Investigation into antiproliferative activity and apoptosis mechanism of new arene Ru(ii) carbazole-based hydrazone complexes.

View Article Online Dalton View Journal Transactions An international journal of inorganic chemistry Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: S. K. Thangavel, M. K. Mohamed Subarkhan, R. Rengan, H. Wang and J. Malecki, Dalton Trans., 2020, DOI: 10.1039/D0DT01476A. This is an Accepted Manuscript, which has been through the Volume 46 Number 10 Dalton 1 P 4 a g M e a s r c 3 h 0 7 2 3 0 - 1 3 7 412 Royal Society of Chemistry peer review process and has been accepted for publication. Transactions Accepted Manuscripts are published online shortly after acceptance, An international journal of inorganic chemistry rsc.li/dalton before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. 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In no event ISSN 0306-0012 shall the Royal Society of Chemistry be held responsible for any errors CDNan o- O dHuM eg tetlM ea llrs u U o r Wc N eyn .I cy CSllit A ce c Tp act hI ai O o erbn n N e s en ( teX a sH l.t +a, bXil i=z eSd, Spea,r Teen)t sulfenyl, selenenyl, or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/dalton Page 1 of 19 PPlleeaassDee addltooo nnnoo Tttr aaanddsjjauucssttti ommnaasrrggiinnss ARTICLE Dalton Transactions Investigations on Antiproliferative Activity and Apoptosis Mechanism of New Arene Ru(II) Carbazole based Hydrazone Received 00th January 20xx, Complexes Accepted 00th January 20xx Thangavel Sathiya Kamatchi,a Mohamed Kasim Mohamed Subarkhan,b Rengan Ramesh,a* DOI: 10.1039/x0xx00000x Hangxiang Wang,b Jan Grzegorz Małeckic Ruthenium complexes with bioactive ligands become the promising substitutes to platinum complexes by showing their t precise actions against various cancers. In the present study, the synthesis of three new arene Ru(II) complexes containing p new carbazole based hydrazone ligands of general formula [(η6-benzene)Ru(L)Cl] (1−3; L = carbazolone benzhydrazone i ligands) and their anticancer properties have been described. The structural characterization of the ligands and their r c ruthenium complexes has been well recognized with the aid of elemental analysis, IR, UV-vis, NMR and HR-MS techniques. The molecular structures of all the three complexes have been elucidated by single crystal X-ray crystallography and reveal s the existence of pseudo-octahedral geometry around ruthenium. In vitro cancer cell growth inhibition property of the u complexes against A549 (lung carcinoma), A2780 (ovarian adenocarcinoma) and non-cancerous 16HBE (human lung n bronchial epithelium) cells were examined by MTT assay. All the complexes display good cytotoxicity on both the cancer a cells than the standard drug cisplatin with low IC values. Remarkably, complex 3, which contains electron-donating 50 M substituent, induces a significant reduction of viability in A2780 cells. The inhibition capacity of the complexes towards A2780 cells proliferation was further confirmed by 5-ethynyl-2-deoxyuridine (EdU) incorporation assay via minimal DNA d synthesis. The result of Acridine orange-Ethidium bromide (AO-EB) fluorescent staining assay establishes that the cytotoxicity of the complexes was mediated by apoptosis in cancer cells. Furthermore, flow cytometry using Annexin V- e FITC / propidium iodide (PI) double staining determines the quantitative discrimination of early apoptosis by the t p externalization of phosphatidylserine. In addition, cell cycle distribution indicates that the complexes block the cell cycle progression in S phase. The outcomes of our investigations witness the promising scope and potency of tailored arene e ruthenium complexes for precise cancer chemotherapy beyond platinum drugs. c c aforementioned criterions with different modes of anticancer A Introduction activities.4 Evidently few Ru(III) drugs, NAMI-A, KP1019 and IT-139 undergone the different phases of clinical trials.5 Nonetheless, the Platinum metal complexes turn out a mainstay in cancer treatments s failure of anti-metastatic NAMI-A in Phase(I) and Phase(I/II) clinical and encompass some of the most powerful and progressive n trials by disease stabilization / partial response and low water chemotherapeutic drugs.1 Despite surgical removal of tumors and solubility of cytotoxic KP1019 stimulated the inventions of other o various therapies scilicet radiation, chemo, immune, hormone, ruthenium drugs and the investigations was diverted to Ru(II) drugs i stem cell, precision medicine etc., shield people to stop dying from t may be one of the alluring hypothetic feature as the activation of c cancer, highly and inevitably used platinum chemotherapy saves Ru(III) pro-drugs to kinetically more reactive Ru(II) drugs by in vivo 50-70 % of all patients’ life2 howbeit with few drawbacks and side a reduction.6 This leads to the conspicuous evolution and consequent effects.3 Mitigating efforts to vanquish the drawbacks triggered the s preclinical studies of direct Ru(II) drugs needless of exploration of more potent metallodrugs concerned with least n activation by intracellular reduction and the two exemplary cases toxicity by cancer cell selectivity, structural diversity, redox activity are poly-pyridyl drug TLD-1433,7 and arene drug complexes of a with amicable biochemical (bio-mimicking ability and ligand general formula, [(η6-arene)Ru(AB)X], arene = benzene, p-cymene r exchange kinetics) properties. Ruthenium complexes imprinted T etc. AB = two N, P monodentate or a NN, ON, CN, OO bidentate; X their promising in vitro anticancer and in vivo antitumor properties = labile ligand(s).4,8 Apropos of arene Ru(II) complexes, RAPTA, in the frontline of anticancer metallotherapeutics by addressing the n RAED, RM175 and UNICAM-1 are the novel complexes in the arena o of arene ruthenium oncotherapy in which all comprise bidentate chelator ligand and a chloride except RAPTA which contain an t a.Centre for Organometallic Chemistry, School of Chemistry, Bharathidasan l University, Tiruchirappalli- 620 024, Tamil Nadu, India. unidentate ligand with two labile chlorides.4,9 Even though RAPTA a [E-mail: ramesh_bdu@yahoo.com and phone No. 0431- 2407053; Fax: 0091-431- complexes are non-toxic to off targets, anti-invasive, anti- D 2407045] metastatic, anti-angiogenic and targeting various biomolecules,10 b.The First Affiliated Hospital; Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, School of Medicine, Zhejiang they are prone to chloride hydrolyses11 thus making it least reactive University, Hangzhou, 310003, PR China.. and jeopardize its further clinical development. c.Department of Crystallography, Institute of Chemistry, University of Silesia, 40- 006, Katowice, Poland. Electronic Supplementary Information (ESI) available: [Details for the materials and methods, tables for crystallographic data, refinement parameters, selected bond lengths, bond angles and hydrogen bonding parameters. Figures illustrating the NMR, HR-MS, stability and cytotoxic studies of the new complexes. CIF for complexes (CCDC numbers 1872212, 1872214, 1916478, 1872211 and 1916479).]. See DOI: 10.1039/x0xx00000x PPlleeaassee ddoo nnoott aaddjjuusstt mmaarrggiinnss .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 2 of 19 ARTICLE Dalton Transactions all the titled complexes exhibited potent and precise anticancer activities by promoting tumor cell apoptosis. Results and discussion Syntheses and characterization A discrete set of three new carbazolone benzhydrazone ligands was synthesized with altered substituents, HL1= (Z)-N'-(2,3,4,9- tetrahydro-1H-carbazol-1-ylidene) benzohydrazide, HL2= (Z)-4- bromo-N'-(2,3,4,9-tetrahydro-1H-carbazol-1-ylidene) t benzohydrazide, HL3= (Z)-4-methoxy-N'-(2,3,4,9-tetrahydro-1H- p carbazol-1-ylidene)benzohydrazide to synthesize their first arene i r By supporting this, Cummings et al. concluded that arene Ru(II) ruthenium complexes (1-3) as portrayed in Scheme 1 in good yields. c Figure 1. Our previous reports on arene Ru(II) hydrazone complexes The non-hygroscopic complexes can be solubilized in various s organic solvents. The structural identities of ligands and their complexes encompassing a hydrophobic spectator arene corresponding complexes were elucidated through elemental u component to stabilize +2 oxidation state and to facilitate lipophilic analyses, IR, UV-vis, NMR, HR-MS and single crystal X-ray diffraction n cellular penetration along with transport, a labile ligand for complex techniques. The IR spectra of ligands showed transmittance bands a activation via ligand exchange and a stable bidentate non-leaving around 1560-1605, 1644-1680 and 3031-3460 cm-1 pro to ν(C=N), M chelating ligand exhibited increased in vitro and in vivo anticancer ν(C=O) and ν(NH ) respectively. The absence ν(C=O) carbazole, hydrazide activities than complexes with an arene, a halide and two and a ν(NH) bands in the IR spectra of the complexes indicated the d unidentate ligands.12 Furthermore, some of the arene Ru complexes possibility of enolisation and the emergence of new ν(C-O) band at with N˄O- bidentate chelating ligands have been reported for 1300 cm-1 attested the binding of the imidolate oxygen after e their excellent anticancer activities.13 Influenced by the facts and deprotonation. Further, the considerable decrement of ν(C=N) t p results, we deliberated to design and synthesize anticancer arene frequency of about 30-70 cm-1 in the complexes demonstrated that ruthenium drug candidates with a labile chloride and biologically another point of attachment of ligand is ylidene nitrogen and e active bidentate chelating ligand with ON fashion (Figure 1) 14 since concluded the N˄O coordination of the ligands to ruthenium c [(η6-arene)Ru(NN)X] type complexes were largely explored.4,7,9a,15 ion. In the UV-vis spectra of the ligands and the complexes, c Among the different types of N˄O- chelating ligands, hydrazones transitions between ligand orbitals were observed as highly A were reckoned as better N˄O- chelating ligands to arene ruthenium intense bands around 240-340 nm. Besides, ruthenium ion to core since of its easy synthesis, tunable structural diversities, facile carbazolone benzhydrazone ligand transitions (MLCT) were s metal chelation and witnessed anticancer activities.16 However, the resonated as medium intense charge transfer bands around n choice of ligand which remains intact with the pro-drug after 390 nm in the complexes. The 1H-NMR spectra of the ligands o activation is imperative because in case of platinum anticancer showed two distinct singlets in the range δ 9.32-11.36 ppm i complexes, the bystander N-donor ammine ligands played a crucial which are attributable to hydrazide and carbazole -NH t role for its anticancer properties.17 Further, it has been chronicled protons. The disappearance of hydrazide -NH signal denoted c that myriad of metal complexes containing N heterocycles exhibited the coordination of imidolate oxygen to Ru ion through a salient anticancer activities as well.18 Moreover, ligands enolisation. The aromatic protons of ligands and complexes s incorporated with minimum one NH functionality in Ru(II) (Figures S1-S6) resonated as multiplets and as well resolved n anticancer complexes facilitate its effectual interaction with DNA doublets / triplets in the range δ 8.17-6.99 ppm. The aliphatic a via hydrogen bonding.19 So it would be interesting to couple a protons of carbazole ring fell as multiplets and well resolved r hydrazone with an N heterocycle to form a new class of a chelating peaks in the range δ 1.98-4.02 ppm. A sharp singlet around δ T ligands. Carbazole, either natural or man-made is a potent N 3.82 ppm is endorsed to methoxy protons of the ligand, HL3 heterocycle and a privileged pharmacopore showcased its and its complex 3. Appearance of sharp singlet at ~ δ 5.50 ppm n significant anticancer activities.20 It is worth to note that, a potent was due to protons of arene moiety. The higher frequency o 5HT3 receptor agonist ondansetron comprising carbazolone motif is shifts of the imidolate carbon (161.8 to 173.1 ppm) and t used for the prevention of nausea in cancer patients caused by ylidene carbons (148.0 to 162.7 ppm) in the complexes relative l radiation and chemotherapies.21 to ligands in 13C NMR spectra (Figures S7-S12) signified the a To test this rationale, we have designed an interesting N˄O coordination to ligands ruthenium ion. The HR-MS D combination of carbazole based hydrazone ligands and successfully spectra displayed peaks for HL1, HL2, HL3, complexes 1, 2 and fabricated their arene Ru(II) complexes (1-3) for the first time. It is 3 at m/z 304.14[M+H]+, 382.05[M+H]+, 334.15[M+H]+, well known from the literature that arene Ru(II) complexes exhibit 482.08[M-Cl]+, 561.99 [M-Cl+2H]+ and 512.09[M-Cl]+ good binding affinity towards various biomolecules.22 Hence, respectively (Figures S13-S18) further confirmed the structural herein, we describe the anticancer property of arene Ru(II) composition of the ligands and complexes. The classical three carbazolone hydrazone complexes along with investigation on (N, O, Cl) legged piano stool geometry with arene seat of apoptosis mechanism by different biochemical assays. Impressively hapticity-6 forming 2 | J. Name., 2020, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Page 3 of 19 PPlleeaassDee addltooo nnnoo Tttr aaanddsjjauucssttti ommnaasrrggiinnss ARTICLE Dalton Transactions t p Scheme 1. Synthetic route to new η6-arene Ru(II) carbazole based hydrazone complexes i r c s u n a M d e t p e c c A pseudo-octahedral architecture of the complexes was s Figure 2. Illustration of ligands HL2, HL3 and complexes 1, 2 and 3 in ORTEP neatness with atom describing scheme. Displacement ellipsoids = - n were drawn at 50 % probability level. All the hydrogen atoms and co-crystallized solvent molecules were excluded for picture clarity. o unequivocally proved by single crystal XRD analysis. Gradual 4.61°; complex 3= -5.77°) angles. The ligands bite the Ru ion i evaporation of compounds in 1:2 mixture of CH 3 CN:CH 2 Cl 2 was through imidolate oxygen and ylidene nitrogen with chelation t resulted the suitable crystals for all the complexes and two angles ∠76.28(7)-76.55(11)° forming a five member chelate c ligands. Figure 2 depicted the crystal structures of HL2, HL3, ring. When the piano stool top was formed by spectator η6- a complexes 1, 2 and 3 in ORTEP style diagrams with atom arene, the three legs were carpentered by a chloride ion and s describing scheme. The crystallographic information and bidentate carbazolone hydrazone ligand being O˄N n selected bond distances together with bond angles were coordinated making the ruthenium ion half sandwiched in a summarized in Tables S1, S2 and S3 respectively. The crystal C3NOCl octahedron. In all the three complexes, the arene ring r systems of HL2 and complex 1 are orthorhombic whereas HL3, and chelate ring were positioned away from the chloride ion T complex 2 and complex 3 are monoclinic. In HL2, there are two which is evident from the bond angles centroid -Ru-Cl = chelate ring independent molecules present in the asymmetric unit. The 90.67-92.03°; centroid -Ru-Cl = 130.04-130.55°. The stool n arene crystal of HL3 was a solvate holding a water molecule. The top benzene is π bonded to Ru ion with average Ru-C bond o structure of HL3 was flat with almost coplanar carbazole and length 2.162-2.178 Å. The distance between benzene centroid t benzhydrazide constituents whereas HL2 constitutes non- and Ru ion is in the range of 1.666-1.669 Å. The bond angles l a planar carbazole and benzhydrazide with dihedral angle and bond lengths were tantamount with few other piano stool 22.45°. The torsions of members of metal chelation ∠N-N-C-O ruthenium complexes with N˄O donor ligands.13d,e,h,23 The D for HL2 = -2.57° and HL3 = 0.90°. Coordination to Ru ion uncoordinated carbazole NH was involved in intramolecular influenced the planarity and torsions of the ligands which were hydrogen bonding with chloride bonded to Ru in all the three reflected in the increased dihedral angles (complex 2=32.56°; complexes. The HL2 and HL3 exhibited intermolecular complex 3=33.20°) between carbazole and benzhydrazide hydrogen bonding interactions with carbonyl oxygen and planes in the complexes and torsions (∠N-N-C-O in complex 2 carbazole NH with neighbouring molecules (Tables S4-S6). Stability studies This journal is © The Royal Society of Chemistry 20xx J. Name., 2020, 00, 1-3 | 3 Please do not adjust margins .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 4 of 19 ARTICLE Dalton Transactions Stability is a very decisive quality measure for a drug that the chelation of ligands to metal ion. All the complexes determines its efficacy, toxicity, safety also governs its showed good cytotoxicity with considerably less IC values on 50 development and clinical success. The aqueous stability of the both the cancerous cells tested. In particular, the complexes complexes was examined by electronic spectroscopy before exhibited a better inhibitory activity on the growth of A2780 the anticancer activity investigations. The complexes were cells than A549 cells. Interestingly, complex 3 which comprise dissolved in 1% DMSO in phosphate buffer (pH-7.4) solution. methoxy substituent showed 3 to 4 fold enhanced activity on Electronic spectra of the complexes were monitored at various both cancer cells over cisplatin.14f,29 This may be due to fact time intervals. All the bands undergone steady decrease in the that the electron releasing –OCH group may enhance the 3 absorbance intensities up to 7 nm red shift on prolongation of lipophilicity of complex 3 which facilitates its permeation time to 72 h without major changes of spectral pattern (Figure through the lipid layer of cell membrane as evidenced from its t S19). The intensity changes were imputable to the process of higher log P value (Table 1).14e,h In spite of this potency, the p exchange of labile chloride ligand by water molecule in complexes exhibit only least toxicity (IC 50 = 60-90 µM) to non- i r aqueous phase. Our observations were analogous to the cancerous 16HBE cells and even less cytotoxic when compared c results of other reported ruthenium complexes containing to anticancer drug cisplatin (IC = 18 µM). The selective 50 s labile chloride ligand.24 anticancer action of the complexes only towards the cancer u cells was validated by the difference in the IC values and 50 selectivity indices of the complexes. Expediently, the titled n Permeation capacity in terms of lipophilicity complexes exhibits better growth inhibitory activity on both a by log P A549 and A2780 cancerous cells than other half sandwich M Lipid cell membrane permeability of drug molecules that have arene Ru(II) complexes ligated with N˄O donor ligands intracellular targets is a key initiative in drug design and reported.8e,13c,e,14c,23,30 The results of MTT assay suggest that d development as it influences the essential pharmacokinetic even a small molecular change can provoke change in e properties importantly absorption, distribution and excretion cytotoxicity and also witness the direct link between of drugs. Literature reports established that there exists a lipophilicity and antiproliferative activity.23 t p direct relationship between permeability and lipophilicity25 and also between lipophilicity and anticancer activity.26 Hence, we Cancer cell antiproliferation by EdU e determined the lipophilicity of all the complexes in terms of incorporation assay c partition coefficient (log P) in n-octanol/water system (Table Owing to its late detection, ovarian cancer becomes the c 1). From the obtained log P values, it is evident that all the second most common cause of gynaecologic cancer death in A complexes possess good lipophilic character when compared women globally. Hence, we focused our attention to examine to cisplatin. Despite the complexes possess similar structural the antitumor activities of the complexes on A2780 cells. Since s features, they differ in their lipophilic character which can be significant cytotoxicity has been observed for the complexes at n correlated to different substituents on the ligands. Unlike their IC in 48 h incubation time, we intended to execute the 50 o complex 2, 3 showed higher lipophilicity implying its rest of investigations on A2780 cells using minimal i transporting ability across the cell membrane which is contrast concentration (1.5 µM) of the complexes for 24 h incubation t to some previous literature that inclusion of halogen atom time with a focus to determine the exact apoptosis ratio. c increases the lipophilicity.27 Further, the obtained log P values Estimating new DNA synthesis is a most suitable method to a for the titled complexes have good conformity with the log P identify changes in cell proliferation and growth. s values of the reported arene ruthenium complexes.28 n a In vitro cancer cell growth inhibition by MTT r assay T Evaluation of anticancer potency of the titled complexes and ligands has been initiated by examining the cancer cell growth n inhibition property by MTT assay (Figures S20&21) and o compared with the standard drug cisplatin. For the t investigation, the two most common cancer cells, A549 (lung l a carcinoma) and A2780 (ovarian adenocarcinoma) were selected. The cytotoxic selectivity of the complexes was also D examined on a 16HBE (human lung bronchial epithelium) non- cancerous cells. The growth inhibitory concentrations IC of 50 the complexes and cisplatin with respect to control groups were determined after 48 h of incubation period and were given in Table 1. Figure 3. (A) A Click-iT EdU assay to quantify the proliferation of A2780 cells The ligands or ruthenium precursors showed no inhibition for 24 h. (B) Quantification of EdU positive cells. on the cancer cell proliferation even up to 25 μM concentrations. Therefore, the observed cytotoxicity is due to 4 | J. Name., 2020, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Page 5 of 19 PPlleeaassDee addltooo nnnoo Tttr aaanddsjjauucssttti ommnaasrrggiinnss ARTICLE Dalton Transactions Table 1. Anticancer potential of ligands, complexes and cisplatin in terms of cancer cell growth inhibition after 48 h of incubation (expressed as IC ± SD in µM)[a]. 50 Compounds A549 cells A2780 cells 16HBE cells Log P 1 (SI) 10 ± 1 (7) 8 ± 0.4 (8) 66 ± 0.3 1.1 ± 0.1 2 (SI) 7 ± 0.5 (13) 6 ± 0.4 (15) 88± 0.6 1.3 ± 0.1 3 (SI) 3 ± 0.5 (27) 3 ± 0.5 (32) 93 ± 0.8 1.7 ± 0.1 Cisplatin (SI) 10± 1(2) 12 ± 0.4 (1) 18 ± 1 -2.2 ± 0.1 HL1 > 25 > 25 > 25 - t HL2 > 25 > 25 > 25 - p HL3 > 25 > 25 > 25 - i [a]The data were derived from the average of three independent experiments and the expressed errors were the corresponding standard deviations. SI (selectivity r index) is defined as IC50 ratio of non-cancerous cells (16HBE (human lung bronchial epithelium) cells) to cancerous cells (A549 (lung carcinoma) or A2780 (ovarian c adenocarcinoma)). s u Consequently, we experimented the activity of the complexes Dual AO-EB fluorescent staining method is a simple yet reliable n on resisting new DNA synthesis by click-iT EdU cell procedure to classify viable, apoptotic and necrotic cells at the a proliferation assay. After the incubation of the complexes, the same time based on the fluorescence emission and cell M cells were treated with EdU. The alkyne terminal motif in EdU morphological features.32 Apoptotic cells show typical incorporates only within the freshly synthesized DNA of morphological features such as cytoplasmic cell shrinkage, d proliferating tumor cells. Hence, the new DNA synthesis was plasma membrane blebbing, trans location of identified by green fluorescent visuals via copper mediated phosphatidylserine (PS) to extracellular side, DNA e click reaction involving the terminal alkyne of EdU and fragmentation and chromatin condensation.33 AO and EB are t p fluorescent azide added to the media (Figure 3A).31 When nucleic acid binding dyes. AO can stain the nucleus of both compared to the untreated cells, the green fluorescent visuals viable and dead cells. In contrast, EB stains only the cells with e were decreased in the treated cells and this effect the injured membranes. Under fluorescence microscope, live c corresponding to inhibition of active DNA synthesis was high cells emit uniform green fluorescence whereas necrotic cells c for complex 3. Tantamount antiproliferative activity was fluoresce uniform orange to red. Apoptotic cells can be A noticed for complexes 1, 2 and cisplatin which are supported visualized as green fluorescent patches due to DNA by the EdU positive cell ratio as shown in Figure 3B. fragmentation and chromatin condensation. In the control s group, the cells are dense and emit uniform green n Cell death by mechanism by Acridine Orange- fluorescence due to highly organized cell structure of live cells. o Ethidium Bromide (AO-EB) fluorescent Complexes 1-3 were incubated with AO-EB stained A2780 staining cells. Since the treatment of the test compounds induced t i One of the therapeutic goals of potent anticancer drugs is to apoptotic cell death by cancer cell membrane disintegration, c target cancer cells and trigger their death by apoptosis EB entered into the cell and produced greenish orange stain by a mechanism. To identify the underlying mechanism of overwhelm AO fluorescence. As shown in Figure 4A&B, and all s significant cytotoxicity and antiproliferative activity of the the three complexes consequentially induced apoptosis. n titled complexes, we have performed AO-EB dual staining Specifically, the amount of live cells was decreased and more a analysis. number of apoptotic bodies was noticed in the experimental r group treated with complex 3 over other test complexes.34 T Quantitative discrimination of apoptosis by n flow cytometry o t l a Apoptosis may be reckoned as an important obstacle for a D damaged cell to become malignant tumors. Since the complexes promote apoptosis induction in cancer cells, flow cytometry using annexin V-FITC / propidium iodide (PI) double staining was carried out for the quantitative discrimination of apoptotic cells. Phosphatidylserine (PS) is a cell cycle signalling phospholipid located inner side of the membrane of a healthy Figure 4. (A) Induction of apoptosis in A2780 cells by AO-EB cell but is reverted to the outer membrane for recognition by assay with complexes 1-3 and cisplatin (1.5 µM concentration) for 24 h. (B) The cell population in the microscopic field. The neighbouring cells at the time of apoptosis. Hence, the statistics are represented as the means ± s.d.; n-5 regions with a total of 1000-1500 cells considered; **p< 0.01 PPlleeaassee ddoo nnoott aaddjjuusstt mmaarrggiinnss .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 6 of 19 ARTICLE Dalton Transactions t p i r c Figure 5. (A) Quantitative discrimination of apoptosis induction s in A2780 cells with complexes 1-3 and cisplatin at 1.5 µM Figure 6. Complexes 1-3 and cisplatin (1.5 µM) were u concentrations for 24 h by flow cytometry analysis. (B) incubated with A2780 cells for 24 h and analyzed by flow Apoptosis ratio in A2780 cells cytometry (A) A2780 cell cycle progression by FACS analysis n (B) Quantification of A2780 cell cycle distribution. a translocation of phosphatidylserine is a morphological M hallmark of apoptosis and can be spotted by its binding with fluorescently labelled annexin V which in turn detected by flow cytometry.35 Further the addition of PI to annexin V stained Experimental section d cells is used to discriminate and concomitantly quantify the Details for materials and methods used were mentioned in ESI, e live cells (lower left quadrant-annexin V(-)/PI(-)), early S3. t apoptotic cells (upper left quadrant-annexin V(+)/PI(-)) and p late apoptotic cells (upper right-quadrant-annexin V(+)/PI(+)) Synthesis of carbazolone-benzhydrazone ligands: e using FACS. As projected in Figure 5A&B, the incubation of c complexes with A2780 cells conspicuously induced apoptosis c General procedure for the preparation of carbazole based (10-13 %).36 It is worth to note that the titled complexes induce A apoptosis even at very low concentrations which is less than hydrazone ligands: their IC . In comparison with control, the cell population was 50 s higher (6-9 %) in annexin V(+)/PI(-) (upper left) quadrant The carbazolone-benzhydrazones (HL1, HL2 and HL3) were n indicating the induction of early apoptosis.37 This effect was synthesized by stirring the equi-molar combination of 2,3,4,9- o ascertained to be high for complex 3 than the rest of the tetrahydro-1H-carbazol-1-one and substituted benzhydrazide in complexes and analogous with the results of MTT, EdU and i ethanol (20 mL) in presence of few drops of conc. HCl at room t AO-EB staining assays. It is to note that the test complexes c temperature for 3 h. The precipitate obtained was recovered by displayed comparatively better apoptotic induction than the a filtration, washed with ethanol/ diethyl ether and was air dried. reported Ru(II) and Ir(III) hand sandwich complexes on A2780 s cells.29b,38 n Analytical and spectroscopic data a (Z)-N'-(2,3,4,9-tetrahydro-1H-carbazol-1-ylidene)benzohydrazide, Cell cycle arrest by flow cytometry r HL1 T Cell cycle proceeds through different phases called as G0-G1, S, and G2-Mitosis. The efficacy of anticancer agent is based on 2,3,4,9-tetrahydro-1H-carbazol-1-one (5.4 mmol, 1 g) and n its capability to disrupt cancer cell cycle in the aforementioned benzohydrazide (5.4 mmol, 0.735 g) were used. HL1 was o phases. The complexes were examined by flow cytometry to precipitated as yellow crystalline solid. Yield: 55 %. M.pt.132 oC. validate its impact on disrupting the A2780 cell cycle. In the t Elemental analysis calculated for C 19 H 17 N 3 O: C, 75.23; H, 5.65; N, l absence of test complexes, 74.8 % of cells were at G0-G1 a 13.85 %. Found: C, 75.12; H, 5.64; N, 13.82 %. UV-visible phase and 18.6 % cells were found at S phase. In the presence (CH CN):λ / nm (ε /dm3 mol-1 cm-1): 243 (17538), 342 (36286). FT- D 3 max of test complexes, the distributions of cells in G0-G1 phase IR ν /cm-1 in KBr: 3458, 3273 (NH); 1644 (C=O); 1576 (C=N): 1H max were decreased to 56.4 % to 56.6 %, 56.5 % and 63.8 % for NMR; δ (400 MHz; CDCl ; 293 K): 10.01 (1H, s, N-H ), 9.32 (1H, H 3 carbazole complexes 1, 2, 3 and cisplatin respectively and increased to s, N-H ), 7.83-7.06 (9H, m, CH ), 2.99 (2H, s, CH ), hydrazide carbazole+hydrazide, 2 42.7 % of (complex 1), 43.3 % (complex 2), 43.4 % (complex 3) 2.64 (2H, s,CH )), 2.10 (2H, s, CH ). 13C{1H} NMR: δ (100 MHz; CDCl ; 2 2 H 3 and 32.2 % (cisplatin) in S phase (Figure 6A&B). Accumulation 293 K): 164.3 (C=O), 148.0 (C=N).ESI-HRMS (CH OH + CH CN) m/z: 3 3 of cells in S phase evidenced that the complexes arrest the cell Calculated for C H N O: 303.1372 [M]+; Observed: 304.1444 19 17 3 cycle progression in S phase.14a,39 [M+H]+. 6 | J. Name., 2020, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Page 7 of 19 PPlleeaassDee addltooo nnnoo Tttr aaanddsjjauucssttti ommnaasrrggiinnss ARTICLE Dalton Transactions (Z)-4-bromo-N'-(2,3,4,9-tetrahydro-1H-carbazol-1- Analytical and spectroscopic data ylidene)benzohydrazide, HL2 [Ru(L1)Cl(arene)] (Complex 1) 2,3,4,9-tetrahydro-1H-carbazol-1-one (5.4 mmol, 1 g) and 4- [(η6-benzene)RuCl ] (0.19 mmol, 0.100 g) and HL1 (0.38 mmol, 22 bromobenzohydrazide (5.4 mmol, 1.161 g) were used. HL2 was 0.115 g) were used. Yield: 63 %. M.pt. 247 °C.Elemental analysis precipitated as pale yellow solid and X-ray quality crystals were calculated for C H ClN ORu: C, 58.08; H, 4.29; N, 8.13 %. Found: C, 25 22 3 grown by slow evaporation of 2:1, CH Cl :CH CN mixture. Yield : 76 57.96; H, 4.28; N, 8.11 %. UV-visible (CH CN): λ / nm (ε /dm3 mol-1 2 2 3 3 max %. M.pt. 126 oC. Elemental analysis calculated for C H BrN O: C, cm-1): 253 (36217), 301(17070), 386 (48380). FT-IR ν /cm-1 in KBr: 19 16 3 max 59.70; H, 4.22; N, 10.99 %. Found: C, 59.55; H, 4.21; N, 10.96 %. UV- 3223 (NH), 1528 (C=N), 1362 (C-O); 1H NMR: δ H (400 MHz; CDCl 3 ; t visible (CH CN):λ / nm (ε /dm3 mol-1 cm-1): 258 (16890), 293 K): 10.87 (1H, s,N-H ), 8.16 (2H, d,3J = 8.0 Hz, CH ), p 3 max carbazole hydrazide 343(44380). FT-IR ν /cm-1 in KBr: 3336, 3054 (NH); 1680 (C=O); 7.63 (H, d,3J = 8.0 Hz,CH ),7.51 (H, d,3J = 8.0 Hz,CH ),7.35 i max carbazole carbazole r 1587 (C=N): 1H NMR; δ (400 MHz; [d ]-DMSO; 293 K):11.33 (1H, s, (3H, m, CH ), 7.19 (2H, dd,3J = 8.0 Hz,CH ),5.50 (6H, s, H 6 hydrazide carbazole c N-H ), 10.95 (1H, s, N-H ), 7.87 (2H, d,3J = 8.0 Hz, CH ), 3.18-3.05 (4H, m, CH ),2.25 (H, s, CH ), 2.15 carbazole hydrazide arene cyclohexane cyclohexane s CH ), 7.72 (2H, d, 3J = 8.0 Hz, CH ), 7.51(1H, d, 3J = 8.0 (H, s, CH ). 13C{1H} NMR: δ (100 MHz; CDCl ; 293 K): 170.9 hydrazide hydrazide cyclohexane H 3 u Hz, CH ), 7.43 (1H, d, 3J = 8.0 Hz, CH ), 7.17 (1H, t, 3J = (C-O-Ru), 162.7 (C=N-Ru), 84.5 (arene).ESI-HRMS (CH OH + CH CN) carbazole carbazole 3 3 8.0, 4.0 Hz, CH ), 7.00 (1H, t, 3J = 8.0 Hz, CH ), 2.81 (4H, m/z: Calculated for C H ClN ORu: 517.0495 [M]+; Observed: n carbazole carbazole 25 22 3 d, 3J = 4 Hz, d, 2CH ), 1.99 (2H, t, 3J = 8.0, 4.0 Hz, CH ). 13C{1H} NMR: 482.0805 [M-Cl]+. a 2 2 δ H (100 MHz; [d 6 ]-DMSO; 293 K): 162.8 (C=O), 152.2 (C=N).ESI-HRMS M (CH OH + CH CN) m/z: Calculated for C H BrN O: 381.0477 [M]+; [Ru(L2)Cl(arene)] (Complex 2) 3 3 19 16 3 Observed: 382.0551 [M+H]+. [(η6-benzene)RuCl ] (0.19 mmol, 0.100 g) and HL2 (0.38 mmol, 22 d 0.145 g) were used. Yield: 69 %. M.pt. 262 °C.Elemental analysis e (Z)-4-methoxy-N'-(2,3,4,9-tetrahydro-1H-carbazol-1- calculated for C H BrClN ORu: C, 50.39; H, 3.55; N, 7.05 %. Found: 25 21 3 t C, 50.26; H, 3.57; N, 7.03 %. UV-visible (CH CN): λ / nm (ε /dm3 ylidene)benzohydrazide, HL3 3 max p mol-1 cm-1): 243 (33767), 303 (18447), 390 (40150). FT-IR ν /cm-1 max e 2,3,4,9-tetrahydro-1H-carbazol-1-one (5.4 mmol, 1 g) and 4- in KBr: 3235 (NH), 1516 (C=N), 1329 (C-O), 743 (C-Br); 1H NMR: c methoxybenzohydrazide (5.4 mmol, 2.059 g) were used. HL3 was δ (400 MHz; CDCl ; 293 K): 10.83 (1H, s, N-H ), 8.02 (H, d,3J = H 3 carbazole precipitated as pale yellow solid and X-ray quality crystals were 8.0 Hz, CH ), 8.03-7.15 (7H, m, CH ), 5.50 (6H, s, c hydrazide carbazole+hydrazide grown from slow evaporation of 2:1, CH Cl :CH CN mixture. Yield : CH ), 4.02-2.22 (6H, m, CH ).13C{1H} NMR: δ (100 MHz; A 2 2 3 arene cyclohexane H 71 %. M.pt.130 oC. Elemental analysis calculated for C H N O : C, CDCl ; 293 K): 172.2 (C-O-Ru), 163.1 (C=N-Ru), 84.5 (arene).ESI- 20 19 3 2 3 72.05; H, 5.74; N, 12.60 %. Found: C, 71.96; H, 5.75; N, 12.58 %. UV- HRMS (CH OH + CH CN) m/z: Calculated for C H BrClNORu: s 3 3 25 21 3 visible (CH 3 CN):λ max / nm (ε /dm3 mol-1 cm-1): 257(25707), 340 594.9600 [M]+; Observed: 561.9902 [M-Cl+2H] +. n (56670). FT-IR ν /cm-1 in KBr: 3423, 3234(NH); 1651 (C=O); 1531 max o (C=N): 1H NMR; δ (400 MHz; [d ]-DMSO; 293 K):11.36 (1H, s,N- [Ru(L3)Cl(arene)] (Complex 3) H 6 i H carbazole ), 10.76 (1H, s, N-H hydrazide ), 7.95 (2H, d, 3J = 8.0 Hz, [(η6-benzene)RuCl 2 ] 2 (0.19 mmol, 0.100 g) and HL3 (0.38 mmol, t CH ), 7.51 (H, d, 3J = 8.0 Hz, CH ), 7.44 (H, d, 3J = 8.0 Hz, 0.127 g) were used. Yield : 74 %. M.pt. 251°C.Elemental analysis c hydrazide hydrazide CH ), 7.20-6.99 (4H, m, CH ), 3.82 (3H, s, OCH ), 2.82 calculated for C H ClN O Ru: C, 57.09; H, 4.42; N, 7.68 %. Found: a hydrazide carbazole 3 26 24 3 2 (4H, s, 2CH ), 1.99 (2H, t, 3J = 8.0, 4.0 Hz, CH ). 13C{1H} NMR: δ (100 C, 56.96; H, 4.41; N, 7.66 %. UV-visible (CH CN): λ / nm (ε s 2 2 H 3 max MHz; [d 6 ]-DMSO; 293 K): 161.8 (C=O), 151.1 (C=N).ESI-HRMS /dm3mol-1 cm-1): 241 (32890), 296 (20533), 387 (50747). FT-IR n (CH OH + CH CN) m/z: Calculated for C H N O : 333.1477 [M]+; ν /cm-1 in KBr: 3226 (NH), 1506 (C=N), 1365 (C-O); 1H NMR: 3 3 20 19 3 2 max a Observed: 334.1546 [M+H]+. δ (400 MHz; CDCl ; 293 K): 10.85 (1H, s, N-H ), 8.09 (2H, d, 3J = H 3 carbazole r 12.0 Hz,CH hydrazide ), 7.60 (H, d, 3J = 8.0 Hz, CH hydrazide ), 7.49 (H, d, 3J = T Synthesis of new Ru(II) arene complexes containing carbazolone- 8.0 Hz, CH ), 7.31-7.12 (2H, m, CH ),6.84 (2H, d, 3J = 8.0 hydrazide carbazole benzhydrazone ligands Hz, CH ),5.47 (6H, s, CH ), 3.82 (3H, s, OCH ), 3.03-3.17 (4H, n carbazole arene 3 General procedure: m, CH cyclohexane ), 2.22 (H, s, CH cyclohexane ), 2.12 (H, s, CH cyclohexane ). o Appropriate carbazolone-benzhydrazone derivative (2 equiv. HL1- 13C{1H} NMR: δ (100 MHz; CDCl ; 293 K): 173.1 (C-O-Ru), 163.7 H 3 t HL3) was dissolved in dichloromethane and added with 2 equiv. of (C=N-Ru), 84.5 (arene).ESI-HRMS (CH OH + CH CN) m/z: Calculated l 3 3 a triethylamine and stirred. To this solution, an equiv. of [(η6- for C H ClN O Ru: 547.0601 [M]+; Observed: 512.0912 [M-Cl]+. 26 24 3 2 D benzene)RuCl ] was charged up was allowed for stirring at room at 22 room temperature for 5 h. TLC was monitored to confirm the Experimental procedure formation of the complex. Then, the solution was concentrated to 5 mL followed by addition of 20 mL of petroleum ether (60-80 oC) Partition coefficients determination results the formation of a reddish brown solid. Slow evaporation of that solid in 2:1, CH Cl :CH CN mixture yielded needle shaped X-ray 2 2 3 The partition coefficient P was examined using “Shake flask” quality crystals for all the three complexes. method as per the previously reported protocol.14a Complexes 1-3 and cisplatin were dissolved in a mixture of n-octanol and water This journal is © The Royal Society of Chemistry 20xx J. Name., 2020, 00, 1-3 | 7 Please do not adjust margins .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 8 of 19 ARTICLE Dalton Transactions and was shacked for 24 h. The mixture was allowed to settle over a plates. After 5 min, the incubating plates were washed with PBS period of half an hour. The resulting two phases were separated three times. Images were obtained from the fluorescence carefully without cross contamination of one solvent layer into microscope (Olympus, IX71) and the cell deaths were measured another. The results were given as the mean values taken from three random fields of the microscope.13f three independent experiments. Finally, the concentration of the Flow cytometry/annexin V-PI staining complexes 1-3 and cisplatin in each phase was analyzed by UV-vis absorption spectroscopy at room temperature. Partition The flow cytometry analysis was examined by using the Apoptosis coefficients for complexes 1-3 and cisplatin were calculated from Detection Kit of fluoresceinisothiocyanate (FITC) (Cell Signalling, the following equation log P = log [(complexes 1-3 and China) in order to confirm the apoptotic ratio of A2780 cells. The cisplatin)oct/( complexes 1-3 and cisplatin)aq]. cells were washed thrice by using trypsin, and resuspended in 1 × binding buffer (500 μL) followed by addition of FITC Annexin V (5 t μL) and PI (10 μL). After 15 min incubation, the samples were p Cell culture analysed by flow cytometry using the BD FACS CantoTM II flow i A549, A2780 and 16HBE cells were obtained from the cell bank of cytometer.13f r c the Chinese Academy of Sciences (Shanghai, China). The A549, Cell cycle analysis A2780 cells were grown in RPMI-1640 (Gibco) supplemented with s A2780 (2 × 104 cells) were seeded onto 48-well plates and hatched 10% fetal bovine serum (FBS; Gibco) and 16HBE cells were grown in at 37 oC under a 5% CO atmosphere for 24 h. Complexes 1-3 and u 2 Dulbeccos modified Eagles medium (DMEM) containing 10% FBS cisplatin (1.5 µM, equiv concentrations) were added to the same n and 1% antibiotics (penicillin). Then, the cells were incubated in a conditions. The cells were fixed using 75% ethanol at 4 oC for 12 h, humid atmosphere with 5% CO at 37 oC. a 2 after 20 min later, incubated with PI (50 μg/mL) for 20 min. The cell M cycle of the samples were analysed by flow cytometry (BD FACS CantoTM II flow cytometer).13f MTT assay d Conclusions A549, A2780 and 16HBE cells were cultured in 96-well plates (4000 e cells/well) and incubated for 24 h at 37 oC. Complexes 1-3 and The metal based drugs are scarce for the second leading human cisplatin were well dissolved in DMSO and the final contents of health problem, cancer. Based on the advantages and preclinical p t DMSO were less than 0.2% (v/v) to avoid the solvent impact to cell evolutions of arene ruthenium anticancer agents, we have viability. Then the cells were treated with various concentrations of synthesized new arene Ru(II) complexes with new carbazole e the complexes 1-3 and cisplatin for 48 h. Experiments were tethered hydrazone ligands. The formation of the ligands, c performed in triplicate and the medium without the complexes complexes and their structural identities were clearly established by c served as control. After 48 h, 30 μL of 3-[4,5-dimethylthiazol-2-yl]- elemental analysis and spectral techniques. Single crystal XRD of A 3,5-diphenyl tetrazolium bromide (MTT) in phosphate buffered the complexes witnesses the κ2-N,O coordinating fashion of ligands saline solution at concentration of 5 mg/mL was added into each and N, O, Cl legged piano stool quasi-octahedral geometry of s well and incubated at 37 oC for 5 h. Then the medium with MTT was ruthenium ion with η6-arene seat. Further, complexes 1-3 exhibited removed and 100 μL of DMSO was added to dissolve the formazan promising cytotoxicity toward the cancerous cells tested than the n crystals formed. The absorbance of each sample was read at 492 free ligands and cisplatin. Especially, complex 3 showed 3 to 4 fold o nm on a microplate reader (Multiskan FC, Thermo Scientific). Cell enhanced activity against A549 and A2780 cells with lower IC 50 i viability was calculated as follows: cell viability (%) = [absorbance of values (3 µM) over that of cisplatin. EdU assay corroborated that t each well / absorbance of control well] × 100. Graph was plotted the complexes exerted the antiproliferative activity by inhibiting the c between % of cell inhibition and concentration of the test active DNA synthesis. AO-EB fluorescent staining and flow a complexes. From this plot, the IC 50 value was calculated. cytometry analysis disclose that all the complexes induce cancer cell s death by apoptosis mechanism. Furthermore, cell cycle analysis n clearly indicates that the complexes arrest the A2780 cell cycle EdU assay a progression in S phase. On the whole, all the complexes presented A2780 (2 × 104 cells) were seeded onto 48-well plates and hatched in this communication displayed considerably good anticancer r at 37 oC under a 5% CO 2 atmosphere for 24 h. Complexes 1-3 and activity even at minimal concentrations. To conclude, arene T cisplatin (1.5 µM, equiv concentrations) were added to the cells ruthenium(II) complexes bearing heterocycle moiety may be the under the same conditions. At the end of the treatment with test suitable platform to invent the next generation anticancer drugs. n compounds, the DNA synthesis was measured using Alexa Fluor 488 o Assay Kit (Invitrogen) by adding Click-iT EdU solution to the culture t and was incubated 2 h at 37 oC. After that, the azide labelled Alexa Conflicts of interest l Fluor 488 was added into 48-well plates and was incubated 25 min a “There are no conflicts to declare”. at dark condition. After 20 min, nuclei was stained with Hoechst D 33342 for 15 min, the cells were photographed using fluorescence microscopy (Olympus, IX71).13f Acknowledgements Acridine Orange-Ethidium Bromide (AO-EB) Staining Dr. T. Sathiya Kamatchi gratefully acknowledges Science and The morphological changes of the cells were examined by biochemical staining, including acridine orange-ethidium bromide Engineering Research Board (SERB) for providing financial (AO-EB) staining. After incubating for 24 h, the cells were seeded at support and for National Post-Doctoral Fellowship a concentration of 1 × 104 onto 24 well plates. Complexes 1-3 and (PDF/2017/001085). Dr. RRM and Dr. TS are also thankful to cisplatin (1.5 µM, equiv concentrations) were added to the same the DST-India (FIST programme) for the use of instrumental 8 | J. Name., 2020, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Page 9 of 19 PPlleeaassDee addltooo nnnoo Tttr aaanddsjjauucssttti ommnaasrrggiinnss ARTICLE Dalton Transactions facilities at the School of Chemistry, Bharathidasan University, Dyson, J. Med. Chem., 2005, 48, 4161-4171; b) C. Gossens, A. Dorcier, P. J. Dyson and U. Rothlisberger, India. Organometallics,2007, 26, 3969-3975; c) C. Scolaro, C. G. Hartinger, C. S. Allardyce, B. K. Keppler and P. J. 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Habtemariam and P. J. Sadler, Nat. Commun., 2015, 6, 6582. 37 Y. Zhao, F. Wang,J. Du, E. Zhang, W. Zheng, Y. Zhang, Z. Wang, Q. Luo, K. Wu and Y. Lin, Metallomics, 2015, 7, 1573-1583. 38 S. Sersen, J. Kljun, K. Kryeziu, R. Panchuk, B. Alte, W. Korner, P. Heffeter, W. Berger and I. Turel, J. Med. Chem., 2015, 58, 3984-3996. 39 a) C. Fan, Q. Wu, T. Chen, Y. Zhang, W. Zheng, Q. Wang and W. Mei, Med. Chem. Commun., 2014, 5, 597–602; b) Q. Wu, C.Fan, 10 | J. Name., 2020, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Page 11 of 19 Dalton Transactions t p i r c s u n a M d e t p Figure 1. Our previous reports on arene Ru(II) hydrazone complexes e c c A s n o i t c a s n a r T n o t l a D .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Dalton Transactions Page 12 of 19 Scheme 1. Synthetic route to new η6-arene Ru(II) carbazole based hydrazone complexes t p i r c s u n a M Chemdraw file d e t p e c c N O A Cl Cl NH HN CH2Cl2/Et3N Ru Ru Ru s Cl Cl RT,Stirring,5h N H N O Cl n R N o i t c a R s R=H(HL1),Br(HL2),OCH3(HL3) R=H(1),Br(2),OCH3(3) n a r T n o t l a D .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Page 13 of 19 Dalton Transactions HL2 HL3 t p i r c s u n a M Complex 1 Complex 2 Complex 3 d Figure 2. Illustration of ligands HL2, HL3 and complexes 1, 2 and 3 in ORTEP neatness with e atom describing scheme. Displacement ellipsoids were drawn at 50 % probability level. All the hydrogen atoms and co-crystallized solvent molecules were excluded for picture clarity. t p e c c A s n o i t c a s n a r T n o t l a D .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A A Untreated Complex 1 Complex 2 B 60 Complex 3 Cisplatin 40 20 0 U ntr e at e d o m pl e x- 1 o m pl e x- 2 o m pl e x- 3 Ci s pl ati n C C C )%( oitar llec evitisop-UdE Dalton Transactions Page 14 of 19 t p i ** r c s u n a M d e t p e c Figure 3. (A) A Click-iT EdU assay to quantify the proliferation of A2780 cells for 24 h. (B) c Quantification of EdU positive cells. A s n o i t c a s n a r T n o t l a D .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Page 15 of 19 Dalton Transactions t p i r c s u n a M d e t p e c c Figure 4. (A) Induction of apoptosis in A2780 cells by AO-EB assay with complexes 1-3 and cisplatin (1.5 µM concentration) for 24 h. (B) The cell population in the microscopic A field. The statistics are represented as the means ± s.d.; n-5 regions with a total of 1000- 1500 cells considered; **p< 0.01 s n o i t c a s n a r T n o t l a D .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Untreated Complex 1 Complex 2 20 Complex 3 Cisplatin 15 10 5 0 U ntr e at e d o m pl e x- 1 o m pl e x- 2 o m pl e x- 3 Ci s pl ati n C C C )%( oitar citotpopa lleC A B ** CTIF-V nixennA Dalton Transactions Page 16 of 19 t p i r c s u n a M d e PI t p e c Figure 5. (A) Quantitative discrimination of apoptosis induction in A2780 cells with c complexes 1-3 and cisplatin at 1.5 µM concentrations for 24 h by flow cytometry A analysis. (B) Apoptosis ratio in A2780 cells s n o i t c a s n a r T n o t l a D .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Untreated Complex 1 Complex 2 80 Complex 3 Cisplatin 60 40 20 0 d 1 2 3 n ntr e at e m pl e x- m pl e x- m pl e x- Ci s pl ati U o o o C C C )%( noitubirtsid elcyc lleC A B G0/G1 phase S phase G2/M phase rebmun lleC Page 17 of 19 Dalton Transactions t p i r c s u n a M d e t p DNA content e c c Figure 6. Complexes 1-3 and cisplatin (1.5 µM) were incubated with A2780 cells for 24 h A and analyzed by flow cytometry (A) A2780 cell cycle progression by FACS analysis (B) Quantification of A2780 cell cycle distribution. s n o i t c a s n a r T n o t l a D .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Dalton Transactions Page 18 of 19 t p i r c s u n a M d e t Graphical Abstract p 193x103mm (96 x 96 DPI) e c c A s n o i t c a s n a r T n o t l a D .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A Page 19 of 19 Dalton Transactions Table of Contents In vitro antiproliferative and apoptosis inducing properties of new arene Ru(II) carbazolone benzhydrazone complexes have been described. t p i r c s u n a M d e t p e c c A s n o i t c a s n a r T n o t l a D .MA 95:70:8 0202/22/7 no mahgnimriB fo ytisrevinU yb dedaolnwoD .0202 yluJ 12 no dehsilbuP View Article Online DOI: 10.1039/D0DT01476A