High in Vitro and in Vivo Tumor-Selective Novel Ruthenium(II) Complexes with 3-(2'-Benzimidazolyl)-7-fluoro-coumarin.

Letter CiteThis:ACSMed.Chem.Lett.XXXX,XXX,XXX−XXX pubs.acs.org/acsmedchemlett High in Vitro and in Vivo Tumor-Selective Novel Ruthenium(II) ‑ ′ fl Complexes with 3 (2 -Benzimidazolyl)-7- uoro-coumarin Qi-Pin Qin, *,† Zhen-Feng Wang, † Xiao-Ling Huang, † Ming-Xiong Tan, *,† Bei-Bei Shi, † and Hong Liang *,‡ † GuangxiKeyLabofAgriculturalResourcesChemistryandBiotechnology,CollegeofChemistryandFoodScience,YulinNormal University, 1303 Jiaoyudong Road, Yulin 537000, PR China ‡ State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai Road, Guilin 541004, PR China * S Supporting Information ABSTRACT: Three novel Ru(II) complexes, namely, (RuCl [La][DMSO] )·H O (Ru1), (RuCl [Lb][DMSO] ) 2 2 2 2 2 (Ru2), and (RuCl [Lc][DMSO] ) (Ru3), which respectively 2 2 contain3-(2′-benzimidazolyl)coumarin(La),3-(2′-benzimida- zolyl)-7-fluoro-coumarin (Lb), and 3-(2′-benzimidazolyl)-7- methoxyl-coumarin(Lc),werefirstdesignedandcharacterized. Ru2showedpotentantitumoractivityagainstNCI-H460cells (IC = 0.30 ± 0.02 μM) and high selectivity between NCI- 50 H460 cancer cells and normal HL-7702 cells. Ru2 induced NCI-H460apoptosisviatelomeraseinhibition,whichinvolvedDNAdamage,cell-cycledistribution,andSphase-proteindown- regulation. However, Ru1 did not demonstrate such effects in NCI-H460 cells, which is undoubtedly associated with the key regulatoryroleofthe7-fluorosubstitutedgroupintheLbligandofRu2.Ru2exhibitedconsiderablyhigheranticancerefficacy (inhibition rate [IR] = 61.3%) compared with cisplatin (IR= 25.5%) in a NCI-H460 xenograft mouse model. Thus, this coumarin Ru(II) compound is a promising Ru2-targeting telomerase anticancer agent. KEYWORDS: 3-(2′-Benzimidazolyl)-7-fluoro-coumarin, Ru(II) complex, telomerase, anticancer activity, cell cycle distribution R u(II) complexes exhibit high water solubility and low Three new coumarin derivatives, Ru(II) complexes Ru1− general toxicity compared with cisplatin and its Ru3, were first synthesized with 2-cyanomethylbenzimidazole derivatives, such as oxaliplatin and carboplatin.1−24 Many as the starting material (Scheme S1). The synthesized La−Lc Ru(II) compounds, such as NAMI-A,5−7 DW1/2,8,9 KP1019 and Ru1−Ru3 were characterized via electrospray mass and KP1339,3,10−13,20 RM175,11−16 RAPTA-T and RAPTA spectrometry (ESI-MS), single-crystal X-ray diffraction, ele- complexes (Figure S1),2,17,18 ([Ru{phen} 2 ] 2 tpphz),4+,19 three mental analysis, and infrared spectroscopy (Figures S1−S10). water-soluble chiral 4-(2,3-dihydroxypropyl)-formamide oxoa- As shown in Figures 1, S11,and S12, the Ru(II) ion in Ru1− porphine Ru(II) complexes,21 Δ-/Λ-(Ru[phen] 2 [p- Ru3 adopted an octahedral environment. The solution MOPIP])2+, and Λ-/Δ-(Ru[phen] 2 [p-HPIP])2+ com- behavior of Ru1−Ru3 (2.0 × 10 −5 M) in Tris-HCl buffer plexes,22,23 display high antitumor activity. Thus, Ru(II/III) (10 mM, pH = 7.35) was further studied by UV−vis compounds must be designed to understand in vitro and in absorption spectra. As shown in Figure S13, the time- vivo apoptosis mechanisms. dependent (0, 24, and 48 h) UV−vis spectra of Ru1−Ru3 Various coumarin derivatives and their metal complexes (2.0 × 10 −5M) suggest that Ru1−Ru3 were stablein10 mM c e a o n xh t m i i v b p i i r l t e al x , a es a w nd i w de i a th n r t a i- n 3 A g - l ( e z 2 h ′ o e - f b im e a n e n z r t i i m a -H c i t d I iv V a i z , t o ie a ly s n . l 2 t ) 5 i c m − o 3 i u 5 c m ro H a b o r i i a w n l, ev a d e n e r t , r i i c v R a a u n t ( i c v I e e I r ) s , Tr T is h -H e C cy l t b o u to ff x e i r ci f t o y r o 4 f 8 La h −L at c, 3 c 7 is- ° R C u . Cl 2 (DMSO) 4 ,cisplatin,and Ru1−Ru3 against human cancer cells (NCI-H460, T-24, SK- (BMC, FBMC, and MBMC) have not been reported, and OV-3,MGC80-3,andA549)andnormalcells(HL-7702)was the detailed antitumor mechanisms of Ru(II) compounds investigated by MTT assays. As shown in Tables S10 and 1, remain unexplored. Thus, we first designed three novel Ru(II) complexes, Ru1−Ru3 did not exhibit cytotoxicity in T-24, SK-OV-3, namely, (RuCl [La][DMSO] )·H O (Ru1), (RuCl [Lb]- MGC80-3, A549, and HL-7702 cell lines. The IC 50 values [DMSO] ) (Ru2 2 ), and (RuCl 2 [Lc][ 2 DMSO] ) (Ru3), 2 which determinedforRu2werelower(IC 50 =0.30±0.02μM)than 2 2 2 contain 3-(2′-benzimidazolyl)coumarin (La), 3-(2′-benzimida- zolyl)-7-fluoro-coumarin (Lb), and 3-(2′-benzimidazolyl)-7- Received: March10,2019 methoxyl-coumarin (Lc), respectively. Furthermore, we Accepted: May21,2019 proposedapossibleinvitroandinvivoanticancermechanism. ©XXXXAmericanChemicalSociety A DOI:10.1021/acsmedchemlett.9b00098 ACSMed.Chem.Lett.XXXX,XXX,XXX−XXX 9102 ,52 yaM no 847:13:12:12 ta ANAIDNI NREHTUOS FO VINU yb dedaolnwoD .89000b9.ttelmehcdemsca/1201.01/iod/gro.sca.sbup//:sptth morf ACS Medicinal Chemistry Letters Letter Figure2.Ru2(0.30μM)andRu1(15.78μM)inhibitedtelomerase Figure 1.Crystalstructuresof Ru2. activityinNCI-H460cellsfor24h.(A)Telomeraseactivityassay.(B andC)Levelsofc-myc/hTRETinRu2(0.30μM)-andRu1(15.78 those for La−Lc, cis-RuCl (DMSO) (RuD), Ru1, Ru3, and μM)-treatedcells. 2 4 cisplatin and followed the order of Ru2 > Ru3 > cisplatin > Ru1. Ru2 demonstrated remarkable antitumor activity against al.21,37 The telomerase inhibition of Ru2 (0.30 μM) may be NCI-H460 cells that was higher than or close to that in differentfromthatofRu1(15.78μM)intheNCI-H460cells. previous reports.5−24,36,37 Therefore, NCI-H460 cells were Telomeraseinhibitionbyeachdrug/compoundmayleadto selectedforfurtherstudyonthecelldeathmechanisminduced cell cycle arrest at the S phase and DNA damage.42−44 As by Ru2 (0.30 μM) and Ru1 (15.78 μM). showninFigureS14,Ru2(0.30μM)-treatedcellsexhibitedan ToexploretheapoptosismechanismofRu2(0.30μM)and increased number of cells (46.98%) at the S phase compared Ru1 (15.78 μM), we performed an ICP-MS (cellular uptake) with the control (36.68%). This phenomenon remarkably study.36−38 Table S11 shows the incubation and treatment of caused DNA damage (Figure 3), consequently increasing the NCI-H460 cells with Ru2 (0.30 μM) and Ru1 (15.78 μM). levelofH2A.Xandcleaved-PARPproteinsanddecreasingthat The concentration of Ru(II) inside these cancer cells and ofcyclinA2andCDK2(FigureS15).Bycontrast,Ru1(15.78 nuclearfractionforRu2([4.58±0.11nmolofRu]/106cells) μM) demonstrated less effect on NCI-H460 cells. at0.30μMwasmorethanthatforRu1([1.56±0.14nmolof Subsequently, we investigated whether Ru2 (0.30 μM) and Ru]/106 cells), oxoaporphine Ru(II) complex ([3.81 ± 0.14 Ru1 (15.78 μM) could induce apoptosis and inhibit the nmolRu]/106cells),andcisplatin([2.08±0.11nmolPt]/106 migration of NCI-H460 cells. Ru2 (0.30 μM, ca. 88.4%) cells).21,37C-myc,telomerase,andhTERTplayacrucialrolein induced the apoptosis (Figure 4) of HeLa cells for 24 h to a cancer cell growth or apoptosis.33,39−50 In the current study, greater extent than oxoaporphine Ru(II) complexes (ca. TRAP-silver staining33,39−41 assays showed that Ru2 (0.30 20.2%−54.7%)21,37 and Ru1 (15.78 μM, ca. 18.9%). Cell μM) exhibited more effective inhibition (53.60%, Figure 2) migration was inhibited by Ru2 (0.30 μM) and Ru1 (15.78 toward telomerase activity compared with Ru1 (6.13%). As μM) by ∼47.1% and 29.4% compared with the control cells, expected, the level of c-myc/hTERT protein in NCI-H460 respectively (Figure S16). Ru2 (0.30 μM) remarkably cellswasreducedinRu2(0.30μM)butwasincreasedinRu1 inhibited NCI-H460 cancer cell migration compared with (15.78μM)comparedwiththecontrol.Apossiblemechanism Ru1 (15.78 μM). of Ru1 (15.78 μM) is the direct activation of the c-myc/ Ru2,whichshowedthehighestsolubilityinsolvent(5%v/v hTERT protein by c-myc before c-myc binds to E-box and DMSO/saline), was used to preliminarily study its safety telomeraseinhibitor,andsuchactivationwasnotrelatedtocell (Figure S17), and ICR mice were treated with possible apoptosis, which was consistent with the reports of Chen et maximal administration values (0.6 mL/20 g) by intra- Table 1. IC Values (μM) of Each Compound against Selected Human Cell Lines a 50 NCI-H460 T-24 SK-OV-3 MGC80-3 A549 HL-7702 La >100 >150 >150 >100 >100 >100 Ru1 15.78±1.02 53.01±1.26 65.02±1.12 91.03±1.01 41.36±0.99 >100 Lb >100 >100 >100 >100 >100 >100 Ru2 0.30±0.02 25.63±1.44 35.69±2.03 68.69±1.15 20.14±0.28 >100 Lc >100 >100 >100 >100 >100 >100 Ru3 10.04±0.73 30.00±1.09 46.25±1.59 88.24±1.79 34.39±1.05 >100 RuD >100 >100 >100 >100 >100 >100 cisplatinb 13.25±1.18 17.03±0.57 15.09±0.91 12.06±1.18 12.36±0.19 17.03±1.06 aIC =mean±SD(standarderrorofthemean,n=5).ThesesixhumancancerandnormalcellsweretreatedwiththeligandsandeachoftheRu 50 complexesfor48 h.bCisplatin(1.0 mM)wasprepared in0.154M NaCl. B DOI:10.1021/acsmedchemlett.9b00098 ACSMed.Chem.Lett.XXXX,XXX,XXX−XXX ACS Medicinal Chemistry Letters Letter In conclusion, we first designed the three novel Ru(II) complexes, namely, Ru1−Ru3 containing 3-(2′- benzimidazolyl)coumarin derivatives. The most active com- pound, Ru2, showed higher cytotoxic potency against NCI- H460 cells (IC = 0.30 ± 0.02 μM) compared with cisplatin 50 (13.25±1.18μM)anddemonstratedhighselectivitybetween NCI-H460 cancer cells and HL-7702 normal cells. Further- more,Ru2accumulatedpreferentiallyinthenuclearfractionof NCI-H460 cells and induced their apoptosis via telomerase inhibition, DNA damage, and cell cycle distribution. The Ru2 exhibited evident priority on antitumor activity than Ru1, whichshouldbehighlyrelatedtothekeyrolesofthe7-fluoro substituted group in Lb ligand of Ru2. An in vivo study suggested that Ru2 exhibited higher anticancer efficacy (IR = 61.3%) compared with cisplatin (IR = 25.5%) in the NCI- H460 xenograft. Therefore, this coumarin Ru(II) compound ■maybeapromisingRu2-targetingtelomeraseanticanceragent. ASSOCIATED CONTENT * S Supporting Information Figure3.EffectsofRu2(0.30μM)andRu1(15.78μM)onthelevel The Supporting Information is available free of charge on the ofH2A.XinNCI-H460cellsfor24h.Thesecellswereincubatedwith ACS Publications website at DOI: 10.1021/acsmedchem- anti-H2A.X (primary antibodies) and Alexa Fluor 488 Goat Anti- lett.9b00098. Rabbit IgG (H+L, green, secondary antibody), stained with DAPI (blue), and examined by immunofluorescence (LeicaTCS-SP5 X-ray crystallization data of Ru1−Ru3. The CCDC confocalmicroscope, Germany, 200× magnification). numbers for Ru1−Ru3 are 1902151−1902153 (ZIP) Experimental procedures, Tables S1−S14, and Figures S1−S16. NMR, ESI-MS, IR, and experiments of Ru1− Ru3 (PDF) ■ AUTHOR INFORMATION Corresponding Authors *Q.-P. Qin: Telephone and Fax: (086) 775-2623650, E-mail: qpqin2018@126.com. *M.-X. Tan: Telephone and Fax: (086) 775-2623650, E-mail: Figure 4. Apoptosis of NCI-H460 cells treated with (c) Ru2 (0.30 μM) and(b) Ru1(15.78 μM)for 24hcomparedwith control(a). mxtan2018@126.com. *H. Liang: Telephone: (086) 773-2120998, Fax: (086) 773- 21209958, hliang@gxnu.edu.cn. peritoneal injection. No apparent body weight decrease (m ORCID start =19.03±0.67g;m =21.33±0.37g)andinjurycondition Qi-Pin Qin: 0000-0001-9596-4512 end (Figures S17 and 5; Tables S12−S14) were observed for Ru2 Ming-Xiong Tan: 0000-0002-5352-4030 (10.0 mg/kg every 2 days [q2z])-treated mice, demonstrating Author Contributions the low systemic toxicity of coumarin Ru(II) compound. The All authors contributed to the writing of this manuscript. in vivo anticancer efficacy of Ru2 (10.0 mg/kg q2d) on NCI- Funding H460 cancer xenograft was analyzed. The NCI-H460 tumor inhibitionrate(IR)was61.3%forRu2(Figure5;TablesS12− We thank the National Natural Science Foundation of China S14), showing a higher anticancer efficiency compared with (Nos. 21867017 and 21761033), the Natural Science cisplatin (IR = 25.5%)37,46,47 and oxoaporphine Ru(II) Foundation of Guangxi (No. 2018GXNSFBA138021), and complex (IR = 53.3%).21,37 the Innovative Team and Outstanding Talent Program of Colleges and Universities in Guangxi (2014-49 and 2017-38) for financial support. Notes T■he authors declare no competing financial interest. ACKNOWLEDGMENTS We acknowledge professors Fu-Ping Huang and Peng-Fei Yao for helping perform the X-ray crystallization data analysis of R■u1−Ru3. ABBREVIATIONS Figure 5. In vivo anticancer activity of Ru2 in mice bearing NCI- H460 xenograft. 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