Hypoxia-Targeting Organometallic Ru(II)-Arene Complexes with Enhanced Anticancer Activity in Hypoxic Cancer Cells.

Article CiteThis:Inorg.Chem.XXXX,XXX,XXX−XXX pubs.acs.org/IC − Hypoxia-Targeting Organometallic Ru(II) Arene Complexes with Enhanced Anticancer Activity in Hypoxic Cancer Cells Jian Zhao, †,‡ Wanchun Li, † Shaohua Gou, *,†,‡ Shuang Li, † Shengqiu Lin, † Qianhui Wei, † and Gang Xu *,†,‡ † PharmaceuticalResearchCenterandSchoolofChemistryandChemicalEngineering,SoutheastUniversity,Nanjing211189,China ‡ Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China * S Supporting Information ABSTRACT: As hypoxia is an important factor to limit chemotherapeutic efficacy in tumors, we herein report three ruthenium(II)−arene complexes containing a hypoxia inducible factor-1α inhibitor (YC-1), which endow the organometallic complexes with potential for hypoxia targeting. In vitro tests showed the resulting complexes had higher anticancer activities in hypoxia than in normoxia against the tested cancer cell lines. Westernblotanalysisrevealedthatcomplexes1−3blockedHIF- 1α protein accumulation under hypoxic conditions. Moreover, these complexes displayed much less cytotoxicity toward the normal human umbilical vein endothelial cell line (HUVEC), indicatingthatcomplexes1−3maybeselectivelycytotoxicforhumancancercelllines.Thesefindingsprovedthatligationwith YC-1 endowed these organometallic ruthenium(II) complexes with potential for hypoxia targeting in addition to enhancing their anticancer activities. ■ INTRODUCTION The therapeutic values of metal-based agents have gained increasing interest since the discovery of cisplatin in 1965.1 Platinum(II) drugs, particularly cisplatin, carboplatin, and oxaliplatin, are now widely used in clinical practice,2−4 which exhibit their activities by covalently binding to DNA and forming stable DNA adducts with guanines and adenine bases.5,6 However, their irreversible binding to DNA and the lack of the selectivity have resulted in severe side-effects.7 Therefore, ruthenium complexes with relatively lower toxicity were considered to be an attractive alternative to platinum drugs.8−16 So far two ruthenium(III) compounds, [IndH]- [trans-Ru(Ind) Cl ] (KP1019, Ind = indazole) and [ImH]- 2 4 [trans-Ru(DMSO)(Im)Cl ] (NAMIA, Im = imidazole), have 4 entered clinical trials,17,18 especially KP1019 and its sodium Figure 1. Representativeanticancer ruthenium(II)complexes. salt KP1339 (Figure 1), which have successfully finished a phase I clinical trial with promising activity.19,20 Besides, organometallic ruthenium(II)−arene complexes also show result in the stabilization and accumulation of hypoxia inducible factor-1α (HIF-1α) protein,37,38 and the resultant great potential for cancer therapy because the coordination ligands in arene−ruthenium(II) complexes can provide more HIF-1αcandimerizewithitsβsubunittoformatranscription factor that binds to hypoxia response elements (HREs) and opportunitytomodulatetheirpharmacologicalpropertiessuch as cellular accumulations and kinetic reactivities.21−29 For activates a number of genes involved in vascular epidermal growth factor (VEGF) and erythropoietin (EPO).39 Thus, examples, [(C H Ph)Ru(en)Cl][PF ] (RM175, en = ethyl- enediamine) an 6 d 5 [(piPrC H Me)Ru( 6 pta)Cl ] (RAPTA-C, pta HIF-1α plays an essential role in tumorigenesis by regulating 6 4 2 the expression of various genes associated with tumor = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]-decane) (Figure 1) are currently at an advanced preclinical stage.19,30−32 metabolism, angiogenesis, metastasis, proliferation, and differ- Hypoxia, caused by the imbalance between oxygen supply and consumption, is an important hallmark of cancer Received: April 18,2018 progression.33−36 The reduced oxygen levels in tumor tissues ©XXXXAmericanChemicalSociety A DOI:10.1021/acs.inorgchem.8b01070 Inorg.Chem.XXXX,XXX,XXX−XXX .)CTU( 84:84:11 ta 8102 ,92 enuJ no ODELOT FO VINU aiv dedaolnwoD .selcitra dehsilbup erahs yletamitigel ot woh no snoitpo rof senilediuggnirahs/gro.sca.sbup//:sptth eeS Inorganic Chemistry Article Scheme 1. Preparation of Ligand L1 and Complexes 1−3 a aReagentsandconditions:(a)KOH,DMF,I,rt,4h;(b)benzylbromide,t-BuOK,THF,rt,4h;(c)(5-formylfuran-2-yl)boronicacid,Pd(PPh), 2 3 4 NaCO,DMF,90°C,12h;(d)NaBH,MeOH,rt,2h;(e)succinicanhydride,EtN,DMF,4h;(f)5-amino-1,10-phenanthroline,HATU,EtN, 2 3 4 3 3 DMF,60°C,12h;(g)[(arene)RuCl],DCM, rt,4h. 2 2 entiation.39 Overexpression of HIF-1α was detected in more et al.54 Thus, the hydrolysis behavior of complexes 1−3 was than 90% of colon, lung, and prostate cancers, but no studied using UV−vis spectroscopy. It was observed that the expression was detected in their corresponding normal hydrolysis was accompanied by a gradual increase of the tissues.40 In addition, HIF-1α overexpression was found to absorptionbandaround275nm,whichwaschosenforkinetic be associated with resistance to treatment and poor patient calculation (Figure 2). The experimental data fitted well to a prognosis in the hypoxic region around solid tumors.38 monoexponential function, and the hydrolysis rate constants Consequently, HIF-1α becomes a compelling drug target for (k) and half-times (t ) of complexes 1−3 were shown in the treatment of tumor angiogenesis and cancer.41−44 Table 1. Obviously, t 1 h / e 2 hydrolysis rate of complex 3 is the 1-Benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole (YC-1) has fastest(t =13.8min)ascomparedwithcomplexes1and2. been reported as an effective HIF-1α inhibitor by stimulation Therelat 1 i / v 2 eorderofthehydrolysisratesofthesecomplexesis of factor inhibiting HIF (FIH)-dependent p300 dissociation 3 > 1 > 2, which is due to the fact that increased electron from HIF-1α,45−47 which can enhance chemosensitivity of density at Ru(II) from the aromatic ligand with more alkyl chemotherapy drugs like cisplatin and sorafenib.48,49 There- groupscanimprovetheCl − labilityandfacilitatethehydrolysis fore, we designed and prepared three organometallic reaction.55,56Thestudyindicatedthatthearenegroupsplayan ruthenium(II) compounds bearing a YC-1 moiety, which are importantroleincontrollingthehydrolysisratesofthearene− expected to target the hypoxic tumor microenvironment and Ru(II) complexes, which may further affect their biological exhibit their anticancer activity through multiple mechanisms activities. so as to improve the therapeutic efficacy of chemotherapeutic In Vitro Cytotoxicity. The cytotoxic activities of d ■ rugs. complexes 1−3 and ligand L1 have been investigated against twohumancancercelllines(HCT-116andA549cancercells) RESULTS AND DISCUSSION and human umbilical vein endothelial cell line (HUVEC) Synthesis and Characterization. Complexes 1−3 were under normoxic or hypoxic conditions together with cisplatin prepared by following the procedure shown in Scheme 1 in as a positive agent. Each complex was measured at eight which YC-1 was obtained as described previously.50 The different concentrations against the tested cell lines, and the resulting arene−ruthenium(II) complexes were characterized corresponding IC 50 values (dose required to inhibit 50% byelementalanalysisand1Hand13CNMRspectraalongwith cellular growth) were determined from dose−survival curves ESI−MSspectrometry(FiguresS1−S11).Allthespectraldata (Figure S12). were compatible with the proposed molecular structures of According to the IC 50 values (Table 2), ligandL1 exhibited complexes 1−3. little cytotoxicity under normoxia, while complexes 1−3 Hydrolysis Studies. Hydrolysis of the organometallic showed dose-dependent inhibition against the tested cell Ru(II)−arene complexes with the type of [(6η-arene)Ru- lines with IC values ranging from 22.8 to 76.3 μM. Notably, 50 (NN)Cl]+ (NN = chelating diamine ligand) is believed as a complex3(22.8±1.2μM)showedcomparablecytotoxicityto key activation step before they bind to the biomolecules,51−53 thatofcisplatin(19.9±0.9μM)inHCT-116cells,whichmay andarelationshipbetweentheaquationrateforarene-−Ru(II) partly attribute to its increased hydrolysis rate. Moreover, all complexesandcytotoxicitywasproposedbyWang andSadler complexes exhibited markedly improved cytotoxic activity B DOI:10.1021/acs.inorgchem.8b01070 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article Figure2.Time-dependentUV−visspectrafortheaquationof0.05mMcomplexes1(a),2(c),and3(e)(95%HO/5%MeOH).Absorbance− 2 timetrace (275 nm)andmonoexponential fit obtainedforthe hydrolysis ofcomplexes 1 (b), 2(d), and 3 (f)at310K. Table 1. Hydrolysis Rate Constants (k) and Half-Times under hypoxia against HCT-116 and A549 cells with HF (t ) of Complexes 1−3 (95% H O/5% MeOH) at 310 K (hypoxiafactor:thetoxicityundernormoxiavshypoxia)values 1/2 2 range from 1.8 to 3.8, indicating that YC-1 can enhance the complex 1 2 3 antiproliferativeactivityoftheRu(II)−arenecomplexesagainst k(10−4s−1) 5.03±0.23 3.89±0.19 8.39±0.29 hypoxic cancer cells. Additionally, significant morphological t (min) 23.0 29.7 13.8 1/2 changes to the HCT-116 cells were observed when they were treated with complex 3 at different concentrations (12.5, 50, and 100 μM) under hypoxia (Figure S13). In contrast, Table 2. Cytotoxicity of the Compounds againstHCT-116 and A549 Cancer Cells under Normoxic and Hypoxic Conditions IC values(μM) 50 HCT-116a A549b HUVECc compound normoxia hypoxia HFd normoxia hypoxia HFd normoxia 1 65.7±0.9** 17.5±1.4 3.8 76.3±3.2** 31.7±2.5** 2.4 129.1±10.6** 2 57.5±2.5** 17.9±0.8 3.2 65.5±4.8** 24.7±0.9* 2.7 92.6±5.7** 3 22.8±1.2* 12.7±0.6** 1.8 36.3±1.6** 15.7±0.6** 2.3 153.2±9.8** L1 112.9±8.6** 53.7±4.5** 2.1 147.1±8.7** 63.8±2.3** 2.3 128.9±11.9** cisplatin 19.9±0.9 19.0±1.3 1.0 20.9±1.5 21.7±1.3 1.0 16.6±1.5 aHumancolorectalcancercellline.bHumannonsmall-celllungcancercellline.cHumanumbilicalveinendothelialcellline.Dataareexpressedas the mean (±SD) for three independent experiments. dHF (hypoxia factor): the toxicity under normoxia vs hypoxia. *p < 0.05 and **p < 0.01 comparedwiththe value ofcisplatin. C DOI:10.1021/acs.inorgchem.8b01070 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article cisplatinshowedsimilarcytotoxicityunderbothnormoxicand hypoxicconditions against thetested cell lines. Notably under hypoxia, the IC values of complexes 1−3 were lower than 50 that of cisplatin against HCT-116 cells, implying that these complexes were more active than cisplatin in hypoxic HCT- 116 cells. Overall, complexes 1−3 showed selective and markedly improved cytotoxic activity against hypoxic cancer cells, proving that the introduction of YC-1 to the Ru(II)− arene complexes is an effective way to enhance anticancer activity in hypoxic cancer cells. Cellular Accumulation. The intracellular ruthenium accumulationofcomplexes1−3inHCT-116cellswasstudied to investigate the possible relationship between cellular accumulation and cytotoxicity. As shown in Figure 3 (Table Figure 4. (a) HCT-116 cells treated with complexes 1−3 under hypoxiafor12hwereexaminedfortheexpressionofHIF-1αproteins using Western blot analysis. (b) Densitometric analysis of the expression of apoptosis-regulated proteinsnormalized withGAPDH. Therelativeexpressionofeachproteinwasrepresentedbythedensity of the protein band/density of GAPDH band. The data are representative of three independent experiments. *p < 0.05 and **p < 0.01comparedwith thevalue of control. staining and flow cytometry assay, with cisplatin as positive control. As demonstrated in Figures 5 and 6, HCT-116 cells exhibited numerous apoptotic cells with condensed or Figure 3. Intracellular accumulation of complexes 1−3 in HCT-116 cellsafter24hofincubationundernormoxicandhypoxicconditions. Data are expressed as the mean (±SD) for three independent experiments.**p < 0.01. S1), the relative order of the cellular accumulation of complexes 1−3 in normoxia is 3 > 1 > 2, which is in accordance with the hydrophobicity of the arene ligands (hexamethylbenzene > cymene > benzene). Thus, the higher accumulation of complex 3 may be another reason for its superior cytotoxicity to complexes 1 and 2. While under hypoxia, the ruthenium contents of complexes 1−3 in HCT- 116 cells dramatically increased, and all these complexes had much higher Ru amount than they did under normoxia, indicating their ability to target hypoxic cancer cells. The resultsshowedthattheintroductionofYC-1canpromotethe cellular accumulation of the Ru(II)−arene complexes in hypoxic cancer cells and lead to more potent anticancer activity for these compounds. Western Blot. In order to determine the effect of these complexes on the expression of HIF-1α under hypoxic conditions, Western blot analysis was applied to detect the HIF-1α protein expression in HCT-116 cells. As shown in Figure 4, the expression of HIF-1α decreased after treatment with complexes 1−3 and L1 that all blocked HIF-1α protein accumulationinadose-dependentmanner.Thisstudyimplied that our metal complexes could effectively inhibit HIF-1α protein accumulation under hypoxic conditions in HCT-116 cells. Figure5.Cellmorphologicalobservationforcellapoptosisinduction Apoptosis Studies. Apoptotic analysis of complexes 1−3 ontheHCT-116cellstreatedwithcomplexes1−3,L1,andcisplatin and L1 against HCT-116 cells was performed under both at30μMfor48h,respectively:(a)normoxiaand(b)hypoxia.Cells normoxicandhypoxicconditionsbytheHoechst33342DNA werestained by Hoechst 33342(size bar = 20μm). D DOI:10.1021/acs.inorgchem.8b01070 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article complexes containing a hypoxia inducible factor-1α inhibitor (YC-1) were first designed and synthesized. In vitro tests showedtheresultingcomplexeshadhigheranticanceractivities inhypoxiathaninnormoxiaagainstthetestedcancercelllines, especiallycomplex3,withmorealkylgroupsthancomplexes1 and 2, exhibiting superior cytotoxicity to cisplatin under a hypoxiccondition.Significantly,thesecomplexesaremuchless cytotoxic than cisplatin against human umbilical vein endothelial cells (HUVEC), suggesting that they have selectivity for cancer cells over normal cells. Both kinetic study on the hydrolysis and cellular accumulation tests indicated that the enhancing cytotoxicity of these complexes under either normoxia or hypoxia matched their hydrolysis rates and cellular accumulations in HCT-116 cancer cells positively.Moreover,complex3wasthemostactivetoinduce apoptosis among the tested compounds under hypoxia, particularly in early stage apoptosis. Western blot analysis revealedthatcomplexes1−3aswellastheligandcontaininga YC-1 moiety blocked HIF-1α protein accumulation in a dose- dependent manner. These findings proved that ligation with YC-1 endowed these organometallic ruthenium(II) complexes with potential for hypoxia targeting in addition to enhancing their anticancer activities. Consequently, our study provides a useful way for further development of ruthenium(II) anticancer agents with selective identification of cancer cells ■and enhanced antitumor activity under hypoxia. EXPERIMENTAL SECTION Materialsand Measurements.All chemicals and solventswere Figure 6. Flow cytometry analysis for apoptosis of HCT-116 cells of analytical reagent grade and used without further purification. inducedbycomplexes1−3,L1,andcisplatinattheconcentrationof [Ru(η6-arene)Cl] (arene = cymene, benzene, and hexamethylben- 30μM for48h:(a) normoxiaand(b) hypoxia. zene)andYC-2 2 w 2 erepreparedaccordingtopreviousreports.57,341H and13CNMRspectraweremeasuredonBrukerAvanceIII-HD600 MHz spectrometer. Mass spectra were recorded an Agilent Q-TOF fragmented nuclei cells, while nuclei of the control cells 6540 spectrometer. Elemental analysis of C, H, and N used a Vario retained the regular round contours, exhibiting that the tested MICRO CHNOS elemental analyzer (Elementar). Hydrolysis study compounds induced more apoptotic cells than the control was performed on a Shimadzu UV2600 instrument equipped with a group.Moreover,underahypoxiccondition,cellstreatedwith thermostatically controlled cell holder. Cancer cells were obtained complex 3 were more condensed and brighter than those in from Jiangsu KeyGEN BioTECH company (China). Cell apoptosis other groups (Figure 5), suggesting the significant cell experiments were measured by flow cytometry (FAC Scan, Becton apoptosis induction of complex 3 on HCT-116 cells. Dickenson) andanalyzed byCell Quest software. The results were further verified by flow cytometry assay. Synthesis of YC-2. To a solution of YC-1 (0.84 g, 2.76 mM) dissolved in DMF (50 mL) were added succinic anhydride (0.55 g, Undernormoxia,theapoptoticratesofHCT-116cellstreated with complexes 1−3 increased as compared with that of the 5 re .5 fl 4 ux m ed M fo ) r a 4 n h d ,a T n E d A th ( e 0 n .5 D 6g M , F 5. w 53 as m re M m ) o . ve T d h u e n r d e e a r ct r i e o d n uc m ed ix p tu r r e e ssu w r a e s . untreated cells (Figure 6). The apoptotic rate of complex 3 The product was purified by column chromatography (silica gel, (44.88%)wascompatibletothatofcisplatin(46.87%),andthe methanol/dichloromethane1:9)togiveYC-2.Yield:1.04g(93.3%). relative order of inducing apoptosis against HCT-116 cells is White solid. Anal. Calcd (%) for C H NO: C 68.31, H 4.98, N 23 20 2 5 cisplatin(46.87%) ≈3(44.88%) >1(35.87%) ≈2(32.54%) 6.93.Found:C68.20,H4.91,N6.79;1HNMR(600MHz,DMSO) > L1 (19.69%), which is in line with the result of the δ2.50−2.52(m,2H),2.57−2.59(m,2H),5.19(s,2H),5.73(s,2H), cytotoxicity assay to some extent. Once hypoxia was 6.71−6.72(d,1H,J=3.4Hz),7.02−7.03(d,1H,J=3.4Hz),7.24− 7.28(m,4H),7.30−7.33(m,2H),7.45−7.47(m,1H),7.76−7.77(d, conducted, both the complexes and their ligand L1 induced 1H,J=8.5Hz),8.10−8.12(d,1H,J=8.2Hz),12.24(s,1H)ppm. animprovedincidenceofearlytolatestageapoptosisinHCT- Synthesis of L1. YC-2 (0.50 g, 1.24 mM), HATU (0.51 g, 1.34 116cellscomparedwiththatinnormoxia.Notably,complex3 mM),TEA(0.14g,1.40mM),andDMF(7.5mL)wereaddedintoa had a higher apoptotic rate than cisplatin, proving that it had 25 mL round-bottom flask. The mixture was stirred at room advantages over cisplatin to induce cell apoptosis under a temperature for 2 h. Then 5-amino-1,10-phenanthroline (0.20 g, hypoxic condition. This further verified the considerable 1.02mM)wasadded,andtheresultingmixturewasstirredat60°C ■antiproliferativeeffectofcomplex3inhypoxicHCT-116cells. for12h.Thesolventwasthenremovedbyevaporationunderreduced pressure. Column chromatography (eluent 30:1 DCM/methanol) CONCLUSION gaveL1.Yield:0.32g(54.0%).Lightyellowsolid.Anal.Calcd(%)for C H NO:C72.28,H4.68,N12.04.Found:C72.20,H4.81,N In view of hypoxia as an important factor to limit chemo- 11 3 . 5 96 2 ; 7 1H 5 N 4 MR (600 MHz, DMSO) δ 2.77−2.79 (m, 2H), 2.88− therapeuticefficacyinclinicalpractice,wepurposedtodevelop 2.90(m,2H),5.24(s,2H),5.70(s,2H),6.74−6.75(d,1H,J=3.4 organometallic ruthenium(II)−arene complexes targeting the Hz),7.01−7.02(d,1H,J=3.3Hz),7.18−7.26(m,4H),7.29−7.31 hypoxic tumor microenvironment so as to enhance their (m,2H),7.39−7.42(m,1H),7.72−7.74(d,1H,J=8.2Hz),7.75− anticancer activity. In this study, three ruthenium(II)−arene 7.77(q,1H,J=4.4Hz),7.84−7.86(q,1H,J=4.4Hz),8.08−8.09 E DOI:10.1021/acs.inorgchem.8b01070 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article (d, 1H, J = 8.2 Hz), 8.18 (s, 1H), 8.45−8.47 (1H, d-d, J = 8.1, 1.4 MTT Assay. Cytotoxicity of complexes 1−3, ligand L1, and Hz),8.69−8.70(d,1H,J=7.4Hz),9.01(m,1H),9.03−9.04(1H,d- cisplatinagainstHCT-116,A549,andHUVECcellswasdetermined d,J= 4.3, 1.6Hz), 9.12−9.13(1H, d-d, J= 4.2,1.5 Hz),10.27 (m, by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl 1H)ppm. tetrazoliumbromide)assay.Cells(5000−10000perwell)withbetter GeneralProcedureforSynthesisofComplexes1−3.Asolutionof vitalitywereseededin96-wellplates.Thecompoundsweredissolved L1(0.29g,0.50mM)inCHCl (10mL)wasaddedtoasuspension by DMF (water for cisplatin) and diluted with medium to various 2 2 of [Ru(arene)Cl] (0.21 mM) in CHCl (15 mL) dropwise. The concentrations(thefinalconcentrationofDMFwaslessthan0.4%). 2 2 2 2 mixture was stirred at room temperature for 8 h. Then the reaction Afterbeingincubatedunderanormoxiccondition(20%O,5%CO, 2 2 mixturewasconcentratedto3mL.Thecrudeproductwasseparated, and75%N)orhypoxiccondition(1%O,5%CO,and94%N)at 2 2 2 2 washedwithEtO(3×10mL)andcoldmethanol(3×10mL),and 37°Cfor72h,cellswerestainedwithMTT(5mg/mL)foranother 2 driedina vacuum-dryer. 5h,andthenthemediumwasthrownawayandreplacedby150mL Complex 1. Yield: 0.23 g (61.3%). Yellow-green powder. Anal. of DMSO. The inhibition of cell growth induced by the tested Calcd(%)forC H ClNORu:C60.88,H4.65,N7.89.Found:C complexeswasdetectedbymeasuringtheabsorbanceofeachwellat 45 41 2 5 4 60.73,H4.74,N7.66.ESI−MS:m/z[M−Cl]+=852.2.1HNMR 570/630nmusingenzymelabelinginstrument.TheIC valueswere 50 (600 MHz, DMSO) δ 0.89−0.91 (t, 6H, J = 6.6 Hz), 2.17 (s, 3H), calculated by SPSSsoftwareafter three parallel experiments. 2.59−2.64(m,1H),2.76−2.78(m,2H),2.95−2.97(m,2H),5.23(s, Cellular Accumulation. HCT-116 cells were seeded in six-well 2H), 5.71 (s, 2H), 6.12−6.15 (m, 2H), 6.36−6.38 (t, 2H,, J = 6.4 platesatadensityof106cells/well.Afterovernightincubation,50μM Hz),6.73−6.74(d,1H,J=3.4Hz),7.02−7.03(d,1H,J=3.4Hz), complexes 1−3 were added, respectively. After incubation under a 7.19−7.26(m,4H),7.30−7.32(m,2H),7.39−7.42(m,1H),7.73− normoxic condition (20% O, 5% CO, and 75% N) or hypoxic 2 2 2 7.75 (d, 1H, J = 8.5 Hz), 8.06−8.11 (m, 2H), 8.18−8.21 (m, 1H), condition(1%O,5%CO,and94%N)at37°Cfor24h,cellswere 2 2 2 8.48(s,1H),8.79−8.80(d,1H,J=8.0Hz),9.27−9.28(d,1H,J=8.0 collectedandwashedthreetimeswithice-coldPBS,thencentrifuged Hz), 9.89 (m, 1H), 10.02 (m, 1H), 11.12 (m, 1H) ppm; 13C NMR at1000rpmfor10minandresuspendedin1mLofPBS.Avolumeof (150 MHz, DMSO-d) δ 18.69, 22.12, 22.14, 29.17, 30.86, 31.23, 100 μL was taken out to determine the cell density. Then the 6 52.44, 58.31, 84.23, 84.39, 86.42, 86.57, 103.23, 104.48, 108.41, remainingcellsweredigestedbyHNO (200μL,65%)at65°Cfor4 3 110.75, 113.28, 120.73, 121.50, 122.14, 126.05, 126.45, 126.85, h.TheRulevelincellswasmeasuredbyICP−MSafterthreeparallel 127.37, 127.73, 128.05, 129.08, 130.12, 133.95, 135.48, 135.65, experiments. 137.76, 138.50, 140.77, 143.18, 145.87, 149.06, 149.59, 155.35, Apoptosis Assessment by Hoechst 33342 Staining. HCT-116 156.60,172.02, 172.61ppm. cellswereseededin24-wellplatesat1×105cells/wellandincubated Complex 2. Yield: 0.21 g (59.6%). Yellow-green powder. Anal. overnight.Cellswereincubatedwith30μMofthetestedcompounds Calcd(%)forC H ClNORu:C59.21,H4.00,N8.42.Found:C under a normoxic condition (20% O, 5% CO, and 75% N) or 41 33 2 5 4 2 2 2 59.03,H4.14,N8.20.ESI−MS:m/z[M−Cl]+=796.2.1HNMR hypoxiccondition(1%O,5%CO,and94%N)at37°Cfor48h. 2 2 2 (600 MHz, DMSO) δ 2.82−2.84 (m, 2H), 3.04 (m, 2H), 5.28 (s, Then the cells were rinsed twice in PBS and stained with Hoechst 2H),5.76(s,2H),6.40(s,6H),6.79−6.80(d,1H,J=3.1Hz),7.06− 33342fluorescentdyefor10mininthedarkat37°C.Cellapoptosis 7.07(d,1H,J=3.1Hz),7.22−7.25(d,1H,J=7.5Hz),7.29−7.38 was examined under the fluorescence microscope with excitation (m,5H),7.44−7.46(m,1H),7.78−7.80(d,1H,J=8.5Hz),8.11(m, wavelength of 330−380 nm, and data were collected from three 1H), 8.13−8.15 (d, 1H, J = 8.2 Hz), 8.23 (m, 1H), 8.50 (s, 1H), independentexperiments. 8.82−8.83(d,1H,J=8.0Hz),9.30−9.31(d,1H,J=8.2Hz),10.03 Apoptosis Analysis by Flow Cytometry. HCT-116 cells were (m,1H),10.17(m,1H),11.18(m,1H)ppm;13CNMR(150MHz, growninasix-wellplateatadensityof2×105cells/wellandcultured DMSO-d) δ 29.19, 31.32, 52.41, 58.29, 87.11, 108.38. 110.71, overnight.Thetestedcomplexeswereadded,whichweredilutedtoa 6 113.29, 120.69, 121.49, 122.11, 125.85, 126.56, 126.65, 127.35, concentrationof30μM.Afterincubationunderanormoxiccondition 127.73, 128.04, 129.07, 130.12, 133.92, 135.46, 135.77, 137.76, (20% O, 5% CO, and 75% N) or hypoxic condition (1% O, 5% 2 2 2 2 138.46, 140.73, 143.33, 146.02, 149.06, 149.57, 155.56, 156.82, CO,and94%N)for48h,cellswerecollectedbycentrifugation(5 2 2 172.03,172.61ppm. min,25°C,2000rpm).Then,thecellswerewashedtwicewithcold Complex 3. Yield: 0.25 g (65.2%). Yellow-green powder. Anal. water and resuspended in binding buffer (10 mM HEPES, 140 mM Calcd(%)forC H ClNORu:C61.64,H4.95,N7.65.Found:C NaCl, 2.5 mM CaCl, pH 7.4). The cells were stained with 5 μL of 47 45 2 5 4 2 61.38,H5.11,N7.53.ESI−MS:m/z[M−Cl]+=880.3.1HNMR Annexin V- FITC and then with 5 μL of propidium iodide (20 μg/ (600MHz,DMSO)δ2.10(s,18H),2.75−2.77(m,2H),2.98−3.00 mL)for15mininthedarkatroomtemperature.Thefluorescenceof (m,2H),5.21(s,2H),5.69(s,2H),6.72−6.73(d,1H,J=2.9Hz), cells was detected by an annexin V-FITC apoptosis detection kit 7.00−7.01 (d, 1H, J = 2.9 Hz), 7.18−7.25 (m, 4H), 7.28−7.31 (m, (Roche) according to the manufacturer’s protocol, and cells were 2H),7.38−7.40(m,1H),7.72−7.73(d,1H,J=8.4Hz),8.08−8.09 quantifiedby system software(CellQuest;BD Biosciences). (m,2H),8.16(m,1H),8.45(s,1H),8.73−8.75(d,1H,J=7.8Hz), Western Blot. HCT-116 cells were grown in a six-well plate at a 9.24−9.27 (m, 2H), 9.37−9.38 (m, 1H), 11.16 (m, 1H) ppm; 13C density of 2 × 105 cells/well and cultured until the cell density NMR (150 MHz, DMSO-d) δ 15.75, 29.19, 31.23, 52.42, 58.29, reached 80%. The solutions of complexes 1−3 and L1 were diluted 6 95.81,108.41.110.75,113.28,120.71,121.50,122.14,126.24,126.27, downinmediatogivetherequiredconcentration(0.1,1,or5μM) 127.06, 127.36, 127.73, 128.05, 129.07, 129.87, 134.05, 135.36, foradditiontothecells,andthecellswereculturedunderanormoxic 135.46, 137.75, 138.05, 140.74, 143.23, 145.92, 149.04, 149.58, condition(20%O,5%CO,and75%N)orhypoxiccondition(1% 2 2 2 153.05,154.24, 172.06,172.62ppm. O, 5% CO, and 94% N) for 12 h at 37 °C. HCT-116 cells were 2 2 2 HydrolysisStudies.Hydrolysisofcomplexes1−3wasrecordedon lysedincelllysisbufferandcollectedbycentrifugationat13000rpm a Shimadzu UV2600 instrument equipped with a thermostatically for 20 min at 4 °C. Proteins from cell lysates were separated by 8− controlledcellholder.TheUV−visspectrawererecordedbyscanning 12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis from185to600nmevery5minat37°C,and275nmwasselected (SDS-PAGE) and transferred onto a polyvinylidine difluoride forthekineticstudy.Thetime-dependentabsorbancewasfittedusing (PVDF) membrane (Amersham Biosciences). The membrane was Origin9.0to give the first order rateconstantk. blockedwithPBSTcontaining5%nonfatdrymilkfor1handfurther Cell Culture. HCT-116 (human colorectal cancer cell line) and incubatedwithmonoclonalantihumanHIF-1αantibody(SantaCruz A549(humannonsmallcelllungcancercellline)weremaintainedin Biotechnology, USA) overnight at 4 °C under gentle shaking. After ahumidifiedatmosphereof5%CO at37°C.Cellswereculturedin that, the membrane was incubated with the secondary antibody 2 RPMI-1640 medium with 10% fetal bovine serum (FBS). All media (1:2000) for 1 h at RT (25 °C). Protein blots were detected with werealsosupplementedwith100mg/mLofpenicillinand100mg/ chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). mLof streptomycin. GAPDH was usedas loadingcontrol. F DOI:10.1021/acs.inorgchem.8b01070 Inorg.Chem.XXXX,XXX,XXX−XXX Inorganic Chemistry Article Statistical Analysis. Differences among samples were calculated single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017, with the two-tailed Student’s t-test using an independent samples t- 46, 5771−5804. test in SPSS 16.0. Differences among groups were considered (12)Hartinger,C.G.;Zorbas-Seifried,S.;Jakupec,M.A.;Kynast,B.; ■statisticallysignificantat P< 0.05. Zorbas, H.; Keppler, B. K. From bench to bedside-preclinical and early clinical development of the anticancer agent indazolium trans- ASSOCIATED CONTENT [tetrachlorobis(1H-indazole)ruthenate(III)](KP1019orFFC14A). * S Supporting Information J. Inorg. Biochem.2006,100,891−904. The Supporting Information is available free of charge on the (13) Han Ang, W.; Dyson, P. J. Classical and non-classical ruthenium-based anticancer drugs: Towards targeted chemotherapy. ACS Publications website at DOI: 10.1021/acs.inorg- Eur. J. Inorg. Chem. 2006, 20,4003−4018. chem.8b01070. (14) Zhao, J.; Zhang, D.; Hua, W.; Li, W.; Xu, G.; Gou, S. 1Hand13CNMRandESI−MSspectraofcomplexes1− Anticancer Activity of Bifunctional Organometallic Ru (II) Arene 3, dose-dependent cell viability curves, and cellular ComplexesContaininga7-HydroxycoumarinGroup.Organometallics accumulation data (PDF) 2018,37, 441−447. ■ (15)Tian, M.;Li,J.;Zhang,S.;Guo,L.;He, X.;Kong,D.;Zhang, ̂ H.;Liu,Z.Half-sandwichruthenium(ii)complexescontainingNN- AUTHOR INFORMATION chelatedimino-pyridylligandsthatareselectivelytoxictocancercells. Corresponding Authors Chem. Commun.2017,53, 12810−12813. *E-mail: sgou@seu.edu.cn. (16)Li,J.;Tian,M.;Tian,Z.;Zhang,S.;Yan,C.;Shao,C.;Liu,Z. *E-mail: xugang@seu.edu.cn. Half-Sandwich Iridium (III) and Ruthenium (II) Complexes ̂ ORCID ContainingPP-ChelatingLigands:ANewClassofPotentAnticancer Agents withUnusual RedoxFeatures. Inorg. Chem. 2018,57, 1705− Shaohua Gou: 0000-0003-0284-5480 1716. Notes (17) Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl, T■he authors declare no competing financial interest. M.; Egger, A.; Berger, W.; Zorbas, H.; Dyson, P. J.; Keppler, B. K. KP1019,anewredox-activeanticanceragent-preclinicaldevelopment ACKNOWLEDGMENTS and results of a clinical phase I study in tumor patients. Chem. WearegratefultotheNationalNaturalScienceFoundationof Biodiversity2008,5,2140−2155. (18) Bergamo, A.; Sava, G. Ruthenium anticancer compounds: China (Grant No. 21601034) and Jiangsu Province Natural Science Foundation (Grant No. BK20160664) for financial mythsandrealitiesoftheemergingmetal-baseddrugs.DaltonTrans. 2011,40, 7817−7823. aids to this work. We also thank the Fundamental Research (19) Riedl, C. A.; Flocke, L. S.; Hejl, M.; Roller, A.; Klose, M. H.; Funds for the Central Universities (Project 2242016K30020 Jakupec, M. A.; Kandioller, W.; Keppler, B. K. Introducing the 4- and 2242017K41025) and Priority Academic Program Phenyl-1, 2, 3-Triazole Moiety as a Versatile Scaffold for the Development of Jiangsu Higher Education Institutions for Development of Cytotoxic Ruthenium (II) and Osmium (II) Arene t■he construction of fundamental facilities are also appreciated. Cyclometalates. Inorg.Chem. 2017,56,528−541. (20)Trondl,R.;Heffeter,P.;Kowol,C.R.;Jakupec,M.A.;Berger, REFERENCES W.; Keppler, B. K. NKP-1339, the first ruthenium-based anticancer (1) Rosenberg, B.; Van Camp, L.; Krigas, T. Inhibition of cell drug on the edge to clinical application. Chem. Sci. 2014, 5, 2925− division in Escherichia coli by electrolysis products from a platinum 2932. electrode.Nature1965,205,698−699. (21) Guerriero, A.; Oberhauser, W.; Riedel, T.; Peruzzini, M.; (2) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. 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