Synthesis of novel anticancer ruthenium-arene pyridinylmethylene scaffolds via three-component reaction.

Accepted Manuscript Synthesis of novel Anticancer Ruthenium-Arene Pyridinylmethylene scaffolds via Three-Component reaction Gajanan Raosaheb Jadhav, Sohini Sinha, Mohit Chhabra, Priyankar Paira PII: DOI: Reference: S0960-894X(16)30366-3 http://dx.doi.org/10.1016/j.bmcl.2016.04.005 BMCL 23767 To appear in: Bioorganic & Medicinal Chemistry Letters Received Date: Revised Date: Accepted Date: 24 October 2015 16 March 2016 5 April 2016 Please cite this article as: Jadhav, G.R., Sinha, S., Chhabra, M., Paira, P., Synthesis of novel Anticancer RutheniumArene Pyridinylmethylene scaffolds via Three-Component reaction, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Synthesis of novel Anticancer Ruthenium-Arene Pyridinylmethylene scaffolds via Three-Component reaction Gajanan Raosaheb Jadhav,† Sohini Sinha,† Mohit Chhabra, Priyankar Paira* Pharmaceutical Chemistry division, School of advanced sciences, VIT University, Vellore-632014 Email: priyankar.paira@vit.ac.in Abstract: A novel three components approach for the synthesis of bioactive Ru−arene pyridinylmethylene complexes has been developed using pyridine carboxaldehyde, amino pyridine and dichloro (p-cymene) ruthenium (II) dimer as starting materials. These scaffolds were screened for their anticancer activity against breast cancer (MCF7) & human Epitheloid Cervix Carcinoma (HeLa) cell line. It was established that compounds [(η6pcymene)RuCl(κ2-N,N-(3,5-dinitro-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4o), [(η6-pcymene)RuCl(κ2N,N-N-(3,5-Dibromo-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4c), [(η6-pcymene)RuCl(κ2-N,N-(3,5dibromo-6-methylpyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4j) & [(η6-pcymene)RuCl(κ2-N,N-3(3Bromo-5-methyl-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4b) were significantly active against both the cell lines. Keywords: Ruthenium dimer, Ruthenium-Arene Pyridinylmethylene scaffolds, three component reactions, anticancer activity, fluorescence profile, toxicity study Usually, the research in drug discovery has been focused on the development of organic molecules as pharmacophores. However, their limited structural diversity makes it difficult for them to access other scaffolds to extent the whole biologically relevant chemical space,1−3 Therefore, investigation of the structurally unique potentially valuable unexplored chemical spaces is highly warranted. Nowadays, transition-metal scaffolds have been developed as potential drug candidates. They can eagerly accommodate higher coordination number and consequently access different molecular geometries which are not possible with pure organic scaffolds4. The first successful transition metal scaffold, cisplatin, was discovered and approved by Food Drug and Administration (FDA) for the treatment of ovarian and testicular cancer.5 ∗ Corresponding author. Tel.: +91-416-2243091; fax: 91-416-2243092; e-mail: priyankar.paira@vit.ac.in Nevertheless, the attempt of fighting drug resistance and rigorous side effects throughout the treatment are the major drawbacks for cisplatin & its congeners.6 Hence the development of complex containing metals other than platinum have been focused in our current research. During the last three decades, ruthenium (II) arene complexes occupy a prominent position among the various metal complexes explored for anticancer activity.7 Moreover, two ruthenium compounds are under advanced clinical evaluation as anticancer drugs named as NAMI-A8 and KP10199. A number of interesting ruthenium (II) complexes bearing a π-bonded arene ligand has already been developed which show promising anticancer activity,10-11 even in cells that had become resistant to cisplatin, for instance Sadler’s compounds containing N,N chelating ligands.12,13 Besides this, some of the Dyson’s RAPTA compounds containing pta ligand have shown antimetastatic activity.14,15 It was also established that some ruthenium compounds bind to DNA more strongly and are less affected by cell repair mechanisms compared to cisplatin.16-18 There are several properties that makes ruthenium complexes are well accepted for medicinal applications such as their rate of ligand exchange, the range of accessible oxidation states, aqueous solubility and stability in biological environment, and the ability of ruthenium to mimic iron in binding to certain biological molecules.19 In addition, a η6–arene moiety stabilizes the oxidation state of metal ion and may assist it’s transportation through cell membrane.20 The three remaining coordination sites can be occupied with other ligands, forming a “piano stool” geometry, typical for the organoruthenium (II) complexes.21,22 The leaving group in metal complex endures easy dissociation which allow the metal ion to coordinate with the target molecule. The reactivity of the complex with different biomolecules was preserved by ancillary bidentate ligand (κ2-N, N-L) through hydrogen bonding interaction. Currently, the metals are coordinated with bioactive organic ligands to improve a biological activity which seems to be a promising strategy in drug discovery.23 In the recent literature, it was established that ruthenium complexes of organic drugs can overcome the resistance developed by the microbe to the organic compound alone.24 Besides this, a combination of ruthenium-arene moiety with different chloroquinoline derivatives led to complexes which exhibited two to five fold more active than chloroquine against drug resistant Plasmodium parasites.25 Recently, we have discovered a series of ruthenium-quinolinol complexes which played as potent antimicrobial agent against various gram positive & gram negative bacteria.24 In addition, Mun Juinn Chow et. al. has reported a series of ruthenium-arene shiff base complexes which showed the low IC50 values in inhibition of cell viability against a breast cancer cell line MCF7, a human ovarian cell line A2780 and A2780cisR26. Despite of encouraging preliminary results, still there are questions about effectiveness of ruthenium complexes. These studies pave the way for the development of ruthenium-arene complexes as the next generation of metal-based anticancer drugs. The multicomponent reactions and combinatorial approaches are impending tools for designing of diversified compound libraries27, 28 for drug screening although they have not been usually applied to metal-based drug discovery. As a consequence, we have spotlighted in the synthesis of some novel organoruthenium scaffolds via multicomponent approach. Herein, we have discovered a simple and efficient approach for the synthesis of novel Ruthenium–arene pyridinylmethylene scaffolds via three component assembly and evaluated in vitro anticancer activity. Our intention was to synthesize a library of novel Ru−arene complexes with ease of purification and operational simplicity via a one pot three-component approach. Initially, we have treated Pyridine-2-carboxaldehyde (1) with 2-aminopyridine (2a) in Methanol. But the formation of imine is irreversible as it hydrolyzed in aqueous medium. Thus, we prompted in multi-component approach for the synthesis of Ruthenium-arene pyridinylmethylene complexes. The reaction involves three components, exclusively, Ru−arene dimer (3), Pyridine-2carboxaldehyde (1), and 2-aminopyridine (2a). Imine condensation between aldehyde and aminopyridine forms a Schiff-base ligand that is predisposed for metal chelation. Such ligands are usually weakly stable in aqueous environment without metal chelation because of hydrolysis of the imine bond. At the outset, dichloro p-cymene Ruthenium (II) dimer (3) was dissolved in methanol, and subsequently, 2 equivalents of Pyridine-2-carboxaldehyde (1) and 2-aminopyridine (2a) was added to the reaction mixture and stirred for 24 hours in ambient temperature. The progress of the reaction was monitored by TLC. A complete color change was observed after 24 hours of reaction. Consequently, the solvent methanol was evaporated and crude product was recrystallized from 5% methanol in diethyl ether via vapor diffusion method. The structure of this complex (4a) was fully characterized by 1H NMR, 13C NMR and ESI-MS. Commonly, in 1H NMR the arene protons in Ru(II)–arene complexes were shifted downfield relative to the corresponding starting Ru(II) dimers. There were also characteristic changes in the splitting pattern of the protons in the arene ligand. Upon ligand coordination, the C2v symmetry of the complex was interrupted. In CDCl3, 4a exhibited two separate doublets (σ 1.08 & 1.28 ppm) for cymene CH in the 1H NMR spectra due to the formation of enantiomers. Similarly, the arene CH protons displayed four separate doublets (σ 5.63-6.07 ppm). The characteristic peak of imine was exhibited as singlet (σ 10.13 ppm) in 1H NMR spectra.29 In 13C NMR spectra, the characteristic peaks of three methyl carbon were exhibited in the range of σ 21-28 ppm. Likewise, the characteristic ruthenium isotopic pattern in mass spectra clearly indicates the quantitative formation of ruthenium complex. Once the reaction condition for the synthesis of 4a was optimized, the scope of this chemical approach was extended for the synthesis of various Ru-arene complexes (4a-r). The structures were confirmed by their 1H NMR, 13C NMR & ESIMS. The scheme of this general reaction was depicted below. (Scheme 1) Cl Ru H O N R1 1 3 R4 R2 2 PF6 N Ru Cl N N R3 Ru Cl Cl NH2 Cl N R1 MeOH, 24 h, r.t. R3 R2 4 a. R1 = H, R2 = H, R3 = H, R4 = H j. R1 = Br, R2 = Br, R3 = H, R4 = Me b. R1 = Br, R2 = Me, R3 = H, R4 = H k. R1 = Cl, R2 = Cl, R3 = H, R4 = H c. R1 = Br, R2 = Br, R3 = H, R4 = H l. R1 = H, R2 = I, R3 = H, R4 = H d. R1 = H, R2 = Br, R3 = H, R4 = H m. R1 = NO2, R2 = Br, R3 = Me, R4 = H e. R1 = Me, R2 = H, R3 = H, R4 = H n. R1 = H, R2 = Br, R3 = Me, R4 = H f. R1 = H, R2 = H, R3 = Me, R4 = H o. R1 = NO2, R2 = NO2, R3 = H, R4 = H g. R1 = OH, R2 = H, R3 = H, R4 = H p. R1 = H, R2 = NO2, R3 = H, R4 = H h. R1 = H, R2 = Me, R3 = H, R4 = H q. R1 = NO2, R2 = Br, R3 = H, R4 = H i. R1 = H, R2 = Cl, R3 = H, R4 = H r. R1 = H, R2 = H, R3 = H, R4 = Me Scheme-1 Possible route for the synthesis of complex 4 The Ru−arene pyridinylmethylene complex (4a-r) assumes the pseudo-octahedral geometry as usual of organoruthenium species, wherein the p-cymene ring is π-bonded to the ruthenium ion. The remaining three coordination sites of ruthenium are occupied by the chlorido ligand and pyridinylmethylene ligand. While DMSO has been used as a co-solvent in biological system, we investigated the stability of the Ru−arene pyridinylmethylene complex (4b) in DMSO-d6 solution using 1H NMR (see ESI†). Screening these solutions along the time, no changes were observed in the NMR signals. The stability of the complex 4b in DMSO in presence of phosphate buffers with pH 8.0 was monitored by UV-Vis spectroscopy over a period of 24 h. The UV-Vis peak profile remains unchanged with the λmax value, thus suggesting that compound is stable in DMSO (Figure 1a). Likewise, in the presence of 0.1 mM GSH, complex 4b was not displaying any shift in UV-Vis spectroscopy, which recommends that compound 4b might be stable in intercellular thiol (Figure 1b). (a) (b) Figure 1. UV-Vis spectrum of (a) 4b (c = 2×10-4 M) in 5% DMSO in phosphate buffer pH = 8 (b) 4b (c = 2×10-4 mol L-1) in 0.1 mM GSH in DMSO. The fluorescence spectra of 4a-r were measured at concentrations of 5x10-7 molL-1 in different solvents. It was observed that compounds are not displaying enough fluorescence in methanol (Figure S1, see ESI†). However, compounds have been displayed excellent fluorescence in DMSO and dichloromethane (Figure 2, see ESI†). The fluorescence excitation wavelengths (λex/nm) and maximum emission wavelength is reported in Table 1. It was observed that compounds 4d, 4g and 4h exhibited comparable fluorescence in both the solvents. Whereas, compounds 4a, 4c and 4i-r exhibited 1.5-2 folds more fluorescence in DMSO than CH2Cl2. Interestingly, compounds 4b and 4e were not showing any remarkable fluorescence in DMSO. Nevertheless, compound 4e is exhibited 1000 folds more fluorescence in CH2Cl2. Likewise, compounds 4b and 4f are exhibited 10 times more fluorescence in CH2Cl2 than DMSO. 4a 4b 4c 4d 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r 600 500 400 300 200 100 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r 650 600 Fluorescence Intensity (A.U) Fluorescence Intensity (A.U) 700 550 500 450 400 350 300 250 200 150 100 50 0 0 -50 300 350 400 450 500 550 600 300 320 340 (a) 380 400 420 440 460 (b) 1600 1400 1200 1000 800 600 400 4j 4k 4l 4m 4n 4o 4p 4q 4r 600 Fluorescence Intensity (A.U) 4a 4b 4c 4d 4e 4f 4g 4h 4i 1800 Fluorescence (A.U) 360 Wavelength (nm) Wavelength (nm) 500 400 300 200 100 200 0 0 300 320 340 360 380 400 420 440 460 300 320 Wavelength (nm) (c) 340 360 380 400 420 440 460 Wavelength (nm) (d) Figure 2. Fluorescence emission spectra of compounds (a) 4a-r in DMSO; 4e was not shown as it has no fluorescence (b) 4h-r in DMSO (c) 4a-i in CH2Cl2 (d) 4j-r in CH2Cl2. Table 1. Spectroscopic data for compounds 4a-r at 298 K in DMSO & CH2Cl2. Compounds 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r λexa (nm) 419 618 290 419 498 320 295 290 290 290 290 290 290 290 290 290 290 290 DMSO λemb (nm) 514 346, 514 514 358 356 360 356 346 348 346 346 346 342 346 346 354 Dichloromethane λexa (nm) λemb (nm) 295 344 320 372 295 356 295 354 295 378 295 358 295 352 295 364 295 346 295 346 295 344 295 344 295 344 295 344 295 346 295 346 295 350 295 364 The cytotoxicity of all the synthesized ruthenium complexes 4a-r (Table 2) was evaluated using standard 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) bioassay beside a panel of cell lines such as human Epitheloid Cervix Carcinoma (HeLa) and human breast carcinoma cell line (MCF-7). These cells were treated with compounds (4a-r) along with cisplatin & doxorubicin as a standard positive control at concentration 1 to 100 µM for 24 h. All experiments were carried out in triplicates, and IC50 was determined from the average of three separate experiments. The inhibition of cell viability of these complexes against HeLa cell line were also evaluated (Figure 3). Most of the ruthenium complexes exhibited higher anticancer profile than cisplatin. However, compounds are slightly less potent than doxorubicin. DMSO was used as control where it was not showing any inhibition of cell. In MCF-7 cells, the IC50 values for ruthenium pyridinylmethylene scaffolds were in the range of 7–25 µM (Table 2). Likewise, in HeLa cells, the IC50 values for these complexes were observed in the range of 7-29 µM. The structure-activity relationship studies revealed that the introduction of electron withdrawing bromo and nitro group at 3rd & 5th position of the pyridine ring (compound 4c & 4o) displayed the best cytotoxicity profile. However, cytotoxicity level is slightly reduced after introducing an electron donating methyl group at 6th position of pyridine ring in compound 4c (compound 4j). It was also observed that compound 4b is more cytotoxic than compound 4d while a methyl group is present at 5th position of pyridine ring in 4b. These results clearly indicate that substitution of electron withdrawing group at 3rd position of pyridine ring is more important than 5th position. It was concluded that electron donating methyl group at 5th position might not affect the cytotoxicity level of compound 4b if 3rd position is already occupied by electron withdrawing bromo group. Compounds 4e, 4f, 4h and 4r exhibited less potency in both the cell lines as electron releasing methyl group is introduced in the pyridine unit of these scaffolds. It was also observed that cytotoxicity levels of 4e & 4f are lower than 4h & 4r. Hence the cytotoxicity will be reduced more by the substitution of electron releasing group at 3rd and 5th positions in the pyridine ring rather than 4th and 6th position. Compound 4g also displayed excellent efficacy against both the cell lines. Hydroxyl group present in the 3rd position of the pyridine ring might be responsible for hydrogen bonding with the target protein. Likewise, compounds 4i, 4k, and 4l was also exhibited exceptional cytotoxicity against both the cell line as electronegative chloro or iodo group was present in the pyridine ring. Cytotoxicity of compounds 4m, 4n, 4p and 4q were maintaining the proper order (4n<4p<4m<4q) as well. Highly electron withdrawing bromo & nitro group at the meta position of pyridine ring might be effective for the generation of reactive oxygen species. 100 90 80 Cell Viability (%) 70 60 50 40 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r 100 90 80 Cell Viability (%) 4a 4b 4c 4d 4e 4f 4g CisPlatin 110 70 60 50 40 30 30 20 20 10 10 0 0 0 20 40 60 80 100 0 Concentration (µM) 20 40 60 80 100 Concentration (µM) (a) (b) Figure 3. Dose-dependent drug efficacy studies for (a) 4a-g and cisplatin (b) 4h-r on cancer cell lines HeLa Table 2. Preliminary MTT cytotoxicity screening of synthesized benzothiazole derivatives at 24 h of drug exposure IC50 (µM)a Compound 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r MCF-7 22.62±0.58 08.25±0.76 07.95±0.45 09.45±0.62 25.12±0.72 25.42±0.64 09.76±0.48 24.26±0.78 10.76±0.79 08.00±0.88 09.46±0.98 08.66±0.43 09.46±0.48 10.66±0.88 07.76±0.88 09.86±0.48 08.56±0.82 23.86±0.72 HeLa 26.32±0.42 17.42±0.85 07.85±1.16 19.6±1.26 28.4±0.18 29.22±1.68 16.20±1.22 27.10±1.60 20.10±0.50 07.96±1.26 17.10±0.54 16.20±0.66 14.30±0.74 18.10±1.90 07.10±1.28 16.22±1.37 10.20±0.80 27.30±1.26 DMSO Cisplatin Doxorubicin 00 9.42±0.52 5.2±0.28 00 16.2±0.48 6.8±0.76 a IC50 is the concentration at which 50% of cells were undergoing cytotoxic cell death due to synthesized compound treatment. The toxicity study of most potent anticancer derivative 4o was carried out using male Sprague Dawley rats. Toxicity profile of test item (4o) was compared with doxorubicin (standard) by single intravenous bolus administration in male Sprague Dawley rats (see ESI†). Test group animals were received a single dose injection of test drug at 10mg/kg in normal saline while the control groups were treated with normal saline only. Likewise, reference group animals were received a single dose injection of doxorubicin at 10 mg/kg in normal saline. Afterwards, the animals in all groups were sacrificed for 48 hours. After 48 h of dosing, blood samples were collected from all the rats for estimation of serum Aspartate aminotransferase (AST) and blood glutathione (GSH). After collecting the blood samples, all the animals were sacrificed by exsanguinations under deep isoflurane anesthesia. Heart was removed to estimate the Thiobarbituric Acid Reactive Substances (TBARS) and glutathione (GSH). The blood GSH levels of the doxorubicin (reference group) and test item (compound 4o) treated animals showed a significant decrease as compared to normal control group (p <0.01). Test item showed a significantly more in blood GSH level as compared to reference (p < 0.01). The AST level was elevated significantly in animals treated with doxorubicin as compared to control group. The level was significantly reduced in test group when compared to reference group (p < 0.01). Likewise, the level of TBARS was significantly elevated in animals treated with doxorubicin alone as compared to corresponding normal control group. While the animals treated with test item showed significantly low in the concentration of TBARS as compared to reference group. It has also been observed that there was a significant decrease in the level of the heart tissue GSH in doxorubicin treated group as compared to normal control animals. However animals treated with test item showed significantly more in the concentration of tissue GSH as compared to group II (p 12 30 10 25 Control Doxorubicin Test item 8 6 4 Heart GSH level Blood GSH level < 0.05) (Figure 4). Control Doxorubicin Test item 20 15 10 5 2 0 0 Control Doxorubicin Control Test item Doxorubicin Test item Treatment Treatment (a) (b) 0.7 5 0.6 4 Doxorubicin Test item 0.4 0.3 Serum AST level Heart TBARS level Control 0.5 Control Doxorubicin 3 Test item 2 0.2 1 0.1 0.0 Control Doxorubicin Treatment (c) Test item 0 Control Doxorubicin Test item Treatment (d) Figure 4. Cardiotoxicity Profile (a) blood GSH level (b) heart GSH level (c) heart TBARS level (d) serum AST level Conclusion In review, a novel, general and effective procedure for the preparation of Ru-arene N-(pyridin-2ylmethylene)pyridin-2-amine complexes in a one-pot sequence was established. This technique allows a great compact of synthetic flexibility and offers the opportunity of synthesizing newer organoruthenium coordination for anticancer screening. The significant features of this methodology are that it works with inexpensive and easily available reactants operating in an environment friendly, mild reaction condition with operational simplicity. Most of the compounds exhibited exceptional cytotoxicity against MCF7 and Hela cell lines compared to cisplatin. Those cytotoxic scaffolds also displayed remarkable fluorescence in DMSO and Dichloromethane which might be useful for theranostic application. Acknowledgement: We are thanking to the VIT University for financial support in our project. We acknowledge to VIT-DST-FIST project for the funding. We also acknowledge to DST-SERB YS scheme for the funding. 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Lett., 2015, 25, 2892-2896. 25 Sanchez-Delgado, R. A.; Navarro, M.; Perez, H.; Urbina, J. A. J. Med. Chem. 1996, 39, 10951099. 26 Mun J. C. Cynthia L. Daniel Y., Giorgia P., Christian G., and Wee H. A. J. Med. Chem., 2014, 57, 6043-6059. 27 Reetz, M. T. Angew. Chem. Int. Ed. 2001, 40, 284-310. 28 Francis, M. B.; Jamison, T. F.; Jacobsen, E. N. Curr. Opin. Chem. Biol. 1998, 2, 422-428. 29 General procedure for the synthesis of compounds 4a-r: Ruthenium (II) p-cymene Dimer (3) (10 mg, 0.016 mmol) was dissolved in 5 ml of methanol in 25 mL RB flask placed on a magnetic stirrer and stirred for 30 min, then Pyridine-2-carboxaldehyde (1) & 2-amino pyridine analogues (2a-r) (2:2 equivalent with respect to 3) was added to reaction mixture and continue the stirring for 24 h at room temperature, color change was observed from deep yellow to reddish brown. We performed the TLC in 1% methanol in ethyl acetate which confirms the product. After completion of the reaction we have evaporated the methanol by using rotator evaporator. The crude product was recrystallized from 5% methanol in diethyl ether and orange to red fine crystal was obtained with 95% yield. [(η6-pcymene)RuCl(κ2-N,N-Pyridin-2-yl-pyridin-2-ylmethylene-amine)]·Cl (4a): Yield: 95 %, Mp: 146-147 °C, Rf (1% MeOH in Ethyl Acetate): 0.26, IR (cm-1): ʋ 553, 748, 762, 829, 997, 1205, 1360, 1391(aromatic C=C stretch), 1632 (-C=N stretch), 1740, 2338, 2804, 2972 (aromatic C-H stretch), 3044 (aromatic C-H stretch), 3119 (aromatic C-H stretch); 1H NMR (CDCl3, 400 MHz): δ 1.09 (d, 3H, J = 6.8 Hz, Cymene CH3), 1.30 (d, 3H, J = 6.8 Hz, Cymene CH3), 2.28 (s, 3H, Cymene CH3), 2.62-2.72 (m, 1H, CH), 5.68 (d, 1H, J = 6.8 Hz, Cymene ArH), 5.78 (d, 1H, J = 5.6 Hz, Cymene ArH), 6.05 (d, 1H, J = 4.8 Hz, Cymene ArH), 6.32 (d, 1H, J = 4.8 Hz, Cymene ArH), 7.09-7.14 (m, 1H, ArH), 7.47-7.52 (m, 1H, ArH), 7.91-8.08 (m, 3H, ArH), 8.27 (d, 1H, J = 6.0 Hz, ArH), 8.62-8.64 (m, 1H, ArH), 9.04 (brs, 1H, ArCH), 10.13 (s, 1H, imine); 13 C NMR (CDCl3, 400 MHz): δ 21.1 (CH3), 21.7 (CH3), 28.7 (CH3), 30.3 (CH), 83.6, 84.3, 85.3, 86.1, 103.7, 105.9, 118.3, 124.7, 129.4, 129.5, 138.5, 138.6, 148.3, 152.4, 156.0, 159.5, 165.6 (C=N); 31P NMR (CDCl3, 400 MHz): δ -153.04 (m, PF6); 19F NMR (CDCl3, 400 MHz): δ -73.05 (6F, PF6); ESI-MS (MeOH): m/z: 454 [M]+; HRMS (ESI): m/z calcd for C21H23N3ClRu+ : 454.0624 [M]+; found: 454.0629 [M]+ [(η6-pcymene)RuCl(κ2-N,N-3(3-Bromo-5-methyl-pyridin-2-yl)-pyridin-2-ylmethyleneamine)]·PF6 (4b): Yield: 90 %, Mp: 143-145 °C, Rf (1% MeOH in Ethyl Acetate): 0.30, IR (cm1 ): ʋ 553, 675, 742, 770, 779, 829, 1057, 1234, 1302, 1381 (aromatic C=C stretch), 1591(-C=N stretch), 1736, 3119 (aromatic C-H stretch); 1H NMR (CDCl3, 400 MHz): δ1.05 (d, 3H, J = 6.65 Hz, Cymene CH3), 1.12 (d, 3H, J = 6.78 Hz, Cymene CH3), 2.21 (s, 3H, Cymene CH3), 2.49 (s, 3H, Cymene CH3), 2.78-2.85 (m, 1H, CH), 5.45 (brs, 1H, Cymene ArH), 5.72 (d, J = 5.52 Hz, 1H, Cymene ArH), 5.82 - 5.91 (m, 2H, Cymene ArH), 7.86 (brs, 1H), 7.99 (brs, 1H, ArH), 8.07 8.19 (m, 2H, ArH), 8.47 (brs, 1H, ArH), 8.61 (s, 1H, ArH), 9.51 (brs, 1H, imine); 13C NMR (CDCl3, 400 MHz): δ 17.8 (CH3), 20.6 (CH3), 21.4 (CH3), 28.7 (CH3), 30.2 (CH), 83.7, 84.1, 85.3, 85.9, 102.8, 106.3, 110.4, 129.5, 129.8, 135.8, 138.6, 143.0, 147.1, 152.2, 155.6, 155.7, 170.0 (-C=N); 31P NMR (CDCl3, 400 MHz): δ -153.00 (m, PF6); 19F NMR (CDCl3, 400 MHz): δ -72.96 (6F, PF6); ESI-MS (MeOH): m/z: 546 [M]+, 548 [M+2]+; HRMS (ESI): m/z calcd for C22H24N3BrClRu+: 545.9886 [M]+; found: 545.9890 [M]+ [(η6-pcymene)RuCl(κ2-N,N-N-(5-bromo-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]·PF6 (4d): Yield: 92 %, Mp: 148-150 °C, Rf (1% MeOH in Ethyl Acetate): 0.28, IR (cm-1): ʋ 555, 692, 717, 739, 770, 822, 1092, 1207, 1306, 1373, 1448 aromatic C=C stretch), 1740 (-C=N stretch), 1970, 3119 (aromatic C-H stretch); 1H NMR (400 MHz, CDCl3) δ1.07 (d, 3H, J = 5.90 Hz, Cymene CH3), 1.12 (d, 3H, J = 6.02 Hz, Cymene CH3), 2.24 (s, 3H, Cymene CH3), 2.56-2.62 (m, 1H, CH), 5.53 (brs, 1H, Cymene ArH), 5.68 (brs, 1H, Cymene ArH), 5.74 (brs, 1H, Cymene ArH), 6.0 (brs, 1H, Cymene ArH), 7.87-7.93 (m, 2H, ArH), 8.09-8.16 (m, 3H, ArH), 8.87 (brs, 1H, ArH), 8.67 (brs, 1H, ArH), 9.58 (s, 1H, imine); 13C NMR (DMSO-d6, 400 MHz): δ18.5 (CH3), 21.7 (CH3), 30.5 (CH3), 30.6 (CH), 83.8, 83.9, 86.4, 87.3, 104.8, 105.4, 121.1, 121.4, 129.6, 131.5, 140.0, 142.3, 149.7, 153.9, 156.3, 158.9, 168.8(-C=N); 31P NMR (CDCl3, 400 MHz): δ -153.02 (m,PF6); 19F NMR (CDCl3, 400 MHz):δ -72.87 (6F, PF6); ESI-MS (MeOH): m/z: 532 [M]+, 534 [M+2]+; HRMS (ESI): m/z calcd for C21H22N3BrClRu+: 531.9729 [M]+; found: 531.9733 [M]+ [(η6-pcymene)RuCl(κ2-N,N-(4-Methyl-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]·PF6 (4f): ° -1 Yield: 94 %, Mp: 146-147 C, Rf (1% MeOH in Ethyl Acetate): 0.27, IR (cm ): ʋ 519, 555, 692, 710, 739, 771, 822, 1304, 1404 (C=C stretch), 1435 (C=C stretch), 1441 (C=C stretch), 1472 (C=C stretch), 1603, 1632, 1668 (-C=N stretch), 2808, 2934, 2970 (aromatic C-H stretch), 3034 (aromatic C-H stretch), 3042 (aromatic C-H stretch), 3101 (aromatic C-H stretch), 3121, 3323, 3642; 1H NMR (CDCl3, 400 MHz): δ 1.05-1.09 (m, 3H, Cymene CH3), 1.29 (d, 3H, J = 7.2 Hz, Cymene CH3), 2.02 (s, 3H, Cymene CH3), 2.26 (s, 3H, Me), 2.62-2.69 (m, 1H, CH),5.32 (d, 1H, J = 5.6 Hz, Cymene ArH), 5.54 (d, 1H, J = 5.6 Hz, Cymene ArH), 5.66 (m, 1H, Cymene ArH), 5.76 (brs, 1H, Cymene ArH), 7.50 (m, 1H, ArH), 7.91-8.05 (m, 4H, ArH), 8.61-8.62 (m, 1H, ArH), 9.04 (s, 1H, ArH), 10.1 (s, 1H, imine CH); 13C NMR (CDCl3, 400 MHz): δ17.8 (CH3), 20.2 (CH3), 20.8 (CH3), 21.1 (CH3), 30.1 (CH), 81.8, 81.9, 84.0, 85.7, 86.3, 103.55, 104.4, 111.1, 113.5, 118.9, 125.8, 130.6, 135.0, 139.0, 139.4, 150.4, 153.6, 159.8 (-C=N); 31P NMR (CDCl3, 400 MHz): δ -152.60 (m, PF6); 19F NMR (CDCl3, 400 MHz): δ -66.35 (6F, PF6); ESIMS (MeOH): m/z: 468 [M]+; HRMS (ESI): m/z calcd for C22H25N3ClRu+: 468.0781 [M]+; found: 468.0786 [M]+ [(η6-pcymene)RuCl(κ2-N,N-(5-Methyl-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]·PF6 (4h): ° -1 Yield: 95 %, Mp: 140-142 C, Rf (1% MeOH in Ethyl Acetate): 0.30, IR (cm ): ʋ 515, 555, 692, 739, 768, 820, 1227, 1389, 1470(aromatic C=C stretch), 1589, 1630(-C=N stretch), 2928, 2968(aromatic C-H stretch), 3319; 1H NMR (DMSO-d6, 400 MHz): δ 0.92-0.95 (m, 6H, Cymene CH3), 2.18 (m, 3H, Cymene CH3), 2.44 (m, 3H, CH3), 2.66-2.73 (m, 1H, Cymene CH), 3.15 (s, 3H, Cymene CH3), 5.71 (d, 1H, J = 5.6 Hz, Cymene ArH), 5.82 (d, 1H, J = 6 Hz, Cymene ArH), 5.88 (d, 1H, J = 6 Hz, Cymene ArH), 6.16 (d, 1H, J = 6 Hz, Cymene ArH), 7.787.90 (m, 2H, ArH), 7.95 (d, 1H, J = 8 Hz, ArH), 8.31-8.51 (m, 3H, ArH), 9.26 (s, 1H, ArH), 9.59 (brs, 1H, imine CH); 13C NMR (DMSO-d6, 400 MHz): δ17.6 (CH3), 18.5 (CH3), 21.6 (CH3), 30.5 (CH3), 30.6 (CH), 84.0, 84.1, 86.5, 87.1, 119.0, 129.3, 131.0, 133.3, 135.5, 139.9, 146.1, 149.0, 154.1, 156.2, 157.8, 167.5(-C=N); ESI-MS (MeOH): 31P NMR (DMSO-d6, 400 MHz): δ 157.36 (m, PF6); 19F NMR (DMSO-d6, 400 MHz): δ -71.08 (6F, PF6); ESI-MS (MeOH): m/z: 468 [M]+; HRMS (ESI): m/z calcd for C22H25N3ClRu+: 468.0780 [M]+; found: 468.0777 [M]+ Graphical Abstract Synthesis of novel Anticancer Ruthenium-arene Leave this area blank for abstract -pyridinylmethylene scaffolds via Three-component info. Reaction Gajanan Raosaheb Jadhav,† Sohini Sinha,† Mohit Chhabra, Priyankar Paira* Pharmaceutical Chemistry division, School of advanced sciences, VIT University, Vellore-632014, Tamilnadu, India