Cytotoxic Ruthenium(II) Complexes of Pyrazolylbenzimidazole Ligands That Inhibit VEGFR2 Phosphorylation.

pubs.acs.org/IC Article Cytotoxic Ruthenium(II) Complexes of Pyrazolylbenzimidazole Ligands That Inhibit VEGFR2 Phosphorylation Ayan Chakraborty,† Souryadip Roy,† Manas Pratim Chakraborty, Shantanu Saha Roy, Kallol Purkait, Tuhin Subhra Koley, Rahul Das, Moulinath Acharya, and Arindam Mukherjee* Downloaded via TSINGHUA UNIV on November 4, 2022 at 00:47:54 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Cite This: Inorg. Chem. 2021, 60, 18379−18394 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: Eight new ruthenium(II) complexes of N,N-chelating pyrazolylbenzimidazole ligands of the general formula [RuII(p-cym)(L)X]+ [where the ligand L is 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole (L1) substituted at the 4 position of the pyrazole ring by Cl (L2), Br (L3), or I (L4) and X = Cl− and I−] were synthesized and characterized using various analytical techniques. Complexes 1 and 3 were also characterized by single-crystal X-ray crystallography, and they crystallized as a monoclinic crystal system in space groups P21/n and P21/c, respectively. The complexes display good solution stability at physiological pH 7.4. The iodido-coordinated pyrazolylbenzimidazole ruthenium(II) p-cymene complexes (2, 4, 6, and 8) are more resistant toward hydrolysis and have less tendency to form monoaquated complexes in comparison to their chlorido analogues (1, 3, 5, and 7). The halido-substituted 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole ligands, designed as organic-directing molecules, inhibit vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation. In addition, the ruthenium(II) complexes display a potential to bind to DNA bases. The cytotoxicity profile of the complexes (IC50 ca. 9−12 μM for 4−8) against the triple-negative breast cancer cells (MDAMB-231) show that most of the complexes are efficient. The lipophilicity and cellular accumulation data of the complexes show a good correlation with the cytotoxicity profile of 1−8. The representative complexes 3 and 7 demonstrate the capability of arresting the cell cycle in the G2/M phase and induce apoptosis. The inhibition of VEGFR2 phosphorylation with the representative ligands L2 and L4 and the corresponding metal complexes 3 and 7 in vitro shows that the organic-directing ligands and their complexes inhibit VEGFR2 phosphorylation. Besides, L2, L4, 3, and 7 inhibit the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and proto-oncogene tyrosine-protein kinase (Src), capable of acting downstream of VEGFR2 as well as independently. Compounds L2, L4, 3, and 7 have a lesser effect on ERK1/2 and more prominently affect Src phosphorylation. We extended the study for L2 and 3 in the Tg(fli1:gfp) zebrafish model and found that L2 is more effective in vivo compared to 3 in inhibiting angiogenesis. ■ INTRODUCTION tion of phase I and entry into phase II clinical trials of the phototherapeutic TLD1433 has further enhanced the role of Ru as a metal of choice for the design of new anticancer agents.18,19 Besides, the organometallic ruthenium(II) pcymene motif, modifiable through mono- or bidentate ligands, and halide coordination provide complexes with excellent antiproliferative activity.20−23 The appropriate choice of mono/bidentate ligands along with the labile halide suitably Metal complexes have occupied an important place in cancer chemotherapy, helping to cure or prolong millions of lives.1 This domination is solely led by the success of platinum(II) (PtII) drugs against various forms of cancer.1−4 Apart from Pt complexes, ruthenium(II/III) (RuII/III), gallium(III) (GaIII), and palladium(II) (PdII) complexes have potency against multiple cancers in clinic or in clinical trials.5−10 Ru complexes are active against various Pt-resistant cancers and exhibit lower side effects and different mechanisms of action compared to Pt drugs.11−13 The Ru drug NAMI-A showed potency against metastasis but did not succeed in clinical trials14,15 but NKP1339/IT-139 and TLD1433 underwent phase I clinical trials against nonsmall cell lung carcinoma16,17 and nonmuscle invasive bladder cancer, respectively. The successful comple© 2021 American Chemical Society Received: September 24, 2021 Published: November 15, 2021 18379 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Article Figure 1. Representative clinically used kinase inhibitors, RuII-based kinase inhibitors, and the RuII complexes from this work. the principal mediator of vascular endothelial growth factor (VEGF)-induced angiogenic signaling through phosphorylation of the tyrosine(s) in the intracellular domain of VEGFR2. The phosphorylation of VEGFR2 sprouts blood vessel growth (angiogenesis) to ensure an uninterrupted supply of oxygen and nutrients to the fast-proliferating cells.35−38 Therefore, targeting VEGFR2 receptors is of importance in anticancer drug design.39−42 Many organic small molecules (Figure 1), including Sunitinib,43 Sorafenib,44,45 Regorafenib46 and Lenvatinib,47 are reported to inhibit VEGFR2 by interfering with the ATP binding pocket and preventing phosphorylation.48−54 Kinases like epidermal growth factor receptor (EGFR) or VEGFR2 are overexpressed in various difficult-to-cure tumors, including triple-negative breast cancers (TNBCs).55 TNBCs are negative for estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2) receptors.55 Thus, the growth of the tumor is not fuelled by hormones like estrogen tunes the target selectivity, stability, and pathway of action of ruthenium(II) p-cymene complexes because of alteration of the steric and electronic features.24,25 Ligand design is a salient aspect to tune the cytotoxicity of metal complexes. In this regard, using organic-directing molecules as ligands to target various cellular events, including impairing protein functions, provides a vital methodology to incorporate multiple pathways of action in a metal complex.26−29 The “organic-directing molecules” can target various organelles or cellular processes because of their ability to interact with the active site of relevant proteins. Their use as metal-complexing ligands provides the scope of introducing new targets, including cellular proteins and enzymes other than DNA, in the generation of cancer chemotherapeutic agents.30−34 Organic-directing molecules have been used to target receptor tyrosine kinases (RTKs). Among the RTKs, vascular endothelial growth factor receptor 2 (VEGFR2), a type II transmembrane RTK overexpressed in many tumors, is 18380 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC compounds are reported. The ligands and complexes synthesized were dried in vacuo and stored in a desiccator in the dark. 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; USB) and all kind of supplements were purchased from Gibco and used as received for cell culture. Synthesis and Characterization. Synthesis of Ligands. Synthesis of Ligands L1and L2. The synthesis of respective substituted pyrazoles is provided in the Supporting Information. 1,2-Diaminobenzene (1 mmol) was dissolved in 1 mL of POCl3 (10 mmol). Then the respective pyrazole (1 mmol) was added portionwise. After 10 min of stirring, 0.33 mmol of triphosgene was added over 20 min. The reaction mixture was refluxed for a further 16 h at 110 °C. Then, the reaction was quenched in ice under hot conditions and neutralized with a NaOH solution. At pH 7, a white precipitate appeared. The precipitate was collected by vacuum filtration, and thin-layer chromatography was performed to confirm the purity. A short column over 60−120 mesh silica gel usinga petroleum ether and ethyl acetate mixture (20% for L1 and a 10−40% gradual increase for L2) provided the pure ligands. Characterization. 2-(1H-Pyrazol-1-yl)-1H-benzo[d]imidazole (L1). Yield: 98%. 1H NMR (500 MHz, DMSO-d6, 298 K): δ 13.08 (s, 1H, BzIm-NH), 8.59 (d, J = 2.2 Hz, 1H, pyz-H), 7.95 (s, 1H, pyzH), 7.58 (s, 1H, BzIm-H), 7.46 (s, 1H, BzIm-H), 7.20 (dd, J = 2.9 Hz, 2H, BzIm-H), 6.66 (s, 1H, pyz-H) (Figure S1). 13C NMR (125 MHz, DMSO-d6, 298 K): δ 146.0, 142.7, 141.7, 133.6, 128.9, 122.2, 118.3, 111.5, 108.9 (Figure S2). ESI-HRMS (MeOH) for [HL1] + ≡ [C10H9N4]+: m/z 185.0821 (calcd m/z 185.0822). 2-(4-Chloro-1H-pyrazol-1-yl)-1H-benzo[d]imidazole (L2). Yield: 83%. 1H NMR (500 MHz, CDCl3, 298 K): δ 8.50 (s, 1H, Pyz-H), 7.69 (s, 1H, Pyz-H), 7.57 (s, 2H, BzIm-H), 7.30 (dd, J = 3.1 Hz, 2H, BzIm-H) (Figure S3). 13C NMR (125 MHz, DMSO-d6, 298 K): δ 145.3, 141.3, 141.1−140.9, 133.6, 126.9, 122.4, 118.4, 112.5, 111.6 (Figure S4). ESI-HRMS (MeOH) for [L2 + Na] + ≡ [C10H7ClN4Na]+: m/z 241.0259 (calcd m/z 241.0251). Synthesis of Ligands L3 and L4. The synthesis of respective substituted pyrazoles is provided in the Supporting Information. 1,2Diaminobenzene (1.0 mmol) was dissolved in 5 mL of dry tetrahydrofuran (THF) at −5 °C under a N2 atmosphere and stirred for 5 min. Carbonyldiimidazole (CDI; 1.2 mmol) in 5 mL of dry dichloromethane (DCM) was added dropwise over 10 min. The offwhite solution turned turbid upon addition, and over time, a white powder precipitated. The precipitate was collected by vacuum filtration after stirring overnight. The precipitate was used for further synthetic steps without any purification. The off-white precipitate of 1,3-dihydro-2H-benzo[d]imidazol-2-one (1 mmol) and the respective substituted pyrazoles (4-pzBr and 4-pzI; 1.2 mmol) was taken into a tube, and 10 equiv of distilled POCl3 was added slowly. The tube was sealed, heated to 110 °C, and kept at that temperature for 24 h. The resultant hot solution was poured over crushed ice, followed by neutralization with a saturated NaOH solution to bring the pH to ca. 7−8. The solid precipitated was filtered, dried, and separated by column chromatography with petroleum ether/ethyl acetate as the eluent. In the case of L3, the initial percentage of ethyl acetate was 5%, which was gradually increased to 20% to complete the elution. To purify L4, the initial percentage of ethyl acetate was 5%, which was gradually increased to 15% to complete the elution. 2-(4-Bromo-1H-pyrazol-1-yl)-1H-benzo[d]imidazole (L3). Yield: 68%. 1H NMR (500 MHz, CDCl3, 298 K): δ 8.54 (d, J = 2.8 Hz, 1H, pyz-H), 7.71 (d, J = 2.7 Hz, 1H, pyz-H), 7.56 (s, 2H, BzIm-H), 7.29 (dt, J = 3.3 Hz, 2H, BzIm-H) (Figure S5). 13C NMR (125 MHz, DMSO-d6, 298 K): δ 145.7, 143.5, 129.5, 122.8, 118.6, 112.8, 96.9 (Figure S6). ESI-HRMS (MeOH) for [L3 + Na] + ≡ [C10H7BrN4Na]+: m/z 284.9750 (calcd m/z 284.9746). 2-(4-Iodo-1H-pyrazol-1-yl)-1H-benzo[d]imidazole (L4). Yield: 63%. 1H NMR (500 MHz, CDCl3, 298 K): δ 8.57 (s, 1H, pyz-H), 7.75 (s, 1H, pyz-H), 7.58 (s, 2H, BzIm-H), 7.29 (dd, J = 3.1 Hz, 2H, BzIm-H) (Figure S7). 13C NMR (125 MHz, DMSO-d6, 298 K): δ 150.3, 146.6, 141.6, 132.8, 122.9−122.7, 121.8, 118.2, 111.2, 109.2 (Figure S8). ESI-HRMS (MeOH) for [L4 + Na] + ≡ [C10H7IN4Na]+: m/z 332.9620 (calcd m/z 332.9608). or progesterone. So, TNBCs do not respond to hormonal therapy or medicines targeting the HER2 receptors. The treatment primarily relies on small-molecule chemotherapy, including Pt drugs, polyadenosine diphosphate−ribose polymerase inhibitors, microtubule disruptors, immune system adjuvants, and kinase inhibitors.56−59 The expression of EGFR and VEGFR2 on the cell surface of various TNBCs promotes tumor invasion and metastasis.55 Thus, inhibition of these proteins and their downstream signaling by small-molecule tyrosine kinase inhibitors is a viable methodology for treatment. During the past 2 decades, various metal complexes (Figure 1) bearing organic-directing groups showed the efficient inhibition of a wide variety of kinases, viz., EGFR, cyclin-dependent kinase (CDK), glycogen synthase kinase 3 beta (GSK 3β), proto-oncogene serine/threonine protein kinase (Pim1), mitogen- and stress-activated protein kinase (MSK1), P21 (RAC1) activated kinase 1 (PAK1), macrophage stimulating 1 (MST1), and myosin light chain kinase (MLCK).34,60−65 However, metal complexes that efficiently inhibit VEGFR2 phosphorylation are scarce.66,67 The design of kinase targeting metal complexes benefits from a ligand that alone can target the ATP binding domain in the kinases, and such metal complexes involve the use of derivatives of clinically relevant motifs.31,66,68 Benzimidazolyl and pyrazolyl compounds are known to be part of a wide variety of clinical drugs with excellent potency, encompassing nonsteroidal antiinflammatory drugs, alkylating agents, anthelmintics, and proton pump inhibitors.69−75 In this work, we present halidosubstituted 2-(1H-pyrazol-1-yl)-1H-benzo[d]imidazole bidentate ligands to design RuII complexes that inhibit VEGFR2 phosphorylation. We have synthesized and characterized four ligands (L1−L4), providing eight RuII complexes (1−8) with variation of a halide on the 4 position of the pyrazole in the bidentate ligand and in coordination to RuII (Figure 1). The eight RuII complexes bear the general molecular formula [RuII(p-cym)(L)X]X, where X = Cl and I. The complexes were characterized by various analytical methods like UV−vis, Fourier transform infrared (FT-IR), 1H, 13C, and HMQC NMR, and high-resolution electrospray ionization mass spectrometry (ESI-HRMS). Complexes 1 and 3 were also characterized by single-crystal X-ray diffraction. The solution stability, cytotoxicity, and VEGFR2 inhibition studies of the corresponding ruthenium(II) p-cymene complexes show that the pyrazolylbenzimidazole ligand systems can act as organicdirecting groups to inhibit VEGFR2 phosphorylation, while RuII is capable of DNA binding, thus improving the cytotoxic efficiency. ■ Article EXPERIMENTAL SECTION Materials and Methods. The chemicals were purchased from multiple commercial sources and used without further purification. The solvents were distilled and dried using standard procedures before use.76 The metal precursor complexes [RuII2(η6-p-cym)2(X)4] were synthesized following literature procedures.77 The solvents used in the spectroscopic measurements were of spectroscopy grade and were purchased from Merck, India. PerkinElmer Lambda 35 and SPECTRUM RX I spectrophotometers were used for UV−vis and FT-IR measurements (in KBr pellets), respectively. The 1H, 13C, and HMQC NMR spectra were recorded using either a 400 MHz JEOL ECS or a 500 MHz Bruker Avance III spectrometer, at room temperature (24−27 °C). The chemical shifts of the relevant compounds are reported in parts per million (ppm). All ESI-HRMS spectra were recorded in electrospray ionization positive mode using a Bruker maXis II TM instrument. Isolated yields of 1H NMR pure 18381 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Article 22.0, 21.8, 19.9 (Figures S18−S20). IR (KBr pellets, cm−1): 3421, 1773, 1524, 1329, 839. UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 305 (17952), 444 (947) (Figure S33). ESI-HRMS (MeOH) for [4 − I − HI]+ ≡ [C20H20ClN4Ru]+: m/z 453.0481 (calcd m/z 453.0420). Anal. Calcd for C20H21ClI2N4Ru: C, 33.94; H, 2.99; N, 7.92. Found: C, 34.10; H, 2.77; N, 8.03. [RuII(p-cym)(L3)Cl]Cl (5). Yield: 92%. 1H NMR (500 MHz, DMSO-d6, 298 K): δ 9.19 (s, 1H, pyz-H), 9.08 (s, 1H, pyz-H), 7.96 (d, J = 7.9 Hz, 1H, BzIm-H), 7.68 (d, J = 7.8 Hz, 1H, BzIm-H), 7.41 (dt, J = 7.3 Hz, 2H, BzIm-H), 6.35 (d, J = 6.0 Hz, 1H, p-cym-H), 6.31 (d, J = 6.1 Hz, 1H, p-cym-H), 6.13 (dd, J = 6.1 Hz, 2H, p-cymH), 2.62 (dt, J = 13.7 Hz, 1H,p-cym-isopropyl-CH), 2.14 (s, 3H pcym-Me), 1.02 (dd, J = 6.9 Hz, 6H, p-cym-isopropyl). 13C NMR (125 MHz, DMSO-d6, 298 K): δ 147.6, 144.7, 139.2, 130.9, 124.2, 123.9, 116.7, 114.1, 104.1, 101.4, 98.1, 83.2, 82.2, 81.6, 80.7, 30.6, 21.8, 18.4 (Figures S21−S23). IR (KBr Pellets, cm−1): 3422, 1691, 1591, 1400, 808. UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 304 (15563), 417 (603) (Figure S33). ESI-HRMS (MeOH) for [5 − Cl − HCl]+ ≡ [C20H20BrN4Ru]+: m/z 496.9896 (calcd m/z 496.9915). Anal. Calcd for C20H21BrCl2N4Ru: C, 42.20; H, 3.72; N, 9.84. Found: C, 41.99; H, 3.64; N, 9.81 [RuII(p-cym)(L3)I]I (6). Yield: 84%. 1H NMR (500 MHz, DMSO-d6, 298 K): δ 9.15 (s, 1H, pyz-H), 8.90 (s, 1H, pyz-H), 7.88 (d, J = 7.8 Hz, 1H, BzIm-H), 7.70 (d, J = 7.5 Hz, 1H, BzIm-H), 7.41 (t, J = 8.0 Hz, 2H, BzIm-H), 6.29 (d, J = 6.0 Hz, 1H, p-cym-H), 6.23 (d, J = 6.1 Hz, 1H, p-cym-H), 6.16 (d, J = 6.0 Hz, 1H, p-cym-H), 6.11 (d, J = 6.0 Hz, 1H, p-cym-H), 2.77 (dd, J = 13.7 Hz, 1H,p-cym-isopropyl-CH), 2.33 (s, 3H, p-cym-Me), 1.05 (t, J = 6.6 Hz, 6H, p-cym-isopropyl). 13 C NMR (125 MHz, DMSO-d6, 298 K): δ 148.7, 144.2, 130.6, 124.13, 116.7, 114.6, 106.3, 100.4, 98.1, 83.0, 82.3, 81.8, 31.3, 22.00, 21.8, 19.9 (Figures S24−S26). IR (KBr pellets, cm−1): 3418, 1773, 1582, 1389, 842. UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 307 (16840), 447 (979) (Figure S33). ESI-HRMS (MeOH) for [6 − I]+ ≡ [C20H21BrIN4Ru]+: m/z 624.8998 (calcd m/z 624.9038). Anal. Calcd for C20H21BrI2N4Ru: C, 31.94; H, 2.81; N, 7.45. Found: C, 31.72; H, 2.71; N, 7.27. [RuII(p-cym)(L4)Cl]Cl (7). Yield: 85%. 1H NMR (500 MHz, DMSO-d6, 298 K): δ 9.12 (s, 1H, pyz-H), 8.98 (s, 1H, pyz-H), 7.97 (d, J = 7.8 Hz, 1H, BzIm-H), 7.68 (d, J = 7.7 Hz, 1H, BzIm-H), 7.42 (dt, J = 7.2 Hz, 2H, BzIm-H), 6.35 (d, J = 5.9 Hz, 1H, p-cym-H), 6.30 (d, J = 6.0 Hz, 1H, p-cym-H), 6.13 (t, J = 7.0 Hz, 2H, p-cym-H), 2.63−2.58 (m, 1H, p-cym-isopropyl-CH), 2.13 (s, 3H, p-cym-Me), 1.02 (dd, J = 6.8 Hz, 6H, p-cym-isopropyl). 13C NMR (125 MHz, DMSO-d6, 298 K): δ 151.7, 144.1, 138.99, 134.9, 124.4, 123.9, 116.8, 113.9, 103.9, 101.4, 83.3, 82.2, 81.6, 80.8, 30.6, 21.8, 18.4 (Figures S27−S29). IR (KBr pellets, cm−1): 3428, 1592, 1455, 1059, 856. UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 305 (14713), 420 (531) (Figure S33). ESI-HRMS (MeOH) for [7 − Cl − HCl]+ ≡ [C20H20IN4Ru]+: m/z 544.9723 (calcd m/z 544.9776). Anal. Calcd for C20H21Cl2IN4Ru: C, 38.98; H, 3.43; N, 9.09. Found: C, 39.31; H, 3.54; N, 8.74. [RuII(p-cym)(L4)I]I (8). Yield: 87%. 1H NMR (500 MHz, DMSO-d6, 298 K): δ 9.09 (s, 1H, pyz-H), 8.81 (s, 1H, pyz-H), 7.89 (d, J = 7.7 Hz, 1H, BzIm-H), 7.71 (d, J = 7.4 Hz, 1H, BzIm-H), 7.43 (t, J = 7.3 Hz, 2H, BzIm-H), 6.30 (d, J = 6.0 Hz, 1H, p-cym-H), 6.23 (d, J = 6.0 Hz, 1H, p-cym-H), 6.16 (d, J = 6.1 Hz, 1H, p-cym-H), 6.11 (d, J = 6.0 Hz, 1H, p-cym-H), 2.80−2.74 (m, 1H, p-cym-isopropyl-CH), 2.32 (s, 3H, p-cym-Me), 1.05 (t, J = 6.4 Hz, 6H, p-cym-isopropyl). 13C NMR (125 MHz, DMSO-d6): δ 149.3, 144.5, 140.5, 131.0, 124.4, 124.1, 123.6, 117.3, 114.3, 106.8, 101.1, 98.0, 83.4, 82.3, 81.3, 31.3, 22.3, 21.8, 19.9 (Figures S30−S32). IR (KBr pellets, cm−1): 3425, 1955, 1591, 1152, 695. UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 308 (17933), 446 (1039) (Figure S33). ESI-HRMS (MeOH) for [8 − I]+ ≡ [C20H21I2N4Ru]+: m/z 672.8870 (calcd m/z 672.8899). Anal. Calcd for C20H21I3N4Ru: C, 30.06; H, 2.65; N, 7.01. Found: C, 30.21; H, 2.71; N, 6.93. X-ray Crystallography. The attempts to crystallize the complexes provided us with good-diffraction-quality single crystals of complexes 1 and 3. The crystals were obtained by layering diethyl ether over a methanol (MeOH) solution of 1 or 3 and keeping them in a Synthesis of Metal Complexes 1−8. General Procedure of the Syntheses of Complexes 1−8. The ligands L1−L4 (0.098 mmol) were added in 15 mL of dry DCM under an inert atmosphere, leading to a suspension. The metal precursor [RuII2(p-cymene)2Cl4] (0.049 mmol; for 1, 3, 5, and 7) or [RuII2(p-cymene)2I4] (0.049 mmol; for 2,4,6, and 8) in 10 mL of dry DCM was added dropwise under a N2 atmosphere to the above suspension. The solution slowly became transparent. By the time the addition was complete, the color had changed from orange to yellow (for 1, 3, 5, and 7) or dark red to orange-red (for 2, 4, 6, and 8). Then turbidity appeared, followed by precipitation. The reaction was left for 24 h to complete and washed five times with diethyl ether (10 mL each) to get rid of excess ligand. The product was collected by vacuum filtration and dried under high vacuum. The purity was confirmed by 1H and 13C NMR, ESI-HRMS, and elemental analysis. [RuII(p-cym)(L1)Cl]Cl (1). Yield: 88%. 1H NMR (500 MHz, DMSO-d6, 298 K): δ 8.96 (d, J = 1.7 Hz, 1H, pyz-H), 8.88 (d, J = 2.9 Hz, 1H, pyz-H), 7.99 (d, J = 7.8 Hz, 1H, BzIm-H), 7.69 (d, J = 7.8 Hz, 1H, BzIm-H), 7.48−7.40 (m, 2H, BzIm-H), 7.06−7.03 (m, 1H, pyz-H), 6.33 (dd, J = 9.6 and 6.2 Hz, 2H, p-cym-H), 6.18 (d, J = 6.0 Hz, 1H, p-cym-H), 6.10 (d, J = 6.0 Hz, 1H, p-cym-H), 2.59 (dd, J = 13.7 Hz, 1Hp-cym-isopropyl-CH), 2.15 (s, 3H, p-cym-Me), 1.03 (d, J = 6.9 Hz, 3H,p-cym-isopropyl), 0.98 (d, J = 6.9 Hz, 3H, p-cymisopropyl). 13C NMR (125 MHz, DMSO-d6, 298 K): δ 148.1, 144.9, 139.1, 130.9, 124.2, 123.8, 116.7, 113.8, 112.1, 103.4, 101.6, 83.6, 82.3, 81.4, 80.3, 30.5, 21.8, 21.7, 18.4 (Figures S9−S11). IR (KBr pellets, cm−1): 3419, 1767, 1595, 1351, 749. UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 298 (21016), 417 (606) (Figure S33). ESIHRMS (MeOH) for [1 − Cl − HCl]+ ≡ [C20H21N4Ru]+: m/z 419.0770 (calcd m/z 419.0810). Anal. Calcd for C20H22Cl2N4Ru: C, 48.99; H, 4.52; N, 11.43. Found: C, 49.19; H, 4.60; N, 11.83. [RuII(p-cym)(L1)I]I (2). Yield: 82%. 1H NMR (500 MHz, DMSO-d6, 298 K): δ 8.92 (d, J = 1.9 Hz, 1H, pyz-H), 8.70 (d, J = 3.0 Hz, 1H, pyz-H), 7.90 (d, J = 7.1 Hz, 1H, BzIm-H), 7.73 (d, J = 7.1 Hz, 1H, BzIm-H), 7.49−7.39 (m, 2H, BzIm-H), 7.08−7.02 (m, 1H, pyz-H), 6.27 (d, J = 6.4 Hz, 2H, p-cym-H), 6.14 (dd, J = 5.0 Hz, 2H, p-cymH), 2.76 (dt, J = 13.8 Hz, 1H, p-cym-isopropyl-CH), 2.34 (s, 3H, pcym-Me), 1.05 (dd, J = 6.9 Hz, 6H, p-cym-isopropyl). 13C NMR (125 MHz, DMSO-d6, 298 K): δ 149.6, 140.2, 130.9, 124.2, 123.9, 117.1, 114.5, 112.7, 106.1, 100.7, 83.4, 82.5, 82.2, 31.6, 22.4, 21.9, 20.3 (Figures S12−S14). IR (KBr pellets, cm−1): 3420, 1767, 1594, 1355, 776. UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 296 (27823), 444 (882) (Figure S33). ESI-HRMS (MeOH) for [2 − I]+ ≡ [C20H22IN4Ru]+: m/z 546.9952 (calcd m/z 546.9933). Anal. Calcd for C20H22I2N4Ru: C, 35.68; H, 3.29; N, 8.32. Found: C, 35.39; H, 3.14; N, 8.24. [RuII(p-cym)(L2)Cl]Cl (3). Yield: 90%. 1H NMR (500 MHz, DMSO-d6, 298 K): δ 9.34 (s, 1H, pyz-H), 9.29 (s, 1H, pyz-H), 8.01 (d, J = 7.8 Hz, 1H, BzIm-H), 7.69 (d, J = 7.7 Hz, 1H, BzIm-H), 7.45 (dd, J = 7.8 Hz, 2H, BzIm-H), 6.37 (d, J = 6.0 Hz, 1H, p-cymH), 6.34 (d, J = 6.1 Hz, 1H, p-cym-H), 6.16 (dd, J = 6.1 Hz, 2H, pcym-H), 2.65 (dt, J = 13.8 Hz, 1H, p-cym-isopropyl-CH), 2.14 (s, 3H, p-cym-Me), 1.06 (d, J = 6.9 Hz, 3H, p-cym-isopropyl), 1.02 (d, J = 6.9 Hz, 3H, p-cym-isopropyl). 13C NMR (125 MHz, DMSO-d6): δ 145.8, 144.9, 139.2, 128.8, 124.2, 123.8, 116.7, 114.2, 113.9, 103.9, 101.4, 83.2, 82.2, 81.6, 80.7, 30.5, 21.8, 18.4 (Figures S15−S17). IR (KBr pellets, cm−1) 3414, 1747, 1595, 1367, 804. UV−vis [MeOH; λmax, nm (ε, M−1 cm−1)]: 304 (11978), 415 (523) (Figure S33). ESIHRMS (MeOH) for [3 − Cl − HCl]+ ≡ [C20H20ClN4Ru]+: m/z 453.0416 (calcd m/z 453.0420). Anal. Calcd for C20H21Cl3N4Ru: C, 45.77; H, 4.03; N, 10.68. Found: C, 45.64; H, 3.94; N, 10.71. [RuII(p-cym)(L2)I]I (4). Yield: 85%. 1H NMR (500 MHz, DMSO-d6, 298 K): δ 9.17 (s, 1H, pyz-H), 8.92 (s, 1H, pyz-H), 7.88 (d, J = 7.8 Hz, 1H, BzIm-H), 7.70 (d, J = 7.5 Hz, 1H, BzIm-H), 7.40 (dd, J = 7.9 Hz, 2H, BzIm), 6.29 (d, J = 6.0 Hz, 1H, p-cym-H), 6.23 (d, J = 6.1 Hz, 1H, p-cym-H), 6.16 (d, J = 6.1 Hz, 1H, p-cym-H), 6.12 (d, J = 6.0 Hz, 1H, p-cym-H), 2.78 (dt, J = 13.6 Hz, 1H, p-cym-isopropyl-CH), 2.34 (s, 3H, p-cym-Me), 1.06 (t, J = 7.1 Hz, 6H, p-cym-isopropyl). 13 C NMR (125 MHz, DMSO-d6, 298 K): δ 146.9, 144.3, 140.1, 128.6, 124.1, 123.8, 116.7, 114.6, 114.1, 106.3, 100.4, 82.9, 82.3, 81.8, 31.3, 18382 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Article Cell Line and Culture Condition. The MDA-MB-231 cells were obtained from NCCS, Pune, India. The cells were grown as an adherent monolayer in a 100 mm Petri dish in a 5% CO2 atmosphere using 1:1 mixture of Dulbecco’s modified Eagle medium (DMEM) and Ham’s F12 nutrient mixture as a culture medium, supplemented with 10% fetal bovine serum (FBS; Gibco) and antibiotics (100 unit mL−1 penicillin and 100 μg mL−1 streptomycin). The cell line was maintained at its logarithmic growth phase before every experiment and seeded when it became 70% confluent. Cell Viability Assay. The inhibitory effect of complexes 1−8 on the growth of the MDA-MB-231 cell line was assessed with the help of MTT assay. In brief, 6 × 103 cells well−1 were seeded in 96-well microplates in a DMEM/F12 culture medium (200 μL) and incubated at 37 °C in a 5% CO2 atmosphere. After 24 h of incubation, the existing medium was replaced by a fresh medium (200 μL). The complexes 1−8 to be tested were dissolved in a minimum volume of DMSO (for cisplatin, we used N,N-dimethylformamide),81 and an appropriate concentration was added in triplicate to the respective wells such that the concentration of DMSO does not exceed 0.2% in each well. After 72 h of incubation with the drug, the drug-containing medium was replaced with a fresh one, followed by the addition of 20 μL of a 1 mg mL−1 MTT solution in 1X PBS (pH 7.2). After incubation with MTT for 3 h at 37 °C, the medium was removed, and the purple crystals of formazan were dissolved in 200 μL of DMSO. The inhibition of the cell growth was evaluated by measuring the absorbance of the drug-treated wells with respect to the untreated controls at 570 nm using a Biotek H1M multimode plate reader. The IC50 values (drug concentrations responsible for 50% growth inhibition of the cells) were calculated by fitting nonlinear four-parameter curves using a dose-response inhibition−variable slope model through GraphPad Prism 5, version 5.03 (Figure S58). The data represented in Table 2 are the mean of three independent experiments, where each concentration was repeated in triplicate. VEGFR2 Inhibition: Western Blot and Immunoprecipitation (IP). To determine the effect of the ligands and corresponding complexes on VEGFR2 signaling in the MDA-MB-231 cell line, we investigated Y1175 phosphorylation of VGEFR2 and the total tyrosine phosphorylation of Src and ERK1/2.82,83 MDA-MB-231 cells were grown in DMEM/F12 (1:1), supplemented with 10% heat-inactivated FBS and antibiotics at 37 °C in 5% CO2. The cells were grown to 80% confluency and then treated with DMSO (0.2%) vehicle control or 5 and 10 μM L2, L4, 3, and 7. After incubation, the cells were activated by 60 nM VEGF165a for 5 min. The activated cells were harvested and lysed with a radio-immunoprecipitation assay lysis buffer [25 mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 1% Nonidet P40, and 1% Triton X-100], supplemented with 0.1 mM vanadate and a protease inhibitor cocktail (5 mg L−1 leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and 2 mM benzamidine). The cell lysate was sonicated and cleared by centrifuging at 500g for 10 min. One fraction of the supernatant was used for VEGFR2 immunoblot; another fraction was incubated with 5 μg of Src antibody and 3.5 μg of ERK1/2 antibody overnight at 4 °C IP. After overnight incubation, it was further incubated with 30 μL of protein G beads for 2 h. The beads were washed four times with icecold PBS, supplemented with 2 mM sodium orthovanadate. The samples were boiled with a Laemmle sample buffer and run in sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE). Following SDS-PAGE, lysates were transferred onto nitrocellulose paper and blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 2 h at room temperature. The blocked membrane was incubated overnight at 4 °C with anti Src, ERK1/2, or VEGFR2 antibody for checking the loading and with anti pY1175 VEGFR2 or anti pY antibody for checking the phosphorylation level, followed by 2 h of incubation with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (Figures 7 and S59). Cell Cycle Analysis. The 2 ×105 MDA-MB-231 cells were seeded in a 6-well plate in a DMEM/F12 culture medium and incubated at 37 °C in a 5% CO2 atmosphere. The medium was renewed after 48 h. Then the IC20 and IC50 concentrations of complexes 3 and 7 were added and incubated for 8 h. After drug exposure, cells were harvested refrigerator in sealed condition for 2 weeks. Suitable yellow-orange transparent single crystals were mounted over a goniometer loop of a SuperNova, Dual, Cu at zero, Eos diffractometer. The data collection temperature was 293(2) K for 1 and 100(10) K for 3 with Cu Kα as the X-ray source (λ = 1.5406 Å). Data reduction was performed with CrysAlisPro 171.37.33c (Agilent Technologies). Finally, the structures were solved using the ShelXT structure solution program78 with intrinsic phasing and refined with the ShelXL78 refinement package using least-squares minimization in Olex2.79 An anisotropic displacement parameter was used for all of the non-hydrogen atoms. All of the hydrogen atoms were calculated and fixed after hybridization of the non-hydrogen atoms (Figures 2 and S34). A few important structural pieces of information and crystallographic parameters are listed in Tables 1 and S1, respectively. The deposition numbers for the crystal structures are CCDC 2087583 (1) and 2087584 (3). The residual electron density of 2.6 Å3 at a distance of 0.9 Å from Ru1 for complex 3 is due to the relatively poor quality of the crystal. Solution Stability. A stability study of complexes 3, 5, 7, and 8 was performed using 1H NMR in dimethyl sulfoxide (DMSO)-d6 and a 10 mM phosphate buffer at pH* 7.4 [pH meter reading without correction for the effects of deuterium on the glass electrode; 3:7 (v/ v)] containing 4 mM NaCl at 37 °C at various time intervals for 72 h (Figures 3 and S35−S38). The halide exchange studies of the iodido complexes 4, 6, and 8 were performed at 27 °C for 24 h similarly except that the NaCl concentration was 130 mM (Figures S39−S41). Determination of the Distribution Coefficient. The distribution coefficient (log Do/w) was determined by following the OECD guidelines80 using the shake-flask method. A known amount of the respective complex (1−8) is solubilized in n-octanol (presaturated with deionized water) and continuously shaken for 6 h on an orbital shaker at 37 °C. Upon completion, the biphasic solutions were centrifuged for 3 min to allow complete phase separation. Then aliquots from each layer were measured separately upon adequate dilution (ca. 10 times diluted) in a UV−vis spectrophotometer using proper dilution to obtain the absorbance below 1 for absorption maxima of ca. 298−308 nm. The concentration of the complexes remaining in the n-octanol phase was determined and subtracted from the originally dissolved concentration, which gave the concentration of the complexes in the water phase. The distribution coefficients (log Do/w) of the complexes were obtained from the concentration ratio of the complexes present in the n-octanol and water phase (Figure 4A). Cellular Accumulation of Ru by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). Around 5 × 105 of the metastatic triple-negative breast carcinoma (MDA-MB-231) cells were seeded in a 100 mm sterile tissue culture Petri dish and grown for 48 h, leading to 80% confluency of the cells. To these cells in the respective wells, 10 μM metal complexes (1−8) were added and incubated for another 12 h. After 12 h, the medium was discarded, and the cells were thoroughly washed with 1X phosphate-buffered saline (PBS; pH 7.2). The drug-treated cells were then harvested by trypsinization, and each set of duplicates was counted accurately, followed by centrifugation to obtain cell pellets. The cell pellets were washed twice by 1X PBS (pH 7.2). Finally, the cells were digested with 200 μL of extra pure [70% (v/v)] nitric acid (Sigma-Aldrich) at 70 °C for 12 h. The digested cell suspension was then diluted to 10 mL using Milli-Q water, and the Ru content in the samples was analyzed on a Thermo Scientific iCAP 7400 ICP-OES instrument. Ru standard solutions were freshly prepared before the experiments to generate the best calibration curve in GraphPad Prism, version 5.03 (Figure 4B). Interaction with the Model Nucleobase 9-Ethylguanine (9EtG). Complexes 3 and 7 were studied with 9-EtG using 1H NMR in DMSO-d6/10 mM phosphate buffer [3:7 (v/v)] at pH* 7.4 containing 4 mM NaCl at 37 °C at various time intervals for a 72 h period. The binding to 9-EtG was confirmed by the downfield chemical shift of H8 of 9-EtG along with analysis of the other required peaks (Figures 6C and S42 and S43). The 9-EtG-bound species were identified by ESI-MS using 1:9 (v/v) MeOH/10 mM phosphate buffer of pH 7.4 and 4 mM NaCl (Figures 6A,B and S54−S57). 18383 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC quickly by trypsinization and washed twice with cold 1X PBS (pH 7.2). The cells were resuspended in 100 μL of cold 1X PBS and fixed with 70% chilled aqueous ethanol overnight at 4 °C. DNA staining was performed by resuspending the cell pellets in a 1X PBS solution containing a PI (55 μg mL−1) and RNase A (100 μg mL−1) solution. The cell suspension was gently mixed with a PI staining solution and incubated at 37 °C for 30 min. Samples were analyzed in a BD Biosciences FACSCalibur or BD LSRFortessa flow cytometer (Figures 8C,D and S60). Detection of Apoptosis: Annexin-V-PE/7AAD Assay. A flow cytometric method was used for apoptosis detection using an Annexin V-PE/7-AAD dual-staining detection kit (BD Pharmigen). The manufacturer’s protocol was followed. Briefly, 5 × 104 cells were seeded in a 35 mm Petri dish with 2 mL of a DMEM/F12 cell culture medium. The cells were incubated for 48 h in a 5% CO2 environment. After that, the medium was renewed, and the cells were treated with an appropriate concentration of the drug solution (IC20 and IC50) of complexes 3 and 7 for 8 h. The cells were harvested with cold 1X PBS containing 0.1 mM EDTA, followed by washing twice with cold 1X PBS. Then the cells were resuspended in a 1X Annexin V binding buffer. The cells were incubated with both Annexin V-PE and 7-AAD in dark conditions for 15 min at 25 °C. Data were collected and analyzed in a BD Biosciences FACS Calibur or a BD LSRFortessa flow cytometer within 1 h of sample preparation (Figures 8A,B and S61). In Vivo Antiangiogenesis on Zebrafish. Animal Husbandry and Breeding. Zebrafish usually start breeding at the onset of light (7 AM). Pairwise breeding was set on a sloping breeding tank filled with water in the evening of the previous day after feeding at 6:30 PM. Four females and two males were kept in the breeding tank to maintain a female/male ratio of 2:1. They frequently bred the next morning when the light appeared at 7 AM. Enough eggs (∼500 eggs) were found at the bottom of the tank. These eggs were collected subsequently using a strainer, transferred into the Petri dish (with an average of 25 embryos/Petri dish) by rinsing the strainer with a 1X E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 10−5% methylene blue), and kept in the incubator at 28.4 °C. Drug Treatment. A transgenic line of zebrafish embryos [Tg(fli1:gfp)] was incubated overnight with drugs (L2, 25 μM; 3, 50 μM) along with the vehicle control DMSO at the 13 somite stage, approximately 14 h postfertilization (hpf) before intersegmental vessels (ISVs), dorsal aorta, duct of Cuvier, and caudal vein were formed. The embryos were treated with specific doses of the drug and transferred into a fresh 1X E3 medium after 12 h of drug incubation. The embryos were treated in the anesthetic Tricaine (ethyl 3aminobenzoate) solution (25× stock solution of Tricaine at 4 mg mL−1 in 20 mM Tris pH 8.8 and brought down to pH 7, aliquoted by 4 mL, and stored at −20 °C) for a few minutes to make them still, followed by taking multiple confocal images at 30 and 48 hpf using a Nikon confocal microscope, and the corresponding images were processed in NIS-Elements viewer 5.21 software (Figure 9). Article NaOH solution to adjust the pH at ca. 7−8 for adequate precipitation. All of the pure ligands (L1−L4) were isolated in good yields (63−98%) after column chromatography. The chlorido complexes (1, 3, 5, and 7) were synthesized from RuII2(p-cym)2Cl4, and the iodido complexes (2, 4, 6, and 8) were synthesized from RuII2(p-cym)2I4 by refluxing the respective bidentate ligands L1−L4 in dry DCM with stirring for 24 h under an inert atmosphere. All metal complexes (1− 8) were characterized by 1H, 13C, and HMQC NMR, FT-IR, and UV−vis spectroscopy along with ESI-HRMS. Complexes 1 and 3 were also characterized by single-crystal X-ray crystallography. The bulk purity of the complexes was determined by elemental analysis. The UV−vis spectra of the complexes showed two different bands at 250−330 nm, corresponding to a π−π* transition in the ligands, and 400− 500 nm, which may correspond to contributions from both charge-transfer and d−d transitions (Figure S33). X-ray Crystallography. Suitable good-quality single crystals of complexes 1 and 3 were obtained by layering their respective methanolic solution with diethyl ether. Complexes 1 and 3 crystallize as a mixture of enantiomers (racemic mixtures) in the monoclinic crystal system in space groups P21/n and P21/c, respectively (Table S1). The singlecrystal structures of 1 and 3 revealed that the RuII center is coordinated by Npz−NBz of the bidentate ligands along with pcymene and chloride. The p-cymene ring is bound to Ru in a η6 fashion. In complex 1, the methyl group of p-cymene is directed toward the bidentate ligand, whereas the isopropyl group is used for for complex 3. Hence, the Ru1−N1 bond length in complex 3 (2.112 Å) is higher than that of complex 1 (2.097 Å; Table 1). The presence of an electronegative chloride atom with a pyrazole ring in complex 3 has no effect on the structure because the Ru1−N3 bond length (2.107 Å) is almost equal to complex 1 (2.108 Å; Table 1). Each unit cell consists of four molecules of 1 and 3 with chloride counteranions (Figure S34) to balance the residual monopositive charge of RuII. The RuII center adopts a pseudooctahedral geometry (Figure 2). The Ru−C bond distances in pcymene range from 2.16 to 2.20 Å for both 1 and 3. The Ru− Cl bond distances in both complexes are ca. 2.40 Å. The bond angles of Npz−Ru−Cl in 1 and 3 (∠N3−Ru1−Cl1) are ca. 85.9° and 85.6°, respectively. The bond angles of ∠NBz−Ru− Cl in both complexes (∠N1−Ru1−Cl1) are ca. 85.4° and ■ RESULTS AND DISCUSSION Synthesis of the N,N-chelating ligands L1−L4 used direct and metal-free amination of the cyclic urea with halo-substituted pyrazoles by refluxing in POCl3 for 16 h (Scheme 1). L1 and L2 were synthesized by a three-component one-pot approach using o-phenylenediamine, triphosgene, and pyrazoles.84 The cyclic urea, an essential intermediate for L3 and L4, was synthesized from o-phenylenediamine and CDI in a dry THF/ DCM mixture at low temperature under an inert atmosphere.85 L3 and L4 were synthesized from the cyclic urea and respective pyrazoles using CDI coupling.86,87 We have employed a onepot coupling method to prepare four ligands (L1−L4)84 as discussed in the Experimental Section. All of the reactions were conducted in a Teflon-cap-sealed glass tube. After completion of the reaction, excess POCl3 was quenched with a saturated Figure 2. ORTEP diagram of complexes 1 and 3. Thermal ellipsoids are drawn at 50% probability. All hydrogen atoms and counteranions were omitted for clarity. 18384 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Article Table 1. Selected Bond Distances (Å) and Angles (deg) for Complexes 1 and 3 as Labeled in Figure 2 1 Ru1−Cl1 Ru1−N1 Ru1−N3 Ru1−C11 Ru1−C12 Ru1−C13 Ru1−C14 Ru1−C15 Ru1−C16 2.404(5) 2.097(2) 2.108(2) 2.202(2) 2.173(2) 2.163(2) 2.193(2) 2.194(2) 2.177(2) 3 N1−Ru1−N3 N1−Ru1−Cl1 N3−Ru1−Cl1 N1−Ru1−C11 N1−Ru1−C12 N1−Ru1−C13 N1−Ru1−C14 N1−Ru1−C15 N1−Ru1−C16 76.05(8) 85.43(5) 85.89(5) 166.83(8) 149.91(8) 113.49(8) 92.54(8) 100.00(8) 128.60(8) Ru1−Cl1 Ru1−N1 Ru1−N3 Ru1−C11 Ru1−C12 Ru1−C13 Ru1−C14 Ru1−C15 Ru1−C16 2.401(2) 2.112(6) 2.107(7) 2.197(10) 2.188(8) 2.185(8) 2.219(8) 2.181(7) 2.182(9) N3−Ru1−N1 N1−Ru1−Cl1 N3 Ru1 Cl1 N1−Ru1−C11 N1−Ru1−C12 N1−Ru1−C13 N1−Ru1−C14 N1−Ru1−C15 N1−Ru1−C16 75.9(2) 86.41(19) 85.67(19) 92.10(3) 101.2(3) 130.8(3) 168.4(3) 148.2(3) 112.2(3) Figure 3. Solution stability of the chlorido complexes 3 and 7 in a 10 mM phosphate buffer (pH* 7.4) with 4 mM NaCl in 30% DMSO-d6. Aquated species are shown by $, and free p-cymene species are shown by @. coordinated ruthenium(II) p-cymene complexes are reported, and the values endorse their high stability in aqueous solution.26,89,90 The iodido complex 8 is more than 95% intact and displays no arene loss or formation of the aquated complex like 7 during the 72 h period at 37 °C (Figure S38).The ruthenium(II) p-cymene complexes 7 and 8, of L4, are the most soluble ones. In the presence of 130 mM NaCl, the chloride exchange initiated rapidly in the iodido complexes 4, 6, and 8 at 27 °C, and after ca. 50% exchange, there is equilibration under the NMR tube conditions and the data show no further change up to the monitored period of 24 h (Figures S39−S41). The ESI-MS studies also support the NMR data that the chlorido coordination is more labile than the iodido coordination. A 200 μM stock solution of the chloridocoordinated complexes 3 and 7 in 1:9 (v/v) MeOH/phosphate buffer of pH 7.4 containing 4 mM NaCl shows a relative decline in the intensity of the molecular-ion peak during the 24 h period (Figures S44−S49). ESI-MS of the iodido complex 8 shows a relatively higher population of the molecular-ion peak during the 24 h time period (Figures S50−S52). However, in a ESI-MS stock solution where the concentration of the sample is low (200 μM), the iodido complex 8 exchanges the 86.4°, respectively. The Ru−N and Ru−Cl distances and N− Ru−Cl angles are in good agreement with similar complexes65,88 and support the deviation from the ideal octahedral geometry. The counteranion chloride is hydrogen-bonded (intramolecular) with N−H of benzimidazole (N−H···Cl), displaying distances of 3.009 and 3.058 Å respectively in 1 and 3 (Figure S34). Solution Stability. The RuII complexes were selectively investigated through 1H NMR for aqueous stability by dissolving in a mixture of 3:7 (v/v) DMSO-d6/10 mM phosphate buffer (pH* 7.4). The chlorido complexes (3, 5, and 7) were studied in the presence of 4 mM NaCl at 37 °C for 72 h, and they aquated immediately under these conditions. After formation of the aquated complexes, there is very little change, showing that the aquated complexes are the major form (ca. 70%) in solution during the 72 h period (Figures 3 and S35, S36, and S37). There is a small amount of arene loss (free p-cymene signals at 2.18, 1.10, and 7.18 ppm)65 starting from 48 h, which is less than 30% even at 72 h (indicated by @ in Figure 3). It must be noted here that we could not identify any peaks corresponding to an unbound ligand or the dinuclear ruthenium(II) p-cymene precursor in solution for any of the complexes. In many cases, the stability constants of N,N18385 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Article Figure 4. (A) Lipophilicity of metal complexes 1−8 in a 1:1 (v/v) noctanol and water mixture at 37 °C. (B) Cellular accumulation study of a RuII complex (10 μM) in MDA-MB-231 cells. coordinated halide with chlorido at both 4 or 130 mM NaCl in the 24 h time period (Figures S50−S53). Overall, the iodido complexes are more stable, but even the chlorido complexes are stable upon immediate monoaquation, and ca. 70% remains in solution even at 72 h. This is unlike our previous report, where the use of the bis(pyrazolylbenzimidazole) ligand of 2,4-dimethylpyrazole with ruthenium(II)-p-cymene showed the chlorido complexes to be more stable.65 The absence of a pyrazole group, the methyl substituents in the pyrazole motif, and the presence of a halide in the pyrazole have altered the electronic parameters, making the ruthenium(II) iodido coordination stronger. Thus, substitution by a halide in the 4 position of the pyrazole did not change the solution stability of the complexes significantly (less than a 10% change in aqueous stability), but it may be argued that the solubility of complexes 7 and 8 of the iodosubstituted L4 is better with respect to the others in the same family presented here. Determination of the Distribution Coefficient. Lipophilicity is an essential parameter for the determination of the drug-likeness of a new molecule.91 According to the comprehensive medicinal chemistry database, pharmacophores generally should have lipophilicity values in the range from −0.4 to +5.6.92 We knew that the chlorido complexes aquate and there is arene loss in a longer period, so it may be argued, what is the merit of the lipophilicity study? Yet, we studied the distribution coefficient (log Do/w) because the equilibration is for a shorter period of time (6 h), and the partition coefficient trend is a good indication of the complex’s ability to traverse across the membrane. The method adopted was the octanol− water shake-flask method as per the literature.92 The data show that the lipophilicity increases with the introduction of halogen in the ligand (L1−L4) and alteration of the halogen coordinated to RuII (Figure 4A), with values increasing from ca. 0.5 in 1 to ca. 2.7 in 8. The data were measured in an unbuffered aqueous medium, but the pH remained between 7.1 and 7.4. The iodido-coordinated complexes (2, 4, 6, and 8) display higher lipophilicity values than their chlorido analogues Figure 5. 3D correlation diagram of the cellular accumulation, lipophilicity, and IC50 values. (1, 3, 5, and 7). The enhancement in the lipophilicity due to the incorporation of iodido coordination correlates well with our earlier results.88,93 Similarly, introducing the halide into the ligand and increasing its size while keeping the chlorido coordination the same shows an increase in the lipophilicity from complexes L1 to L4 (1 < 3 < 5 < 7). The presence of 4iodopyrazole in L4 makes the respective metal complex 7 much more lipophilic than the iodido-coordinated 2. Complex 8 has both the iodido-coordinated RuII and iodo-containing L4, displaying the highest lipophilicity of 2.64 ± 0.05 among the eight complexes. Cytotoxicity Studies. In vitro cytotoxicity of all four ligands (L1−L4) and metal complexes (1−8) against the TNBC cell line MDA-MB-231 showed improvement in a cytotoxic dose of the metal complexes with the introduction of halides in their corresponding ligand compared to the metal complexes of an unsubstituted pyrazolylbenzimidazole ligand. A TNBC cell line was chosen because of its reluctance to respond to many therapeutic measures other than chemotherapy because of the absence of three primary targeting receptors (estrogen, progesterone, and HER2).94 The TNBCs are known to have a very high relapse rate.95 The ligands were designed as antiangiogenesis agents, so we also measured their standalone toxicity. The IC50 of L1 is greater than 100 μM, and the IC50 of L2−L4 is greater than 70 μM, suggesting lower in vitro toxicity of the ligands (Figure S58). However, we do not see more than 75% killing by the ligands alone even at higher doses (200 μM), so we could not determine the IC50 using the Table 2. In Vitro Anticancer Activity of Complexes 1−8 in the Cancer Cell Line MDA-MB-231 under Normoxic Conditions after 72 h of Incubation with the Complex complexes IC50 ± SD (μM) a 1 2 3 4 5 6 7 8 CDDPb >75 >50 13.0 ± 1.6 9.7 ± 0.5 11.9 ± 0.7 9.5 ± 0.2 10.1 ± 2.2 9.0 ± 1.8 14.1 ± 0.5 IC50 ± SD is determined by MTT assay in normoxia (∼15% O2). IC50 values were calculated by nonlinear four-parameter curve fitting in a doseresponse inhibition−variable slope model using GraphPad Prism. SD = standard deviation. The indicative plots are provided in Figure S58. The data presented are the mean of at least three independent experiments; each concentration was assayed in triplicate in a single experiment. The statistical significance (P) of the data is >0.001 to <0.05. bCDDP = cisplatin. a 18386 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Article Figure 6. Interaction of complexes 3 and 7 with 9-EtG (2.5 equiv) in a 10 mM phosphate buffer containing 4 mM NaCl (pH* 7.4) shown in both ESI-MS and 1H NMR: (A) 9-EtG-bound species with complex 3 recorded right after 5 min; (B) 9-EtG-bound species with complex 7 recorded right after 5 min. (C) 1H NMR of complexes 3 and 7 with 9-EtG at different time intervals showing that the Ru center binds with 9-EtG. lipophilicity and better stability, enabling better internalization of intact complexes in cells. The approximate 5-fold increase in the cytotoxicity of 3 compared to 1 suggests that the introduction of a halide in the 4 position of the pyrazole in L2 has a strong impact on the cytotoxic efficacy, justifying the synthesis and study of various halide-substituted ligands and their corresponding complexes. Complex 8, which has 4iodopyrazole in the ligand (L4) instead of an unsubstituted pyrazole and Ru−I coordination, is at least 8 times more toxic than 1 with the unsubstituted pyrazole and Ru−Cl curve-fitting process, and only the lower range is provided. Certainly, the results show that the ligand is not as toxic in vitro compared to their corresponding RuII complexes (Table 2). The solution stability study of the chlorido analogues (3, 5, and 7) suggests immediate aquation, and it is mostly the aquated complexes that are responsible for the cytotoxicity. Most of the iodido complexes (2, 4, 6, and 8) exhibit higher toxicity than their respective chlorido analogues (1, 3, 5, and 7). The major factors in the increase of the cytotoxicity of the iodido analogues may be the combination of higher 18387 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Article Figure 7. Effect of L2, L4, 3, and 7 on the kinase activity in the MDA-MB-231 cell line. (A) Schematic representation of the VEGFR2 signaling pathway (left panel) and structural drawings of L2, L4, 3, and 7 (right panel). (B and C) Representative Western blot strips of total VEGFR2, Y1175-phosphorylated VEGFR2, ERK1/2, and Src, and phosphorylated ERK1/2 and Src after treatment with various concentrations of L2, L4, 3, and 7. The bar plots represent the quantification of Y1175 phosphorylation of VEGFR2 and the total phosphorylation level of immunoprecipitated Src and ERK1/2, indicating the concentration. coordination. Complex 2 with L1 and Ru−I coordination is also poor in cytotoxicity. Thus, 1 and 2, with no halide in the ligand, have poor cytotoxic profiles compared to the rest of the family, which have either a Cl, Br, or I in the ligand by using a 4-halo-substituted pyrazole. While Ru−I coordination increased the stability, introducing an iodo in the ligand improved the cytotoxicity (Table 2). The ruthenium(II) pcymene complex 3, having 4-chloropyrazole and a Ru−Cl bond, displays toxicity similar to that of the 4-iodopyrazolebearing L4-coordinated Ru−Cl or Ru−I complexes 7 and 8. The use of 4-bromopyrazole (5 and 6) also led to improvement in the cytotoxicity profile compared with 1 and 2. However, there is only a marginal difference in the cytotoxicity of 5 and 6 compared with the iodido-substituted 7 and 8. Thus, although the introduction of the halide in the unsubstituted pyrazole increased the cytotoxicity, the change of 18388 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Article Figure 8. In vitro mechanistic analysis against MDA-MB-231 using flow cytometry: induction of apoptosis by complexes 7 (A) and 3 (B) in a dosedependent (IC20 and IC50) manner; cell cycle analysis of complexes 7 (C) and 3 (D) in a dose-dependent (IC20 and IC50) manner. Figure 9. In vivo antiangiogenic activity of L2 and 3 on Tg(Fli1:gfp) zebrafish at different time points. The white arrows demonstrate the GFPexpressing areas of interest (duct of Cuvier, dorsal aorta, ISVs, and caudal vein) in the vehicle control (0.2% DMSO), while the red arrows dictate the affected parts in treated samples. Scheme 1. Synthetic Scheme of the Ligands L1−L4 and the RuII Complexes 1−8 18389 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Article VEGFR2 inhibition.65,100 Nevertheless, the inhibitory activity is suggesting that the ligand design has successfully rendered pyrazolylbenzimidazole-based ruthenium(II) p-cymene complexes, inhibiting VEGFR2 phosphorylation. The inhibition of phosphorylation was also found for other related kinases capable of acting downstream of VEGFR2 and independently, viz., ERK1/2 and Src.101,102 L2, L4, 3, and 7 exhibit less effect on ERK1/2 and more prominently affect Src phosphorylation. Thus, the results show that L2, L4, 3, and 7 inhibit VEGFR2 phosphorylation and affect the downstream Src signaling pathway more prominently (Figures 7 and S59). Treatment of the MDA-MB-231 cells with complexes 3 and 7 shows that they arrest the cell cycle in the G2/M phase at IC20 and IC50 values (Figures 8C,D and S60) and induce apoptosis (Annexin V-PE/7-AAD dual staining assay). The late apoptotic cell population increases compared to the control for IC50 doses of 3 and 7, respectively, upon 8 h of drug exposure (Figures 8A,B and S61). In vivo Antiangiogenesis on Zebrafish. The zebrafish (Danio rerio) made an appearance as an ideal vertebrate model to study numerous biological processes in the past decade because their semitransparent nature facilitates visualization and they have ca. 70% similarity with the human genome. Their high fecundity rates and the possibility for external fertilization and rapid growth and development ascribe them to an important embroyological model. Vascular studies on zebrafish provide insight into vasculogenesis and angiogenesis,103 and their semitransparent nature allows excellent optical clarity.104 In order to find out whether our in vitro inhibition of VEGFR2 phosphorylation translates to antiangiogenic activity in vivo, we used transgenic Tg(fli1:gfp) zebrafish expressing the GFP on endothelial cells during development as a tool to anticipate developing blood vessels. Chloride-conjugated ligand L2 and its corresponding chlorido metal complex 3 were chosen as representative compounds for this experiment because complexes 3−8 showed cytotoxicity in a similar range and L2 and 3 also showed good activity against phosphorylation of VEGFR2. Developing embryos treated for 12 h with either L2 (25 μM) or 3 (50 μM) at 14 hpf, followed by drug removal, showed that at 30 hpf there is no formation of the duct of Cuvier, defects in the formation of the ISVs, caudal vein, and development of dorsal aorta in comparison with development with the vehicle control (0.2% DMSO). However, the effect is more prominent in L2-treated zebrafish than 3. There is a partial reversal of the effect at 48 hpf after drug removal, but a persistent effect has been observed in various areas in the caudal vein and ISVs. We also noticed that the mortality rate is zero because there was no death in the experiments, taking (25 × 3) zebrafish at given doses. Thus, the ligands are mostly responsible for the antiangiogenic activity of the complexes based on our in vitro and in vivo findings. the halide from chloride to iodide in the 4 position of the pyrazole showed less dose improvement in cytotoxicity profile. Thus, complexes 4 and 6−8 exhibit similar IC50 values in the range of 9−10 μM but are more toxic than cisplatin against the TNBC cell MDA-MB-231.96 Cellular Accumulation. We investigated the cellular ruthenium uptake to compare the internalization, lipophilicity, cytotoxicity, and stability, with a variation of the halide in the ligand and in coordination to the ruthenium(II) p-cymene. ICP-OES measured the Ru content (nmol of Ru per 106 cells) in MDA-MB-231 cells after 16 h of treatment with 10 μM 1− 8. The cellular Ru uptake data (Figure 4B) correlate well with the lipophilicity and cytotoxicity (Figure 5). There is an overall increase in cellular accumulation because of the introduction of halogens, which increase the bulk in the ligand system and coordinate with RuII. Thus, complex 8 has the highest accumulation, and complex 7 is the next closest, but 7 has poorer solution stability and is marginally less cytotoxic than 8. Our observation is supported by similar results from earlier works with halogen-substituted hydroxyquinoline-based metal complexes.97,98 Complexes 4 and 6 are quite efficient despite their low cellular accumulation and almost similar in cytotoxicity to 7 and 8. Pathways of Cytotoxicity. Interaction with the Model Nucleobase 9-EtG. RuII/III complexes interact with both nucleobases and proteins.28,29,97 We used complexes 3 and 7 as representatives to investigate the DNA interaction with the model nucleobase 9-EtG. The cytotoxicity data of the metal complexes emphasized the importance of halide substitution in the pyrazolylbenzimidazole ligand system. We found that the toxicities of complexes 3−8 are similar. In addition, ligands L2 and L4 also showed better toxicity than the other two ligands, so complexes of the corresponding ligands 3 and 7, which aquate well, were taken for the 9-EtG binding studies. We observed that the complexes rapidly bind to 9-EtG (2.5 equiv) within 5−10 min of addition, confirmed by 1H NMR (Figure 6C) and ESI-HRMS (Figure 6A,B). A substantial downfield shift of the H8 proton was observed in 1H NMR after 10 min of addition (Figures 6C and S42 and S43). This downfield shift confirms that 9-EtG binds to RuII in the complex.98 The ESIHRMS studies showed the 9-EtG-bound adduct for 3 and 7 at m/z 632.1263 (calcd 632.1222) and 724.0592 (calcd 724.0578), respectively, after 24 h of incubation with 9-EtG (Figures S54−S57). VEGFR2 Inhibition: Effect on the Kinase Signaling Pathway. The RuII complexes demonstrate 9-EtG binding capability and much higher cytotoxicity than the ligands. The ligands were designed as the organic-directing groups to inhibit VEGFR2 phosphorylation. The effect of the ligands and metal complexes on phosphorylation of the tyrosine, Y1175, showed the signature of inhibition of VEGFR2 signaling activity.99 Activation of VEGFR2 by VEGF165a in the MDA-MB-231 cell line, followed by treatment with two doses (5 and 10 μM) of L2, L4, 3, and 7, showed that the ligands and corresponding complexes inhibit the phosphorylation at Y1175. The ligands are responsible for inhibiting VEGFR2 phosphorylation, and the quantitation data from Western blots suggest that L2 and L4 display similar activities, which are higher than their corresponding metal complexes 3 and 7 (Figures 7 and S59). It is important to note here that the ligands have IC50 values greater than 70 μM but they inhibit VEGFR2 phosphorylation at 5−10 μM. Thus, the cellular proliferation under in vitro conditions may not strongly depend on the ■ CONCLUSIONS The organometallic Ru(II) complex of N,N-chelating pyrazolylbenzimidazole ligands inhibit phosphorylation of VEGFR2 in VEGF165a-activated MDA-MB-231. The iodidocoordinated complexes (2, 4, 6, and 8) are more resistant toward aquation at physiological conditions (pH 7.4, 4 mM Cl−). The complexes display the potential ability to bind to DNA through the RuII center, whereas the ligands assist VEGFR2 inhibition. Complexes 3, 7 and their respective ligands (L2 and L4) inhibit the VEGFR2 signaling pathway, 18390 https://doi.org/10.1021/acs.inorgchem.1c02979 Inorg. Chem. 2021, 60, 18379−18394 Inorganic Chemistry pubs.acs.org/IC Tuhin Subhra Koley − Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur 741246, India Rahul Das − Department of Biological Sciences and Centre for Advanced Functional Materials, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur 741246, India; orcid.org/0000-0001-7841-8229 Moulinath Acharya − National Institute of Biomedical Genomics, Kalyani 741251, India downregulating phosphorylated VEGFR2, ERK1/2, and Src. Inhibition by the ligand is observed at nontoxic in vitro doses in comparison to the metal complex. The time-dependent 9EtG binding via 1H NMR shows that DNA may be a target for the metal complexes, providing a reason for their higher cytotoxicities. The in vivo studies on the Tg(fli1:gfp) zebrafish model show that antiangiogenic activity is observed in zebrafish treated with specific doses of L2 and 3 and the effect is more distinct in zebrafish treated with L2 than 3. In fact, 3-treated zebrafish show rather poor effects on the antiangiogenic activity. The scope of further functionalization of the halo derivatives of pyrazolylbenzimidazole ligands provides an excellent platform to tailor more potent VEGFR2-inhibiting metal complexes in future. ■ Complete contact information is available at: https://pubs.acs.org/10.1021/acs.inorgchem.1c02979 Author Contributions † These authors contributed equally to this paper. Author Contributions ASSOCIATED CONTENT The project was designed by A.M. A.C. performed the synthesis, characterization, NMR data for solution stability, lipophilicity, ICP-OES, cell cycle, and apoptosis. S.R. performed all of the NMR data for solution stability at 37 °C, 9-EtG reactivity, in vitro cytotoxicity data, cell cycle, and apoptosis, treated cells for immunoblots, helped in the in vivo zebrafish experiments, coordinated the manuscript writing, and verified the cell cycle and apoptosis data carried out by A.C. K.P. performed single-crystal X-ray crystallography and solved the structures. T.S.K. completed and verified the ESI-MS stability studies. M.P.C. carried out all of the kinase assays and coordinated manuscript writing, supervised by R.D. S.S.R. performed the in vivo experimentation on zebrafish under the supervision of M.A. A.M. supervised the overall work. All authors have approved the final version of the manuscript. * Supporting Information sı The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c02979. NMR spectra (1H and 13C) of ligands (Figures S1−S8) and complexes (Figures S9−S32; 1H, 13C, and HMQC), UV−vis spectra of all of the complexes (Figure S33), lattice packing and crystallographic parameter table (Figure S34 and Table S1), hydrolysis and 9-EtG binding of the complexes (1−8) by 1H NMR (Figures S35−S43) and ESI-MS (Figures S44−S57), IC50 plots of all complexes and ligands (Figure S58), uncropped Western blot (Figure S59), and flow cytometric determination of the cell cycle and apoptosis (Figures S60 and S61) (PDF) Notes The authors declare no competing financial interest. ■ Accession Codes CCDC 2087583 and 2087584 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. ■ Article ACKNOWLEDGMENTS We earnestly acknowledge SERB for financial support via Project EMR/2017/002324. We also thank IISER Kolkata for infrastructural and financial support. The authors acknowledge NIBMG Kalyani for providing intramural support to the NIBMG Zebrafish facility. A.C. thanks UGC, S.R. thanks Inspire, M.P.C. thanks CSIR, K.P. thanks UGC, and T.S.K. thanks IISER Kolkata for providing their research fellowships. The authors thank Tamal Ghosh for his help with flow cytometry. AUTHOR INFORMATION Corresponding Author Arindam Mukherjee − Department of Chemical Sciences and Centre for Advanced Functional Materials, Indian Institute of Science Education and Research (IISER) Kolkata, Mohanpur 741246, India; orcid.org/0000-0001-9545-8628; Email: a.mukherjee@iiserkol.ac.in ■ REFERENCES (1) Ndagi, U.; Mhlongo, N.; Soliman, M. E. Metal complexes in cancer therapy - an update from drug design perspective. Drug Des., Dev. Ther. 2017, 11, 599−616. (2) Florea, A.-M.; Buesselberg, D. Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers 2011, 3, 1351−1371. (3) Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7 (8), 573−584. (4) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The Next Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016, 116 (5), 3436− 3486. 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