleotide residue). Samples of DNA modified by 1, 2, or cisplatin and analyzed further by biophysical or biochemical methods were prepared in NaClO 4 (10 mm) at 37 C. After 24 h of the reaction of DNA with the complex, the samples were precipitated in ethanol and dissolved in the medium necessary for a particular analysis, and the r b value in an aliquot of this sample was checked by FAAS. In this way, all analyses performed in cell-free media described in this article were performed in the absence of unbound (free) platinum complex. Bifunctional platinum compounds, which coordinate base residues in DNA, form various types of interstrand and intrastrand cross-links and monofunctional adducts. Considerable evidence suggests that the antitumor efficacy of bifunctional platinum compounds is the result of the formation of these lesions, but their relative efficacy remains unknown. Therefore, we decided to quantitate first the interstrand cross-linking efficiency of complexes 1 and 2 in linearized puc19 plasmid. This plasmid DNA was linearized by EcoRI (EcoRI cuts only once within puc19 plasmid) and globally modified by 1, 2, or cisplatin. The samples were analyzed for the interstrand cross-links by agarose gel electrophoresis under denaturing conditions [17]. The intensity of the more slowly migrating band increased with increasing level of the platination (Fig. 4). The radioactivity associated with the individual bands in each lane was measured to obtain estimates of the fraction of non-cross-linked or crosslinked DNA under each condition. The DNA interstrand cross-linking efficiency of complexes 1 and 2 was 5 %. This implies that interstrand cross-links represent only a minor portion of the adducts formed in DNA by complexes 1 and 2, i.e., in this respect, complexes 1 and 2 behave like the parent cisplatin, whose interstrand crosslinking efficiency is 6 % under identical conditions (Fig. 4) [17]. Previous studies [18 20, 24] have shown that under the appropriate conditions thiourea quantitatively reacts with DNA monoadducts of bifunctional Pt II complexes without displacing platinum from the DNA. In other words, during the initial period of the reaction of DNA with bifunctional Pt II complexes, when a significant proportion of the molecules are bound monofunctionally, the other coordination site can be blocked by thiourea [20]. This is so because, although the Pt N bond has higher thermodynamic stability than the Pt S (thioether) bond, thioether sulfur is kinetically more favorable than the guanine nitrogen when binding to Pt II drugs. Thus, incorporation of [ 14 C]thiourea into DNA under controlled conditions [18] (see Materials Fig. 4 The formation of interstrand cross-links by 1, 2, and cisplatin in puc19 DNA. Autoradiograms of the denaturing 1 % agarose gels of linearized DNA which was 3 0 -end-labeled; the interstrand crosslinked DNA appears as the top bands (ICLs) migrating on the gels more slowly than the single-stranded DNA (ss) (contained in the bottom bands). Plasmid linearized by EcoRI was not modified (lane C), modified by complex 1 (lane 1), modified by complex 2 (lane 2), or modified by cisplatin (lane 3). The r b value was

90 586 J Biol Inorg Chem (2013) 18: and methods ) was used to quantitate platinum DNA monofunctional adducts of complexes 1 and 2. The adducts in which complexes 1 and 2 still possessed free coordination sites not involved in binding to DNA (which could be blocked by thiourea, presumably in monofunctional lesions) were quantitated after 24 h incubation of complex 1 or complex 2 with CT DNA at r b = by incorporation of [ 14 C]thiourea. The complete protocol is described in Materials and methods, but it should be emphasized that these experiments were performed in the absence of any free (unbound to DNA) platinum complex. The proportions of free coordination sites in 1, 2, and cisplatin not involved in the binding to DNA calculated in this manner were 12, 10, and 2 %, respectively. The resulting data indicate that after 24 h of the reaction of DNA with complexes 1 and 2, approximately 88 and 90 % of adducts were already bifunctional cross-linked adducts, whereas the reactions of DNA with cisplatin resulted in 98 % bifunctional adducts, in agreement with previously published results [7]. Reactions with glutathione Pt II compounds have a strong thermodynamic preference for binding to sulfur-donor ligands, such as thiolates [25, 26]; hence, before Pt II drugs reach the DNA in the nucleus of tumor cells, or even after they bind to DNA, they may still react with various sulfur-containing compounds [27, 28]. These reactions are generally believed to play a role in mechanisms underlying tumor resistance to platinum compounds, their inactivation, and side effects. Therefore, interest in the interactions of platinum antitumor drugs with sulfur-containing molecules of biological significance has recently increased markedly [28]. In this work, we investigated, using UV absorption spectrophotometry, irreversible binding of GSH to complexes 1 and 2 in comparison with cisplatin following the procedure outlined by Dabrowiak et al. [21], which was slightly modified as described in Materials and methods. Complexes 1 and 2 were incubated with GSH at a ratio of thiol to drug of 500:1, which represents the physiologically relevant value [21]. Figure 5 shows the UV absorbance (at 260 nm) of the platinum complexes and GSH as a function of time, with the absorbances of GSH and the platinum complex alone subtracted. To establish the rate of the initial reaction with respect to the platinum complex, each difference curve was fitted by nonlinear regression (GraphPad Prism) to the following equation: I d = C?A 1 exp(-b 1 t)?a 2 exp(-b 2 t) (A 1, A 2, b 1, b 2, and C are constants, and t is the time of the reaction). The initial slope (S in ) was calculated as -(A 1 b 1? A 2 b 2 )[21]. S in values of ± and ± min -1 were calculated for reaction of Fig. 5 UV absorbance associated with the reaction of 1, 2, and cisplatin with glutathione (GSH). The absorbance at 260 nm is shown as a function of time for a 240-min incubation at 37 C of the platinum complexes at a concentration of 30 lm with GSH (15 mm) in a medium of NaCl (4.6 mm) plus NaClO 4 (100 mm, ph 6.0) in the dark at 37 C. The curves represent the absorbances (260 mm) of solutions containing the platinum complex plus GSH from which absorbances of GSH and the platinum complex alone were subtracted. Curve 1 complex 1, curve 2 complex 2, curve 3 cisplatin GSH with complexes 1 and 2, respectively, and ± min -1 was calculated for the reaction of GSH with cisplatin. Thus, complexes 1 and 2 reacted with GSH with a similar rate as cisplatin. DNA repair synthesis by human cell extract DNA repair is another key factor which significantly reduces the number of platinum adducts on DNA in cells and thereby may significantly contribute to their biological activity [29]. Therefore, the DNA repair efficiency in puc19 plasmid (2,686 bp) globally modified by 1, 2, or cisplatin at r b = 0.07 was tested using cell-free extract of repair-proficient HeLa S3 cells. Untreated plasmid pbr322 (4,361 bp) was also included in each reaction to monitor damage-independent nucleotide incorporation. Repair activity was monitored by measurement of the amount of radiolabeled nucleotide incorporated. The incorporation of radioactive material was corrected for the relative DNA content in each band, determined by densitometry of EtBrstained gel. As illustrated in Fig. 6, damage-induced DNA repair synthesis determined for the plasmid modified by complex 1 or complex 2 was lower than that found for cisplatin at the same level of modification (60 or 45 %, respectively, of that found for the plasmid modified by cisplatin; Fig. 6b). Thus, DNA repair appears to be a factor contributing to the elevated toxicity in tumor cells of 1 and 2 compared with cisplatin. Cytotoxicity in cisplatin-resistant tumor cells The results described so far indicate that cellular accumulation and DNA repair may be important factors 123

91 J Biol Inorg Chem (2013) 18: A2780cisR human ovarian carcinoma cells (with acquired cisplatin resistance), which are resistant to cisplatin through a combination of decreased uptake, enhanced DNA repair/tolerance, and elevated GSH levels [30, 31]. The tumor cell line was incubated for 24 or 72 h with the platinum compounds and the cell survival in the culture treated with the platinum compounds was evaluated as described in Materials and methods. The IC 50 values (mean ± SD of three independent experiments) obtained for 1, 2, and cisplatin after 24 h were 2.0 ± 0.4, 3.6 ± 0.6, and 26.0±3.1 lm, respectively; the IC 50 values obtained after 72 h were 0.6±0.1, 0.7±0.1, and 14.8±0.8 lm, respectively. These results show that both complex 1 and complex 2 were markedly more active than cisplatin in the cisplatin-resistant cell line A2780cisR. Discussion Fig. 6 In vitro DNA repair synthesis assay. DNA repair of puc19 modified with cisplatin or complexes 1 and 2 at r b = 0.07 was mediated by the extract from repair-proficient HeLa S3 cells. Unmodified pbr322 was also used as an internal control. Repair efficiency is given by the radioactivity of incorporated [a- 32 P]dATP normalized to the relative amount of DNA in the band determined from the ethidium bromide (EtBr)-stained gel. a Results of a typical experiment. Top panel: autoradiographic image of the same gel showing radiolabel incorporation. Bottom panel: EtBr-stained gel showing migration of undamaged control plasmid (pbr322, top bands) and platinated puc19 (bottom bands). C control, unmodified puc19 plus unmodified pbr322; cispt puc19 modified with cisplatin plus unmodified pbr322; 1 puc19 modified with complex 1 plus unmodified pbr322; 2 puc19 modified with complex 2 plus unmodified pbr322. b Incorporation of [a- 32 P]dATP into unmodified or platinated puc19 plasmid. For all quantifications representing the mean values of two separate experiments, incorporation of radioactive material is normalized to the relative DNA content in each band determined from the relative densities of the bands on the EtBrstained gel. The radioactivity associated with incorporation of [a- 32 P]dATP into DNA modified with cisplatin was arbitrarily set to 100 %. Values shown in the graph are the means (± SD) of two separate experiments, each conducted in duplicate responsible for differences in toxicity of 1, 2, and cisplatin in tumor cell lines. To further support this thesis, we tested the effects of complexes 1 and 2 on viability of cancer cells which acquired resistance to cisplatin also owing to reduced cellular accumulation and enhanced DNA repair. Such tumor cells should be more sensitive to complexes 1 and 2 in comparison with cisplatin. Therefore, the cytotoxicity of complexes 1 and 2 was also determined in We have demonstrates in this work that complexes 1 and 2 are quite toxic to ovarian tumor cells, with IC 50 values that were significantly better than those observed for cisplatin in the cisplatin-sensitive cell line A2780. Thus, these results complement those obtained in a previous study [4] showing that complexes 1 and 2 exhibit promising cytotoxicity with regard to the HOS (osteosarcoma), MCF7 (breast adenocarcinoma), and LNCaP (prostate adenocarcinoma) human cancer cell lines. In this work we investigated factors which might be involved in the mechanism underlying the cytotoxic effects of complexes 1 and 2 and compared these factors with those involved in the mechanism underlying the cytotoxic effects of the most frequently studied anticancer metallodrug, cisplatin. Cisplatin is known to exert its cytotoxic effect by inducing apoptosis [32 34]. To investigate further the mechanism of cell death induced by complexes 1 and 2, the levels of apoptosis and necrosis were quantified. The results (Fig. 2) indicate that complexes 1 and 2 induce cell death by apoptosis in A2780 cells with considerably higher efficiency than cisplatin and that apoptosis markedly prevailed over necrosis. Activation of cell cycle checkpoints is a general cellular response after exposure to cytotoxic agents. Previous studies have indicated that cisplatin and other platinum agents predominantly inhibit cell cycle progression at the S and/or G 2 phase [13, 35 37]. Our studies performed in the cell line with wild-type p53 status show differences between complexes 1 and 2 and cisplatin at the level of cell cycle regulation (Fig. 3). Nuclear debris from apoptotic or necrotic cells is observed as a sub-g 1 population for cells treated with complexes 1 and 2, but the sub-g 1 population is markedly lower for cisplatin-treated cells (Fig. 3), and this is consistent with a significantly higher level of DNA 123

92 588 J Biol Inorg Chem (2013) 18: fragmentation due to apoptosis in comparison with cisplatin in A2780 cells (Fig. 2a). Moreover, although cisplatin blocks A2780 cells in the G 2 phase at concentration as low as 3 lm, complexes 1 and 2 induced a somewhat smaller block in A2780 cells only at 5 lm concentration. The differences between complexes 1 and 2 and cisplatin at the level of cell cycle regulation imply a molecular mechanism of action of the cis-dichloridoplatinum(ii) complexes containing 7-azaindole halogeno derivatives distinct from that of cisplatin. The results of this work also show that using bulkier ligands such as 7-azaindole halogeno derivatives gives rise to increased cellular accumulation and higher cytotoxicity. In addition, the amount of platinum found on the DNA of A2780 cells incubated with complexes 1 and 2 for 5 or 24 h was higher than that found in cells treated with cisplatin (Table 1), which correlates with their cytotoxicity (see Cytotoxicity ) and cellular accumulation (Table 1). DNA may therefore be a potential target also for these cytotoxic cisplatin analogues containing halogeno-substituted 7-azaindoles, although we cannot rule out the possibility that nuclear DNA may not be the only target. The finding that complexes 1 and 2 are capable of delivering platinum to DNA in the cell nucleus prompted us to examine the binding of complexes 1 and 2 to DNA in a cell-free medium. The resulting DNA damage triggers downstream effects, including the inhibition of replication and transcription, and cell cycle arrest [1, 2]. The DNA binding experiments performed in this work in a cell-free medium indicated that modification reactions result in the irreversible coordination of complexes 1 and 2. In addition, transcription mapping experiments [4], determination of monofunctional adducts, and determination of the interstrand cross-linking efficiency of complexes 1 and 2 (Fig. 4) suggest that several aspects of the DNA binding mode of complexes 1 and 2 are similar to those of the parent cisplatin. However, it cannot be excluded that identical types of DNA adducts of 1, 2, and cisplatin can distort the DNA conformation differently and can be processed by cellular components differently. Thus, the distinct differences in cell killing observed for complexes 1 and 2 and cisplatin may be associated with processes at the DNA level. DNA adducts of cisplatin inhibit DNA replication to an extent that slows cell cycle progression through the S phase but allows cells to accumulate in the G 2 phase [38]. Efficient repair of the cross-links and replicative bypass across the adducts by translesion DNA polymerases contribute to the survival of cisplatin-treated cells [39]. However, the low accumulation of cells in the G 2 phase and the still significant cell killing observed in A2780 cells treated with complex 1 or complex 2 suggests that inhibition of DNA synthesis by adducts of these agents is more lethal to the cells than are DNA adducts of cisplatin. The results of this work also demonstrate that complexes 1 and 2 react with GSH with a rate similar to that of cisplatin (Fig. 5). Both clinical and preclinical studies have shown that cells with an elevated level of GSH (more than 10 mm) may be resistant to cisplatin and its analogues [40 43]. Further studies are warranted to determine whether the different cytotoxicity of complexes 1 and 2 and cisplatin is attributable to different reactions with sulfurcontaining compounds. Recent clinical studies suggest that high levels of expression of proteins associated with nucleotide excision repair of cisplatin DNA adducts result in tumor resistance and, ultimately, are responsible for the low efficacy of classic platinum-based regimens [44, 45]. Nucleotide excision repair system most efficiently recognizes and removes irreversible DNA adducts that severely distort and destabilize double-helical DNA. Thus, on the basis of the findings of our experiments in cell-free systems, the adducts produced by complexes 1 and 2 should be poorer substrates for nucleotide excision repair than the adducts of cisplatin. The relative resistance to DNA repair would explain why complexes 1 and 2 show major pharmacological advantages over cisplatin in the ovarian cancer cell line. The repair synthesis assay in randomly modified plasmid performed in this study (Fig. 6) demonstrates that the presumably most cytotoxic and major adducts formed by complexes 1 and 2 are repaired considerably less efficiently than the damage caused by cisplatin, which may potentiate toxic effects of this class of Pt II compounds in tumor cells. The toxicity of 1, 2, and cisplatin in A2780cisR cells suggests that complexes 1 and 2 are capable of circumventing resistance to the parent metallodrug induced by alterations in cellular accumulation and DNA repair. Hence, the latter two factors appear to be responsible for differences in the toxicity of complexes 1 and 2 and cisplatin in tumor cells. The work reported here demonstrates that rational chemical design can be applied to cis-dichloridoplatinum(ii) complexes to achieve potent cancer cell cytotoxicity. It is notable that halogeno-substituted 7-azaindole nonleaving groups can play a major role in controlling the chemical and biological properties, such as cellular accumulation, effects at the level of cell cycle regulation, propensity for DNA adduct repair, binding to DNA, and reactivity toward sulfur-containing nucleophiles. The results of this work reinforce the idea that direct analogues of conventional cisplatin-containing halogeno-substituted 7-azaindoles offer much promise for the design of novel therapeutic agents. 123

93 J Biol Inorg Chem (2013) 18: Acknowledgments This research was supported by the Czech Science Foundation (grant P301/10/0598) and the Ministry of Education of the Czech Republic (grant LH13096). The research of T.M., and J.P. was also supported by the student project of Palacky University in Olomouc (grant PrF ). J.K. s research was also supported by the Operational Program Education for Competitiveness European Social Fund (CZ 1.07/2.3.00/ ) of the Ministry of Education, Youth and Sports of the Czech Republic. P.S. and Z.T. acknowledge funding from the Operational Program Research and Development for Innovations European Regional Development Fund (CZ.1.05/2.1.00/ ) and the Operational Program Education for Competitiveness European Social Fund (CZ.1.07/2.3.00/ ) of the Ministry of Education, Youth and Sports of the Czech Republic. References 1. Kelland L (2007) Nat Rev Cancer 7: Wang D, Lippard SJ (2005) Nat Rev Drug Discov 4: Fojo T, Farrell N, Ortuzar W, Tanimura H, Weinstein J, Myers TG (2005) Crit Rev Oncol Hematol 53: Starha P, Travnicek Z, Popa A, Popa I, Muchova T, Brabec V (2012) J Inorg Biochem 115: Reedijk J (2003) Proc Natl Acad Sci USA 100: Johnson NP, Butour J-L, Villani G, Wimmer FL, Defais M, Pierson V, Brabec V (1989) Prog Clin Biochem Med 10: Jamieson ER, Lippard SJ (1999) Chem Rev 99: Fuertes MA, Castilla J, Alonso C, Perez JM (2003) Curr Med Chem 10: Brabec V, Palecek E (1970) Biophysik 6: Brabec V, Palecek E (1976) Biophys Chem 4: Reardon JT, Vaisman A, Chaney SG, Sancar A (1999) Cancer Res 59: Bugarcic T, Novakova O, Halamikova A, Zerzankova L, Vrana O, Kasparkova J, Habtemariam A, Parsons S, Sadler PJ, Brabec V (2008) J Med Chem 51: Kisova A, Zerzankova L, Habtemariam A, Sadler PJ, Brabec V, Kasparkova J (2011) Mol Pharm 8: Moser C, Lang SA, Kainz S, Gaumann A, Fichtner-Feigl S, Koehl GE, Schlitt HJ, Geissler EK, Stoeltzing O (2007) Mol Cancer Ther 6: Egger AE, Rappel C, Jakupec MA, Hartinger CG, Heffeter P, Keppler BK (2009) J Anal At Spectrom 24: Farrell N, Qu Y, Feng L, Van Houten B (1990) Biochemistry 29: Brabec V, Leng M (1993) Proc Natl Acad Sci USA 90: Boudny V, Vrana O, Gaucheron F, Kleinwächter V, Leng M, Brabec V (1992) Nucleic Acids Res 20: Eastman A (1983) Biochemistry 22: Eastman A (1986) Biochemistry 25: Dabrowiak JC, Goodisman J, Souid AK (2002) Drug Metab Dispos 30: Hagrman D, Goodisman J, Dabrowiak JC, Souid AK (2003) Drug Metab Dispos 31: Clodi K, Kliche KO, Zhao SR, Weidner D, Schenk T, Consoli U, Jiang SW, Snell V, Andreeff M (2000) Cytometry 40: Page JD, Husain I, Sancar A, Chaney SG (1990) Biochemistry 29: Lempers ELM, Inagaki K, Reedijk J (1988) Inorg Chim Acta 152: Lempers ELM, Reedijk J (1990) Inorg Chem 29: Reedijk J (1999) Chem Rev 99: Wang X, Guo Z (2007) Anticancer Agents Med Chem 7: Brabec V, Kasparkova J (2009) In: Hadjiliadis N, Sletten E (eds) Metal complex DNA interactions. Wiley, Chichester, pp Kelland LR, Barnard CFJ, Mellish KJ, Jones M, Goddard PM, Valenti M, Bryant A, Murrer BA, Harrap KR (1994) Cancer Res 54: Kelland LR, Sharp SY, ONeill CF, Raynaud FI, Beale PJ, Judson IR (1999) J Inorg Biochem 77: Wang GD, Reed E, Li QQ (2004) Oncol Rep 12: Perez J-M, Montero EI, Quiroga AG, Fuertes MA, Alonso C, Navarro-Ranninger C (2001) Metal Based Drugs 8: Sedletska Y, Giraud-Panis M-J, Malinge J-M (2005) Curr Med Chem Anticancer Agents 5: Ormerod M, O Neill C, Robertson D, Kelland L, Harrap K (1996) Cancer Chemother Pharmacol 37: Siddik ZH (2003) Oncogene 22: Liskova B, Zerzankova L, Novakova O, Kostrhunova H, Travnicek Z, Brabec V (2012) Chem Res Toxicol 25: Sorenson CM, Eastman A (1988) Cancer Res 48: Kartalou M, Essigmann JM (2001) Mutat Res 478: Akiyama S, Chen ZS, Sumizawa T, Furukawa T (1999) Anticancer Drug Des 14: Chen G, Hutter KJ, Zeller WJ (1995) Cell Biol Toxicol 11: Brabec V, Kasparkova J (2005) Drug Resist Updates 8: Kelland LR (2000) Drugs 59: Weaver D, Crawford E, Warner K, Elkhairi F, Khuder S, Willey J (2005) Mol Cancer 4: Fujii T, Toyooka S, Ichimura K, Fujiwara Y, Hotta K, Soh J, Suehisa H, Kobayashi N, Aoe M, Yoshino T, Kiura K, Date H (2008) Lung Cancer 59:

94 PŘÍLOHA 4 Štarha, P.; Hošek, J.; Vančo, J.; Dvořák, Z.; Suchý, P.; Popa, I.; Pražanová, G.; Trávníček, Z. Pharmacological and molecular effects of platinum(ii) complexes involving 7-azaindole derivatives PLoS ONE 9 (2014) e90341

95 Pharmacological and Molecular Effects of Platinum(II) Complexes Involving 7-Azaindole Derivatives Pavel Štarha 1, Jan Hošek 1,Ján Vančo 1, Zdeněk Dvořák 2, Pavel Suchý Jr 3, Igor Popa 1, Gabriela Pražanová 3, Zdeněk Trávníček 1 * 1 Department of Inorganic Chemistry, Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University, Olomouc, Czech Republic, 2 Department of Cell Biology and Genetics, Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacký University, Olomouc, Czech Republic, 3 Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Brno, Czech Republic Abstract The in vitro antitumour activity studies on a panel of human cancer cell lines (A549, HeLa, G-361, A2780, and A2780R) and the combined in vivo and ex vivo antitumour testing on the L1210 lymphocytic leukaemia model were performed on the cis- [PtCl 2 (naza) 2 ] complexes (1 3) involving the 7-azaindole derivatives (naza). The platinum(ii) complexes showed significantly higher in vitro cytotoxic effects on cell-based models, as compared with cisplatin, and showed the ability to avoid the acquired resistance of the A2780R cell line to cisplatin. The in vivo testing of the complexes (applied at the same dose as cisplatin) revealed their positive effect on the reduction of cancerous tissues volume, even if it is lower than that of cisplatin, however, they also showed less serious adverse effects on the healthy tissues and the health status of the treated mice. The results of ex vivo assays revealed that the complexes 1 3 were able to modulate the levels of active forms of caspases 3 and 8, and the transcription factor p53, and thus activate the intrinsic (mitochondrial) pathway of apoptosis. The pharmacological observations were supported by both the histological and immunohistochemical evaluation of isolated cancerous tissues. The applicability of the prepared complexes and their fate in biological systems, characterized by the hydrolytic stability and the thermodynamic aspects of the interactions with cysteine, reduced glutathione, and human serum albumin were studied by the mass spectrometry and isothermal titration calorimetric experiments. Citation: Štarha P, Hošek J, Vančo J, Dvořák Z, Suchý Pô Jr, et al. (2014) Pharmacological and Molecular Effects of Platinum(II) Complexes Involving 7-Azaindole Derivatives. PLoS ONE 9(3): e doi: /journal.pone Editor: Heidar-Ali Tajmir-Riahi, University of Quebect at Trois-Rivieres, Canada Received December 16, 2013; Accepted January 31, 2014; Published March 6, 2014 Copyright: ß 2014 Štarha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors acknowledge funding from the Czech Science Foundation (GAČR P207/11/0841), Operational Program Research and Development for Innovations - European Regional Development Fund (CZ.1.05/2.1.00/ ), Operational Program Education for Competitiveness - European Social Fund (CZ.1.07/2.3.00/ ) of the Ministry of Education, Youth and Sports of the Czech Republic, Palacký University in Olomouc (PrF_2013_015), and University of Veterinary and Pharmaceutical Sciences Brno (IGA VFU 82/2012/FaF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * zdenek.travnicek@upol.cz Introduction Cisplatin is a simple platinum(ii) coordination compound that is used world-wide for the treatment of various types of cancer [1,2]. The discovery of its antitumour effect on human cancer cells in 1960s [3,4] and its consequent approval by the Food and Drug Administration for the therapeutic use in 1978 represent an important milestone in the field of both bioinorganic and medicinal chemistry. Representing a relatively uncomplicated leading compound, hundreds and hundreds of platinum(ii) complexes were prepared using diverse strategies how to modify the structure and biological action of cisplatin. Two basic approaches focused either on the substitution of the leaving groups, i.e. two chlorides (e.g. in carboplatin [5] or nedaplatin [6]), or on the substitution of two NH 3 molecules within the cisplatin molecule by different N-donor ligands (e.g. in oxaliplatin [7] or lobaplatin [8]) represent the most promising ways leading towards clinically useful platinum(ii) compounds. Nevertheless, none of these compounds avoided completely both of the main disadvantages related with the platinum-based drugs application, i.e. the resistance (acquired or intrinsic), and negative and dose-limiting side effects (nephrotoxicity, neurotoxicity, myelosuppression etc.) [1,2,9]. Since 1978, the development of new cisplatin-inspired bioactive complexes seemed many times as a lost cause, nevertheless the preparation of several novel highly-active compounds proved that there is still a room for the improvement of pharmacological properties of antitumour platinum complexes [10]. One of the possible future research directions was shown in the case of picoplatin [11]. A rational replacement of one NH 3 molecule in the cisplatin molecule by one relatively simple heterocyclic N-donor ligand (2-methylpyridine) led to the lower interaction with sulphurcontaining biomolecules (e.g. glutathione) resulting in lower inactivation of the substance and, as a consequence of this, in a notable ability to overcome resistance of various tumour types to the action of cisplatin and oxaliplatin [11 13]. Although picoplatin failed in the clinical trials on non-small-cell lung carcinoma due to the continued progression of the disease and showing several drawbacks (e.g. neutropenia, thrombocytopenia or vomiting), it is currently undergoing clinical trials as therapeutic for colorectal and prostate cancer [14]. Bearing this in mind, we aimed to find a simple, planar and well-coordinating N-donor heterocycle, whose incorporation into PLOS ONE 1 March 2014 Volume 9 Issue 3 e90341

96 Anticancer Pt(II) Complexes with 7-Azaindole the cisplatin molecule, instead of one or both NH 3 molecules, could bring in the similar effect on the antitumour properties as in the case of picoplatin. Thus, we chose 7-azaindole and its halogeno derivatives, which fulfil the mentioned requirements [15 17]. Moreover, the biological perspective of 7-azaindole moiety, as recently proved on various 7-azaindole derivatives reported as having notable biological properties, such as anticancer activity [18], inhibition of kinases (e.g. tropomyosin-related [19] or Abl and Src kinases [20]) or antiviral effect [21], has to be taken into account as well. Recently, we prepared and thoroughly characterized the platinum(ii) dichlorido complexes involving 7-azaindole or its halogeno derivatives 3-chloro-7-azaindole (the complex 1 in this work), 3-iodo-7-azaindole (2 in this work) and 5-bromo-7- azaindole (3 in this work) (see Figure 1) and screened them for their in vitro antitumour activity against HOS osteosarcoma, MCF7 breast carcinoma and LNCaP prostate carcinoma human cancer cell lines with IC 50 equalled mm [22,23]. In addition, the results of mechanistic studies confirming their analogous mechanism of action to cisplatin and significantly higher cell-uptake, intracellular transport and DNA platination, resulting in the higher in vitro effectiveness as compared with cisplatin were recently reported [23,24]. Following the previous promising results of in vitro studies, we were determined to perform an advanced study of in vitro cytotoxicity on an extended panel of human cancer cell lines (A549, HeLa, G-361, A2780 and cisplatin-resistant A2780R), together with the in vivo and ex vivo studies on L1210 lymphocytic leukaemia model complemented by the histological and immunohistochemical investigation on the cancerous tissues and studies of expression of caspases 3 and 8, p53 and VEGF-A, i.e. the proteins associated with the tumour growth progression and induction of apoptosis. In this paper, we present the results of thorough biological testing, extended by the data regarding the applicability of the compounds and the stability of their solutions in water containing media. Materials and Methods Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health. The protocol was approved by the Expert Committee on the Protection Figure 1. General structural formula of the studied platinum(ii) complexes. R 3 = Cl for 1, I for 2 and H for 3;R 5 = H for 1, H for 2 and Br for 3. doi: /journal.pone g001 of Animals Against Cruelty at the University of Veterinary and Pharmaceutical Sciences Brno (Permit Number: 42/2011). To minimize the suffering of laboratory animals, the number of pharmacological interventions was limited to the necessary minimum and the animals were observed regularly for any signs of unnecessary suffering from the symptoms of the tumor progression. All animals showing at least one of the following symptoms - weight loss higher than 50% of the initial weight, symptoms of acute toxicity caused by the tested compounds, and excessive volume of the tumors hindering the mobility or social communication of the animals, had to be sacrificed immediately by cervical dislocation. However, no such cases occurred within the whole 21 days of pharmacological testing. The animal tissues for the ex vivo experiments were taken post mortem, immediately after all animals were sacrificed by the cervical dislocation. Chemicals and Biochemicals K 2 [PtCl 4 ], 3-chloro-7-azaindole (3Claza), 3-iodo-7-azaindole (3Iaza), 5-bromo-7-azaindole (5Braza) and solvents (acetone, methanol, ethanol, diethyl ether, N,N9-dimethylformamide, water) were purchased from the following commercial sources - Sigma Sigma-Aldrich Co. (Praha, Czech Republic), Acros Organics Co. (Pardubice, Czech Republic) and Fisher-Scientific Co. (Pardubice, Czech Republic). The complexes cis-[ptcl 2 (3Claza) 2 ] (1), cis- [PtCl 2 (3Iaza) 2 ] (2) and cis-[ptcl 2 (5Braza) 2 ] (3) (Figure 1) were synthesized by the reactions of water solution of K 2 [PtCl 4 ] with two molar equivalents of naza dissolved in ethanol, and characterized as reported previously [23]. The RPMI 1640 medium and penicillin-streptomycin mixture were purchased from Lonza (Verviers, Belgium). Phosphatebuffered saline (PBS), fetal bovine serum (FBS), phorbol myristate acetate (PMA), Auranofin (98%#), erythrosin B, and Escherichia coli 0111:B4 lipopolysaccharide (LPS) were purchased from Sigma- Aldrich (Steinheim, Germany). Cell Proliferation Reagent WST-1 was obtained from Roche (Mannheim, Germany). Instant ELISA Kits (ebioscience, Vienna, Austria) were used to evaluate the production of TNFa and IL-1b. NMR Spectroscopy 1 H, 13 C and 195 Pt NMR spectra and two dimensional correlation experiments ( 1 H 1 H gs-cosy, 1 H 13 C gs-hmqc, 1 H 13 C gs-hmbc; 1 H 15 N gs-hmbc; gs = gradient selected, COSY = correlation spectroscopy, HMQC = heteronuclear multiple quantum coherence, HMBC = heteronuclear multiple bond coherence) of the DMF-d 7 solutions were measured at 300 K on a Varian 400 device at MHz ( 1 H), MHz ( 13 C), MHz ( 195 Pt) and MHz ( 15 N). 1 H and 13 C spectra were adjusted against the signals of tetramethylsilane (Me 4 Si). 195 Pt spectra were calibrated against potassium hexachloroplatinate (K 2 PtCl 6 ) in D 2 O found at 0 ppm. 1 H 15 N gs-hmbc experiments were obtained at natural abundance and calibrated against the residual signals of DMF adjusted to 8.03 ppm ( 1 H) and ppm ( 15 N). The splitting of proton resonances in the reported 1 H spectra is defined as s = singlet, d = doublet, t = triplet, br = broad band, dd = doublet of doublets, m = multiplet. Stability study: the DMF-d 7 solutions of 1 3 were studied by 1 H NMR after 6 h, 24 h and 1 week and by all the above-mentioned NMR experiments after two weeks of standing at laboratory temperature, and by 1 H NMR after 15 min and 1 h of heating at 100uC. Hydrolysis study: the complexes 1 and 3 (ca. 20 mg) were dissolved in 0.5 ml of DMF-d 7 and 0.25 ml of distilled water were added. The mixture was heated to 50 or 100uC for 3 h and white PLOS ONE 2 March 2014 Volume 9 Issue 3 e90341

97 Anticancer Pt(II) Complexes with 7-Azaindole solid formed. The product was removed and dissolved in DMF-d 7. The purity of the hydrolyzed products was.95%. The Interaction and Stability Studies of 1 Evaluated by the ESI-MS The 10 mm methanolic solution (final concentration) of the selected complex cis-[ptcl 2 (3Claza) 2 ] (1) was mixed with the equivalent volume of L-cysteine (Cys) and reduced glutathione (GSH) dissolved in water in the physiological concentrations (the final concentration of 260 mm for Cys, and 6 mm for GSH, respectively) [25]. The mixture was sealed and kept at 25uC for a month. During this period (immediately after the preparation, 1 h, 12 h and 1 month after the preparation), the small amounts of the reacting mixture (usually 20 ml) were analyzed by the FIA-ESI- MS method in both the positive and negative ionization mode. The mobile phase composed of 90% methanol and 10% of 10 mm ammonium acetate was pumped at the rate of 0.2 ml/ min by the quaternary pump of Dionex Ultimate 3000 HPLC System. The samples were injected (50 ml) into the flow of the mobile phase by the autosampler of the HPLC from the same make. The parameters of Thermo LCQ Fleet mass spectrometer were set as follows: API Source voltage 4.5 kv, sheath gas flow rate 40 (arb units), aux gas flow rate 20 (arb units), capillary temperature 275uC, RF lens voltage V, lens V, lens V, gate lens V, multipole1 offset V, front lens V. The similar FIA-ESI-MS experiment was arranged to test the stability of the selected complex 1 in a water/ methanol solution (10 mm, 1:1 v/v) over the same time period as mentioned above. ITC Experiments All the isothermal titration calorimetry (ITC) measurements were performed at 30uC using a VP-ITC device (MicroCal Inc.). The studied solutions (2.5 mm HSA, mm GSH, mm Cys, 1.0 mm cisplatin and 1.0 mm 1) in a water/dmf mixture (1:1 v/v) were degassed prior to the titration. The experiments (titration of mm Cys with 1.0 mm solution of 1, titration of mm GSH with 1.0 mm solution of 1, titration of 2.5 mm HSA with 1.0 mm solution of 1, titration of mm Cys with 1.0 mm solution of cisplatin, titration of mm GSH with 1.0 mm solution of cisplatin and titration of 2.5 mm HSA with 1.0 mm solution of cisplatin) were carried out uniformly: the biomolecule (HSA, GSH or Cys) was titrated with 1 (or cisplatin)by 25 injections of 10 ml each with interval between injections being 300 s. The blank experiments were performed using the same conditions without appropriate biomolecule in cell, where only the water/dmf (1:1 v/v) mixture was poured. The blank experiment data were subtracted and the data were fitted (one-site, two-site or sequential binding model) using the MicroCal Origin software version 7.0. Cell Culture and In Vitro Cytotoxicity Testing In vitro cytotoxic activity of 1 3 and the clinically used platinumbased drug cisplatin was determined by an MTT assay in Human Negroid Cervix Epitheloid Carcinoma (HeLa; ECACC No ), Human Caucasian Malignant Melanoma (G- 361; ECACC No ), Human Ovarian Carcinoma (A2780; ECACC No ), Human Ovarian Carcinoma Cisplatin-resistant Cell Line (A2780R; ECACC No ) and Human Caucasian Lung Carcinoma (A549; ECACC No ) cancer cell lines purchased from European Collection of Cell Cultures (ECACC). The cells were cultured according to the ECACC instructions and they were maintained at 37uC and 5% CO 2 in a humidified incubator. The human cancer cells were treated with 1 3 and cisplatin (applied up to 50 mm) for 24 h, using multi-well culture plates of 96 wells. In parallel, the cells were treated with vehicle (DMF; 0.1%, v/v) and Triton X- 100 (1%, v/v) to assess the minimal (i.e. positive control) and maximal (i.e. negative control) cell damage, respectively. The MTT assay was measured spectrophotometrically at 540 nm (TECAN, Schoeller Instruments LLC). The data were expressed as the percentage of viability, when 100% and 0% represent the treatments with DMF and Triton X- 100, respectively. The cytotoxicity data from the cancer cell lines were acquired from three independent experiments (conducted in triplicate) using cells from different passages. The IC 50 values were calculated from viability curves. The results are presented as arithmetic mean6sd. In Vivo Antitumour Activity The testing of in vivo antitumour activity was carried out according to the previously published procedure using the female DBA/2 SPF mice as experimental animals [26]. In this specific case, the animals were housed in the Sealsafe NEXT IVC Blue Line Housing System (Tecniplast, Italy) to ensure the best possible experimental conditions and eliminate the risk of possible intergroup cross-contamination. Due to the lack of toxicological data and in order to eliminate the excessive use of laboratory animals, the experimental setup, involving the use of the same dose for all the platinum(ii) complexes (3 mg/kg) was used. The animals were weighed daily and observed several times a day for the signs of tumour progression, changes in behaviour, sudden death, and sacrificed if their body weight decreased below 50% of the starting weight or if other severe toxicological problems were seen. The experimental data, describing the survival of the experimental animals, were expressed as the percentage of mean survival time, %T/C defined as the ratio of the mean survival time of the treated animals (T) divided by the mean survival of the untreated control group (C). There were no deaths attributable to acute toxicity of the tested compounds. Histological and Histochemical Evaluations The tissue samples, obtained by the dissection of the sacrificed animals were divided into two parts, the first one was kept below 280uC for further evaluation by the methods of molecular biology, and the second one was processed by the histological procedures as described previously [26]. The paraffin embedded preparations were stained by the standard hematoxylin and eosin staining. The immunohistochemical detection of the Caspases 3 and 8, tumour necrosis factor-alpha (TNF-a) and transcription factor p53 expression were achieved by the use of appropriate antibodies, selective for the mouse species, obtained from AbCam (Cambridge, UK). The quantification of the tissue expression of different proteins in the samples was semi-quantitative, based on the percentage of the areas containing the detected proteins in the view field of at least three different samples. The scale from 0 to 4 was used, where 0 = protein was not detected, 1 = up to 25%, 2 = up to 50%, 3 = up to 75% and 4 = up to 100% of the area contains the detected protein within the view field. The median value from at least three observations was used to evaluate the expression of selected proteins and other histological observations. Protein Expression Analysis Frozen tumour samples were homogenized by a bench blender IKA DI25 (IKA-Werke, Germany) in the presence of a lysis buffer [50 mm Tris-HCl ph 7.5, 1 mm EGTA, 1 mm EDTA, 1 mm sodium orthovanadate, 50 mm sodium fluoride, 5 mm sodium PLOS ONE 3 March 2014 Volume 9 Issue 3 e90341

98 Anticancer Pt(II) Complexes with 7-Azaindole pyrophosphate, 270 mm sucrose, 0.5% (v/v) Triton X-100]. Subsequently, the blended samples were centrifuged at g at 4uC for 15 min. The supernatants were collected and the protein concentration was measured by the Brandford s method using the Brandford reagent (Amresco, USA) according to a manufacturer s manual. The measured samples were stored at 280uC for the following experiments. The concentration of VEGF-A was evaluated by ELISA using VEGF-A Platinum ELISA (ebioscience, Austria). The amount of produced VEGF-A was normalized according to the total protein concentration. The expression of caspase 3, caspase 8 and p53 were evaluated by Westernblot and immunodetection. Lysates were mixed with a denaturing loading dye [250 mm Tris-HCl ph 6.8, 5% (v/v) b- mercaptoethanol, 10% (w/v) SDS, 30% (v/v) glycerol, 0.04% (w/ v) bromophenol blue], heated for 5 min at 70uC and loaded into a 12% polyacrylamide denaturing gel. The final protein amount was 50 mg per well. After protein separation in the gel, they were transferred on the supported nitrocellulose membrane 0.2 mm (Bio-Rad, USA) and the proteins were visualized by Ponceau S dye (Sigma-Aldrich, USA) for loading control. Then, the membrane was blocked by 5% (w/v) BSA at TBST [10 mm Tris-HCl ph 7.5, 150 mm NaCl, 0.1% (v/v) Tween-20]. The following primary and secondary antibodies [conjugated with horseradish peroxidase (HRP)] were used for immunodetection: caspase 3 (ebioscience, USA) in the dilution of 1:1000, caspase 8 (Abcam, UK) in the dilution of 1:1000 and p53 (Abcam, UK) in the dilution of 1:1000, goat anti-mouse antibody in the dilution of 1:3000 (Sigma-Aldrich, USA), goat anti-rabbit antibody in the dilution of 1:3000 (Sigma-Aldich, USA). HRP was detected by Amplified Opti-4CN kit (Bio-Rad, USA). One sample from the control group was loaded into all gels to avoid inter-gel variability. The amount of the proteins was determined by densitometric analysis using AlphaEasy FC software (Alpha Innotech, USA) and was correlated according to the control sample. Zymography Samples were prepared by the same way as for immunodetection. Zymography of MMPs was performed in the gelatin impregnated gel as was described previously [27]. For the analysis, 30 mg of proteins obtained from lysates were loaded in a gel. The intensity of the digested regions was calculated by AlphaEasy FC software (Alpha Innotech, USA) for densitometric analysis. The results were normalized according to 1% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, USA), which was used in each gel as a standard control. Statistical Evaluation The significance of the differences between the results was assessed by the ANOVA analysis, followed by Tukey s post-hoc test for multiple comparisons, with p,0.05 considered to be significant (QC Expert 3.2, Statistical software, TriloByte Ltd.). Results and Discussion Synthesis and General Properties The studied complexes cis-[ptcl 2 (3Claza) 2 ] (1), cis- [PtCl 2 (3Iaza) 2 ] (2) and cis-[ptcl 2 (5Braza) 2 ] (3) (Figure 1) were prepared by the one-step reactions of 7-azaindole halogenoderivatives (naza) with K 2 [PtCl 4 ]at50uc as described previously [23], yielding the products of high chemical purity as demonstrated by 1 H, 13 C, 15 N and 195 Pt NMR data, with isomeric purity. 95% (based on 1 H NMR data; see Figure 2A). Stability Studies The NMR spectra of the studied complexes in DMF-d 7 did not show any difference after two weeks at laboratory temperature and even after 15 min at 100uC. We observed a slight increase of transisomer portion (see the data in File S1 labelled as 1t, 2t, and 3t, which correspond to the trans-isomers of the complexes 1, 2, and 3, respectively; the NMR data for the corresponding cis-isomers were already published in our previous work [23]) after 1 h at 100uC (Figure 2). In addition, the hydrolytic stabilities of the complexes 1 and 3 were investigated by means of 1 H NMR spectroscopy in the mixture of DMF-d 7 /H 2 O (2:1 v/v). We did not detect any 1 H NMR spectra changes even after one week at laboratory temperature. However, the set of new signals was detected at the spectra of the complexes heated in the mentioned DMF-d 7 / H 2 O mixture to 50uC or 100uC (see the data in File S1 labelled as 1hand 3hfor the hydrolytic products of the complexes 1, and 3, respectively). These changes are most probably connected with the hydrolysis of the studied complexes in the mentioned watercontaining system, since the chemical shifts of the new signals do not correspond to either cis- (NMR data given in [23] or transisomer (the NMR data are given in File S1) of the studied complexes or to the signals of free naza molecules. Probably, these signals may be associated with the formation of the hydrolytic products/species, such as [Pt(naza) 2 (OH) 2 ] or [Pt(naza) 2 (H 2 O) 2 ] 2+. In addition, the similar ESI mass spectrometry experiment was arranged to test the stability of the complex 1 in water-containing solution, i.e. in a water/methanol mixture (10 mm, 1:1 v/v) in this case. The attention was drawn towards the possible formation of intermediates which could (at least theoretically) facilitate the interaction of the prepared complexes with target biomolecules (e.g. parts of the proteasome or DNA), and indeed, a very slow time-dependent accrual in the relative intensity of ions corresponding to hydrolysis products (Figure 3), dominantly the species at m/z , together with m/z corresponding to the ionic species [M-H-2Cl+2OH] 2, and [M-H-2Cl+2OH+ CH 3 OH] 2, respectively, was evident in the mass spectra obtained in the negative ionization mode. In comparison to that, the inverse dependence of intensity on the time was found for another type of intermediate observed at m/z , corresponding (according to mass and isotopic distribution) to the ionic species [M-L] 2 ; L=3Claza. ESI-MS Interaction Studies of 1 with Cysteine and Reduced Glutathione In an effort to describe the reactivity of the prepared complexes with the major sulphur-containing biomolecules found in the human plasma, the methanolic solution of the complex 1, as a representative example, was mixed with the mixture of L-cysteine (Cys) and reduced glutathione (GSH) in water in the physiological concentrations. The macroscopic appearance (a colourless solution without any traces of precipitation) of the solution stayed the same during the whole duration of the experiment. The analysis of mass spectra (and that applies to all measured data) did not reveal the evidence that the prepared complexes could be able to interact with physiological levels of main sulphur-containing biomolecules (Cys and GSH). We did not detect any species whose mass corresponds to the adduct of the fragments or hydrolysis products of the studied platinum(ii) complex with Cys and/or GSH even one month after the preparation of the mixture. In fact, only the primal pseudomolecular ion at m/z , corresponding (according to mass and isotopic distribution) to the ionic species [M-H-2Cl+CH 3 OH+5H 2 O] +, was clearly present in the mass spectra of the analysed mixture (see Figure S1 in File S1). PLOS ONE 4 March 2014 Volume 9 Issue 3 e90341

99 Anticancer Pt(II) Complexes with 7-Azaindole Figure 2. Part of the 1 H NMR spectra of cis-[ptcl 2 (5BrHaza) 2 ] (3). The spectra show the N1 H signal of cis- (left) and trans-isomers (right) as observed at different times and temperatures: (A) fresh solution at laboratory temperature; (B) after two weeks at laboratory temperature; (C) after 15 min at 100uC; (D) after 1 h at 100uC. doi: /journal.pone g002 Figure 3. The ESI-MS spectra of the complex 1. The studied complex was dissolved in water/methanol solution (10 mm, 1:1 v/v) and measured 12 h (A) and 1 month after the preparation (B) using the negative ionization mode. doi: /journal.pone g003 PLOS ONE 5 March 2014 Volume 9 Issue 3 e90341

100 Anticancer Pt(II) Complexes with 7-Azaindole ITC Interaction Studies with Cysteine, Reduced Glutathione and Human Serum Albumin The ITC experiments for the representative complex 1 and cisplatin were performed to demonstrate their ability to interact with Cys, GSH and human serum albumin (HSA) as well as differences between their interaction. The interaction with biomolecules, such as the above-mentioned reduced glutathione or cysteine, is one of the crucial features of antitumour effective platinum(ii) complexes [28]. Since any covalent interactions of the complex 1 with GSH and Cys were not observed by means of ESI-MS, some kind of interactions of the studied complexes with GSH were detected by Uv-Vis spectroscopy in our previous work [24]. Therefore, we decided to use ITC as a very sensitive tool for the investigation and thermodynamic characterization of the interaction (unnecessary covalent from the principle of ITC) with various biomolecules [29,30]. We performed analogical experiments for the representative complex 1 and cisplatin to demonstrate the differences between both platinum(ii) dichlorido complexes. An interaction of the complex 1 with both the cysteine and GSH was observed by ITC (see Figure S2 in File S1). Low solubility of the complex 1 in the medium used did not allow us to improve the data quality (in the case of more soluble cisplatin, we used the same conditions to make the obtained results on both compounds comparable). The data were fitted by two- (GSH) and three-site (cysteine) sequential binding model to give the following results: K 1 = ( )610 5 M 21, DH 1 = kcal/mol, DS 1 = 37.6 cal mol 21 K 21, K 2 = ( )610 4 M 21, DH 2 = kcal/mol, DS 2 = 1.87 cal mol 21 K 21, K 3 = ( )610 4 M 21, DH 3 = kcal/mol, DS 3 = 107 cal mol 21 K 21 for titration of cysteine and K 1 = ( )610 4 M 21, DH 1 = kcal/ mol, DS 1 = 18.4 cal mol 21 K 21, K 2 = ( )610 4 M 21, DH 2 = kcal/mol, DS 2 = 36.7 cal mol 21 K 21 for GSH. In the case of cisplatin, the best-fitted results were obtained by three- (GSH) and four-site (cysteine) sequential binding model with the following results: K 1 = ( )610 5 M 21, DH 1 = kcal/mol, DS 1 = 50.7 cal mol 21 K 21, K 2 = ( ) M 21, DH 2 = kcal/mol, DS 2 = 30.9 cal mol 21 K 21, K 3 = ( )610 4 M 21, DH 3 = kcal/mol, DS 3 = 169 cal mol 21 K 21, K 4 = ( )610 4 M 21, DH 4 = kcal/mol, DS 4 = 2271 cal mol 21 K 21 for titration of cysteine and K 1 = ( )610 5 M 21, DH 1 = kcal/ mol, DS 1 = 14.9 cal mol 21 K 21, K 2 = ( )610 4 M 21, DH 2 = kcal/mol, DS 2 = 29.6 cal mol 21 K 21, K 3 = ( )610 4 M 21, DH 3 = kcal/mol, DS 3 = cal mol 21 K 21 for GSH. A comparison of the data obtained on 1 and cisplatin indicated different non-covalent interactions of these platinum(ii) dichlorido complexes with cysteine and GSH (see Figure S2 in File S1), but due to the above-mentioned reasons regarding solubility we were unable to compare the systems from thermodynamic point of view. Serum proteins, including human serum albumin (HSA), are well-known to perform transport intracellular and delivery of the platinum(ii) metallodrugs to the tumour tissues [31,32]. It has been also proved that an interaction of some platinum(ii) complexes with albumin could result in enhancement of antitumour activity [33]. Therefore the study of a complex (1 and cisplatin in the case of this work) interaction with HSA should be carried out for complete description of biological properties of the studied substance. Again, we used ITC as a sensitive and capable thermodynamic characterization method suitable for the description of a platinum complex interaction with serum albumin (see Figure S2 in File S1). The data for the complex 1 were fitted to a two-site model resulting in the following thermodynamic parameters: n 1 = , K 1 = ( )610 4 M 21, DH 1 = kcal/mol, DS 1 = cal mol 21 K 21, n 2 = , K 2 = ( )610 7 M 21, DH 2 = kcal/mol, DS 2 = cal mol 21 K 21 (for cisplatin the data K 1 = ( )610 3 M 21, DH 1 = kcal/mol, K 2 = ( )610 4 M 21, DH 2 = kcal/mol, DS 2 = 1.07 kcal mol 21 K 21, K 3 = ( )610 3 M 21, DH 3 = kcal/mol were obtained by a three-site sequential binding model). In other words, the ITC results indicated two different binding sites of the complex 1 on albumin and different type of non-covalent interaction of the complex 1 in comparison with cisplatin. Similar results, but with only one-fold difference between the binding constants K 1 and K 2, were reported for (to the best of our knowledge) the only platinum complex, whose interaction with HSA was studied by ITC [34]. The presumption that the difference in K n could be caused by the conformation changes of HSA after binding of the complex to the first binding site, has to be taken into account as well. In Vitro Antitumour Activity The complexes 1 3 were studied for their antitumour activity in vitro against lung carcinoma A549, cervix epithelial carcinoma HeLa, malignant melanoma G-361, ovarian carcinoma A2780 and cisplatin-resistant ovarian carcinoma A2780R human cancer cell lines, commonly used in the antitumour activity testing of platinum(ii) complexes [35 38]. The results are given in Figure 4 and Table 1. The complexes 1 3 exceeded the antitumour activity in vitro of cisplatin against all the employed cancer cell lines as they were found to be 3.6-, 2.5- and 5.3-times (A549), 2.2-, 2.0- and 2.3- times (HeLa), 1.7-, 1.1- and 5.7-times (G-361), 4.6-, 5.0- and 6.7- times (A2780) and 10.0-, 9.6- and 12.9-times (A2780R) more effective than cisplatin. The complex 3 was the most active substance determined by the in vitro experiments with IC 50 values lower than those of cisplatin as well as both the complexes 1 and 2. The complexes 1 and 3 were significantly more antitumour active in vitro (ANOVA, p,0.05) against all the cell lines as compared with cisplatin, while the same conclusion can be made for the complex 2 only against the A549, A2780 and A2780R cell lines (Figure 4, Table 1). These results indirectly proved that the studied platinum(ii) complexes with 7-azaindoles are able to overcome intrinsic resistance to cisplatin on the A549, A2780 and A2780R (1 3), and HeLa and G-361 (1, 3) human cancer cell lines in vitro. To support this statement, we used a comparison of logic 50 (see Table S1 in File S1) of the complexes 1 3 and cisplatin, particularly the differences between the mean logic 50 observed for individual substances on all cancer cell lines reported in this work and in [23], and logic 50 of the individual substances against the concrete cell line (Figure 5A), and the differences between the mean logic 50 obtained on individual human cancer cell lines and logic 50 of the individual complex (Figure 5B), to demonstrate the sensitivity or resistance of the cancer cell to the action of the studied complexes in comparison with the others including cisplatin [39,40]. The antitumour activity in vitro of the complexes 1 3 can be evaluated also by means of the resistance factors, since the substances were tested on both cisplatin-sensitive (A2780) and resistant (A2780R) ovarian carcinoma cell lines (Figure 4). The resistance factors, calculated as IC 50 (A2780R)/IC 50 (A2780), equal 1.04 (1), 1.17 (2), 1.17 (3) and 2.25 (cisplatin), which show on the ability of the complexes 1 3 to avoid also the acquired type of cancer cell resistance against cisplatin. This feature of the studied complexes is in good agreement with the above-discussed ability of the studied complexes to overcome an interaction with the sulphur-containing biomolecules, which is well-known as one of PLOS ONE 6 March 2014 Volume 9 Issue 3 e90341

101 Anticancer Pt(II) Complexes with 7-Azaindole Table 1. In vitro antitumour activity given as IC 50 6 S.D. in mm of the complexes 1 3 and cisplatin. Cell line Cisplatin Reference A * * * This work HeLa * * This work G * * This work A * * * This work A2780R * * * This work MCF * * [23] HOS * * * [23] LNCaP [23] Asterisks (*) symbolize significant difference (p,0.05) in in vitro antitumour activity of 1 3 as compared to cisplatin. doi: /journal.pone t001 Figure 4. The in vitro antitumour activity testing results. The data were obtained by the MTT assay against human lung carcinoma A549, cervix epithelial carcinoma HeLa, malignant melanoma G-361, ovarian carcinoma A2780 and cisplatin-resistant ovarian carcinoma A2780R cell lines for 1 3 and cisplatin. The cells were exposed to the compounds for 24 h. Measurements were performed in triplicate and each cytotoxicity experiment was repeated three times. The given IC 50 6S.D. (mm) values represent an arithmetic mean. The asterisk (*) denotes a significant difference (p,0.05) between the studied complexes and cisplatin (A); the plot of resistance factors calculated as IC 50 (A2780R)/IC 50 (A2780) for 1 3 and cisplatin (B). doi: /journal.pone g004 the crucial factors decreasing the resistance of various tumours to the action of platinum-based drugs [11 13]. To conclude the in vitro antitumour activity testing of the complexes 1 3 on the human cancer cell lines, it has to be stated that they were recently tested for their antitumour activity in vitro against three others human cancer cell lines (i.e. eight human cancer cell lines in total), namely breast adenocarcinoma MCF7 (IC 50 = 3.4, 8.0, and 2.0 mm for 1 3, respectively), osteosarcoma HOS (IC 50 = 3.8, 3.9, and 2.5 mm for 1 3, respectively) and prostate adenocarcinoma LNCaP (IC 50 = 3.3, 3.8, and 1.5 mm for 1 3, respectively) [23]. Two of the studied complexes (1, 3) have significantly higher in vitro antitumour activity (p,0.05) than cisplatin against all eight human cancer cell lines, while the complex 2 only on the A549, A2780, A2780R and HOS cells. Although the testing of the platinum complexes against five or more human cancer cell lines is quite common in the literature [40 42], the markedly higher antitumour effect than control (cisplatin in this work as well as in most of similarly focused paper) obtained on all the cell lines may be considered as unique to the best of our knowledge. In vivo Antitumour Activity The in vivo antitumour activity of the complexes 1 3 and the reference drug cisplatin was tested on the mouse model of lymphocytic leukemia (L1210) using the complexes as therapeutic agents in the 7-day dosing schedule (3 mg/kg, i.p.) following the previous 10-day initiation period in which the primary tumours formed (examined by palpation). The percentages of mean survival time, %T/C defined as the ratio of the mean survival time of the treated animal groups (T) divided by the mean survival time of the untreated control group (C), were calculated and are shown in Table 2 and Figure 6. It should be noted that despite the relatively large differences of the absolute %T/C values between Figure 5. The results of in vitro antitumour activity on logarithmic scale. The plot representing the differences from the mean logic 50 obtained for the individual compounds (1, 2, 3 and cisplatin) on eight human cancer cell lines reported in this work (A549, HeLa, G-361, A2780 and A2780R) and in [23] (MCF7, HOS and LNCaP), where the positive values show the sensitivity of the particular cell line, while the negative ones relate to the resistance of the cancer cell line to the action of the platinum(ii) complex (A) and the differences from the mean logic 50 obtained on the individual human cancer cell lines reported in this work (A549, HeLa, G-361, A2780 and A2780R) and in [23] (MCF7, HOS and LNCaP) (B). doi: /journal.pone g005 the cisplatin group and the others experimental groups (1 3), none of these results proved to be significantly different (p,0.01). This is not a surprising result, because to reach the similar results of %T/ C between two antitumour active compounds, they usually have to share also a portion of similarity in toxicological, pharmacodynamic, and pharmacokinetic parameters. It seems likely, that this was not a case in the described experiment. Even if the tested complexes showed no significant extension of the mean survival time in comparison to the control group, the decision about the overall antitumour activity of tested complexes is much more complex process. That is why we chose a multiparametric semiquantitative approach to evaluate the overall antitumour PLOS ONE 7 March 2014 Volume 9 Issue 3 e90341

102 Anticancer Pt(II) Complexes with 7-Azaindole activity of both the prepared complexes and cisplatin, used as a standard. Other parameters, which were determined post mortem, comprise the weight of the tumour tissue and its weight ratio to the whole body weight (see Table 2). Both these parameters proved the aggressive cytotoxic action of cisplatin (elimination of more than 50% of tumour tissues, as compared to control group), while the effect of the complexes 1 3 was much more gradual (the complexes 2 and 3 caused the 2%, and 8% decrease of tumour tissue weight, respectively). The overall health status observations made during the whole time of the experiment showed that the animals in the groups treated by the complexes 1 3 showed no signs of toxicity or changes of normal behavior up to the 18 th day of the experiment (in average the last day of the experiment), while the animals treated with cisplatin showed all known side effects of this therapy, i.e. loss of weight, fatigue, loss of appetite, various types of aberrant behavior. With respect to the given results we can conclude that the lower toxicity of the complexes 1 3 and less aggressive effect against tumour cells have to be considered as more beneficial for the treated animals than far more aggressive action of cisplatin. In addition to the macroscopic observations, much more information was obtained from the methods that evaluated the mechanisms of cytotoxicity, starting from the histological and histochemical observations, followed by the ELISA and Westernblot determination of the expression of selected proteins. Macroscopic and Histological Observations All the animals implanted with the cell line L1210 expressed significant tumour infiltration of the abdominal cavity, peritoneal infiltration and well circumscribed tumours in the place of application, which were identified as anaplastic lymphoma. In all the animals, these tumours proliferated in the visceral fatty tissue and in the gonadal area, and formed well circumscribed focuses. Tumour cell dissemination of the diffuse type was found in GALT (gut-associated lymphoid tissue), the proliferation of the tumour tissues was found in the ileocaecal area and mesocolon. This type of tumour was identified according the WHO classification as diffuse B-lymphoma [43]. The infiltration by tumour cells was observed in several different organ preparations, but no metastatic focuses were identified. This observation is in agreement with the unchanged level of MMPs (see Figure S3 in File S1), which are responsible, at least in part, for metastasis formation [44]. Evaluation of Cellular Processes and Semiquantitative Immunohistochemical Detection of Selected Proteins in Tumour Tissue Samples The results of antiproliferative or pro-apoptotic activity, and necrosis-induction and other physiopathological processes, as determined by the histological and histochemical methods, induced by the tested compounds are summarized in Table 3. As a parameter that indicated the overall state of tumour regression, the reactivity index was calculated as the ratio of atypical mitoses to the apoptoses in one view field of a microscope at 2006 magnification. The reactivity index demonstrates quite well the reactivity of the tumour tissue to the chemotherapy and its possible outcomes, i.e. the probability of further regression of the tumour tissues or, on the other hand, the possibility to restore the earlier rates of its proliferation leading to the impending death. The semi-quantitative evaluation of the tumour reactivity revealed, as expected from the lowering of the tumour tissue weight, the highest antiproliferative activity in the cisplatin-treated group of animals with the reactivity index of The relatively low mitotic activity was accompanied by massive necroses and hemorrhage, which might be responsible for the induction of the inflammatory response documented by the higher detection of the pro-inflammatory cytokine TNF-a. The induction of the proinflammatory cellular responses was recently associated also with its nephrotoxicity [45]. The reactivity index calculated for the complex 3-treated group, which did not correlate proportionally with the decrease of tumour tissue mass, was found to be very positive and perspective. Nevertheless, its absolute value of 1.71 indicates that the complex 3 stimulates all the cellular signs of tumour regression, even if its onset of action is in comparison with cisplatin significantly slower. In the complex 1- and 2-treated groups, there were also found signs of tumour regression (reactivity index was 3.69, and 3.67, respectively), in this case, however, their impact on the reduction of the tumour tissue mass was insignificant. Other parameters semi-quantitatively determined in the tissue samples, are in agreement with the above mentioned overall evaluation on the basis of the reactivity index and their incidence correlate quite well with its values. These preliminary results could serve as the basis for further in vivo studies of the most promising compound (i.e. the complex 3) on the solid tumour models. ELISA and Westernblot Determination of the Expression of Selected Proteins A tumour blood supplementation is very important for its growth. Hence, the vascular endothelial growth factor A (VEGF- A) was quantified in tumour lysates. No significant change was observed for this cytokine, only the complex 1 slightly decreased its amount in comparison with the untreated samples (see Figure S4 in File S1). A level of the initiator caspase 8 (Casp-8), effector caspase 3 (Casp-3), and regulatory protein p53 was also measured. The complex 1 significantly decreased the amount of the active form of Casp-8 (p18) by the factor of 1.8 in comparison with the untreated control tumours (Figure 7A). The complex 2 also reduced p18, but only 1.2-times without any statistical significance. Table 2. Selected parameters obtained from the pharmacological part of the in vivo L1210 antitumour activity model. Compound Control Cisplatin Body weight6 S.E. (g) Weight of the cancer tissues 6 S.E. (g) % of tumour tissues6 S.E Mean survival time 6S.E. (days) % T/C doi: /journal.pone t002 PLOS ONE 8 March 2014 Volume 9 Issue 3 e90341

103 Anticancer Pt(II) Complexes with 7-Azaindole Figure 6. The Kaplan-Meier curves depicting the percentage of survival of animals in the individual groups. doi: /journal.pone g006 Table 3. Selected parameters obtained from the histological and immunohistochemical analyses of the in vivo antiproliferative assay. Compound Control Cisplatin Necrosis Hemorrhage Mitotic Index Caspase Caspase p TNF-a Total Score Reactivity Index doi: /journal.pone t003 On the other hand, the complex 3 and cisplatin slightly increased the level of active Casp-8 by the factor of 1.3. In comparison to cisplatin, the complexes 1 and 2 showed significant diminution of the Casp-8 level by the factor of 2.4, and 1.5, respectively. The complex 3 was the only compound which was able to significantly increase the level of the active form of the effector Casp-3 (Figure 7B), concretely 1.5-times in comparison to the untreated tumours. The complex 2 and cisplatin only weakly raised its amount by the factor of 1.2 and the complex 1 did not have any effect. The expression pattern for p53 was similar to Casp-8. The complex 1 significantly decreased the level of p53 by the factor of 1.8 in comparison to the untreated tumours (Figure 7C). The moderate diminution of p53 level was also detected for the complex 2 (1.4- times). An increase of the p53 level was observed in the case of the complex 3 (1.4-times) and cisplatin (1.6-times). Finally, all the groups treated by the platinum(ii) complexes showed lower mitotic index (MI; lower MI indicates tumour-static or tumour-toxic activity of the given complexes), which indicates tumour regression, in comparison with untreated control, in the order of its absolute value: cisplatin,3,2,1. In other words, the obtained results showed on different action of the complexes 1 and 2 as compared with the complex 3 and cisplatin in accordance with the above-mentioned results of both in vitro and in vivo studies. It is a well-established fact that antitumour active platinum(ii) complexes covalently bind to the DNA molecule [46]. This mode of action was, together with notably higher cell-uptake and DNA platination as compared with cisplatin, also proved within the mechanistic studies performed very recently on the herein reported complexes with 7-azaindoles [23,24]. Platination of DNA molecule induces DNA damage which results in a cell cycle arrest or in apoptosis [47]. This effect is usually caused by the stabilization and activation of the transcription factor p53, which recognizes damaged DNA and is able to trigger the intrinsic (mitochondrial) apoptotic pathway in the case of massive DNA damage [48]. This mechanism is in concordance with our results, since we detected non-significantly higher amounts of p53 in tumours treated by both the most in vivo active compounds - cisplatin and the complex 3. On the other hand, the complex 1 significantly decreased the p53 expression, but the amount of active effector Casp-3 remained unchanged. This effect could be caused by the activation of Casp-3 by p53-independent pathways, e.g. via p73 activation [49]. The complexes 1 and 2 also attenuated activation of the initiator Casp-8, which might indicate the triggering of apoptosis via an intrinsic pathway rather than extrinsic pathway [50]. Higher amounts of active Casp-3 after the complex 3 and cisplatin treatment correspond with TNF-a positivity observed in histological preparations. It has been described that macrophages surrounding cancer cells are able to produce antitumour cytokines, e.g. TNF-a and FasL, after cisplatin treatment in vitro [51]. These cytokines subsequently trigger apoptosis via activation of TNF-a and FasL receptors. The higher production of TNF-a, and the related inflammatory reaction related with it, is probably responsible for significantly elevated necrosis and hemorrhage after cisplatin and the complex 3 treatments. PLOS ONE 9 March 2014 Volume 9 Issue 3 e90341

104 Anticancer Pt(II) Complexes with 7-Azaindole Figure 7. The results of the studies of Casp-8, Casp-3 and p53 expression. (A): The amount of the active form of Casp-8 (p18) was detected by Westernblot and immunodetection. The results are presented as mean 6 S.E. ** significant difference in comparison to untreated cells (p,0.01), # significant difference in comparison to cisplatin-treated cells (p,0.05),### significant difference in comparison to cisplatin-treated cells (p,0.001). (B): the amount of the active form of Casp-3 (p17) was detected by Westernblot and immunodetection. The results are presented as mean 6 S.E. ** significant difference in comparison to untreated cells (p,0.01). (C): Amount of p53 was detected by Westernblot and immunodetection. The results are presented as mean 6 S.E. ** significant difference in comparison to untreated cells (p,0.01),### significant difference in comparison to cisplatin-treated cells (p,0.001). doi: /journal.pone g007 Conclusions In conclusion, this work represents a substantial contribution to the broad and systematic study of cisplatin-derived complexes involving the 7-azaindole derivatives (naza) for the identification of notable biological activities and molecular mechanisms interconnected with these activities. The presented data elaborate previous promising results of in vitro cytotoxicity screening carried out on a series of cis-[ptcl 2 (naza) 2 ] complexes (1 3) and underlying mechanisms based on the interaction of the complexes with DNA. In this paper, we extended the scope of in vitro cytotoxicity testing to the expanded panel of human cancer cell lines (A549, HeLa, A2780 and cisplatin-resistant A2780R, and G-361) and found significant antitumour activity in vitro (the IC 50 values were as low as 1 mm level), which surpassed that of cisplatin considerably. The complexes 1 3 also proved to be able to avoid the acquired resistance of the A2780R cell line to cisplatin. The in vitro References 1. Kelland L (2007) The resurgence of platinum-based cancer chemotherapy. Nature Rev Cancer 7: studies, which strengthened the evidence in the perspective of advanced testing of biological activities, were followed by in vivo and ex vivo trials based on the mouse L1210 lymphocytic leukemia model. The pharmacological observations have been complemented by the histological and immunohistochemical evaluations of isolated cancerous tissues and the expression of selected key proteins, associated with the tumour growth progression and induction of apoptosis (i.e. caspases 3 and 8, p53, VEGF-A were determined by ELISA and Western blot methods). The complexes 1 3 (applied at the same dose as cisplatin) showed that their effect on the reduction of cancerous tissues volume is markedly lower than that of cisplatin, however, they also proved to cause less serious adverse effects on the healthy tissues and the health status of the treated mice. The results of ex vivo assays revealed that above all others, the complex 3 was also able to effectively modulate the levels of active forms of caspases 3 and 8, and the transcription factor p53, and thus activate the intrinsic (mitochondrial) pathway of apoptosis. These results, unequivocally confirming the highest antitumour effect of the complex 3, were supported both by the histological and immunohistochemical observations. The overall positive results of antitumour activity testing were supported by the results of stability and interaction studies, which indicated the ability of the tested complexes to resist the formation of covalent adducts with low molecular sulfur-containing biomolecules in the biologically applicable media. The presented results unambiguously demonstrate that there is still a room for improvement of pharmacological properties of antitumour cis-dichloridoplatinum(ii) complexes using the synthetic approach, based on the substitution of carrier ligands and that the 7-azaindole might serve as a viable basis for such studies. Supporting Information File S1 Supporting information. The 1 H, 13 C, 15 N and 195 Pt NMR data assigned to trans-[ptcl 2 (naza) 2 ] complexes (1t, 2t, 3t) detected as an impurity of the studied cis-[ptcl 2 (naza) 2 ] complexes, the 1 H, 13 C, 15 N and 195 Pt NMR data for the products of hydrolysis of 1 and 3 in DMF-d 7 /H 2 O mixture (1 hand 3h). Figure S1. The ESI-mass spectrum of the mixture of the complex 1 with cysteine and glutathione measured one month after the mixing of the components. Figure S2. ITC results showing the heat released during the titration of cysteine, GSH and HSA by 1 or cisplatin and the binding isotherms. Figure S3. The effect of 1 3 and cisplatin on MMPs secretion in isolated tumours. Figure S4. The effect of 1 3 and cisplatin on VEGF-A secretion in isolated tumours. Table S1. The values of logic 50 (mm) for the complexes 1 3 and cisplatin. (DOCX) Acknowledgments We thank Mr. Alexandr Popa for his assistance with the preparation of the complexes and Mrs. Kateřina Kubešová for help with in vitro cytotoxicity testing. Author Contributions Conceived and designed the experiments: PŠ JH JV ZD ZT. Performed the experiments: PŠ JH JV PS IP GP. Analyzed the data: PŠ JH JV ZD ZT. Wrote the paper: PŠ JH JV ZT. 2. Kelland LR, Farrell NP (2000) Platinum-Based Drugs in Cancer Therapy. Humana: Totowa. PLOS ONE 10 March 2014 Volume 9 Issue 3 e90341

105 Anticancer Pt(II) Complexes with 7-Azaindole 3. Rosenberg B, VanCamp L, Krigas T (1965) Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. Nature 205: Rosenberg B, VanCamp L, Trosko JE, Mansour VH (1969) Platinum compounds - a new class of potent antitumour agents. Nature 222: Harrap KR (1985) Preclinical studies identifying carboplatin as a viable cisplatin alternative. Cancer Treat Rev 12: Akaza H, Togashi M, Nishio Y, Miki T, Kotake T, et al. (1992) Phase II study of cis-diammine(glycolato)platinum, 254 S, in patients with advanced germ-cell testicular cancer, prostatic cancer, and transitional-cell carcinoma of the urinary tract. Cancer Chemother Pharmacol 31: Kidani Y, Inagaki K, Iigo M, Hoshi A, Kuretani K (1978) Anti-tumor activity of 1,2-diaminocyclohexaneplatinum complexes against sarcoma-180 ascites form. J Med Chem 21: McKeage MJ (2001) Lobaplatin: a new antitumour platinum drug. 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106 PŘÍLOHA 5 Štarha, P.; Trávníček, Z.; Popa, I.; Dvořák, Z. Synthesis, characterization and in vitro antitumor activity of platinum(ii) oxalato complexes involving 7-azaindole derivatives as coligands Molecules 19 (2014)

107 Molecules 2014, 19, ; doi: /molecules OPEN ACCESS molecules ISSN Article Synthesis, Characterization and in Vitro Antitumor Activity of Platinum(II) Oxalato Complexes Involving 7-Azaindole Derivatives as Coligands Pavel Štarha 1, Zdeněk Trávníček 1, *, Igor Popa 1 and Zdeněk Dvořák Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, CZ Olomouc, Czech Republic; s: pavel.starha@upol.cz (P.S.); igor.popa@upol.cz (I.P.) Regional Centre of Advanced Technologies and Materials, Department of Cell Biology and Genetics, Faculty of Science, Palacký University, Šlechtitelů 11, CZ Olomouc, Czech Republic; zdenek.dvorak@upol.cz * Author to whom correspondence should be addressed; zdenek.travnicek@upol.cz; Tel.: ; Fax: Received: 7 July 2014; in revised form: 16 July 2014 / Accepted: 17 July 2014 / Published: 25 July 2014 Abstract: The platinum(ii) oxalato complexes [Pt(ox)(naza) 2 ] (1 3) were synthesized and characterized by elemental analysis (C, H, N), multinuclear NMR spectroscopy ( 1 H, 13 C, 15 N, 195 Pt) and electrospray ionization mass spectrometry (ESI-MS); naza = 4-chloro-7- azaindole (4Claza; 1), 3-bromo-7-azaindole (3Braza; 2) or 4-bromo-7-azaindole (4Braza; 3). The prepared substances were screened for their in vitro antitumor activity on the osteosarcoma (HOS) and breast adenocarcinoma (MCF7) human cancer cell lines, where 2 showed moderate antitumor effect (IC 50 = 27.5 μm, and 18.3 μm, respectively). The complex 2 was further tested on a panel of six others human cancer cell lines, including the malignant melanoma (G361), cervix carcinoma (HeLa), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), lung carcinoma (A549) and prostate adenocarcinoma (LNCaP). This substance was found to be moderate antitumor effective against G361 (IC 50 = 17.3 μm), HeLa (IC 50 = 31.8 μm) and A2780 (IC 50 = 19.2 μm) cell lines. The complex 2 was also studied by NMR for its solution stability and by ESI-MS experiments for its ability to interact with biomolecules, such as cysteine, glutathione or guanosine 5'-monophosphate.

108 Molecules 2014, Keywords: platinum(ii) complexes; oxalato complexes; 7-azaindole derivatives; multinuclear NMR; antitumor activity 1. Introduction Platinum carboxylates represent a notable group of transition metal complexes, which have been used for the treatment of various types of cancer for many years [1 3]. Various carboxylate anions involved in the structures of the clinically used or studied platinum-based metallotherapeutics can be mentioned, particularly the cyclobutane-1,1-dicarboxylate dianion (involved in carboplatin), glycolate dianion (involved in nedaplatin), lactate dianion (involved in lobaplatin), malonate dianion (involved in heptaplatin), acetate anion (involved in satraplatin) or neodecanoate anion (involved in aroplatin) [4 9]. One of the carboxylate-based leaving groups is the oxalate dianion involved in the well-known substance oxaliplatin clinically used mainly for the treatment of colorectal tumours [10]. In other words, the platinum(ii) oxalato complexes are, thanks to the mentioned oxaliplatin, biologically relevant and worth studying group of compounds. Search for novel antitumor active platinum complexes in terms of novel carrier (i.e., N-donor ligands) and leaving (i.e., carboxylates) ligands is one of the crucial challenge of modern bioinorganic chemistry [11 13], although those involved in clinically used drugs are still substantial part of this research, as exemplified by the NH 3 carrier ligands involved in original platinum-based drug cisplatin, as well as in currently studied picoplatin [14]. As for the biologically effective platinum oxalato complexes, it is quite interesting that although these complexes can be considered, thanks to clinically used anticancer drug oxaliplatin [15], as biologically perspective group of compounds [16], not many papers dealing with such compounds in connection with their biological effect have been reported in last five years [17 24]. Our research group reported the platinum(ii) oxalato complexes involving variously substituted N6-benzyladenine derivatives [17 19], whose the most effective representatives showed IC 50 (HOS) = 3.6 µm ([Pt(ox)(L 1 )]; L 1 =2-chloro-N6-(2-methoxybenzyl)-9-isopropyladenine) and IC 50 (MCF7) = 3.6 µm ([Pt(ox)(L 2 )]; L 2 =2-chloro-N6-(2,4-dimethoxybenzyl)-9-isopropyladenine). Utku et al. described the platinum(ii) oxalato complexes with 2-phenylbenzimidazole ligand and their antibacterial and antifungal activity [20]. The work of Silva et al. dealt with the [Pt(ox)(L 3 )] (L 3 symbolizes a long-chain aliphatic diamine) and their cytotoxic effect against A549, B16-F1, B16-F10 and MDA-MB-231 cancer cells and BHK-21 and CHO non-cancer cells. The obtained results proved four reported oxalato complexes as less active against the named cancer cells (IC 50 (A549) = µm, IC 50 (B16-F1) = µm, IC 50 (B16-F10) = µm, IC 50 (MDA-MB-231) = µm) as compared with cisplatin (IC 50 (A549) = 2.7 µm, IC 50 (B16-F1) = 3.5 µm, IC 50 (B16-F10) = 4.2 µm, IC 50 (MDA-MB-231) = 1.4 µm) [21]. Other platinum(ii) oxalato complexes involved 1,2- diaminocyclohexane with variously monoalkyl-substituted nitrogen atom (L 4 ) [22]. These substances were studied for their in vitro cytotoxicity against HepG-2, MCF7, A549 and HCT-116 human cancer cell lines. The antitumor effect of the most effective complex, expressed as IC 50 = 3.7, 8.8, 18.4, and 2.1 µm, respectively, against the mentioned cells, exceed both cisplatin and oxaliplatin used as standards in this study. The biological perspective of the oxalate anion, used as O-donor ligand in the

109 Molecules 2014, biologically active transition metal complexes, can be also demonstrated on the palladium(ii) oxalato complexes with N6-benzyladenin-based N-donor ligands, which in many cases showed noticeable antitumor activity against human cancer cell lines in many cases [19,25,26]. The herein described platinum(ii) oxalato complexes with variously substituted 7-azaindole halogeno-derivatives (1 3; Scheme 1), namely 4-chloro-7-azaindole (4Claza; the complex 1), 3-bromo-7-azaindole (3Braza; the complex 2) and 4-bromo-7-azaindole (4Braza; the complex 3), follow recently reported analogues involving differently substituted 7-azaindoles (3-chloro-7-azaindole, 3Claza; 3-iodo-7-azaindole, 3Iaza; 5-bromo-7-azaindole, 5Braza) [24]. Although the mentioned recently reported platinum(ii) oxalato complexes with 3Claza, 3Iaza and 5Braza N-donor ligands did not show any biological effect on osteosarcoma HOS, breast carcinoma MCF7 and prostate carcinoma LNCaP human cancer cell lines (these complexes were poorly soluble in the medium used), we decided to study, whether substitution variability of the 7-azaindole moiety within the platinum(ii) oxalato complexes may led to the formation of antitumor active compounds. Scheme 1. The synthesis and schematic representation of the prepared [Pt(ox)(naza) 2 ] complexes (1 3) given with the atom numbering scheme of the used 7-azaindole derivatives. R 3 =H and R 4 =Cl for 4Claza and [Pt(ox)(4Claza) 2 ] (1); R 3 =Br and R 4 =H for 3Braza and [Pt(ox)(3Braza) 2 ] (2) and R 3 =H and R 4 =Br for 4Braza and [Pt(ox)(4Braza) 2 ] (3). 2. Results and Discussion 2.1. General Properties The platinum(ii) oxalato complexes [Pt(ox)(naza) 2 ] (1 3; Scheme 1) were prepared by a one-step synthetic procedure using the bis(oxalato)platinate(ii) salt as a starting compound [17]. The composition of the products was proved by the results of elemental analysis. The electrospray ionization mass spectra obtained in the positive mode (ESI+) of the studied complexes contained the molecular peaks detected at m/z (calc ; 5%; {[Pt(ox)(4Claza) 2 ]+H} + for 1), m/z (calc ; 40%; {[Pt(ox)(3Braza) 2 ]+H} + for 2) and m/z (calc ; 25%; {[Pt(ox)(4Braza) 2 ]+H} + for 3), their adducts with different cations, namely m/z (calc ; 30%; {[Pt(ox)(4Claza) 2 ]+K} + for 1), m/z (calc ; 50%; {[Pt(ox)(4Claza) 2 ]+Na} + for 1), m/z (calc ; 100%; {[Pt(ox)(3Braza) 2 ]+K} + for 2), m/z (calc ; 15%; {[Pt(ox)(3Braza) 2 ]+Na} + for 2), m/z (calc ; 100%; {[Pt(ox)(4Braza) 2 ]+K} + for 3) and m/z (calc ; 55%; {[Pt(ox)(4Braza) 2 ]+Na} + for 3). The peak of the released N-donor ligand was found at m/z (calc ; 5%; {4Claza+H} +

110 Molecules 2014, for 1), m/z (calc ; 20%; {3Braza+H} + for 2) and m/z (calc ; 15%; {4Braza+H} + for 3). The pseudomolecular peaks of the {[Pt(ox)(naza) 2 ] H} species were detected by means of ESI (electrospray ionization in the negative mode) mass spectrometry at m/z (calc ; 60%; {[Pt(ox)(4Claza) 2 ] H} for 1) and m/z (isomeric complexes 2 and 3; calc ; 100%; {[Pt(ox)(3Braza) 2 ] H} for 2, and: calc ; 85%; {[Pt(ox)(4Braza) 2 ] H} for 3). The ESImass spectra of the studied compounds also contain the peaks whose mass correspond to the {[Pt(ox)(naza)] H} fragment (434.0 m/z, calc ; 30%; {[Pt(ox)(4Claza)] H} for 1; m/z, calc ; 30%; {[Pt(ox)(3Braza)] H} for 2; m/z, calc ; 65%; {[Pt(ox)(4Braza)] H} for 3) and {naza H} ligand (151.0 m/z, calc ; 10%; {4Claza H} for 1; m/z, calc ; 15%; {3Braza H} for 3; m/z, calc ; 20%; {4Braza H} for 3). The complexes 1 3, as well as the starting compounds naza and K 2 [Pt(ox) 2 ] 2H 2 O, were studied by means of multinuclear and 2D NMR spectroscopy. All the 1 H, 13 C and 15 N signals of free naza molecules were unambiguously detected in the spectra of corresponding platinum(ii) complexes and assigned by means of the below-mentioned 2D NMR experiments. The significantly different 15 N-NMR coordination shift values of the N1 ( Δδ = ppm) and N7 ( Δδ = ppm) atoms clearly proved the coordination of naza molecules through the N7 atoms. The 13 C-NMR chemical shifts of the C11 and C12 atoms of the oxalate dianion (detected at ppm) and the 195 Pt chemical shifts, which equal (1), (2) and (3), correlate well with the values of the formerly reported platinum(ii) oxalato complexes with 7-azaindole [23] or its derivatives [24] NMR and ESI-MS Stability and Interaction Studies 1 H and 195 Pt NMR spectroscopy (solution of 2 in DMF-d 7 and DMF-d 7 /H 2 O mixture, 9:1 v/v) and electrospray ionization mass spectrometry (ESI-MS; solution of 2 in methanol and water/methanol mixture, 1:1 v/v) (the presence of the organic solvents ensured the solubility of the studied complex, because carrying out of the experiments in water was prevented by limited solubility of the mentioned complex in water) were used to investigate the behaviour of the representative complex 2 in the mentioned organic or water-containing solvents. As it is generally accepted for the antitumor active platinum(ii) complexes, hydrolysis is a crucial step within the mechanism of action, which lead to the replacement of leaving groups (i.e., the oxalato ligands) and formation of the activated and more reactive aqua- and/or hydroxidoplatinum(ii) species, probably with formulas cis-[pt(h 2 O) 2 (3Braza) 2 ] 2+ and/or cis-[pt(oh) 2 (3Braza) 2 ] in our case [1,2,27,28]. The hydrolysis of the platinum(ii) oxalate complexes should be connected with opening of the PtO 2 C 2 ring and/or substitution of the oxalate dianion by two H 2 O or OH species as ligands, both resulting in the change of inner coordination sphere (resulting in new peaks in mass spectra) and electron density within the initial complex, which is known to provide different 1 H and 195 Pt NMR chemical shifts [27,28]. Since we did not observe any new signals in both the 1 H and 195 Pt NMR spectra (Figure 1), it can be concluded that the complex 2 is stable and do not undergo any changes within the structure during 5 days in DMF-d 7 as well as in the DMF-d 7 /H 2 O mixture. Similarly it was found that the complex is stable and did not show any change in the composition from the mass spectrometry point of view, because its mass spectra (methanol solutions) recorded after 12 h did not contain any novel peaks as compared with the spectra obtained on the fresh solution of 2.

111 Molecules 2014, Figure 1. Time dependent 195 Pt NMR spectra on the representative complex 2 dissolved in DMF-d 7 /H 2 O mixture (9:1 v/v) showing on the stability of the complex. In the case of water/methanol mixture solution of 2, we detected several new peaks in the mass spectra in comparison with the spectra of 2 dissolved in pure methanol, but their isotopic distribution did not correspond to that of platinum-containing species (Figure 2). In other words, no new platinumcontaining species was found in the mass spectra recorded on the water/methanol solution of 2, which, as in the case of NMR, proved that no hydrolytic processes proceeded under the experimental condition used. Thus it can be said that we did not get any evidence of the hydrolysis usually involved within the mechanism of action of the cytotoxic active platinum(ii) complexes including the clinically used platinum(ii) oxalate complex oxaliplatin [1,2,29]. On the other hand, it is known that the oxaliplatin hydrolysis in water (leading to diaqua-species) and under in vivo conditions has different course, since the latter one provides the carbonato or phosphato adducts (instead of the above mentioned diaqua ones), which consequently enhance the reactivity of such species towards nucleophiles including nucleobases [30 32]. It means that although we did not observed any processes usually associated with the action of cytotoxic platinum(ii) complexes (see Section 2.3. for the in vitro cytotoxicity of 1 3) under experimental conditions, the cytotoxic action itself is not excluded with respect to different conditions in the cells (cytosol involving various ions) as compared with those used in the herein discussed NMR and ESI-MS experiments. ESI-MS was also used to study the ability of 2 to interact with sulphur-containing biomolecules (cysteine (cys) and reduced glutathione (GSH)) or guanosine 5'-monophosphate (GMP) in water/methanol mixture (1:1 v/v) (again, the presence of the organic solvent ensured the solubility of the studied complex). It is well-known that ability of the cytotoxic platinum(ii) complexes to interact with the intracellular sulphur-containing compounds correlates with their activity as well as with the resistance of the respective tumours in terms of inactivation of the platinum(ii) species and their removing from the cell [1,29]. With respect to this phenomena, ability of the studied platinum(ii) complexes to interact with the sulphur-containing biomolecules should be investigated by relevant techniques. In the case of this work, we studied an interaction of 2 with the mixture of cys and GSH in water/methanol mixture. We did not observe any adducts of 2 (or its fragments formed during ionization) with cysteine or reduced glutathione assignable to the species formed by their interaction (Figure 2), which corresponds to the above-described reluctance of 2 to undergo hydrolysis. The only exception from this statement is very weak peak of the {[Pt(cys)(ox)(3Braza) 2 ]+H} + species (Figure 2, inset) detected in the ESI+ spectra of the studied complex 2 at m/z (calcd m/z), which most probably contains a ring-opened product of the interaction with a monodentate bound oxalate dianion.

112 Molecules 2014, Figure 2. The ESI+ mass spectra (recorded on fresh solutions and after 12 h) for the stability in methanol/water mixture, and for the interaction of the representative complex 2 with the mixture of cysteine and reduced glutathione (cys+gsh), and guanosine 5'-monophosphate (GMP), given together with the spectra of the fresh solutions of the individual reactants, i.e., the complex 2, cys+gsh mixture and GMP. * stands for {cys+h} +, for {GSH+H} +, for {H 2 GMP+H} +, {HNaGMP+H} + and {Na 2 GMP+H} +, for {3Braza+H} + and for {[Pt(ox)(3Braza) 2 ]+H} + and {[Pt(ox)(3Braza) 2 ]+Na} + ; Inset: Part of the ESI+ mass spectrum of the mixture of the complex 2 with cys+gsh (recorded 12 h after the preparation) showing the peak of the {[Pt(cys)(ox)(3Braza) 2 ]+H} + species, together with the simulated isotopic distribution labeled as the green triangles. The mechanism of action itself of the clinically used antitumor active platinum(ii) complexes is based on the covalent binding of activated platinum(ii) species to the nuclear DNA molecule of the tumour cells [33], which is also expected for most of the platinum(ii) complexes having cytotoxic effect. A simple model to study the ability of the platinum(ii) complexes to bind DNA molecule is based on binding reactions with various nucleobase-based compounds such as GMP employed in this work. However, we have to state that we did not detect any species whose mass and isotopic distribution would correspond with those of adduct of the studied complex, its fragments or its hydrolysis products with GMP (Figure 2) In Vitro Cytotoxicity The complexes 1 3 were screened for their in vitro antitumor activity against two types of the human cancer cell lines - osteosarcoma (HOS) and breast adenocarcinoma (MCF7) (Table 1). In the

113 Molecules 2014, case of 1 and 3, the testing was limited by low solubility of the compounds, which can be expressed as <1.0 μm. Interestingly, the solubility of 2 was much higher (>50.0 μm) in the medium used. This compound showed the moderate in vitro anticancer activity, concretely 27.5 ± 3.4 μm against HOS and 18.3 ± 3.6 μm against MCF7 cells, which is comparable effect with that of cisplatin (IC 50 (HOS) = 25.4 ± 8.5 μm, IC 50 (MCF7) = 18.1 ± 5.1 μm) (Table 1, Figure 3). It has to be mentioned, that we also tried to compare the results of 2 with another platinum-based drug, oxaliplatin, which involve the same living group in its structure as the studied complexes 1 3. However, such comparison is limited by the fact that oxaliplatin did not show any cytotoxic effect on both cell lines up to the 50.0 μm concentration (IC 50 (HOS) > 50.0 μm, IC 50 (MCF7) > 50.0 μm). Nevertheless, it can be stated that the complex 2 exceeded the in vitro antitumor activity of oxaliplatin on HOS and MCF7 human cancer cell lines. Table 1. The results of the in vitro antitumor activity of the studied platinum(ii) oxalato complexes (1 3), cisplatin (CDDP) and oxaliplatin (OXA) against osteosarcoma (HOS), breast adenocarcinoma (MCF7), malignant melanoma (G361), cervix carcinoma (HeLa), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), lung carcinoma (A549) and prostate carcinoma (LNCaP) human cancer cell lines, as obtained by an MTT assay on the cells exposed to the compounds for 24 h. HOS MCF7 G361 HeLa A2780 A2780R A549 LNCaP 1 >1.0 >1.0 nt nt nt nt nt nt ± ± ± ± ± 3.7 >50.0 >50.0 > >1.0 >1.0 nt nt nt nt nt nt CDDP 25.4 ± ± ± ± ± ± 9.6 > ± 1.5 OXA >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 With respect to the results obtained on HOS and MCF7, the complex 2 was tested against next six human cancer cell lines, namely malignant melanoma (G361), cervix carcinoma (HeLa), ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R), lung carcinoma (A549) and prostate adenocarcinoma (LNCaP). It has been observed that its in vitro antitumor activity equals IC 50 = 17.3 ± 3.1 μm (G361; 5.8 ± 2.4 μm for cisplatin, >50.0 μm for oxaliplatin), IC 50 = 31.8 ± 6.2 μm (HeLa; 39.9 ± 4.6 μm for cisplatin, >50.0 μm for oxaliplatin), IC 50 = 19.2 ± 3.7 μm (A2780; 21.8 ± 3.9 μm for cisplatin, >50.0 μm for oxaliplatin) and IC 50 > 50.0 μm (A2780R, A549 and LNCaP; 32.0 ± 9.6, >50.0 and 3.8 ± 1.5 μm for cisplatin, respectively, >50.0 μm for oxaliplatin) cell lines (Table 1, Figure 3). The antitumor activity of 2 against G361, HeLa, A2780, A2780R, A549 and LNCaP can be evaluated as moderate and slightly higher on A2780 and HeLa in comparison with clinically used platinum-based therapeutic cisplatin and on G361, A2780 and HeLa as compared with another platinum-based drug oxaliplatin. As it is mentioned above, 1 3 follow recently reported [24] analogous oxalato complexes involving different types of 7-azaindole halogeno-derivatives, concretely 3-chloro-7-azaindole (3Claza), 3-iodo- 7-azaindole (3Iaza) and 5-bromo-7-azaindole (5Braza), which, similarly to 1 and 3, did not show any effect against both HOS and MCF7 cell lines up to the concentration of 10.0, 25.0, and 0.5 μm, respectively. Concerning all six platinum(ii) oxalato complexes with different 7-azaindole derivatives together, it can be said that the biological activity, in terms of bioavailability, is strongly affected by

114 Molecules 2014, the position of halogeno-substituent of the 7-azaindole moiety, because the solubility of the complexes with 3Claza, 3Braza and 3Iaza ( μm) is significantly higher than that of the complexes involving the 7-azaindole derivatives substituted in the position 4 (4Claza and 4Braza) or 5 (5Braza), whose solubility did not exceed 1.0 μm in the medium used. Figure 3. The in vitro antitumor activity of the complex 2 and cisplatin (CDDP) on osteosarcoma (HOS), breast adenocarcinoma (MCF7), malignant melanoma (G361), cervix carcinoma (HeLa) and ovarian carcinoma (A2780) human cancer cell lines. Asterisk (*) symbolizes significant difference (p < 0.05) in in vitro antitumour activity of 2 as compared to cisplatin; nt = not tested. 3. Experimental Section 3.1. Materials and Methods Potassium tetrachloridoplatinate(ii) (K 2 [PtCl 4 ]), potassium oxalate monohydrate (K 2 (ox) H 2 O), 4-chloro-7-azaindole (4Claza), 3-bromo-7-azaindole (3Braza), 4-bromo-7-azaindole (4Braza), cisplatin, oxaliplatin, cysteine (cys), reduced glutathione (GSH), guanosine 5'-monophosphate disodium salt (GMP) and solvents were purchased from Sigma-Aldrich Co. (Prague, Czech Republic) and Acros Organics Co. (Pardubice, Czech Republic) and used as received. General methods: Elemental analysis was performed on a Flash 2000 CHNS Elemental Analyzer (Thermo Scientific, Waltham, MA, USA). Electrospray ionization mass spectrometry (ESI-MS): Mass spectra were obtained on fresh methanol solutions and after 2 h and 12 h by an LCQ Fleet ion trap mass spectrometer using both the positive (ESI+) negative (ESI ) mode electrospray ionization technique (Thermo Scientific, QualBrowser software, version 2.0.7, Thermo Fischer Scientific, Waltham, MA, USA). The 10 µm (final concentration) solution of 2 in methanol was mixed together with the same volume of water (hydrolysis studies; presence of methanol ensured the solubility of the studied complex, because carrying out of the experiments in water was prevented by limited solubility of the mentioned complex in water), water solutions of the mixture of GSH (6 µm) and cys (260 µm) or water solution of GMP. 20 µl of the mixtures was analysed by means of flow injection analysis/mass spectrometry (FIA/ESI-MS) in both the positive and negative ionization modes 0 h, 2 h and 12 h after the preparation.

115 Molecules 2014, NMR spectroscopy: 1 H, 13 C and 195 Pt NMR spectra and two dimensional correlation 1 H- 1 H gs-cosy, 1 H- 13 C gs-hmqc, 1 H- 13 C gs-hmbc and 1 H- 15 N gs-hmbc experiments (DMF-d 7 solutions) were performed at 300 K on a Varian 400 device (Santa Clara, CA, USA) at MHz ( 1 H), MHz ( 13 C), MHz ( 195 Pt) and MHz ( 15 N); gs = gradient selected, COSY = correlation spectroscopy, HMQC = heteronuclear multiple quantum coherence, HMBC = heteronuclear multiple bond coherence. 1 H and 13 C spectra were adjusted against SiMe 4, while 195 Pt spectra were calibrated against K 2 [PtCl 6 ] in D 2 O found at 0 ppm. 1 H- 15 N gs-hmbc experiments were obtained at natural abundance and calibrated against the residual signals of DMF (8.03 ppm for 1 H, ppm for 15 N). The splitting of proton resonances in the reported 1 H-NMR spectra is defined as s = singlet, d = doublet, t = triplet, br = broad band, m = multiplet. The coordination shift (Δδ; ppm) is calculated as Δδ = δ complex δ ligand. The stability studies of the DMF-d 7 and DMF-d 7 /H 2 O (9:1 v/v) solutions of 2 was carried out by means of 1 H and 195 Pt NMR after 1, 2, 3, 4 and 5 days of standing at laboratory temperature Synthesis of Complexes 1 3 A solution of 1.0 mmol of 4Claza (for 1), 3Braza (for 2) or 4Braza (for 3) in 10 ml of hot (50 C) ethanol was slowly poured into the solution of K 2 [Pt(ox) 2 ] 2H 2 O (0.5 mmol) in 10 ml of hot (50 C) distilled water. The reaction mixtures were stirred at 50 C for two days. The products, which formed, were filtered off, washed (5 ml of distilled water and 5 ml of ethanol) and dried at 40 C (Figure 1). The described syntheses followed a procedure reported in our recent works for analogous complexes with different 7-azaindoles [23,24]. Bis(4-chloro-7-azaindole)-κN7}(oxalato-κ 2 O,O )platinum(ii) (1, C 16 H 10 N 4 Cl 2 O 4 Pt) Light grey solid; yield 80%; 1 H-NMR (400.0 MHz, DMF-d 7 ): δ/δδ = 13.41/1.34 (br, NH-1), 8.71/0.49 (d, J = 6.4, CH-6), 7.98/0.30 (d, J = 3.6, CH-2), 7.37/0.17 (d, J = 6.3, CH-5), 6.79/0.21 (d, J = 3.6, CH-3) ppm. 13 C-NMR (100.6 MHz, DMF-d 7 ): δ/δδ = (C-11,12), 148.2/ 1.6 (C-7a), 146.6/3.0 (CH-6), 138.4/3.8 (C-4), 129.3/1.9 (CH-2), 122.2/3.1 (C-3a), 117.5/1.9 (CH-5), 100.5/2.2 (CH-3) ppm. 15 N-NMR (40.5 MHZ, DMF-d 7 ): δ/δδ = 145.6/3.4 (NH-1), 154.6/ (N-7) ppm. 195 Pt NMR (86.0 MHz, DMF-d 7 ): δ = ppm. ESI MS (30 ev): m/z = (M+Na), (M H), (M 4Claza H), (4Claza+H), (4Claza H). Anal. Calc.: C, 32.7%; H, 1.7%; N, 9.5%. Found: C, 32.8%; H, 1.6%; N, 9.6%. Bis(3-bromo-7-azaindole)-κN7}(oxalato-κ 2 O,O )platinum(ii) (2, C 16 H 10 N 4 Br 2 O 4 Pt) Light grey solid; yield 75%; 1 H-NMR (400.0 MHz, DMF-d 7 ): δ/δδ = 13.49/1.32 (br, NH-1), 8.82/0.48 (d, J = 5.7, CH-6), 8.11/0.23 (d, J = 8.0, CH-4), 8.09/0.31 (s, CH-2), 7.31/0.10 (m, CH-5) ppm. 13 C-NMR (100.6 MHz, DMF-d 7 ): δ/δδ = (C-11,12), 147.5/3.2 (CH-6), 147.0/ 0.9 (C-7a), 130.6/4.0 (CH-4), 128.0/2.1 (CH-2), 122.6/3.3 (C-3a), 118.0/1.4 (CH-5), 89.3/1.6 (C-3) ppm. 15 N-NMR (40.5 MHZ, DMF-d 7 ): δ/δδ = 143.5/2.5 (NH-1), 159.4/ (N-7). 195 Pt NMR (86.0 MHz, DMF-d 7 ): δ = ppm. ESI MS (30 ev): m/z = (M+H), (M H), (M 3Braza H), (3Braza+H), (3Braza H). Anal. Calc.: C, 28.4%; H, 1.5%; N, 8.3%. Found: C, 28.4%; H, 1.5%; N, 8.3%. Bis(4-bromo-7-azaindole)-κN7}(oxalato-κ 2 O,O )platinum(ii) (3, C 16 H 10 N 4 Br 2 O 4 Pt) Light grey solid; yield 80%; 1 H-NMR (400.0 MHz, DMF-d 7 ): δ/δδ = 13.40/1.31 (br, NH-1), 8.60/0.47 (d, J = 6.3, CH-6),

116 Molecules 2014, /0.30 (d, J = 3.5, CH-2), 7.51/0.16 (d, J = 6.2, CH-5), 6.72/0.21 (d, J = 3.5, CH-3) ppm. 13 C-NMR (100.6 MHz, DMF-d 7 ): δ/δδ = (C-11,12), 147.3/ 1.6 (C-7a), 146.3/2.9 (CH-6), 129.4/1.9 (CH-2), 128.0/4.1 (C-3a), 124.6/3.2 (C-4), 120.6/1.8 (CH-5), 102.1/2.2 (CH-3) ppm. 15 N-NMR (40.5 MHZ, DMF-d 7 ): δ/δδ = 145.5/3.1 (NH-1), 155.4/ (N-7). 195 Pt NMR (86.0 MHz, DMF-d 7 ): δ = ppm. ESI MS (30 ev): m/z = (M+H), (M H), (M 4Braza H), (4Braza+H), (4Braza H). Anal. Calc.: C, 28.4%; H, 1.5%; N, 8.3%. Found: C, 28.3%; H, 1.5%; N, 8.4% In Vitro Cytotoxicity Testing Breast adenocarcinoma (MCF7; ECACC No ), osteosarcoma (HOS; ECACC No ), malignant melanoma (G361; ECACC No ), cervix epitheloid carcinoma (HeLa; ECACC No ), A2780 ovarian carcinoma (ECACC No ), A2780R cisplatin-resistant ovarian carcinoma (ECACC No ), lung carcinoma (A549; ECACC No ) and prostate adenocarcinoma (LNCaP; ECACC No ) cancer cell lines were purchased from European Collection of Cell Cultures (ECACC; Prague, Czech Republic). In vitro cytotoxicity was determined by an MTT assay against MCF7 (the complexes 1 3), HOS (1 3), G361 (2), HeLa (2), A2780 (2), A2780R (2), A549 (2) and LNCaP (2) human cancer cell lines. The cells were maintained in a humidified incubator (37 C, 5% CO 2 ). The cells were treated with 1 3 or standards (cisplatin, oxaliplatin) at the 0.01, 0.1, 1.0, 5.0, 25.0 and 50 μm concentrations for 24 h, using multi-well culture plates of 96 wells. In parallel, the cells were treated with vehicle (DMF; 0.1%, v/v) and Triton X-100 (1%, v/v) to assess the minimal (i.e., positive control) and maximal (i.e., negative control) cell damage, respectively. The MTT assay was measured spectrophotometrically at 540 nm (TECAN, Schoeller Instruments LLC). The data were expressed as the percentage of viability, when 100% and 0% represent the treatments with DMF and Triton X-100, respectively. The cytotoxicity data from the cancer cell lines were acquired from three independent experiments (conducted in triplicate) using cells from different passages. The IC 50 values (µm) were calculated from viability curves. The results are presented as arithmetic mean ± SD. The significance of the differences between the results was assessed by the ANOVA analysis, followed by Tukey s post-hoc test for multiple comparisons, with p < 0.05 considered to be significant (QC Expert 3.2, Statistical software, TriloByte Ltd., Pardubice, Czech Republic). 4. Conclusions This work describes three new platinum(ii) oxalato complexes [Pt(ox)(naza) 2 ] (1 3) and evaluates their in vitro cytotoxicity on the selected human cancer cell lines. The testing revealed the complex 2 (involving 3Braza) as in vitro antitumor active against HOS, MCF7, G361, HeLa and A2780 with IC µm. Because the complex 2 differs from 1 (involving 3Claza) and 3 (involving 4Braza) in the nature or position of the 7-azaindole moiety substituent, it can be concluded that 3Braza represents a very perspective N-donor ligand, which could be used as a carrier ligand involved into platinum(ii) complexes with different leaving group than oxalato one.

117 Molecules 2014, Acknowledgments The authors thank the Czech Science Foundation (GAČR P207/11/0841), Operational Program Research and Development for Innovations European Regional Development Fund (CZ.1.05/2.1.00/ ) of the Ministry of Education, Youth and Sports of the Czech Republic, and Palacký University in Olomouc (PrF_2013_015 and PrF_2014_009). The authors also thank Bohuslav Drahoš and Ján Vančo for help with ESI-MS experiments, Radka Křikavová for assistance with NMR experiments, and Alexandr Popa for collaboration on the syntheses. Author Contributions Conceived and designed the experiments: PŠ, ZT, IP, ZD. Performed the experiments: PŠ, ZT, IP, ZD. Analyzed the data: PŠ, ZT, IP, ZD. Wrote the paper: PŠ, ZT. Conflicts of Interest The authors declare no conflict of interest. References 1. Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7, Kelland, L.R.; Farrell, N.P. Platinum Based Drugs in Cancer Therapy; Humana Press: Totowa, NJ, USA, Gielen, M.; Tiekink, E.R.T. Metallotherapeutic Drugs and Metal-Based Diagnostic Agents; John Wiley & Sons, Ltd.: Chichester, UK, Harrap, K.R. Preclinical studies identifying carboplatin as a viable cisplatin alternative. Cancer Treat. Rev. 1985, 12, Akaza, H.; Togashi, M.; Nishio, Y.; Miki, T.; Kotake, T.; Matsumura, Y. Yoshida, O.; Aso, Y. Phase II study of cis-diammine(glycolato)platinum, 254-S, in patients with advanced germ-cell testicular cancer, prostatic cancer, and transitional-cell carcinoma of the urinary tract. Cancer Chemoth. Pharm. 1992, 31, McKeage, M.J. Lobaplatin: A new antitumour platinum drug. Expert Opin. Inv. Drugs 2001, 10, Kim, D.K.; Kim, G.; Gam, J.; Cho, Y.B.; Kim, H.T.; Tai, J.H.; Kim, K.H.; Hong, W.S.; Park, J.G. Synthesis and antitumor activity of a series of [2-substituted-4,5-bis(aminomethyl)-1,3-dioxolane] platinum(ii) complexes. J. Med. Chem. 1994, 37, Kelland, L.R.; Abel, G.; McKeage, M.J.; Jones, M.; Goddard, P.M.; Valenti, M.; Murrer, B.A.; Harrap, K.R. Preclinical antitumor evaluation of bis-acetato-ammine-dichloro-cyclohexylamine platinum(iv): An orally active platinum drug. Cancer Res. 1993, 53, Dragovich, T.; Mendelson, D.; Kurtin, S.; Richardson, K.; Von Hoff, D.; Hoos, A. A Phase 2 trial of the liposomal DACH platinum L-NDDP in patients with therapy-refractory advanced colorectal cancer. Cancer Chemoth. Pharm. 2006, 58,

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120 PŘÍLOHA 6 Štarha, P.; Trávníček, Z.; Dvořák, Z.; Radošová-Muchová, T.; Prachařová, J.; Vančo, J.; Kašpárková, J. Potentiating effect of UVA irradiation on anticancer activity of carboplatin derivatives involving 7-azaindoles. PLoS ONE (2015) akceptovaný manuskript

121 1 2 3 Potentiating effect of UVA irradiation on anticancer activity of carboplatin derivatives involving 7-azaindoles Pa el Šta ha 1, )de ěk T á íček 1, *, )de ěk D ořák 2, Te eza Radošo á-mu ho á 3, Jitka P a hařo á 4, Já Va čo 1, Ja a Kašpá ko á Regional Centre of Advanced Technologies and Materials & Department of Inorganic Chemistry, Fa ulty of S ie e, Pala ký U iversity, Olo ou, Cze h Repu li 2 Regional Centre of Advanced Technologies and Materials & Department of Cell Biology and Genetics, Faculty of S ie e, Pala ký U iversity, Olomouc, Czech Republic 3 Ce tre of the Regio Ha á for Biote h ologi al a d Agri ultural Resear h & Department of Biophysics, Faculty of Science, Pala ký U iversity, Olomouc, Czech Republic 4 Department of Biophysics, Faculty of S ie e, Pala ký U iversity, Olo ou, Cze h Republic * Corresponding author zdenek.travnicek@upol.cz (ZT)

122 21 A stra t The moderate-to-high in vitro cytotoxicity against ovarian A2780 (IC 50 = µm, p ostate LNCaP IC 50 = µm a d p ostate PC-3 (IC 50 = µm human cancer cell lines of the platinum(ii) cyclobutane-1,1'-dicarboxylato complexes Pt(cbdc)(naza) 2 ] (1 6; cbdc = cyclobutane-1,1'-dicarboxylate(2-); naza = halogenosubstituted 7-azaindoles), derived from the anticancer metallodrug carboplatin, are reported. The complexes containing the chloro- and bromo-substituted 7-azaindoles (1, 2, and 4 6) showed a significantly higher (p < 0.05) cytotoxicity against A2780 cell line as compared to cisplatin used as a reference drug. Addition of the non-toxic o e t atio. µm of L-buthionine sulfoximine (L-BSO, an effective inhibitor of -glutamylcysteine synthase) markedly increases the in vitro cytotoxicity of the selected complex 3 against A2780 cancer cell line by a factor of about 4.4. The cytotoxicity against A2780 and LNCaP cells, as well as the DNA platination, were effe ti el e ha ed UVA light i adiatio max = 365 nm) of the complexes, with the highest phototoxicity determined for compound 3, resulting in a 4-fold decline in the A2780 cells viability from 25.1% to 6.1%. The 1 H NMR and ESI-MS experiments suggested that the complexes did not interact with glutathione as well as their ability to interact with guanosine monophosphate. The studies also confirmed UVA light induced the formation of the cis-[pt(h 2 O) 2 (cbdc`)(naza)] intermediate, where cbdc` represents monodentate-coordinated cbdc ligand, which is thought to be responsible for the enhanced cytotoxicity. This is further supported by the results of transcription mapping experiments showing that the studied complexes - 2 -

123 43 44 preferentially form the bifunctional adducts with DNA under UVA irradiation, in contrast to the formation of the less effective monofunctional adducts in dark Keywords: Carboplatin; Platinum(II) complexes; Cyclobutane-1,1'-dicarboxylato; 7- Azaindole; Cytotoxicity; Phototoxicity

124 49 I trodu tio Although the well-known story of platinum-based anticancer metallotherapeutics have slowly reached their second half-century, the application, development and research is still one of the leading branches of bioinorganic chemistry [1 3]. However, there is still room for improvement with regard to the therapeutic effects of platinum-based antitumor active complexes, combined with the suppression of negative side-effects (e.g. nephrotoxicity, neurotoxicity or myelosuppression) and/or ability to overcome both the intrinsic or acquired resistance of various tumor cells against chemotherapeutics [2 5]. One of the current approaches for reaching the aforementioned objectives is based on the irradiation at selected wavelengths of light and converting the initially inactive drugs into significantly enhanced cytotoxics [6-8]. Recently, the considerably increased ability of the 2 nd generation platinumbased anticancer drug carboplatin to bind to the DNA upon UVA irradiation, resulting in increased cytotoxicity, was reported [8]. Carboplatin represents a complex, whose composition offers the possibility of facile derivatization. The approach is based on the derivatization of the cyclobutane- 1,1'-dicarboxylate(2-) (cbdc) (e.g. the diammineplatinum(ii) complexes with the furoxan-substituted cyclobutane moiety [9]), while the second involves the replacement of the NH 3 carrier-ligands (e.g. with adenine-based N-donor ligands [10]). In this work, the second approach was applied to yield a series of cyclobutane- 1,1'-dicarboxylatoplatinum(II) complexes where both the NH 3 ligands are substituted by various 7-azaindoles (naza). 7-Azaindole was recently used as a suitable N-donor carrier ligand of various types of antitumor active platinum(ii) complexes, and a - 4 -

125 number of dichlorido [11 13], mixed-ligand [14,15] and oxalato [11] complexes have been reported to date. The herein presented complexes represent a logical step towards the extension of the group of dichlorido and oxalato platinum(ii) complexes, involving the analogical halogeno-derivatives of 7-azaindole, recently developed by our research group [11 13]. In the case of dichlorido complexes, considerably high in vitro cytotoxicity (with IC 50 values up to 0.6 µm) was found against various human cancer cell lines (ovarian A2780, breast MCF7, osteosarcoma HOS, lung A549, cervical HeLa, malignant melanoma G361 and prostate LNCaP). These cisplatin analogues complexes also successfully overcame an acquired resistance to cancer cells (ovarian carcinoma model) and effectively reduced the tumor tissues volume during the in vivo experiments on mice (L1210 lymphocytic leukemia model), while showing less serious negative side-effects on the healthy tissues as compared with cisplatin [13]. The above-mentioned positive findings, regarding the in vitro and in vivo anticancer activities of platinum(ii) complexes bearing 7-azaindole monodentate ligands, motivated us to study the carboplatin analogues involving the mentioned N- donor ligands (Fig. 1), their cytotoxicity on selected human cancer cell lines and mechanisms of their action under normal conditions and upon UVA light irradiation, using the set of advanced analytical and biological methods Fig. 1. Structural formula of the studied complexes [Pt(cbdc)(naza) 2 ] (1 6). The formula is given together with their atom numbering scheme; R 3, R 4, R 5 = Cl, H, H (3- chloro-7-azaindole (3Claza), involved in complex 1); Br, H, H (3-bromo-7-azaindole (3Braza), 2); I, H, H (3-iodo-7-azaidole (3Iaza), 3); H, Cl, H (4-chloro-7-azaindole - 5 -

126 95 96 (4Claza), 4); H, Br, H (4-bromo-7-azaindole (4Braza), 5); and H, H, Br (5-bromo-7- azaindole (5Braza), 6) Materials a d Methods 99 Chemicals and Biochemicals The reagents (K 2 [PtCl 4 ], 3Claza, 3Braza, 3Iaza, 4Claza, 4Braza and 5Braza, cyclobutane-1,1'-dicarboxylic acid, NaOH, AgNO 3 ), solvents (N,N - dimethylformamide (DMF), acetone, methanol, diethyl ether) and other chemical (reduced glutathione (GSH), guanosine 5'-monophosphate disodium salt hydrate (GMP)) were supplied by Sigma-Aldrich (Prague, Czech Republic) and Acros Organics (Pardubice, Czech Republic). Calf thymus DNA (CT DNA; 42% G:C, mean molecular mass of approximately 20,000 kda) was isolated as previously described [16]. Sephadex G-50 (coarse) was from Sigma-Aldrich (Prague, Czech Republic). MTT, 3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, was from Calbiochem (Darmstadt, Germany). RPMI 1640 medium, fetal bovine serum, trypsin/edta, and Dul e o s odified Eagle s ediu e e f o PAA Pas hi g, Aust ia. Ge ta i i was from Serva (Heidelberg, Germany) General Method for the Synthesis of The platinum(ii) cyclobutane-1,1'-dicarboxylato complexes, [Pt(cbdc)(naza) 2 ] (1 6; naza = 3Claza for 1, 3Braza for 2, 3Iaza for 3, 4Claza for 4, 4Braza for 5 and 5Braza for - 6 -

127 ), were prepared by well-established Dhara`s method [17]. Briefly, 1.0 mmol (415 mg) of K 2 [PtCl 4 ] was dissolved in 15 ml of deionized water at room temperature and KI (830 mg; 5.0 mmol) was added. The solution turned black during 1 h of stirring at room temperature and then 2.0 mmol of naza dissolved in 15 ml of methanol were poured in. The mixture was stirred overnight at room temperature and the obtained yellow solid, i.e. cis-[pti 2 (naza) 2 ] (yields 90%), was filtered off and washed with deio ized ate L a d etha ol L, d ied a d sto ed i desi ato over silica gel. The obtained platinum(ii) diiodido complexes (0.5 mmol) were dissolved in DMF (5 ml) and silver(i) cyclobutane-1,1'-dicarboxylate (0.5 mmol) was added into the solution. The mixtures were stirred at room temperature and in the dark for 48 h. The formed AgI precipitate was collected a d ashed ith DMF ml). Deionized water (25 ml) was poured into the filtrate and the obtained white precipitate was removed by filtration and washed successively with deionized water L, etha ol L a d dieth l ethe L. The p odu ts of [Pt(cbdc)(naza) 2 ] (1 6; Fig. 1) were dried in desiccator over silica gel and stored without any further purification. Characterization data for 1 6 are given in Supporting Information (S1 Text) Physical Measurements A combustion analysis (C, H, N) was performed using a Flash 2000 CHNS Elemental Analyzer (Thermo Scientific). Electrospray ionization mass spectroscopy (ESI-MS) of the methanol solutions was performed on an LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific; QualBrowser software, version 2.0.7) in both the positive (ESI+) - 7 -

128 and negative (ESI ) ionization modes. Infrared spectra were recorded on a Nexus 670 FT-IR (Thermo Nicolet) using the ATR technique in the cm 1 region. The NMR spectra ( 1 H, 13 C, 1 H 1 H gs-cosy, 1 H 13 C gs-hmqc and 1 H 13 C gs-hmbc; gs = gradient selected, COSY = correlation spectroscopy, HMQC = heteronuclear multiple quantum coherence, HMBC = heteronuclear multiple bond coherence) were acquired on the DMF-d 7 solutions at 300 K on either a JEOL JNM-ECA600II spectrometer at MHz ( 1 H) and MHz ( 13 C) (complexes 1, 4 and 6) or a Varian 400 spectrometer at MHz ( 1 H) and MHz ( 13 C) (complexes 2, 3 and 5). Proton and carbon spectra were calibrated against the residual DMF-d 1 6 H NMR (8.03, 2.92 and 2.75 ppm) and 13 C NMR (163.15, and ppm) signals. The symbols s (singlet), d (doublet), t (triplet), qui (quintet), br (broad signal) and m (multiplet) is used for the splitting of the proton resonances Solution Stability Studies Stability of the studied complexes in DMF-d 7 (1 6) and DMF-d 7 /H 2 O mixture (1:1, v/v; 5) was monitored by 1 H NMR spectroscopy (Varian 400 MHz device) after 24 h (both solutions) and 14 days (only DMF-d 7 solutions) of standing at room temperature under ambient light Methods of Biological Testing Cell Culture and In Vitro Cytotoxicity Testing In vitro cytotoxicity of the complexes 1 6, cisplatin and carboplatin was tested by an MTT assay against ovarian carcinoma A2780 (ECACC No ), prostate - 8 -

129 carcinoma LNCaP (ECACC No ) and prostate carcinoma PC-3 (ECACC No ) human cancer cells obtained from European Collection of Cell Cultures (ECACC), as described in our previous works [11,13]. The cell lines were maintained in a humidified incubator ( C, 5% CO 2 ). The cells were treated with 1 6, cisplatin and carboplatin at the M o e t atio s usi g 96-well culture plates. The cells were treated in parallel with vehicle (DMF; 0.1%, v/v), and Triton X-100 (1%, v/v) to assess the minimal (100% of cell viability), and maximal (0% of cell viability) cell damage, respectively. The exposure time was 24 h. The MTT assay was used to determine the cell viability by the spectrophotometric measurements of the solubilized dye at 540 nm (TECAN, Schoeller Instruments LLC). Analogical in vitro cytotoxicity experiments were performed in the case of complex 3 on the A2780 cells with the addition of L-buthionine sulfoximine (L-BSO), which as i depe de tl added to ea h ell to gi e the. µm fi al o e t atio of L- BSO. µm o e t atio of L-BSO is known to be non-toxic and optimal for the experiments focusing on the modulation of anticancer active transition metal complexes) [18]. These experiments were performed with two negative controls (DMF and. µm L-BSO), and no statistically different results were obtained between the controls. The IC 50 values (compound concentrations that produce 50% of cell growth inhibition; µm±sd e e a ui ed three independent experiments (each conducted in triplicate) performed on the cells from different passages. The statistical evaluation (p < 0.05 were considered as significant) of the obtained data was carried out by ANOVA using QC Expert 3.2 statistical software (TriloByte Ltd.)

130 UVA Light Irradiation A LZC-4V photoreactor (Luzchem, Ottawa, ON, Canada) employed with a temperature controller was used for irradiation (4.3 mw cm -2 ; max = 365 nm) of the DNA samples in cell-free media and using the UVA tubes, as described previously [8] Platination of DNA in Cell-free Media CT DNA (0.2 mg ml 1 ) was mixed with 1 6 or, for comparative purposes, with carboplatin in NaClO 4 (10 mm) and immediately irradiated (UVA, max = 365 nm) for 3 h at C i the da k a d the kept fo additio al h u de the e tio ed conditions, similarly as described in [8].. The r i value was 0.08 (r i = the molar ratio of free platinum complex to nucleotide phosphates at the onset of incubation with DNA). After the incubation, the samples were quickly filtered using a Sephadex G-50 column to remove free (unbound) platinum. The platinum content in these DNA samples (r b, defined as the number of the molecules of platinum complex coordinated per nucleotide residue) was determined by flameless atomic absorption spectrometry (FAAS) In Vitro Phototoxicity A2780 and LNCaP cells were seeded in 96-well tissue culture plates in 100 L medium in the absence of antibiotics at a density of 5,000 cells per well and placed in the incubator for 24 h, as previously reported [8]. The solutions of 2, 3, 4 or 5 in the medium (100 L) were added. The cells were incubated for 24 h. After that the cells were washed out, and the medium containing the platinum(ii) complex was

131 replaced by a drug-free medium in the absence of antibiotics, followed by 20 min irradiation with UVA or sham irradiation. After additional 24 h, cell viability was evaluated by an MTT (vide supra) Stability and Interaction Studies after UVA Light Irradiation The 1 H NMR spectra (Varian 400 MHz device) of the selected representative complex 5, its mixture with two molar equivalents of GSH (5+GSH), and its mixture with two molar equivalents of guanosine monophosphate (5+GMP) in DMF-d 7 /H 2 O mixture (1:1, v/v) were recorded right after the UVA irradiation (20 min) after 24 h of standing at room temperature under ambient light. In the case of irradiated 5 itself (i.e. without GSH or GMP) the 1 H NMR spectrum was also recorded after 96 h of standing at room temperature under ambient light. The ESI mass spectra in both the positive and negative ionization modes were recorded using all the mentioned solutions (i.e. 5, 5+GSH and 5+GMP) 24 h after UVA irradiation by the ThermoFinnigan LCQ Fleet Ion Trap mass spectrometer (Thermo Scientific) Transcription Mapping of DNA Adducts In Vitro Linear psp73kb/hpai DNA was incubated with the selected platinum complexes 3 or 5 so that the DNA samples with the platinum(ii) complex were irradiated with UVA fo i ith su se ue t i u atio fo additio al. h i the da k at C, or, alternatively, the DNA samples were incubated with the platinum(ii) complexes in the da k at C fo h. Afte i u ation, the samples were precipitated with ethanol to remove unbound complex and the obtained solid (pellet) was dissolved in

132 M NaClO 4. The aliquots of the samples were used to determine level of Pt bound to DNA (r b, defined as the number of molecules of the platinum(ii) complex bound per nucleotide residue) by using FAAS and spectrophotometric determination of DNA at 260 nm. Transcription of the linear psp73kb/hpai DNA treated with the complexes with DNA-dependent T7 RNA polymerase, followed by the electrophoretic analysis of transcripts were carried out according to the manufacturer (Promega Protocols and Applications, 43 46, 1989/90) recommended protocols, as described previously [19]. The DNA concentration used in this assay (relative to the monomeric nucleotide content) as µm Interstrand DNA Cross-linking in a Cell-free Medium Linear psp73kb DNA/EcoRI (2455 bp) was mixed with 3 or 5 and immediately irradiated with UVA for 30 min with subsequent incubation for additional 4.5 h in the da k at C [8]. Alternatively, the DNA was incubated with the platinum(ii) o ple es i the da k at C fo h. Afte i u atio, the sa ples e e precipitated to remove free, unbound platinum complex, dissolved in 0.01 M NaClO 4 and the r b in the aliquots of these samples was estimated by FAAS and spectrophotometric determination of DNA at 260 nm. DNA in the remaining part of sa ples as -end-labeled by means of the Klenow fragment of DNA polymerase I in the presence of [ - 32 P]dATP. The labeled samples were evaluated for DNA interstrand cross-links according to the previously published procedures by electrophoresis under denaturing conditions on alkaline agarose gel (1%) [19,20]. After the electrophoresis had been completed, the intensities of the bands

133 corresponding to single strands of DNA and interstrand cross-linked duplex were quantified. The frequency of interstrand cross-links was calculated as ICL/Pt (%) = XL/4918 r b (the DNA fragment contained 4918 nucleotide residues), where ICL/Pt (%) is the number of interstrand cross-links per adduct multiplied by 100, and XL is the number of interstrand cross-links per molecule of the linearized DNA duplex, and was calculated assuming a Poisson distribution of the interstrand cross-links as XL = - ln A, where A is the fraction of molecules running as a band corresponding to the non-cross-linked DNA Fluorescence Quenching Experiments Fluorescence measurements of systems consisting of ethidium bromide (EtBr) and CT DNA with addition of platinum complexes 3 or 5 were carried out at a 546 nm excitation wavelength,, and the emitted fluorescence was analyzed at 590 nm. These measurements were performed on a Varian Cary fluorescence spectrophotometer usi g a ua tz ell. The fluo es e e i te sit as easu ed at C in 0.4 M NaCl to avoid secondary binding of EtBr to DNA [21,22]. The concentrations were 0.01 mg ml for DNA and 0.04 mg ml for EtBr, which corresponded to the saturation of all sites of EtBr in DNA [21]

134 275 Results 276 Chemistry A series of six complexes of the general formula Pt(cbdc)(naza) 2 ] (1 6; Fig. 1) was prepared in ca. 40% yields (relating to K 2 [PtCl 4 ]) and their chemical purity (>95%) was checked by combustion analysis (see S1 Text) and by 1 H NMR spectroscopy (S1 Fig). All the corresponding 1 H and 13 C signals (with the appropriate integral intensities) of coordinated naza and cbdc ligands were detected in the spectra (S1 Fig). An N7 coordination mode of the naza ligands was clearly proved for the complexes 1 6 from the calculated 1 H and 13 C NMR coordination shifts (S1 Table). All the complexes were found to be stable in DMF-d 7 over 14 days (no changes were detected in the 1 H NMR spectra). In the case of the selected complex 5 dissolved in the DMF-d 7 /H 2 O mixture (1:1, v/v), a new set of signals corresponding to 4Braza ligand (e.g. N1 H signal at ppm or C6 H signal 8.19 ppm) was detected after 24 h of standing at room temperature under ambient light (Fig. 2B). The chemical shifts detected were different from those of free 4Braza molecule in the same solvent (e.g. N1 H signal at ppm) Fig H NMR stability studies. Time-dependent (A and C - fresh solutions; B and D - after 24 h of standing at room temperature under ambient light) 400 MHz 1 H NMR spectra (N1 H region of 4Braza) as observed before (A and B) and after (C and D) UVA i adiatio i, max = 365 nm) of complex 5 dissolved in the DMF-d 7 /H 2 O solution (1:1, v/v)

135 The ESI-MS spectra, measured in both the positive (ESI+) and negative (ESI ) ionization mode, of all the studied complexes contained the molecular peaks, i.e. { Pt(cbdc)(naza) 2 ]+H} +, and { Pt(cbdc)(naza) 2 ] H}, respectively (S3 Fig.). Moreover, the adducts with sodium ions, { Pt(cbdc)(naza) 2 ]+Na} +, as well as the { Pt(cbdc)(naza)] H} and {naza+h} + spectra as well. species were identified in the appropriate 305 Biological activities testing 306 In Vitro Cytotoxicity The in vitro cytotoxicity of the prepared complexes 1 6 (applied within the concentration range of M, depe di g o the solu ilit i ate containing medium) can be generalized as high against the A2780 ovarian carcinoma cancer cell line (IC 50 = 4.7. µm a d ode ate i the ase of LNCaP IC 50 = µm a d PC-3 (IC 50 = µm p ostate a i o a a e ell li es Ta le 1). All the complexes exceeded the activity of the reference drug cisplatin against A2780, with the most effective complex 6 being ca. 4.6-fold more cytotoxic. Except for compound 3, the in vitro cytotoxicity of the studied complexes against the A2780 cells was significantly higher (ANOVA, p < 0.05) as compared to cisplatin. In the case of LNCaP and PC-3, the evaluation was limited by the fact that IC 50 of cisplatin could not be obtained, because it is higher than the highest applied concentration, above which the compounds are generally considered to be ineffective (i.e. IC 50 >. µm. Complex 4 was found to be the most cytotoxic on LNCaP and PC-3 cell lines (Table 1)

136 The studied complexes also exceeded the in vitro cytotoxicity of carboplatin, which was found to be non-to i up to the. µm o e t atio agai st all th ee a e cell lines used (Table 1) Table 1. In vitro antitumor activity of 1 6, cisplatin and carboplatin against A2780, LNCaP and PC-3 cancer cell lines. The results of the in vitro antitumor activity testing of 1 6, cisplatin and carboplatin against human ovarian (A2780) and prostate (LNCaP and PC-3) cancer cell lines. Cells were treated with the tested compounds for 24 h, measurements were performed in triplicate, and cytotoxicity experiment was repeated in three different cell passages. Data are expressed as IC 50 ± SD M. Complex A2780 LNCaP PC-3 1. ±. *. ±.. ±. 2. ±. * >20.0 a. ±. 3. ±. >50.0 a. ±. 4. ±. *. ±.. ±. 5. ±. *. ±.. ±. 6. ±. *. ±.. ±. Carboplatin >50.0 a >50.0 a >50.0 a Cisplatin. ±. >50.0 a >50.0 a asterisk (*), significantly different values (p < 0.05) between 1 6 and cisplatin; a) IC 50 were not reached up to the given concentration The in vitro cytotoxicity of the selected representative complex 3 was found to be sig ifi a tl highe i the p ese e of. µm L-BSO, an effective inhibitor of glutathione synthesis, as the IC 50 value determined for the A2780 cell line equalled

137 to. ±. µm. This eans about 4.4-times enhancement in the antiproliferative activity as compared to the same complex without added L-BSO Phototoxicity in Cell Cultures The effect on the cell viability determined for each tested compound was significantly (p 0.05) higher when the complex was applied in combination with UVA irradiation, as compared to the reference sample (in the dark) (Fig. 3). Importantly, the control cells (with and without UVA exposure) grew at the same rate Fig. 3. Cytotoxic activity of the complexes 2 5. Cytotoxic activity of 2 5 at their 10 µm o e t atio s agai st the A top pa el a d LNCaP otto pa el ell lines. Viability of the untreated, sham-irradiated cells was taken as 100%. The asterisk (*) denotes significant difference (p < 0.05) between the irradiated and sham irradiated cells DNA Binding in Cell-free Media Samples of double-helical CT DNA were incubated with the complex at r i value of 0.08 in 0.01 M NaClO 4 at C a d su se ue tl di ided i to t o pa ts. O e pa t was irradiated with UVA light ( max = 365 nm, 4.3 mw cm -2 ) immediately after addition of the complex; the other (control) sample was kept in the dark. After 5 h of incubation, the samples were assayed for platinum content bound to DNA, as described above, by FAAS. The amount of platinum bound to DNA in the samples,

138 which were kept in the dark, ranged from 22 to 42% (Table 2). In contrast, after 5 h of continuous UVA irradiation, the platination of DNA increased ca. 2 3-fold, as compared to the samples incubated in the dark (Table 2) Table 2. DNA binding of 1 6 in cell free media. Data are expressed as percentage of plati u ou d to DNA to total plati u i o e. Data ep ese t the ea ± SD of three independent experiments. Complex dark UVA 1 ± ± 2 ± ± 3 ± ± 4 ± ± 5 ± ± 6 ± ± Carboplatin ± ± Stability Studies after the UVA Irradiation The 1 H NMR spectrum of the complex [Pt(cbdc)(4Braza) 2 ] (5) dissolved in DMFd 7 /H 2 O (1:1, v/v) was recorded before and after 20 min of UVA irradiation ( max = 365 nm; 4.3 mw cm -2 ). Before the irradiation, 5 showed one signal at ppm, corresponding to the N1 H atom of the coordinated 4Braza ligand, while two new N1 H signals were detected at and ppm after the irradiation (Figs. 2 and 4). The 1 H NMR spectrum of the starting complex 5 (before the irradiation) showed, as assumed, one quintet (C13 H 2 ) and one triplet (C11 H 2, C12 H 2 ) at 1.89, and

139 ppm, respectively (S4 Fig.). One new C13 H 2 quintet (2.08 ppm) was detected in the 1 H NMR spectrum of the irradiated sample. The 1 H NMR spectroscopy performed on the irradiated sample did not show any new signals nor any change in the integral intensities, as compared with the fresh irradiated solution, after 24 h (Fig. 2), but at 96 h lower intensity of the signal at ppm and one new signal at ppm were detected. Additional 1 H NMR experiments were performed on the irradiated solution of 5 (96 h after the irradiation) spiked with free 4Braza dissolved in the same solution (DMF-d 7 /H 2 O, 1:1, v/v), leading to marked intensity increase of the signal at ppm, which suggested that the signal at ppm belongs to the 4Braza molecule released from the studied complex (Fig. 4) Fig. 4. UVA irradiation effect on the composition of the complex 5. Time-dependent (after 24 (A) or 96 h (B and C) of standing at room temperature under ambient light) 400 MHz 1 H NMR spectra (N1 H region of 4Braza) as observed in the DMF-d 7 /H 2 O (1:1, v/v) solutions of complex 5 (A and B), and complex 5 spiked with free 4Braza (C), with UVA irradiation (20 min, max = 365 nm), respectively The ratio of the integral intensities, showing on the portion of the rearranged/decomposed complex, of the N1 H signal of the parent compound and both the newly emerged signals is 1.00 (13.02 ppm) to 0.65 (12.35 ppm) to 1.06 (11.82 ppm), which indicates that the amount of the decomposed complex is ca 63% after 20 min UVA irradiation. The ESI+ and ESI mass spectra of complex 5 before the irradiation contained the {[Pt(cbdc)(4Braza) 2 ]+H} + (733.2 m/z), {[Pt(cbdc)(4Braza) 2 ] H} (731.2 m/z) and

140 {[Pt(cbdc)(4Braza) 2 ]+Na} + (755.2 m/z) peaks of the starting complex or their adducts with the Na + ion, as well as the peaks of the {[Pt(cbdc)(4Braza)] H} (533.6 m/z) and {4Braza+H} + (197.1 m/z) species (S5 Fig.). All these peaks were detected also in the spectra of the irradiated sample, but with significant changes in intensities. 401 Concretely, the peak of the {4Braza+H} + fragment showed about 5-fold higher relative abundance in the ESI+ mass spectrum after the irradiation, which corresponded with markedly higher intensity of the peak of the {[Pt(cbdc)(4Braza)] H} species with one 4Braza molecule released (S5 Fig.) Photoreaction with Biomolecules (GSH and GMP) Analogous 1 H NMR and ESI-MS experiments, as described above for complex 5, were performed also for the mixtures of 5 with GSH (symbolized as 5+GSH) or GMP (5+GMP) in DMF-d 7 /H 2 O (1:1, v/v). The 1 H NMR spectra of 5+GSH, both before and after the irradiation, contained the characteristic GSH signals, such as triplet at 8.68 ppm and doublet at 8.56 ppm of the N H hydrogen atoms of glycine, and cysteine part of the GSH molecule, respectively. As for 4Braza, the N1 H region of the 1 H NMR spectrum (before irradiation) does not contain any new signals, as discussed above. As for the 1 H NMR spectrum of the irradiated 5+GSH mixture, only the same two new signals (i.e. at and ppm) in the N1 H region of 4Braza as in the case of the irradiated starting material itself, and no new signals in the region of the N H hydrogen atoms of glycine and cysteine were observed. Furthermore, any peaks whose mass corresponded to the adducts of GSH and 5 or fragments were detected in the ESI+ and ESI spectra of 5+GSH, both before and after irradiation (S5 Fig.)

141 Regarding the 5+GMP mixture, the signals of the coordinated 4Braza as well as C8 H signal of free GMP (at 8.37 ppm) were clearly detected in the 1 H NMR spectra before the irradiation. The UVA irradiation caused several changes: the signals (e.g ppm for C6 H; N1 H signals were not detected) of the released 4Braza ligand were detected and one new signal was observed very close to the C8 H signal of free GMP (at 8.31 ppm) with the integral intensity about three times lower compared to the C6 H signal of 4Braza of the starting material in the mixture (S6 Fig.). The mass spectra of the mixtures containing the GMP also showed the new peaks (in addition to those discussed above, corresponding to the {4Braza+H} +, {[Pt(cbdc)(4Braza) 2 ]+H} +, {[Pt(cbdc)(4Braza) 2 ]+Na} +, {[Pt(cbdc)(4Braza) 2 ] H}, {[Pt(cbdc)(4Braza)] H} species) of the {GMP Na+2H} +, {GMP+H} +, {GMP 2Na+H} and also those corresponding to the {[Pt(cbdc)(4Braza)(GMP)] Na+2H} +, 433 {[Pt(cbdc)(4Braza)(GMP)]+H} +, and {[Pt(cbdc)(4Braza)(GMP)] 2Na+H} species at , 408.3, 362.4, 920.2, 942.3, and m/z, respectively (S5 Fig. and S6 Fig.) Characterizations of DNA Adducts Formed in Dark and under the UVA Irradiation Besides the DNA binding capacity, the important factor which modulates the cytotoxicity of platinum compounds is the nature of the conformational changes induced in DNA. In order to determine the nature of DNA adducts formed by Pt(cbdc)(naza) 2 ] complexes in the dark and under the UVA irradiation, several biochemical and biophysical methods have been applied. As model compounds,

142 complexes 3 and 5 have been selected since they exhibited the highest phototoxic effects Transcription Mapping of Platinum DNA Adducts Experiments on in vitro RNA synthesis by T7 RNA polymerase were carried out using a linear psp73kb/hpai DNA fragment, treated with complex 3 or 5 in dark or under the UVA irradiation conditions (see Materials and Methods). The major stop sites produced by the templates treated with 5 either in dark or under the irradiation are shown in Fig. 5 (lanes 5-dark and 5-UV). These stop sites were similar to those produced by cisplatin (Fig. 5, lane cisplatin), i.e. appeared mainly at GG or AG sites - the preferential DNA binding sites for this metallodrug [23]. The stop sites produced by transplatin (shown for comparative purposes) were less regular and appeared mainly at single G and C sites - the preferential DNA binding sites for this platinum complex [24]. Importantly, the efficiency to block the RNA polymerases differed significantly for the adducts formed by 5 in the dark and the adducts formed upon UVA irradiation. The adducts formed by 5 (at r b = 0.003) under irradiation conditions were much more effective in inhibiting the RNA synthesis compared to the adducts formed by 5 in the dark at the same or even higher level of platination (r b = and 0.01) [cf. lanes 5-UV (0.003), 5-dark (0.003) and 5-dark (0.01) in Fig. 5. Similar results were obtained also for complex 3 (not shown) Fig. 5. Inhibition of RNA synthesis by 5, cisplatin and transplatin. Inhibition of RNA synthesis by T7 RNA polymerase on the psp73kb/hpai fragment modified by 5 under the irradiation or in the dark, cisplatin or transplatin. Autoradiogram of 6%

143 polyacrylamide/8 M urea gel. Lanes: Control UVA, unmodified template irradiated with UVA; Control dark, non-irradated unmodified template; A, U, G, C, chain terminated marker RNAs; 5-UV (0.003), the template modified at r b = by irradiated 5; 5-dark (0.01), the template modified at r b = 0.01 by 5 in the dark; 5-dark (0.003), the template modified by 5 at r b = in the dark; cisplatin (0.01), the template modified at r b = 0.01 by cisplatin; transplatin (0.01), the template modified at r b = 0.01 by transplatin Interstrand DNA Cross-links Bifunctional platinum compounds, which coordinate to the base residues in DNA, form various types of interstrand and intrastrand cross-links. Such cross-links in the target DNA are important factors involved in the DNA damaging action of the genotoxic agents. Therefore, we have quantified the interstrand cross-linking efficiency of 3 or 5 when photoactivated or in the dark using linear psp73kb/ecori DNA. The DNA samples were treated with complex 3 or 5 in dark or under the UVA irradiation conditions as described above. Samples were analyzed by agarose gel electrophoresis under denaturing conditions. The interstrand cross-linked DNA appears in the autoradiogram as the top bands (Fig. 6A), as it migrates more slowly than the single-strand DNA (the bottom bands). The frequencies of interstrand crosslinks formed by photoactivated 3 and 5 e e ±, a d ± %, espe ti el. Interestingly, the modification of DNA in dark resulted in the absence of the slowly migrating bands, indicating that these samples contained no detectable interstrand cross-linking, although the levels of platination (r b ) were similar to those in the irradiated samples

144 Fig. 6. Formation of interstrand cross-links and dependence of ethidium bromide (EtBr) fluorescence. A. The formation of interstrand cross-links by complex 5 under the irradiation with UVA (lanes 1 5) or in the dark (lanes 6 10). Lanes: 1, control, untreated DNA (incubated under irradiation conditions); 2 5, r b = , , and , respectively; 6, control, untreated DNA (incubated in the dark); 7 10, r b = , , and , respectively. B. Dependence of ethidium bromide (EtBr) fluorescence on r b for DNA modified by irradiated 5 (squares) or by 5 i the da k t ia gles. Data a e a e age ± SD fo th ee i depe de t experiments. Data for cisplatin (dashed line) and monofunctional dienplatin (dotted line) recorded under identical experimental conditions are taken from the literature [25] Characterization of DNA Adducts by Fluorescence Experiments Ethidium bromide (EtBr), as a fluorescent probe, can be used to characterize the DNA binding of small molecules, such as platinum antitumor drugs and to distinguish the bifunctional from monofuntional DNA adducts of platinum complexes [21,22]. Binding of EtBr to DNA by intercalation is blocked in a stoichiometric manner by the formation of bifunctional adducts of a series of platinum complexes, including cisplatin, which results in a loss of fluorescence intensity. However, DNA binding of monofunctional complexes such as dienplatin (chloridodiethylenetriamineplatinum(ii) chloride) results only in small decrease of the fluorescence intensity [25]. Modification of DNA by 3 or 5 under irradiation conditions resulted in a decrease of EtBr fluorescence (shown in Fig. 6B for 5) similar

145 to that caused by cisplatin at equivalent r b values. On the contrary, the decrease caused by the adducts of 5 formed in the dark was only slightly higher at equivalent r b values than that induced by monodentately DNA-binding dienplatin (having only one leaving ligand). The analogous results were obtained also for the complex Dis ussio The studied Pt(cbdc)(naza) 2 ] complexes (1 6; Fig., p epa ed Dha a s method [17], represent the derivatives of the clinically used platinum-based drug carboplatin involving 7-azaindole (naza) derivatives as N-donor carrier ligands. The coordination mode of the naza ligands in the studied complexes was determined to be through the N7 atom as in the previous works reported in the X-ray structures of the platinum(ii) dichlorido [11,26] and oxalato [26] complexes with the analogous naza ligands. The same coordination mode was clearly proven also for the herein reported complexes 1 6 by the 1 H and 13 C NMR coordination shifts (S1 Table). The prepared complexes showed high (against ovarian carcinoma cells) or moderate (against both the prostate carcinoma cancer cell lines) in vitro cytotoxicity (Table 1). In comparison to the recently studied dichlorido complexes (IC 50 = µm agai st A a d.. µm agai st LNCaP ells [11]) with analogous N-donor ligands, the complexes studied in this work (1 6) are less effective against the mentioned human cancer cell lines. One of the hot-topics in the field of platinum bioinorganic and medicinal chemistry is the preparation of agents having good stability and no or very low cytotoxic effect (so called prodrugs) and study their activation towards biologically

146 active species [27]. Obviously, the studied complexes were not found to be inactive, but their cytotoxicity was still markedly lower as compared to their dichlorido analogues [11,13], which meant that there is still room to improve the biological activity of the studied carboplatin-analogues. To reach this goal, we used two of several possible strategies which can be applied to increase the biological activity of cytotoxic transition metal complexes, the first one was based on the addition of L- BSO (a selective inhibitor of -glutamylcysteine synthase), effectively blocking the inactivation of the complexes by GSH conjugation, and the second one using the photoactivation of the studied compounds by UVA light. The complex 3 showed ca. 4.4-fold enhancement of in vitro cytotoxicity when it as ad i ist ated to the A ells togethe ith. µm L-BSO. As L-BSO is a wellk o i hi ito of -glutamylcysteine synthase, it has a profound effect on the mechanism of action of the cytotoxic transition metal complexes having either cisplatin-like or redox processes modulating mechanism of action [18,28,29]. In other words, decreasing of the GSH cellular levels, caused by the L-BSO addition, affects the cytotoxicity of the complexes inactivated by a GSH-mediated cellular detoxification (e.g. cisplatin in the cisplatin-resistant cancer cell lines) as well as the cytotoxicity of the complexes, whose biological effect is mediated through the cellular redox processes. Since it has been proved by the 1 H NMR and ESI-MS experiments that the studied complexes do not interact with GSH, the increase of the biological effect of 3 on the A2780 cancer cells could come from the modulation of other cellular redox pathways. Iit is known for carboplatin that its in vitro cytotoxicity could be enhanced by the UVA irradiation [8]. That is why we decided to study the UVA irradiation effect on

147 the biological profile of the herein studied carboplatin derivatives. We chose A2780 and LNCaP cells, which were exposed to 2 5 for 24 h, followed by 20 min irradiation with UVA or sham irradiation. We observed that upon UVA irradiation, the in vitro cytotoxicity of the tested substances increased markedly, as compared with the experiments performed in dark (Fig. 3). These promising results motivated us to further perform more detailed molecular biological and biophysical studies to uncover the mechanistic aspects responsible for the considerable biological activity of the studied complexes. The studied compounds were stable in DMF-d 7 over 14 days, as judged by 1 H NMR spectra. On the other hand, analogical experiments performed in the DMF-d 7 /H 2 O mixture (1:1, v/v) provided one new N1 H signal of the 4Braza N-donor ligand (11.94 ppm) after 24 h (Fig. 2). The chemical shift value differs from that of free 4Braza (11.82 ppm), which suggested (together with the fact that only one new signal was detected - release of the 4Braza molecule from complex 5 would have to led to at least two new signals) that the mentioned N1 H signal belongs to the 4Braza ligand coordinated in the species having different composition from that of the starting material. Speculatively, the mentioned species could contain an open six-membered PtO 2 C 3 ring (formed by the central atom and bidentate-coordinated cbdc ligand within the starting complex 5) and could correspond to the composition cis- [Pt(4Braza) 2 (cbdc`)(h 2 O)] (cbdc` = monodentate-coordinated cbdc ligand), which was both experimentally [30,31] and theoretically [32] proven for carboplatin, but we did not get any evidence for this process (e.g. from ESI-MS performed on this sample) in the case of herein reported carboplatin derivatives

148 With an intention to better understand the composition and behavior of the studied complexes (represented by the selected complex 5) before and after the UVA irradiation, as well as in the presence of GSH, GMP or genomic DNA, we decided to perform the stability and interaction studies ( 1 H NMR, ESI-MS) of 5 and its mixtures with GSH (one of the major reducing sulfur-containing agents of human plasma with known coordination affinity towards Pt(II) atom, representing both the transport opportunities and some ways of inactivation of Pt(II) anticancer drugs) or GMP (well-known model system of the target binding site on DNA molecule attacked by the cytotoxic platinum(ii) complexes). In the case where the studied complex was irradiated by UVA light ( max = 365 nm) for 20 min, two new N1 H signals of 4Braza were detected in the 1 H NMR spectra at and ppm together with the signal of the starting material at ppm, while the signal at ppm (as detected for the unirradiated complex 5) was not found (Fig. 2). One of these signals (11.82 ppm) belongs to the free 4Braza molecule, as proved by the 1 H NMR experiments with the irradiated solution of 5 spiked with the solution of free 4Braza ligand (Fig. 4). This is also consistent with the results of ESI-MS, where the intensity of the peaks of the {[Pt(cbdc)(4Braza)] H} and {4Braza+H} + fragments, whose formation is directly associated with a release of the N-donor ligand from the parent complex, markedly increased after the UVA irradiation (S5 Fig.). The second new peak (12.35 ppm) split into two (12.35 and ppm) after longer than 24 h standing at room temperature under ambient light (Fig. 4). We believe that this change is caused by rearrangement or isomerization (e.g. cis-to-trans transformation) of the species formed after the irradiation, and not by the formation of the new species with different composition, because the overall integral intensity

149 of these two signals is in the same ratio to the other two N1 H peaks at and ppm, as in the case of the spectrum recorded 24 h after the irradiation. Further, this observation indirectly proved the opening of the six-membered PtO 2 C 3 chelate ring, without which the above mentioned isomerization would not be possible. Opening of this chelate ring was also indicated by the 1 H NMR results, because the chemical shift of one new C13 H 2 quintet has been detected in the 1 H NMR spectrum of the irradiated sample at 2.08 ppm (S4 Fig.) differing from both the complex 5 and free H 2 cbdc molecule. With respect to the described findings, it can be assumed that the composition of the platinum-containing species formed from complex 5 after the UVA irradiation corresponds to cis-[pt(h 2 O) 2 (cbdc`)(4braza)] (N1 H signal of 4Braza at ppm), which partially rearranges with time to trans- [Pt(H 2 O) 2 (cbdc`)(4braza)] (12.18 ppm). Unfortunately, no direct evidence for these statements from the mass spectra of the irradiated sample was found, probably due to decomposition of the mentioned aqua species connected with the Pt OH 2 bond cleavage under electrospray ionization conditions. Photoreaction with GSH does not lead to the formation of adducts of GSH with complex 5 or fragments, as judged by the 1 H NMR and ESI-MS experiments (S5 Fig.). Although the changes in the NMR and mass spectra before and after the irradiation were detected, they correspond only to irradiation (as discussed above for the original complex) and not to interaction with this sulfur-containing biomolecule. The statement that GSH does not interact with complex 5 or with the species formed from complex 5 after UVA irradiation can be proved also by the fact that no new N1 H signals of 4Braza were observed in the 1 H NMR spectra, as compared to the 1 H

150 NMR spectra of 5 alone (as discussed above). Further, the platinum-containing adducts with GSH were not found by the ESI-MS before and after irradiation (S5 Fig.). On the other hand, GMP showed the ability to interact with Pt(II) atom in the representative complex 5, since the 1 H NMR spectra of the irradiated 5+GMP mixture contained one new signal of the C8 H hydrogen atom of GMP at 8.31 ppm, as well as the signals of free 4Braza released from the parent complex (S6 Fig.). Although one could expect the coordination of GMP to the Pt(II) atom (as known for carboplatin [30]), it could not be unambiguously judged based on the proton NMR spectra, because no new set of signals of 4Braza, coordinated within the GMPcontaining species, was detected. Still, even if we could think about the substitution of both the 4Braza molecules involved in the starting complex by two GMP molecules to be possible, this process would be firstly not very probable, and secondly it was directly disproved by ESI-MS results showing the peaks assignable to the {[Pt(cbdc)(4Braza)(GMP)] Na+2H} +, {[Pt(cbdc)(4Braza)(GMP)]+H} + and {[Pt(cbdc)(4Braza)(GMP)] 2Na+H} species (S5 Fig..). It has to be noted, that these peaks were identified only in the spectra of the irradiated samples, contrary to the peaks at , and m/z of the species whose mass corresponds to {[Pt(cbdc)(4Braza) 2 (GMP)] Na+2H} +, {[Pt(cbdc)(4Braza) 2 (GMP)]+H} + and {[Pt(cbdc)(4Braza)(GMP)] 2Na+H}, respectively, which were detected in the appropriate spectra even before the UVA irradiation of the studied 5+GMP mixture, which indicates that these adducts formed only as a consequence of the electrospray ionization process. Since DNA is the major pharmacological target of the antitumor platinum drugs [2,33,34] it was also of great interest to examine whether the enhanced cytotoxicity

151 of the complexes correlates with DNA binding of the photoactivated derivatives, similarly as in the case of carboplatin [8]. The initial experiments were aimed to quantify the binding of 1 6 and carboplatin to mammalian DNA in cell-free media. The results proved that the amount of platinum bound to DNA was markedly enhanced due to the UVA irradiation (after 5 h) as compared to the samples incubated in dark (Table 2). The results of DNA binding of carboplatin in dark and under continuous irradiation with UVA are in good agreement with the previously published data [8], confirming that less than 5% of carboplatin is bound to DNA in the sample which was kept in the dark. This is in contrast to the increased level of DNA-platination in the sample irradiated with UVA, so that more than 50% of platinum from carboplatin was bound after 5 h (Table 2). Notably, under comparable conditions, the amount of molecules of carboplatin bound to DNA was lower than that of molecules of 1 6. Recent works have shown that the transcription on DNA templates modified by bidentate adducts of platinum complexes can be prematurely terminated at the level or in the proximity of such adducts, while the monofunctional DNA adducts of platinum complexes were unable to terminate the RNA synthesis [19,35,36]. So, the considerably different efficacy of DNA adducts formed by 3 or 5 in dark and under the irradiation conditions (at the same level of DNA platination) to inhibit RNA polymerase is consistent with different frequency of mono- and bifunctional adduct formed by these complexes in dark and under the irradiation conditions. Thus, the results of transcription mapping experiments (Fig. 5) support the hypothesis that under irradiation conditions, complexes 3 and 5 preferentially form the bifunctional adducts with DNA, capable of effective

152 termination of RNA synthesis by RNA polymerases. On the other hand, in dark, the formation of less effective monofunctional adduct prevails. The results of transcription mapping experiments are in good agreement with the characterization of DNA adducts formed by 3 or 5 in the dark and under irradiation conditions estimated by the EtBr fluorescence quenching (Fig. 6B). These results show that 3 and 5 form in the dark the DNA adducts which resemble, from the viewpoint of their capability to inhibit EtBr fluorescence, those formed by monofunctional platinum complexes. Notably, the DNA adducts formed by 3 and 5 under irradiation conditions inhibited EtBr fluorescence to the same extent as bifunctional cisplatin. Hence, the fluorescent analysis is consistent with the idea and supports the postulate that the major DNA adducts formed by 3 or 5 in dark are mainly monofunctional lesions. In contrast, under comparable conditions (at the same level of DNA platination), but under the irradiation conditions, 3 and 5 form on DNA mainly bifunctional adducts similar to those formed by cisplatin. The latter conclusion is also reinforced by the observation (Fig. 6A) that 3 or 5 formed a significant amount of bifunctional interstrand cross-links under the irradiation of DNA (even slightly higher than cisplatin at the same level of platination [19]) whereas 3 or 5 formed under comparable conditions no such bifunctional lesions in the dark in DNA. In addition, the results characterizing the monofunctional binding of 3 or 5 to highly polymeric double-helical DNA are consistent with the formation of a ringopened species [Pt(cbdc`)(naza) 2 (H 2 O)], containing monodentate cbdc`, in the dark. The bifunctional cross-links are probably formed as a consequence of the photoactivation by UVA and very likely occur as a consequence of the reaction of

153 DNA with bifunctional cis-[pt(h 2 O) 2 (cbdc`)(naza)] active intermediate (containing two easily exchangeable H 2 O ligands). The structure of the such bifunctional product was suggested on the basis of 1 H NMR and ESI-MS characterization of the UVA-irradiated solutions of complex 5 (vide supra). To conclude the presented work, we prepared and characterized a series of carboplatin derivatives, involving the halogeno-substituted 7-azaindoles as the N- donor carrier ligands. The in vitro cytotoxicity of the prepared complexes against A2780 human ovarian carcinoma cell line was markedly (ca. 4.4-times) increased by the addition L-BSO. Based on the results of the detailed 1 H NMR and ESI-MS studies carried out on the starting complex 5 and its mixtures with biomolecules GSH or GMP, as well as on the results of DNA-platination, it is obvious that UVA irradiation (20 min, max = 365 nm) led to the release of one 4Braza ligand and hydrolysis of one of the Pt O bonds between central Pt(II) atom and the chelating cbdc dianion. Additionally, the UVA irradiation led to subsequent formation of the activated species (most probably cis-[pt(h 2 O) 2 (cbdc`)(naza)]) and resulted in markedly higher cytotoxicity of 5 against A2780 ovarian carcinoma and LNCaP prostate adenocarcinoma human cancer cell lines, as compared with sham-irradiated samples. Moreover, DNA binding of the studied complexes is markedly enhanced by the irradiation, as was proven on both chemical (ability to interact with GMP) and biological (higher CT DNA platination) experimental levels. Thus, in connection with the acquired results we have reason to believe that the complexes 1 6 could represent suitable candidates for use in photoactivated cancer chemotherapy

154 726 A k owledge e ts The authors thank to Ms. Kateři a Ku ešo á fo help ith the toto i it testi g, D. Radka Křika o á fo pe fo i g NMR e pe i e ts a d D. Bohusla D ahoš fo performing ESI-MS experiments Refere es Rosenberg B, VanCamp L, Krigas T. Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. Nature. 1965; 205: Kelland L. The resurgence of platinum-based cancer chemotherapy. Nature Rev Cancer. 2007; 7: Barry NPE, Sadler PJ. Exploration of the medical periodic table: towards new targets. Chem Commun. 2013; 49: Kelland LR, Farrell NP. Platinum-Based Drugs in Cancer Therapy. Humana: Totowa; Siddik ZH. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene. 2003; 22: Smith NA, Sadler PJ. Photoactivatable metal complexes: from theory to applications in biotechnology and medicine. Phil Trans R Soc. 2013; A 371: Mackay FS, Woods JA, Heringova P, Kasparkova J, Pizarro AM, Moggach SA, et al. A potent cytotoxic photoactivated platinum complex. Proc Natl Acad Sci USA. 2007; 104:

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159 833 Supporti g I for atio S1 Text. The characterization data ( 1 H and 13 C NMR, elemental analysis, FTIR and ESI-MS) for 1 6. S1 Fig. 1 H NMR, 13 C NMR, 1 H 1 H gs-cosy, 1 H 13 C gs-hmqc and 1 H 13 C gs-hmbc spectra of 5. The 1 H-NMR (up left), 13 C-NMR (up right), 1 H 1 H gs-cosy (middle left), 1 H 13 C gs-hmqc (middle right) and 1 H 13 C gs-hmbc (down) spectra obtained on the solution of 5 in DMF-d 7 ; the chemical shift values are given in Experimental section in the main text. S1 Table. The 1 H and 13 C NMR coordination shifts ( al ulated as δ = δ complex δ ligand ; ppm) of the prepared complexes. The 1 H and 13 C NMR oo di atio shifts al ulated as = complex ligand ; ppm) of the prepared complexes. S2 Fig. ESI+ mass spectrum of 5. ESI+ mass spectrum (0 800 m/z range) of the methanolic solution of the complex 5 (A) and its part between 720 and 765 m/z showing the molecular peak (together with isotopic distribution) and its adduct with sodium ion observed experimentally (B) and calculated (C). S3 Fig. Time-dependent 1 H NMR spectra before and after UVA irradiation of 5. Time-dependent (fresh solution and after 24 h) 400 MHz 1 H NMR spectra as observed before and after UVA irradiation (20 min, 365 nm) of the complex 5 dissolved in the DMF-d 7 /H 2 O solution (1:1, v/v). S4 Fig. ESI+ and ESI mass spectra of 5 and its mixtures with GSH or GMP with or without UVA irradiation. ESI+ (left) and ESI (right) mass spectra ( m/z range) of the complex 5 and its mixtures with GSH or Na 2 GMP (dissolved in the DMF

160 d 7 /H 2 O, 1:1, v/v) as detected on the samples with or without irradiation (20 min, 365 nm) 24 h after preparation. stands for {4Braza+H} +, fo {GMP Na+2H} +, {GMP+H} + or {GMP 2Na H}, fo {GS SG+H} + or {GS SG+Na} +, fo {[Pt d 4Braza) 2 ]+H} +, {[Pt(cbdc)(4Braza) 2 ]+Na} + or {[Pt(cbdc)(4Braza) 2 ] H}, for {[Pt(cbdc)(4Braza)] H}, a d fo {[Pt d 4Braza)(GMP)] Na+2H} +, {[Pt(cbdc)(4Braza)(GMP)]+H} + {[Pt(cbdc)(4Braza)(GMP)] 2Na H}. or S5 Fig. 1 H NMR spectrum after UVA irradiation of the mixture of 5 and GMP. 400 MHz 1 H NMR spectrum as observed after UVA irradiation (20 min, 365 nm) of the mixture of the complex 5 and GMP dissolved in the DMF-d 7 /H 2 O solution (1:1, v/v). 865 S6 Fig. ESI mass spectrum of the {[Pt(cbdc)(4Braza)(GMP)] 2Na+H} species Experimental (up) and simulated (down) mass spectrum isotope distribution of the {[Pt(cbdc)(4Braza)(GMP)] 2Na+H} species detected in the ESI mass spectrum of the complex 5 and GMP mixture dissolved in the DMF-d 7 /H 2 O solution (1:1, v/v). The fresh mixture was irradiated (20 min, 365 nm) and the spectrum was recorded 24 h after preparation. S7 Fig. Impact of UVA irradiation of platinum(ii) carboxylato complexes with 7- azaindoles as carrier ligands on their cytotoxicity

161 PŘÍLOHA 7 Štarha, P.; Stavárek, M.; Tuček, J.; Trávníček, Z. 4-Aminobenzoic acid-coated maghemite nanoparticles as potential anticancer drug magnetic carriers: a case study on highly cytotoxic cisplatin-like complexes involving 7-azaindoles Molecules 19 (2014)

162 Molecules 2014, 19, ; doi: /molecules Article OPEN ACCESS molecules ISSN Aminobenzoic Acid-Coated Maghemite Nanoparticles as Potential Anticancer Drug Magnetic Carriers: A Case Study on Highly Cytotoxic Cisplatin-Like Complexes Involving 7-Azaindoles Pavel Štarha 1, Martin Stavárek 1, Jiří Tuček 2 and Zdeněk Trávníček 1, * 1 2 Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, Olomouc CZ 77146, Czech Republic; s: pavel.starha@upol.cz (P.Š.); martin.stavarek01@upol.cz (M.S.) Regional Centre of Advanced Technologies and Materials, Department of Experimental Physics, Faculty of Science, Palacký University, 17. listopadu 12, Olomouc CZ 77146, Czech Republic; jiri.tucek@upol.cz * Author to whom correspondence should be addressed; zdenek.travnicek@upol.cz Tel.: ; Fax: Received: 23 December 2013; in revised form: 17 January 2014 / Accepted: 23 January 2014 / Published: 28 January 2014 Abstract: This study describes a one-pot synthesis of superparamagnetic maghemite-based 4-aminobenzoic acid-coated spherical core-shell nanoparticles (PABA@FeNPs) as suitable nanocomposites potentially usable as magnetic carriers for drug delivery. The PABA@FeNPs system was subsequently functionalized by the activated species (1* and 2*) of highly in vitro cytotoxic cis-[ptcl 2 (3Claza) 2 ] (1; 3Claza stands for 3-chloro- 7-azaindole) or cis-[ptcl 2 (5Braza) 2 ] (2; 5Braza stands for 5-bromo-7-azaindole), which were prepared by a silver(i) ion assisted dechlorination of the parent dichlorido complexes. The products 1*@PABA@FeNPs and 2*@PABA@FeNPs, as well as an intermediate PABA@FeNPs, were characterized by a combination of various techniques, such as Mössbauer, FTIR and EDS spectroscopy, thermal analysis, SEM and TEM. The results showed that the products consist of well-dispersed maghemite-based nanoparticles of 13 nm average size that represent an easily obtainable system for delivery of highly cytotoxic cisplatin-like complexes in oncological practice.

163 Molecules 2014, Keywords: maghemite; nanoparticles; magnetic; platinum complexes; 7-azaindole derivatives; drug delivery 1. Introduction The platinum(ii) complexes, such as cisplatin, oxaliplatin or carboplatin, are well-established anticancer chemotherapeutics used worldwide for the treatment of various types of cancer [1]. Application of these drugs causes several negative side-effects, such as nephrotoxicity, neurotoxicity or myelosuppression, which represent a permanent incentive for bioinorganic chemists to find novel non-platinum drugs (e.g., ruthenium complexes) or platinum complexes with diminished side-effects or to study the possibilities of the targeted drug delivery of the known as well as novel cytotoxic platinum complexes to the tumour tissues. The latter option, i.e., targeted drug delivery, offers various approaches, as reviewed e.g., in [2 4]. One of them is based on the use of magnetic nanoparticles (NPs) coated with a suitable shell, comprising e.g., organic molecules of the same entity, that is able to interact with the drug [5,6]. A distribution of such systems within the organism suffering with cancer could be affected by an external magnetic field, whose application concentrates the drug into the tumour tissue. Among all the magnetic nanoparticles of transition metals and their oxides, iron oxide-based nanosystems hold a paramount position in various medical fields due to their promising properties including magnetic (e.g., superparamagnetism, strong magnetic response and saturation under small applied magnetic fields, excellent heating performance in the frequencies of the alternating magnetic field safe for humans) and biochemical (e.g., very low toxicity, biocompatibility, biodegradability) features [7,8]. To date, they have been successfully employed as negative contrast agents in magnetic resonance imaging, for cell labelling and separation, drug delivery, and as functional components in magnetically-assisted hyperthermia for cancer treatment. Once iron oxide nanoparticles are functionalized with suitable bioactive substances, the resulting system then performs both the diagnostic and therapeutic actions, giving birth to a novel branch of medicine known as theranostics [9]. Many iron oxide-based (both maghemite or magnetite) nanocarriers designed and fabricated for magnetic drug delivery purposes have been reported in the literature to date, involving various synthetic approaches and the use of different organic-coating layers [5,6,10 19]. Regarding the nanocomposites with maghemite-based core-shell NPs functionalized with platinum-based drugs (i.e., structurally similar to those reported in this work), to the best of our knowledge only two such works have been reported to date. Both of them report maghemite-based NPs with dechlorinated cisplatin bound to 4-oxo-4-(3- (triethoxysilyl)propylamino)butanoic acid (OTPBA) [20,21]. These systems showed remarkable, timedependent in vitro cytotoxicity against MCF7, HeLa, A549 and A549R human cancer cell lines, which is comparable with that of cisplatin after 72 h, and moreover, they can be simultaneously used as MRI contrast agents. Several other works have dealt with similar magnetite-based NPs suitable for magnetic drug delivery and studied cisplatin as the model drug as follows: Deng and Lei reported the Fe 3 O 4 /SiO 2 cores with PEG PLA shell (PEG = polyethylene glycol, PLA = polylactic acid) and loaded cisplatin [22]. A similar system, but with a PLA shell, was studied by Devi et al., who focused on

164 Molecules 2014, loading and release properties of cisplatin [23]. Ashjari et al. published the preparation of cisplatinfunctionalized magnetite NPs with biodegradable poly(lactic-co-glycolic acid) (PLGA) with different morphological properties of the resulting composite [24]. Several other studies focused on the analogical systems (magnetite core and cisplatin as the functionalizing agent) differing in organic shells, such as folate acid- [25], squalene- [26], carboxymethylcellulose- [27], dextrane- [28] or poly(ethyl-2-cyanoacrylate)- [29] based layer. Finally, Li et al. studied the effect of cisplatin-loaded magnetite-based NPs on multidrug resistance and its mechanism [30]. Although all of the mentioned nanosystems have to be considered as universal in terms of functionalization by the platinum complexes, it has to be noted that to the best of our knowledge only one work [31] has reported iron oxide-based NPs functionalized with platinum complexes other than the mentioned cisplatin. In particular, the magnetite-silica composite nanoparticles were investigated as carriers of a photoactive platinum diimine complex. In an effort to research the possibilities of targeted delivery of the recently reported highly in vitro cytotoxic cisplatin-like complexes involving 7-azaindole derivatives investigated by our team [32 34], we developed a novel system based on easily obtainable magnetic nanoparticles. They consist of maghemite-based 4-aminobenzoic acid (PABA)-coated core-shell nanoparticles (PABA@FeNPs) functionalized by the activated (i.e., dechlorinated) platinum(ii) complexes bearing various 7-azaindoles (the complexes 1 and 2, whose activated form is symbolized as 1* and 2*; Figure 1a). The obtained systems 1*@PABA@FeNPs and 2*@PABA@FeNPs (Figure 1b) were thoroughly characterized by relevant techniques including Mössbauer and FTIR spectroscopy, simultaneous TG/DTA thermal analysis, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS). The resulting nanocomposites, which were found to be 13.0 ± 2.1 nm of size with acceptable dispersibility, are of high potential from the magnetic drug delivery point of view. Figure 1. (a) The structural formulas of highly cytotoxic cis-[ptcl 2 (3Claza) 2 ] (1; 3Claza = 3-chloro-7-azaindole) and cis-[ptcl 2 (5Braza) 2 ] (2; 5Braza = 5-bromo-7- azaindole), and the corresponding activated diaqua species symbolized as 1* and 2* used for the interaction with PABA@FeNPs; (b) the proposed composition of the studied 1*@PABA@FeNPs and 2*@PABA@FeNPs based on the maghemite core coated with 4-aminobenzoic acid and functionalized with 1* or 2*; and (c) photos of the obtained aqueous suspensions of PABA@FeNPs (up) and 2*@PABA@FeNPs (down) without (left) and with (right) an external magnetic field.

165 Molecules 2014, Results and Discussion 2.1. Preparation and Properties of The maghemite-based nanoparticles (FeNPs) coated with 4-aminobenzoic acid were prepared be a method using a mixture of the Fe(III) and Fe(II) salts (FeCl 3 6H 2 O and FeCl 2 4H 2 O in this work), which was mixed together with PABA in deionized water under atmospheric conditions. NH 4 OH was finally added to the mixture which resulted in the formation of the maghemite-based PABA@FeNPs nanoparticles. It has to be noted that the usual preparation of maghemite-based NPs involves magnetite NPs (prepared from the mixture of Fe(III) and Fe(II) salts under nitrogen [35,36]) and subsequently oxidized by e.g., diluted nitric acid [20,21]. Since we aimed to prepare maghemitebased NPs, we used a different approach we did not perform the syntheses under nitrogen but under atmospheric conditions, which was found to be sufficient for the magnetite oxidation to occur without addition of any other oxidizing agent. The presence of the organic layer coating the maghemite NPs within the obtained PABA@FeNPs was proved by the FTIR spectra recorded in the 400 4,000 cm 1 region (Figure 2 and Figure S1) as well as by the thermal analysis (Figure S2) [35,37]. The FTIR spectrum of PABA@FeNPs contained the characteristic peak of maghemite at ca. 550 cm 1 clearly assignable to the (Fe O) vibration, as well as a series of peaks, whose positions in the spectrum correlates with those of free PABA molecule (see Figure S1 and Experimental Section). The results of the FTIR spectroscopy also indirectly proved that PABA is bonded to γ-fe 2 O 3 within the PABA@FeNPs nanocomposite through the deprotonated carboxyl group, because there is only one major peak in the region characteristic for this functional group with a band centred at 1,603 cm 1, as compared with four in total peaks found in the spectrum of free PABA at 1,571, 1,597, 1,623 and 1,660 cm 1 (Figure S1). Figure 2. FTIR spectra of the maghemite nanoparticles coated with 4-aminobenzoic acid (PABA@FeNPs; red line), the parent complex 2 involving 5-bromo-7-azaindole (blue line), and the resulting system with the activated complex (2*) bound on the maghemite nanoparticles coated with 4-aminobenzoic acid (2*@PABA@FeNPs).

166 Molecules 2014, The hybrid systems exhibited, as expected, a strong attraction to the magnetic field (Figure 1c) and good stability in solution (no macroscopic changes, e.g., colour or magnetism, after one weak of standing in the fridge) and in solid state (usable with no changes of their properties after more than two months of standing in the fridge) Functionalization of PABA@FeNPs by Platinum(II) Complexes We assumed a bonding of the platinum(ii) complexes through Pt N bonds formed between the Pt(II) atom of the activated species and the amino group of PABA. As previously reported, PABA with a substituted carboxyl group (which simulates the bonding of PABA to the maghemite NPs through the mentioned functional group) binds to platinum through the amino group [38 40]. Further, the X-ray structures of several platinum(ii) complexes involving aniline or its variously substituted derivatives, such as 4-alkylanilines (e.g., [41,42]) are described in the literature. These facts proved PABA is a suitable coating agent for magnetic therapeutic/theranostic systems functionalized with platinum-based agents. The initial cis-dichloridoplatinum(ii) complexes of the composition cis-[ptcl 2 (naza) 2 ] (1 and 2), which were recently described in the literature by our team as having significant antitumor properties [33,34], were activated by their reactions with a stoichiometric amount of silver(i) nitrate, resulting in dechlorination and formation of the diaquaplatinum(ii) species cis-[pt(h 2 O) 2 (3Claza) 2 ] 2+ (1*) and cis-[pt(h 2 O) 2 (5Braza) 2 ] 2+ (2*). The activated species, involving the labile Pt aqua bonds which represent suitable sites for consequent formation of the mentioned Pt N bonds with PABA, were allowed to interact in acetone with the PABA@FeNPs nanoparticles for 48 h. The final systems (1*@PABA@FeNPs and 2*@PABA@FeNPs), which showed strong attraction to the external magnetic field (Figure 1c), were magnetically isolated, purified and stored in the fridge. The presence of the platinum(ii) species within the obtained maghemite-based nanocomposite was proved by FTIR spectroscopy (Figure 2) as well as by EDS spectroscopy (Figure 3f). FTIR spectroscopic experiments were performed with the aim to better characterize the studied systems. The detailed analysis of FTIR spectra revealed that bands observed in the spectrum of 1*@PABA@FeNPs at 785, 1,018, 1,099, 1,277, 1,338, 1,516, 1,602, 2,906 and 3,107 cm 1 and in the spectrum of 2*@PABA@FeNPs at 700, 883, 946, 1,267, 1,341, 1,473, 1,603 and 3,082 cm 1 were not detected in the spectrum of the PABA@FeNPs system. Moreover, the positions of these bands correlate well with those detected in the spectra of the complexes 1 and 2 (Figure 2 for the complex 2 and 2*@PABA@FeNPs), thus showing on the presence of the platinum(ii) 7-azaindole species within the resulting nanocomposites. An interpretation of the far-ftir spectra recorded at cm 1 provides indirect proof of the covalent bonding between the platinum(ii) species and PABA@FeNPs within the studied nanosystems 1*@PABA@FeNPs and 2*@PABA@FeNPs. In particular, the performed far-ftir experiments regarding 2, 2* and 2*@PABA@FeNPs indicated changes in inner coordination spheres in the vicinity of the central platinum(ii) atom, i.e., the changes going from a PtCl 2 N 2 donor set (the starting complex 2; two (Pt Cl) maxima at 336 and 345 cm 1 ), through a PtN 2 O 2 donor set (the dechlorinated complex 2*; two (Pt O) maxima at 322 and 332 cm 1 ) to a PtN 4 one (the resulting system 2*@PABA@FeNPs; no bands detected in the region mentioned for both the (Pt Cl) or (Pt O) vibrations), as depicted in Figure S3. In other words, although we did not detect the vibrations connected with the anticipated

167 Molecules 2014, Pt N bonds between the platinum(ii) species and amino group of PABA, we assume that these vibrations are overlapped (analogically to those at ca 520 cm 1 assignable to Pt N bonds between the central Pt(II) atom and 7-azaindole rings) by a wide and intensive band belonging to (Fe O). The (Fe O) vibration was, together with a band centred at 579 cm 1 assignable to the vibrations connected with the deformation of the 7-azaindole moiety [43], the only band detected in the discussed far-ftir spectrum of 2*@PABA@FeNPs.TEM images of the prepared 1*@PABA@FeNPs and 2*@PABA@FeNPs systems provided the relevant information regarding the shape, size, and uniformity of the resulting NPs (Figures 3a,b). The systems were found to be spherical, core-shell well-dispersed composites with an average size of 13.0 ± 2.1 nm. SEM was used to investigate the surface morphology of the prepared maghemite-based NPs (Figure 3c,e). A comparison of the SEM images depicted for PABA@FeNPs (Figure 3c) and 2*@PABA@FeNPs (Figure 3e) did not show any noticeable difference between their properties, since both systems were detected by SEM (as well as by the above-mentioned TEM) as having a spherical shape of individual NPs, which agglomerated together to the structure without any specific shape. Figure 3. TEM images of 2*@PABA@FeNPs {(a) with 100 nm resolution and (b) with 50 nm resolution}, SEM images of PABA@FeNPs (c) and 2*@PABA@FeNPs (e) given with their EDS patterns (d, f). Simultaneous TG/DTA thermal analysis revealed a considerable difference between the weight losses of PABA@FeNPs and those involving functionalized platinum(ii) complexes (represented by 2*@PABA@FeNPs; Figure S2). PABA@FeNPs did not show any weight increase in the C range (after the loss of water physically adsorbed on the the prepared NPs), which is known to be

168 Molecules 2014, connected with an oxidation of Fe 2+ ions, which indirectly proved the chemical composition of the magnetic core (maghemite). The system was thermally stable between 114 and 148 C, when its thermal degradation (connected with an oxidation of the organic layer coating the maghemite core) started and continued to 460 C (total weight loss equals 8.0%). The product of the thermal decomposition, most probably γ-fe 2 O 3 as previosly reported for maghemite NPs [44], did not show any weight change up to 530 C, however, an exothermic effect unambiguously assignable to the γ-fe 2 O 3 to α-fe 2 O 3 conversion was detected on the DTA curve with the maximum at 488 C (Figure S2). A considerably different weight loss (19.8%) as well as TG-curve shape (a continual decomposition) was found for 2*@PABA@FeNPs indirectly proving the presence of the platinum(ii) species within the resulting system (Figure S2) Mössbauer Spectroscopy The 57 Fe Mössbauer spectra of the samples studied are depicted in Figure 4, while the values of the Mössbauer hyperfine parameters, derived from the fitting of the recorded Mössbauer spectra, are listed in Table 1. Figure 4. Room-temperature Mössbauer spectra of (a) PABA@FeNPs and (b) 2*@PABA@FeNPs; doublet - assigned to the Fe 3+ relaxation component, singlet - asigned to the Fe 3+ superparamagnetic component. Table 1. Values of the Mössbauer hyperfine parameters, derived from the fitting of the room-temperature Mössbauer spectra of PABA@FeNPs and 2*@PABA@FeNPs, where δ is the isomer shift, ΔE Q is the quadrupole splitting and RA is the spectral area of the individual spectral components. Sample Component δ ± 0.01 ΔE Q ± 0.01 RA ± 1 (mm/s) (mm/s) (%) Assignment PABA@FeNPs Doublet Fe 3+ relaxation component Singlet Fe 3+ superparamagnetic component 2*PABA@FeNPs Doublet Fe 3+ relaxation component Singlet Fe 3+ superparamagnetic component

169 Molecules 2014, The room-temperature Mössbauer spectrum of both the and sample shows only relaxation components (i.e., singlet and doublet, see Figures 4a,b, respectively) with the values of the Mössbauer hyperfine parameters (see Table 1) typical of high-spin Fe(III) atom in iron(iii) oxides [45]; there is no indication of presence of the Fe 2+ valence state. Thus, the nanoparticles in both samples are solely of -Fe 2 O 3 origin. This is expected in connection with their small size with a large surface area securing their complete oxidation. On the timescale of the Mössbauer technique, all the nanoparticles in both assemblies behave in a superparamagnetic manner at room temperature. The doublet component belongs to the nanoparticles with thermally fluctuating superspins having relaxation times much smaller than the characteristic measurement time (τ m ) of the Mössbauer spectroscopy, while the presence of a singlet corresponds to those nanoparticles the superspin of which thermally fluctuates between the energetically favored orientations with a relaxation time close to τ m. The PABA@FeNPs and 2*@PABA@FeNPs systems would show superparamagnetic features in the magnetization measurements at room temperature, however, their magnetic characteristics will be significantly driven by finite-size and surface effects (manifested, for example, by a smaller saturation magnetization or lack of saturation and reduced magnetic response under small applied magnetic fields). 3. Experimental 3.1. Materials and Methods The starting materials K 2 [PtCl 4 ], 3-chloro-7-azaindole (3Claza), 5-bromo-7-azaindole (5Braza), iron(iii) chloride hexahydrate (FeCl 3 6H 2 O), iron(ii) chloride tetrahydrate (FeCl 2 4H 2 O), p-aminobenzoic acid (PABA), 25% NH 4 OH, silver nitrate (AgNO 3 ) and solvents were supplied by Sigma-Aldrich Co. (Prague, CzechRepublic) and Acros Organics Co. (Pardubice, CzechRepublic), and used as received. The platinum(ii) complexes cis-[ptcl 2 (3Claza) 2 ] (1) and cis-[ptcl 2 (5Braza) 2 ] (2) (Figure 1a) were prepared as described in our recent paper [33]. Transmission electron microscopy (TEM) was carried out on a JEOL 2010 microscope (200 kv, 1.9 Å point-to-point resolution). A drop of high-purity water with the ultrasonically dispersed samples was placed onto a holey-carbon film supported by a copper-mesh TEM grid and dried in air at room temperature. Scanning electron microscopy (SEM) was performed, together with energy-dispersive X-ray (EDS) spectroscopy, by a Hitachi 6600 FEG microscope (5 15 kev accelerating voltage; the dried samples were placed on an aluminum holder equipped with double-sided adhesive carbon tape). The 57 Fe Mössbauer spectra of the studied samples were recorded at room temperature employing a Mössbauer spectrometer operating at the constant acceleration mode and equipped with a 50 mci 57 Co(Rh) source. The isomer shift values are related to α-fe at room temperature. The Mössbauer spectra were fitted with the MossWinn software program; prior to fitting, the signal-to-noise ratio was enhanced by a statistically based algorithm [46]. Infrared spectra ( cm 1 and cm 1 regions) were recorded on a Nexus 670 FT-IR (Thermo Nicolet, Waltham, MA, USA) using the ATR technique. Simultaneous thermogravimetric (TG) and differential thermal (DTA) analyses were performed using an Exstar TG/DTA 6200 thermal analyzer (Seiko Instruments Inc., Chiba, Japan) from room temperature to 650 C (5.0 C min 1 ) in dynamic air atmosphere (50 ml min 1 ).

170 Molecules 2014, Nanoparticles 4-Aminobenzoic acid (PABA; 0.62 g, 4.5 mmol) was, due to its poor solubility at laboratory temperature, dissolved in hot (80 C) deionized water (75 ml) and then, FeCl 3 6H 2 O (1.17 g; 4.3 mmol) dissolved in deionized water (5 ml) was added. The mixture was stirred at 80 C for 30 min and then FeCl 2 4H 2 O (0.86 g; 4.3 mmol) dissolved in deionized water (2 ml) was added to the solution. After 30 min of stirring at 80 C the last reagent (10 ml of 25% NH 4 OH) was slowly added. The solution turned dark black as the PABA@FeNPs formed. The suspension was intensively stirred at 80 C for 60 min. Finally, nanoparticles were magnetically isolated and washed with deionized water (3 20 ml) and acetone (3 20 ml). Part of the final ferrofluid suspension of PABA@FeNPs (Figure 1c) was stored under degassed acetone in the fridge, while the rest of the product was dried with nitrogen gas and in desiccator over silica gel, and stored in the fridge. PABA@FeNPs: FTIR (ν ATR /cm 1 ): 552vs, 783w, 1,179m, 1,395vs, 1,493m, 1,603s, 3,219s, 3,334s Synthesis of 1*@PABA@FeNPs and 2*@PABA@FeNPs The complexes 1 (110 mg; 0.2 mmol) and 2 (130 mg; 0.2 mmol) were dissolved in acetone (10 ml) and two molar equivalents of AgNO 3 dissolved in a minimum volume of deionized water were added. The mixture was stirred at laboratory temperature in the dark for 24 h. After that, AgCl was removed by filtration and washed with acetone (3 1 ml) to produce the filtrate containing the solution of the dechlorinated complexes of the composition cis-[pt(h 2 O) 2 (3Claza) 2 ] 2+ (1*), and cis-[pt(h 2 O) 2 (5Braza) 2 ] 2+ (2*) (Figure 1a). PABA@FeNPs (0.5 ml of the acetone suspension involving 100 mg of PABA@FeNPs) was poured in and the mixture was stirred for 48 h to produce the final systems 1*@PABA@FeNPs and 2*@PABA@FeNPs (Figures 1b,c). These products were magnetically isolated, washed with acetone (3 20 ml), dried (nitrogen and then in desiccator over silica gel) and stored in the fridge. 1*@PABA@FeNPs: FTIR (ν ATR /cm 1 ): 550vs, 785w, 1,018m, 1,099w, 1,179m, 1,208m, 1,277s, 1,338s, 1,398vs, 1,516m, 1,602s, 1,694w, 2,906s, 3,107s. 2*@PABA@FeNPs: FTIR (ν ATR /cm 1 ): 547vs, 700w, 883w, 946w, 1,017w, 1,101w, 1,177m, 1,267vs, 1,341s, 1,400vs, 1,473s, 1,500s, 1,603m, 1,646s, 1,694w, 3,082s. For far-ftir spectra of 2, 2*, PABA@FeNPs and 2*@PABA@FeNPs see Supplementary Materials. 4. Conclusions A simple approach was applied to obtain magnetic 4-aminobenzoic acid-coated maghemite nanoparticles with good stability in solution, with high magnetic response to the external magnetic field and showing superparamagnetic behaviour as proved by the Mössbauer spectroscopy experiments at room temperature. The systems were designed to be able to bind highly cytotoxic platinum(ii) complexes involving 7-azaindole derivatives represented by the diaquaplatinum(ii) species 1* and 2* prepared by a Ag(I) ion-assisted activation from the initial highly cytotoxic dichlorido complexes. The incorporation of the platinum(ii) species was proved by relevant techniques (FTIR, EDS), while a combination of the microscopic techniques (SEM, TEM) showed the obtained core-shell nanocomposites as having the spherical shape and an average size of 13 nm in diameter. Although we are aware of the fact that other important properties of the reported systems (e.g., release of the

171 Molecules 2014, complex, in vitro cytotoxicity, in vitro toxicity or MRI experiments), should by also elucidated (the results will be a subject of our forthcoming studies), we have reason to believe that the prepared systems fulfil the basic requirements (magnetism, size, dispersibility or functionalization with therapeutically active substance) for the nanoparticles used in the field of theranostic nanomedicine. Supplementary Materials Supplementary materials can be accessed at: Acknowledgments The authors gratefully thank the Czech Science Foundation (GAČRP207/11/0841), Operational Program Research and Development for Innovations-European Regional Development Fund (CZ.1.05/2.1.00/ ), Operational Program Education for Competitiveness-European Social Fund (CZ.1.07/2.3.00/ ) of the Ministry of Education, Youth and Sports of the Czech Republic and Palacký University in Olomouc (PrF_2013_015). The authors would like to thank Jana Gáliková and Klára Šafářová for performing FTIR, and TEM and SEM experiments, respectively. Author Contributions Conceived and designed the experiments: PŠ, ZT. Performed the experiments: MS, PŠ, JT. Analyzed the data: PŠ, JT, ZT. Wrote the paper: PŠ, JT, ZT. Conflicts of Interest The authors declare no conflict of interest. References 1. Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7, Butler, J.S.; Sadler, P.J. Targeted delivery of platinum-based anticancer complexes. Curr. Opin. Chem. Biol. 2013, 17, Zhang, Y.; Chan, H.F.; Leong, K.W. Advanced materials and processing for drug delivery: The past and the future. Adv. Drug Deliv. Rev. 2013, 65, Goncalves, A.S.; Macedo, A.S.; Souto, E.B. Therapeutic nanosystems for oncology nanomedicine. Clin. Transl. Oncol. 2012, 14, Chomoucka, J.; Drbohlavova, J.; Huska, D.; Adam, V.; Kizek, R.; Hubalek, J. Magnetic nanoparticles and targeted drug delivering. Pharmacol. Res. 2010, 62, Kievit, F.M.; Zhang, M. Surface Engineering of iron oxide nanoparticles for targeted cancer therapy. Acc. Chem. Res. 2011, 44, Gupta, A.K.; Gupta, M. Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 2005, 26,

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175 PŘÍLOHA 8 Štarha, P.; Smola, D.; Tuček, J.; Trávníček, Z. Efficient synthesis of a maghemite/gold hybrid nanoparticle system as a magnetic carrier for the transport of platinum-based metallotherapeutics Int. J. Mol. Sci. 16 (2015)

176 Int. J. Mol. Sci. 2015, 16, ; doi: /ijms Article OPEN ACCESS International Journal of Molecular Sciences ISSN Efficient Synthesis of a Maghemite/Gold Hybrid Nanoparticle System as a Magnetic Carrier for the Transport of Platinum-Based Metallotherapeutics Pavel Štarha 1, David Smola 1, Jiří Tuček 2 and Zdeněk Trávníček 1, * 1 Regional Centre of Advanced Technologies and Materials, Department of Inorganic Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, Olomouc CZ-77146, Czech Republic; s: pavel.starha@upol.cz (P.S.); david.smola01@upol.cz (D.S.) 2 Regional Centre of Advanced Technologies and Materials, Department of Experimental Physics, Faculty of Science, Palacký University, 17. listopadu 12, Olomouc CZ-77146, Czech Republic; jiri.tucek@upol.cz * Author to whom correspondence should be addressed; zdenek.travnicek@upol.cz; Tel.: ; Fax: Academic Editor: Bing Yan Received: 11 December 2014 / Accepted: 13 January 2015 / Published: 16 January 2015 Abstract: The preparation and thorough characterization of a hybrid magnetic carrier system for the possible transport of activated platinum-based anticancer drugs, as demonstrated for cisplatin (cis-[pt(nh3)2cl2], CDDP), are described. The final functionalized mag/au LA CDDP* system consists of maghemite/gold nanoparticles (mag/au) coated by lipoic acid (HLA; LA stands for deprotonated form of lipoic acid) and functionalized by activated cisplatin in the form of cis-[pt(nh3)2(h2o)2] 2+ (CDDP*). The relevant techniques (XPS, EDS, ICP-MS) proved the incorporation of the platinum-containing species on the surface of the studied hybrid system. HRTEM, TEM and SEM images showed the nanoparticles as spherical with an average size of 12 nm, while their superparamagnetic feature was proven by 57 Fe Mössbauer spectroscopy. In the case of mag/au, mag/au HLA and mag/au LA CDDP*, weaker magnetic interactions among the Fe 3+ centers of maghemite, as compared to maghemite nanoparticles (mag), were detected, which can be associated with the non-covalent coating of the maghemite surface by gold. The ph and time-dependent stability of the mag/au LA CDDP* system in different media, represented by acetate (ph 5.0), phosphate (ph 7.0) and carbonate (ph 9.0) buffers and connected with

177 Int. J. Mol. Sci. 2015, the release of the platinum-containing species, showed the ability of CDDP* to be released from the functionalized nanosystem. Keywords: nanoparticles; maghemite; magnetic; cisplatin; drug delivery 1. Introduction Targeted drug delivery involving nanoparticles (e.g., iron oxides or gold) is currently generally accepted as a suitable and prospective alternative pathway in conventional anticancer chemotherapy using platinum-based metallotherapeutics [1 3]. The basic advantage is that the targeted delivery could reduce the amount of drug administrated to the body, and as a consequence of this, the negative side effects (e.g., nephrotoxicity, neurotoxicity or myelosuppression) can be partially eliminated [4]. Additionally to tumor therapy, the magnetic iron oxide-based nanoparticles also offer diagnostic (e.g., MRI) potential [5 7]. On the other hand, the gold nanoparticles are known to show a surface plasmon resonance usable in photothermal therapy or diagnostics [8 11]. Hybrid systems, consisting of gold and iron oxide nanoparticles, offer a combination of these properties within one nanosystem [12], which has been of a great interest in the field of drug delivery agents [13]. In particular, the coating of the magnetic iron oxide nanoparticles with the biocompatible precious metal, gold, prevents not only the chemical and enzymatic degradation of the iron oxide core, but also seems to be suitable for further binding of various types of compounds (especially sulfur-containing ones) and functionalization [14,15]. There are plenty of works, as shown below, reporting gold/iron oxide-based nanoparticles available for further derivatization and/or functionalization of gold. Surprisingly, only one of them describes functionalization with platinum-based species, namely cisplatin [13]. In this case, the activated cisplatin was bound to the thiolated polyethylene glycol (PEG) linker (the thiol part of the linker was lipoic acid (HLA), also used in this work). In vitro cytotoxicity of these nanoparticles was found to be more than 100-times higher against both the cisplatin-sensitive and resistant human ovarian carcinoma cancer cell lines (A2780, A2780/cp70) as compared to cisplatin. It can be highlighted that the herein reported mag/au LA CDDP* nanoparticles represent a synthetically more easily and quickly obtainable system (there is no need for the preparation of the thiolated PEG linker), which is (very important from the pharmacological point of view) able to bind even more platinum(ii) species (as discussed below). Regarding other maghemite/gold nanoparticle systems, those layered by differently-thiolated PEG were reported as a promising MRI contrast diagnostic agent for malignant tumors [16]. Similar systems, but with anti-podoplanin antibody bound to PEG on the surface of the studied nanoparticles, were shown as suitable MR imaging agents in evaluating lymphangiogenesis in breast cancer cells in vivo [17]. Fan et al. prepared gold-coated maghemite core-shell nanoparticles that highly specifically targeted SK-BR-3 human breast cancer cells through the S6 aptamer bound on the surface, which exhibit SK-BR-3 cells targeting themselves [18]. Modification of the surface by fibrinogen resulted in interesting cell targeting properties, representing another possible application of gold-coated iron oxide core-shell nanoparticles [19]. Similar nanoparticles functionalized with different agents (PEG, glucose or fluorochrome) did not show any in vitro cytotoxicity against the cervix carcinoma HeLa cancer cell line [20]. Interesting possibilities within this field of study are hybrid nanosystems with an iron oxide

178 Int. J. Mol. Sci. 2015, core and a gold shell separated by an intermediate layer (e.g., polyethyleneimine) [21] or dumbbell-like nanocomposites consisting of a gold nanoparticle bound together with an iron oxide one, where the gold part can be functionalized by the sulfur-containing molecules or linkers [22]. In this study, we deal with the preparation and characterization of potential theranostic maghemite/gold nanoparticles layered with a simple sulfur-containing carboxylic acid (lipoic acid) and directly functionalized by the platinum metallotherapeutic-based species, such as the herein used activated cisplatin. The reported data represent an optimized and easily realized pathway leading to gaining of the discussed biocompatible nanosystem, which may serve as a necessary base for further biological studies. 2. Results and Discussion 2.1. Preparation and Functionalization The synthesis of the magnetite nanoparticles, maghemite nanoparticles, iron oxide/gold nanoparticles system, as well as the iron-oxide/gold nanoparticle hybrid system layered by organic sulfur-containing carboxylic acid is well-established in the literature [13,23 25]. In the case of this work, the freshly prepared magnetite nanoparticles were gently oxidized by diluted nitric acid to produce the maghemite nanoparticles (mag), which are macroscopically pronounced by the color change from black to brown. The coating of maghemite by gold was carried out by the slow addition of a 1% HAuCl4 water solution, again connected with a color change of the nanoparticles (mag/au) to dark purple (further addition of the HAuCl4 solution led to the formation of gold nanoparticles, observable as a purple/pink color above the mag/au nanoparticles). The prepared mag/au nanoparticles kept their magnetic properties (being attracted by the external magnet) and are stable in the tetramethylammonium hydroxide (TMAOH) solution for at least two weeks (there are no color changes of the nanoparticles themselves nor the solution above the nanoparticles). As is frequently described in the literature [13,25], gold nanoparticles can easily interact with sulfur-containing molecules. We let a slight excess of lipoic acid (HLA) react with mag/au, resulting in mag/au HLA nanoparticles. Within the final step of the synthetic procedure, including the crucial functionalization, the mag/au HLA nanoparticles interacted with the activated platinum-based drug, cisplatin, i.e., with cis-[pt(nh3)2(h2o)2] 2+ species (CDDP*) prepared by the reaction of cisplatin (cis-[pt(nh3)2cl2], CDDP) with two molar equivalents of AgNO3 in the dark [26], to give the final product of the mag/au LA CDDP* system (Figure 1). Regarding the yield of the synthesis, we got 155 mg of mag/au LA CDDP*, but this cannot be expressed as a percent, because every synthetic intermediate was partly used for synthesis and partly stored or used for characterization. Regarding the lipoic acid used, it has to be stated that it was previously used, for example, for the modification of the gold seeds of the iron oxide magnetic core, where lipoic acid was subsequently modified by the interaction with iminodiacetic acid, which finally bound copper(ii) ions [27], or lipoic acid was used as the thiol part of the thiolated polyethylene glycol (PEG) linker layering the gold-coated maghemite nanoparticles and binding the activated cisplatin as functionalizing platinum-based species [13]. In comparison to the latter work, here, we applied a different approach resulting in the magnetic gold-coated maghemite nanoparticles layered by lipoic acid itself, with its carboxylic group ready for the covalent binding of the biologically-active platinum-based species, such as activated

179 Int. J. Mol. Sci. 2015, cisplatin. Interestingly, the use of the lipoic acid itself (this work), instead of the LA PEG thiolated polyethylene glycol linker [13], not only simplified and accelerated the synthesis, but also provided the nanosystems with a higher Pt content (as discussed below). Figure 1. The reaction pathway leading to the preparation of the mag/au LA CDDP* nanoparticle system Characterization HRTEM, TEM and SEM Microscopy The prepared mag/au LA CDDP* nanosystems are of a uniform spherical shape (average size of 12.2 ± 1.9 nm), as proven by the HRTEM, TEM and SEM techniques applied to characterize their morphology and dispersibility (Figure 2). Any impurity (e.g., crystalline lipoic acid or CDDP*) was not microscopically detected within the studied product. The intermediates, mag, mag/au and mag/au HLA, showed similar morphological properties compared with the discussed mag/au LA CDDP*. The dispersibility of all of the intermediates and the final functionalized nanoparticles was comparable, as well, with no agglomeration observed after the individual synthetic steps. Figure 2. HRTEM (left; 10 nm size bar), TEM (top right; 50 nm size bar) and SEM (bottom right; 500 nm size bar) images, as obtained for the studied functionalized mag/au LA CDDP* nanoparticles.