Chengken Chen,Chunmei Gao,Zigao Yuan*,Yuyang Jiang**

a Department of Chemistry,Tsinghua University,Beijing 100084,China

b The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology,The Graduate School at Shenzhen,Tsinghua University,Shenzhen 518055,China

c College of Chemistry and Chemical Engineering,Shenzhen University,Shenzhen 518060,China

d Department of Pharmacology and Pharmaceutical Sciences,School of Medicine,Tsinghua University,Beijing 100084,China

e Shenzhen Kivita Innovative Drug Discovery Institute,Shenzhen 518055,China

Key words:Platinum compounds Bioreductive Nitrophenylalkanoic group Cellular uptake Antitumor

ABSTRACT Cisplatin is one of the most successful antitumor agents,yet also restricted by its poor cellular uptake and low selectivity.Since 3-(2-nitrophenyl)propionic acid(NPPA)has been reported as a bioreductive prodrug moiety,herein we combined NPPA with cisplatin(compound 1)to improve its lipophilicity and targetability and then to improve the antitumor outcomes.In addition,compound 2 possessing 3-phenyl propionic acid(PPA)was also synthesized as a comparison to test the influence of the NPPA to the cytotoxicity,since PPA was not a bioreductive moiety.Bioevaluations showed that 1 displayed more potent antitumor potency than cisplatin and 2,suggesting Pt(II)complexes possessing NPPA groups may be a good strategy for future platinum drug discovery.

Platinum-based drugs,such as cisplatin,carboplatin and oxaliplatin,are used w orldw ide in clinical cancer therapy(Fig.1A)[1,2].As one of the most successful anticancer drugs,cisplatin can be taken up by cells and bond with DNA to form DNA adducts,causing DNA damage and resulting in cell death,so it is used to fi ght against a w ide spectrum of solid neoplasms like ovarian,testicular,bladder,colorectal,lung,and head and neck cancers[3].However,due to the high activity of chloride ions,platinum(II)complexes are instable and easily combined with nucleophiles such as glutathione,metallothioneins and other sulfur-containing amino acids,leading to drug resistance[4–6].Moreover,cisplatin has low lipophilicity,which leads to its low uptake.In addition,cisplatin can cause serious adverse effects including dose-limiting nephrotoxicity,ototoxicity,as well as nausea and vomiting.Although some strategies have been explored to develop novel platinum drugs(e.g.,polynuclear Pt(II)complexes,Pt(IV)prodrugs)with reduced toxicities and improved stability[7–11],it is still necessary to develop new approaches for platinum drugs.

Due to the rapid grow th of cancer cells and the abnormal angiogenesis within them,solid tumors usually cause a hypoxic microenvironment,which could be practically targeted by bioreductive prodrugs[12–15].Up to now,several typesof bioreductive prodrugshave been developed,including nitro groups,quinones,N-oxides and transition metals(Fig.1B)[16–19].Nitrophenylalkanoic acid has been recognized as a good bioreductive prodrug moiety[20],its derivatives are nontoxic or weakly toxic in normal tissues,but decompose under convertible reduction and release antitumor agents[21,22].Complexes containing nitrophenylalkanoic groups show high selectivity to hypoxic cells[23,24].For example,bioreductive paclitaxel prodrug(NPPA-PTX),which is stable in PBS and rat plasma as well as in the blood circulation,can be completelyconverted to theactive PTXin hypoxic tumor tissuesand then exhibits its antitumor activity[25].Furthermore,researches haveproposed that theactivation processtendsto be triggered in an acidic environment,which suggests that it is more likely to be reduced at a low p H in tumor tissues[26].

Fig.1.(A)Chemical structures of the clinically used platinum drugs.(B)Chemical structures of bioreductive prodrugs.

Considering the aforementioned information,and in continuation of our interest in the development of new anticancer compounds[27–31],herein we intend to combine nitrophenylalkanoic acid and cisplatin to develop novel bioreductive Pt(II)drug and compound 1 containing 3-(2-nitrophenyl)propionic acid(NPPA)was constructed.Besides,to assess the advantage of bioreductive reactivation for antitumor potency,compound 2 containing 3-phenyl propionic acid(PPA)was also synthesized as a control(Fig.2A).We proposed that the nitrophenylalkanoic ester group in compound 1 w ould be helpful to improve lipophilicity and hypoxia-targeting ability,thereby enhancing the cellular uptake and improving antitumor potency compared to that of cisplatin.

The synthesis of target compounds 1 and 2 was show n in Fig.2B.Firstly,3-(2-nitrophenyl)propionic acid(NPPA)was synthesized according to the reported method[32].Secondly,1 and 2 were synthesized via the combination of cis-[Pt(NH3)2(H2O)2]2+with NPPA sodium or PPA sodium.NMRspectroscopy has been applied to characterize the structures of Pt(II)complexes 1 and 2.As show n in Fig.3,it is obvious that the chemical shifts of H1 and H2 on NPPA are 3.04 and 2.60 ppm respectively,but they are 2.99 and 2.35 ppm in complex 1,respectively,which indicated that NPPA has been added to the Pt(II)anion[33].Also,the appearance of NH3signal and the disappearance of OH signal on NPPA further con fi rmed the structure of complex 1.HPLC was used to measure the purity of complexes 1 and 2(Figs.S7 and S11 in Supporting information).All the electrospray ionization mass(ESI-MS)spectra of the complexes(Figs.S6 and S10 in Supporting information)gave main peaks corresponding to[M+Na]+or[M?H]?ions,which composed of a few isotopic peaks ow ing to the presence of platinum isotopes,suggesting the successful synthesis of 1 and 2.

Fig.3.1H NMR of complexes NPPA and its product 1.

ESI-MSis a highly sensitive method to investigate the binding ability of compounds and DNA.As we know,the preferred DNA binding site of cisplatin isthe N7 position of guanine bases[34,35].To investigate w hether 1 could display the similar DNA binding activity as cisplatin,the binding of 1 to 2'-deoxyguanosine(2'-d G)as a DNA analogue was analyzed by ESI-MS,with cisplatin as a positive control.In the presence of nitroreductase(NTR)and NADPH only Pt/2'-d G-bisadduct could be found after 24 h(Fig.S12 in Supporting information).In comparison,both Pt/2'-d G-bisadduct and Pt/2'-d G-monoadduct were observed in 2'-deoxyguanosine with cisplatin or 1 after 2 days(Figs.S13 and S14 in Supporting information),which indicated the ability of cisplatin and 1 binding to DNA and supported that guanine bases were the target position of cisplatin and 1,and also suggest that NTR can accelerate the decomposition of 1 and improve the binding ability with DNA.

The proposed activation mechanism of compound 1 in cells was show n in Fig.4.Compound 1 can be taken up by cells via active transport and passive permeability[36,37],then the nitro groups of 1 are reduced by reductase such as NTR and gradually generate hydroxylamine or amino derivatives[25,38,39].The nitrogen atom of hydroxylamine or amino derivative can attack itscarbonyl group,ultimately releasing cis-[Pt(NH3)2(H2O)2]2+through intramolecular cyclization.To investigate w hether the reductive products were generated,we dissolved compound 1 in w ater and then treated it with NTRand NADPHat 37?Cfor 24 h.The resulting massspectrum from LC-MS(Figs.S15 and S16 in Supporting information)showed the appearance of reductive products(3,4-dihydro-2(1H)-quinolinone and its hydroxylamine derivative).Notably,we also found the signalof cis-[Pt(NH3)2(H2O)2]2+,indicating thesuccessful activation of compound 1 by NTR.

Fig.4.Possible activation mechanism of compound 1 before and after it enters the cells.

Table 1 Cytotoxicity of compound 1 and 2 against several cancer cell lines.

The cytotoxicity of compounds 1 and 2 against several cancer cells was evaluated by the MTTmethod with cisplatin and NPPA as controls.The IC50values(the concentration of compounds which causes death in 50%of cells)were show n in Table 1.After 72 h incubation,NPPA displayed hardly cytotoxicity against all tested cells,while 1 and 2 showed better or similar cytotoxicity against all tested cancer cells compared to cisplatin.In particular,1 was about 6.4 and 3.1 times more effective than cisplatin against Hep G2 and HCC827 cells,respectively.Although 1 and 2 had higher IC50values compared to that of cisplatin against A549 cells,they showed better cytotoxicity against A549/DDP cells than cisplatin with RF values of 0.91 and 1.33 compared to 2.90,respectively.As a w hole,the cytotoxicity of 1 is more potent than 2,which indicated that the bioreductive activity may contribute much to the cytotoxicity,as 2 can only be hydrolyzed but not bioreducible.Compound 2 displayed better anti-proliferative activity than cisplatin,which might attribute to the improved lipophilicity compared to cisplatin by introducing phenylpropionic acid group.All these results indicated the success of our design.

In order to certify that our compounds had improved lipophilicity compared with cisplatin,the Pt contents of cisplatin,1 and 2 in all tested cells were analyzed using ICP-MSafter 6 h or 12 h of incubation.As show n in Fig.5 and Table S1(Supporting information),the Pt contents of 1 had a high value(51.42 ng/106cells)in HCC827 cells after 6 h,and had a great increase after 12 h.In comparison,cisplatin showed a lower value in HCC827 cells after 6 h,and reach the value of over 50 ng/106cells after 12 h.Also,the Pt content of 1 in Hep G2 cells is 3.0 times higher than that of cisplatin after 6 h,and can keep in a high level after 12 h.It meant 1 can be quickly taken up by HCC827 and Hep G2 cells than cisplatin.In addition,the platinum contents of all three complexes in A549 and A549/CDDP were kept in a low value,which indicated a low availability against these tw o cells.

Fig.5.Platinum contents of cisplatin,1 and 2 in cancer cells after 6 h and 12 h.

Fig.6.Representative fl ow cytometric diagrams of apoptotic Hep G2 cells after 72 h treatment with cisplatin and 1. Q1 represents the necrotic or damaged dead cells,Q2 represents apoptosis cells in later stage,Q3 represents apoptosis cells in early stage,and Q4 represents live cells.

In order to evaluate w hether compound 1 could induce apoptosis like cisplatin does,we then conducted an Annexin-V/PI binding assay.Hep G2 cells were treated with 1 for 72 h at different concentrations,with cisplatin as a positive control.As show n in Fig.6,a few(5.77%)apoptotic cells were present in the negative control group(DMSO).The apoptosis population rose to 37.0%,77.9%and 81.6%after treatment with 1 at 1 m mol/L,5 m mol/L and 10 m mol/L,respectively.However,cisplatin only induced 23.1%,36.0%and 63.2%apoptosis populations at 1 m mol/L,5 m mol/L and 10 m mol/L,respectively.It meant 1 is a more potent proapoptotic agent than cisplatin.

In this study,we proposed a novel strategy by introducing the bioreductive group NPPAinto Pt(II)compounds,and then designed and synthesized tw o target compounds.Compound 1 showed better cytotoxicity,higher cellular uptake and more potent proapoptotic potency in Hep G2 cells than that of cisplatin and 2,indicating that Pt(II)complexes containing bioreductive groups can be an alternative strategy for novel platinum drug development,and compound 1 represents a good lead compound for further antitumor drug discovery.

[13_TD DIFF]8IF]Acknow ledgment

The authors w ould like to thank Shenzhen Sci&Tech Bureau(Nos.JCYJ20160301153959476 and JCYJ20160324163734374).

Appendix A.Supp lementary data

Supplementary data associated with this article can be found,in the online version,at https://doi.org/10.1016/j.cclet.2018.04.010.