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Synthesis, antineoplastic and cytotoxic activities of some mononuclear Ru(II) complexes.

PMID: 20128638
Journal of Enzyme Inhibition and Medicinal Chemistry, 2010; 25(4): 513–519 Journal of Enzyme Inhibition and Medicinal Chemistry 2010 Original article 00 Synthesis, antineoplastic and cytotoxic activities of some mononuclear Ru(II) complexes 00 513 Sreekanth Thota1, Subhas S. Karki2, K. N. Jayaveera3, Jan Balzarini4, and Erik De Clercq4 519 1Department of Pharmaceutical Chemistry, S.R. College of Pharmacy, Ananthasagar, Warangal, Andhra Pradesh, India, 2Department of Pharmaceutical Chemistry, KLES College of Pharmacy, Rajajinagar, Bangalore, Karnataka, India, 3Department of Chemistry, JNTU College of Engineering, Ananthapur, Andhra Pradesh, India, and 4Rega Institute for Medical Reasearch, Katholieke Universiteit Leuven, Leuven, Belgium abstract A series of mononuclear Ru(II) complexes of the type [Ru(S)(K)]2+, where S = 1,10-phenanthroline/2,2’-bipyridine 2 and K = 4-OH-btsz, 4-CH-btsz, 3,4-di-OCH-btsz, 4-OH-binh, 4-CH-binh, 3,4-di-OCH-binh, were prepared and 3 3 3 3 characterized by elemental analysis, FTIR, 1H-NMR, and mass spectroscopy. The complexes displayed metal–ligand charge transfer (MLCT) transitions in the visible region. These ligands formed bidentate octahedral ruthenium complexes. The title complexes were evaluated for their in vivo anticancer activity against a transplantable murine tumor cell line, Ehrlisch’s ascites carcinoma (EAC), and in vitro cytotoxic activity against human cancer cell lines Molt 4/C and CEM and murine tumor cell line L1210. The ruthenium complexes showed promising biological 8 activity especially in decreasing tumor volume and viable ascites cell counts. Treatment with these complexes prolonged the life span of mice bearing EAC tumors by 10–52%. In vitro evaluation of these ruthenium complexes revealed cytotoxic activity from 0.21 to 24 µM against Molt 4/C, 0.16 to 19 µM aginst CEM, and 0.75 to 32 µM 8 against L1210. Keywords: Ruthenium complexes; anticancer; isonicotinyl hydrazones; thiosemicarbazones Introduction and their antitumor property was done at the beginning The success of cisplatin and related platinum complexes as of the 1980s with the compounds fac-[RuCl(NH)] and 3 3 3 anticancer agents has stimulated a search for other active cis-[RuCl(NH)]Cl6, preceded by the discovery in the 1970s 2 3 4 transition metal complexes, and ruthenium in particular has that ruthenium red possesses antitumor properties7,8. Since ENZ attracted research1. Metal complexes of ruthenium contain- then, compounds such as trans-(IndH)[Ru(ind)Cl] (Ind = 2 4 ing nitrogen and oxygen donor ligands are found to be effec- indazole), mer-[Ru(terpy)Cl] (terpy = 2,2’-terpyridine)9–11, 3 435935 tive catalysts for oxidation, reduction, hydrolysis, and other [Ru(dmso)Cl] (dmso = dimethyl sulfoxide)12, ImH[Ru(im) 4 2 organic transformations2. The coordination environment Cl]13, ImH[Ru(im)-Cl]14, and ImH[Ru(im)(dmso)Cl]15 5 2 4 4 around ruthenium plays a key role in stabilizing its different (NAMI-A) (im = imidazole) have also become well-known 28 June 2009 oxidation states and hence dictates the redox properties of antitumor agents. the control atoms3,4. Although the mechanism of action of ruthenium com- Ruthenium compounds are regarded as promising alter- pounds is not fully understood, it is thought that, for certain 11 September 2009 natives to platinum compounds, and offer many approaches species, it is similar to that of platinum drugs16,17. NAMI-A to innovative metallopharmaceuticals. The compounds are has high selectivity for solid tumor metastasis and low host 22 September 2009 known to be stable and have predictable structures both in toxicity at pharmacologically active doses18, and it was the the solid state and in solution. The tuning of ligand affini- first ruthenium compound to enter clinical trials19. It has a ties is accompanied by a steadily increasing knowledge of remarkably low general toxicity20,21 and shows marked effi- 1475-6366 the biological effects of ruthenium compounds1,5. The cacy against metastases22,23. It does not affect primary tumor first systematic investigation of ruthenium compounds growth24,25 and does not exhibit cytotoxicity against tumor 1475-6374 Address for Correspondence: Sreekanth Thota, (Ph.D), Assistant Professor, Department of Pharmaceutical Chemistry, S.R. College of Pharmacy, Ananthasagar, Warangal-506371, Andhra Pradesh, India. Tel: +91 0870 2425520. E-mail: sri_237@yahoo.co.in © 2010 Informa UK Ltd (Received 28 June 2009; revised 11 September 2009; accepted 22 September 2009) 10.3109/14756360903357577 ISSN 1475-6366 print/ISSN 1475-6374 online © 2010 Informa UK Ltd DOI: 10.3109/14756360903357577 http://www.informahealthcare.com/enz 41/81/60 no lavaL fo ytisrevinU yb moc.erachtlaehamrofni morf dedaolnwoD yrtsimehC lanicideM dna noitibihnI emyznE fo lanruoJ .ylno esu lanosrep roF 514 Sreekanth Thota et al. cells in vitro. A related ruthenium(III) compound, indazolium 4-OH-btsz Yield 56%, m.p. 224–225°C (lit., 226°C). IR trans[tetrachlorobis (1H-indazole) ruthenate(III)], KP101926, (KBr) cm−1: 3469–3320 (NH and NH), 3200–2700 (O-H), 3133 2 has also entered clinical trials, since it was found to exhibit (C-H), 1610 (N-H), 1328 (C=S). Calcd. for CHNOS: C, 49.21; 8 9 3 antiproliferative activity in vitro in human colon carcinoma H, 4.64; N, 21.52. Found C, 49.20; H, 4.62; N, 21.28%. λ nm max cell lines27. (MeOH): 242, 321, 398. 1H NMR (DMSO-d): δ = 12.6 (1H, s), 6 In comparing the general toxicity of ruthenium com- 11.24 (1H, s), 8.07 (1H, s), 7.99 (1H, s), 7.89 (1H, s, -OH), 7.73 pounds with platinum drugs, ruthenium has lower toxic- (2H, d, J = 8.6 Hz), 6.95 (2H, d, J = 8.6 Hz). ity, which has been attributed to the ability of ruthenium 4-CH-btsz Yield 79%, m.p. 160–162°C (lit., 160–161°C). 3 compounds to specifically accumulate in cancer tissues. The IR (KBr) cm−1: 3416–3321 (NH and NH), 3151 (C-H), 1615 2 higher specificity of these compounds for their targets may (N-H), 1325 (C=S). Calcd. for CH NS: C, 55.93; H, 5.74; 9 11 3 be linked to selective uptake by the tumor compared with N,21.74. Found C, 55.87; H, 5.62; N, 21.53%. λ nm (MeOH): max healthy tissue28,29 and selective activation by reduction to 234, 325, 389. 1H NMR (DMSO-d): δ = 11.41 (1H, s), 8.10 (1H, 6 cytotoxic species within the tumor30. s), 7.98 (1H, s), 7.78 (1H, s), 7.71 (2H, d, J = 8.9 Hz), 6.98 (2H, Ruthenium compounds with bidentate ligands show d, J = 8.9 Hz), 1.64 (3H, s, CH). 3 intercalation properties with DNA31. The Ru(II) com- 3,4-di-OCH-btsz Yield 56%, m.p. 194–195°C (lit., 195°C). 3 pounds are kinetically more reactive than Ru(III)32. We have IR (KBr) cm−1: 3406–3320 (NH and NH), 3133 (C-H), 1610 2 reported that Ru(II) compounds bearing thiosemicarbazides, (N-H), 1332 (C=S). Calcd. for C H NOS: C, 50.19; H, 5.47; 10 13 3 2 8-h ydroxyquinolines, and 4-substituted thiopicolinanalides N,17.56. Found C, 50.21; H, 5.61; N, 17.43%. λ nm (MeOH): max have in vivo anticancer and in vitro antibacterial activity33–35. 239, 331, 395. 1H NMR (DMSO-d): δ = 11.32 (1H, s), 8.16 (1H, 6 Recently, we have reported that Ru(II) compounds bearing s), 8.02 (1H, s) 7.97 (1H, s) 7.51 (1H, d), 7.13 (1H, dd, J = 8.6 Hz), isatin thiosemicarbazones and chloro-fluoro-phenyl imino 6.94 (1H, d, J = 8.3 Hz), 3.81 (3H, s, -OCH), 3.78 (3H, 3 methyl phenol have in vivo anticancer and in vitro cytotoxic s, -OCH). 3 activity36. In this work, we describe the synthesis and char- acterization of some ruthenium complexes, their in vitro General procedure for preparing substituted benzyl cytotoxic activity against human cancer cell lines Molt 4/C isonicotinyl hydrazones (r-binh) 8 and CEM and murine tumor cell line L1210, and their in vivo A mixture of substituted benzaldehyde (1 mmol) and isoni- anticancer activity against transplantable murine tumor cell azid (1 mmol) in 100 mL of ethanol was refluxed for 3 h and line EAC (Ehrlisch’s ascites carcinoma). left overnight. The solid that separated was filtered and dried. The crude solid was purified by recrystallization from alcohol to give crystals. Materials and methods 4-OH-binh Yield 65%, m.p. 287–288°C (lit., 287°C). IR Chemistry (KBr) cm−1: 3328(NH), 3180–2750 (O-H) 3148 (C-H),1683 AR grade solvents were obtained from S.D. Fine-Chem, (C=O), 1615 (N-H). Calcd. for C H NO: C, 60.22; H, 5.05; 13 11 3 2 Mumbai, and E. Merck, Mumbai. Puriss grade reagents were N,16.21. Found C, 60.17; H, 5.03; N, 16.07%. λ nm (MeOH): max obtained from Fluka and E. Merck. 233, 315, 391. 1H NMR (DMSO-d): δ = 11.52 (1H, s), 11.27 6 Hydrated ruthenium trichloride was purchased from Loba (1H, s), 8.03 (1H, s, O-H), 7.78 (2H, d, J = 8.7 Hz), 6.95 (2H, d, Chemie, Mumbai, and used as received. Ultraviolet (UV)- J = 8.7 Hz), 7.76 (2H, d, J = 8.4 Hz), 6.87 (2H, d, J = 8.4 Hz). visible spectra were recorded on a Jasco spectrophotometer. Fourier transform infrared (FTIR) spectra were recorded in Preparation of cis-[bis(S)dichlororuthenium(II)] KBr powder on a Jasco V410 FTIR spectrometer by the dif- cis-[Ru(S) Cl ]37 (where S = 2,2’-bipyridine/1, 2 2 fuse reflectance technique. 1H/13C-nuclear magnetic reso- 10-phenanthroline) nance (NMR) spectra were measured in CDCl and dimethyl RuCl.HO, 1g (2.5 mmol) and ligand S (5 mmol) were refluxed 3 3 2 sulfoxide (DMSO)-d on Bruker Ultraspec 500 MHz/AMX in 50 mL dimethylformamide (DMF) for 3 h under a nitrogen 6 400 MHz/300 MHz spectrometers. The reported chemical atmosphere. The reddish brown solution slowly turned pur- shifts were against that of tetramethylsilane (TMS). Fast ple and the product precipitated in the reaction mixture. The atom bombardment (FAB) mass spectra were recorded on solution was cooled overnight at 0°C. A fine microcrystalline a Jeol JMS600 spectrometer with meta-nitrobenzylalcohol mass was filtered off. The residue was repeatedly washed with (mNBA) matrix. Substituted thiosemicarbazones were pre- 30% LiCl solution and finally recrystallized from the same. pared according to the literature method. The product was dried and stored in a vacuum desiccator over PO for further use (yield 75%). 2 5 General procedure for preparing substituted benzyl thiosemicarbazones (r-btsz) General procedure for preparing -[Ru(S) (K)Cl ] (where 2 2 A mixture of substituted benzaldehyde (1 mmol) and S = 1,10-phenanthroline (Ru 1)/2,2’-bipyridne (Ru 2); thiosemicarbazide (1 mmol) in 100 mL of ethanol was refluxed where K = 4-OH-btsz, 4-CH -btsz, 3,4-di-OCH -btsz, 3 3 for 3 h and left overnight. The solid that separated was filtered 4-OH-binh, 4-CH -binh, 3,4-di-OCH -binh) 3 3 and dried. The crude solid was purified by recrystallization To the black microcrystalline cis-bis(S)dichlororuthenium(II) from alcohol to give crystals. {cis-Ru(S)Cl} (2 mmol), excess of ligand (r-btsz and r-binh) 2 2 41/81/60 no lavaL fo ytisrevinU yb moc.erachtlaehamrofni morf dedaolnwoD yrtsimehC lanicideM dna noitibihnI emyznE fo lanruoJ .ylno esu lanosrep roF Activities of mononuclear Ru(II) complexes 515 (2.5 mmol) was added and refluxed in ethanol under a nitro- (m, 2H), 9.41 (s, 1H), 8.87–8.83 (m, 2H), 8.71 (s, 1H, br), gen atmosphere. The initial colored solution slowly changed 8.53–8.51 (m, 1H), 8.46–8.34 (d, J = 5.7 Hz, 3H), 8.31–8.24 (m, to brownish orange at the end of the reaction, which was 4H), 8.01 (s, 2H, br, NH), 7.91–7.85 (m, 4H), 7.59–7.45 (dd, 2 verified by TLC on silica plates. Then the excess of ethanol 1H, J = 8.2, 8.1 Hz), 7.51–7.42 (m, 2H), 7.23 (d, J = 8.3 Hz, 2H, was distilled off and to the remaining solution was added br), 6.95 (d, 1H, J = 8.5 Hz), 6.13 (s, 1H). FAB-MS (mNBA): silica gel (60–120 mesh). The product was purified by column 725 [Ru(phen) (4-CH-btsz)]2+(Cl)−; 654 [Ru(phen) (4-CH- 2 3 2 2 3 chromatography using silica gel as the stationary phase and btsz)]2+; 474 [Ru(phen) (4-CH-btsz)]2+; 462 [Ru(phen)]. 3 2 chloroform–methanol as the mobile phase. Ru 6 44%, black crystals, IR (KBr) cm−1: 3409–3219 Ru 1 46%, black crystals, IR (KBr) cm−1: 3402–3329 (NH (NH & N-H), 3035 (C-H), 1615 (N-H), 1327 (C=S). Calcd. for 2 2 & N-H), 3210–2700 (O-H) 3036 (C-H), 1611 (N-H), 1328 (C=S). C H ClNRuS: C, 51.41; H, 3.98; N, 14.47. Found C, 50.98; H, 29 27 2 7 Calcd. for C H ClNORuS: C, 52.81; H, 3.43; N, 13.48. Found 3.79; N, 14.35%. 1H NMR (DMSO-d): δ ppm: 10.01 (m, 1H), 32 25 2 7 6 C, 52.26; H, 3.39; N, 13.32%. 1H NMR (DMSO-d): δ ppm: 8.82–8.76 (m, 2H), 8.70 (d, 1H, J = 5.6 Hz), 8.61 (d, 1H, J = 8.0 Hz), 6 10.02 (d, J = 5.1 Hz, 1H), 9.03 (s, 1H), 8.91 (d, J = 4.9 Hz, 1H), 8.43 (d, 1H, J = 8.0 Hz), 8.06–8.00 (m, 3H, 7.79–7.73 (m, 2H), 8.84 (t, J = 8.6 Hz, 2H), 8.63 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 8.4 7.65–7.59 (m, 2H), 7.46 (d, 1H, J = 5.6 Hz), 7.31–7.22 (m, 3H), Hz, 1H), 8.34–8.20 (m, 6H), 8.15–8.08 (m, 2H), 7.91 (d, J = 5.0 7.19–7.16 (mt, 3H, J = 12.0 Hz ), 6.97 (d, 2H, J = 12.0 Hz ), Hz, 1H), 7.81–7.75 (m, 2H), 7.68–7.64 (s, 1H, O-H), 7.49–7.45 6.22 (s, 2H, br, NH), 1.61(s, 3H, -CH) FAB-MS (mNBA): 677 2 3 (m, 1H), 6.91 (s, 2H, br, NH), 6.73 (d, J = 14.6 Hz, 2H), 6.13 (s, [Ru(bpy) (4-CH-btsz)]2+(Cl)−; 606 [Ru(bpy) (4-CH-btsz)]2+; 2 2 3 2 2 3 1H). FAB-MS (mNBA): 727 [Ru(phen) (4-OH-btsz)]2+(Cl)−; 452 [Ru(bpy) (4-CH-btsz)]2+; 413 [Ru(bpy)]. 2 2 3 2 656 [Ru(phen) (4-OH-btsz)]2+; 475 [Ru(phen) (4-OH-btsz)]2+; Ru 9 46%, black crystals, IR (KBr) cm−1: 3418–3226 2 462 [Ru(phen)]. (NH & N-H), 3042 (C-H), 1608 (N-H), 1339 (C=S). Calcd. 2 2 Ru 2 42%, black crystals, IR (KBr) cm−1: 3401–3238 (NH for C H ClNORuS: C, 52.91; H, 3.76; N, 12.71. Found C, 2 34 29 2 7 2 & N-H), 3200–2700 (O-H) 3041 (C-H), 1621 (N-H), 1344 (C=S). 52.87; H, 3.68; N, 12.42%. 1H NMR (DMSO-d): δ ppm: 10.09 6 Calcd. for C H ClNORuS: C, 49.48; H, 3.68; N, 14.43. Found (d, J = 5.2 Hz, 1H), 8.98 (d, J = 5.6 Hz, 1H), 8.80 (t, J = 8.8 Hz, 28 25 2 7 C, 49.24; H, 3.59; N, 14.32%. 1H NMR (DMSO-d): δ ppm: 10. 2H), 8.68 (d, J = 8.6 Hz, 1H), 8.51 (d, J = 8.6 Hz, 1H), 8.40–8.20 6 (d, J = 4.9 Hz, 1H), 9.15 (s, 1H), 8.90 (d, J = 5.0 Hz, 1H), 8.72–8.42 (m, 6H), 8.11–8.03 (m, 2H), 7.88 (d, J = 5.0 Hz, 1H), 7.83–7.77 (m, 5H), 8.12–7.98 (m, 2H), 7.82–7.53 (m, 3H), 7.45–7.32 (m, (m, 2H), 7.67–7.63 (m, 1H), 7.46–7.42 (m, 1H), 6.98 (s, 2H, 2H), 7.22–7.16 (m, 1H), 7.09–6.99 (m, 2H), 6.92–6.72 (m, 3H), br, NH), 6.75 (d, J = 14.9 Hz, 2H), 3.68 (s, 3H, -OCH), 3.62 2 3 6.61 (s, 2H, br, NH), 6.34–6.13 (m, 2H). FAB-MS (mNBA): 679 (s, 3H, -OCH), FAB-MS (mNBA): 771 [Ru(phen) (3,4-di- 2 3 2 [Ru(bpy) (4-OH-btsz)]2+(Cl)−; 608 [Ru(bpy) (4-OH-btsz)]2+; OCH-btsz)]2+(Cl)−; 700 [Ru(phen) (3,4-di-OCH-btsz)]2+; 2 2 2 3 2 2 3 452 [Ru(bpy) (4-OH-btsz)]2+; 413 [Ru(bpy)]. 521 [Ru(phen) (3,4-di-OCH-btsz)]2+; 461 [Ru(phen)]. 2 3 2 Ru 3 44%, black crystals, IR (KBr) cm−1: 3318 (N-H), Ru 10 43%, black crystals, IR (KBr) cm−1: 3406–3217 3200–2700 (O-H), 3041 (C-H), 1601 (N-H), 1681 (C=O). Calcd. (NH & N-H), 3025 (C-H), 1612 (N-H), 1322 (C=S). Calcd. for 2 for C H ClNORu: C, 57.43; H, 3.49; N, 12.67. Found C, C H ClNORuS: C, 49.79; H, 4.01; N, 13.55. Found C, 49.56; 37 27 2 7 2 30 29 2 7 2 57.26; H, 3.34; N, 12.32%. 1H NMR (DMSO-d): δ ppm: 10.01 H, 3.95; N, 13.42%. 1H NMR (DMSO-d): δ ppm: 10.02 (d, J = 6 6 (d, J = 5.1 Hz, 1H), 9.02 (s, 1H), 8.87 (d, J = 5.6 Hz, 1H),8.64 5.0 Hz, 1H), 8.73–8.72 (d, J = 5.4 Hz, 1H,), 8.63–8.41 (m, 5H), (d, J = 8.3 Hz, 1H), 8.46 (d, J = 8.6 Hz, 1H), 8.37–8.19 (m, 6H), 8.10–8.03 (m, 3H), 7.88–7.70 (m, 6H), 7.46 (d, J = 4.9 Hz, 2H), 8.13–8.07 (m, 2H), 7.93 (d, J = 5.1 Hz, 2H), 7.84–7.78 (m, 2H), 7.39–7.12 (m, 3H), 6.94 (s, 2H, br, NH), 3.76 (s, 3H, -OCH), 2 3 7.64–7.60 (s, 1H, O-H), 7.46–7.43 (m, 2H), 7.38–7.32 (m, 2H), 3.69 (s, 3H, -OCH), FAB-MS (mNBA): 723 [Ru(bpy) (3,4-d i- 3 2 6.93 (s, 2H, br, NH), 6.77 (d, J = 15.2 Hz, 2H), 6.11 (s, 1H). OCH-btsz)]2+(Cl)−; 652 [Ru(bpy) (3,4-di-OCH-btsz)]2+; 496 2 3 2 2 3 FAB-MS (mNBA): 773 [Ru(phen) (4-OH-binh)]2+(Cl)−; 702 [Ru(bpy) (3,4-di-OCH-btsz)]2+; 413[Ru(bpy)]. 2 2 3 2 [Ru(phen) (4-OH-binh)]2+; 521 [Ru(phen) (4-OH-binh)]2+; 2 462 [Ru(phen)]. Antineoplastic activity 2 Ru 4 44%, black crystals, IR (KBr) cm−1: 3312 (N-H), Albino swiss mice (18–20 g body weight) were maintained 3200–2700 (O-H), 3041 (C-H), 1615 (N-H), 1675 (C=O). Calcd. in identical laboratory conditions and given standard food for C H ClNORu: C, 54.62; H, 3.72; N, 13.52. Found C, pellets (Hindustan Lever Ltd, Bombay, India) and water ad 33 27 2 7 2 53.89; H, 3.55; N, 13.28%. 1H NMR (DMSO-d): δ ppm: 9.98. (d, libitum. LD values of the synthesized compounds were 6 50 J = 4.9 Hz, 1H), 9.18 (s, 1H), 8.91 (d, J = 5.3 Hz, 1H), 8.74–8.44 determined according to the literature38. All compounds (m, 5H), 8.11–7.97 (m, 2H), 7.93–7.89 (m, 2H), 7.80–7.51 (m, were dissolved in 10% DMSO solution. The animals were 3H), 7.46–7.22 (m, 2H), 7.21–7.15 (s,1H, O-H), 7.10–7.01 (m, divided into 15 groups each containing 12 mice. Group I was 2H), 6.94–6.72 (m, 3H), 6.63 (s, 2H, br, NH), 6.36–6.15 (m, the vehicle control group (5mL/kg body weight, i.p.) and 2 2H). FAB-MS (mNBA): 725 [Ru(bpy) (4-OH-binh)]2+(Cl)−; group II was the EAC control group (2 × 106 EAC cells/mouse, 2 2 654 [Ru(bpy) (4-OH-binh)]2+; 498 [Ru(bpy) (4-OH-binh)]2+; i.p.). Group III were treated with the standard drug cisplatin 2 413 [Ru(bpy)]. (2 mg/kg body weight). All the compounds were administered 2 Ru 5 44%, black crystals, IR (KBr) cm−1: 3414–3224 (i.p.) at a dose of 2 mg/kg body weight in groups IV–XV, respec- (NH & N-H), 3032 (C-H), 1632 (N-H), 1331 (C=S). Calcd. for tively. Mice were treated with the compounds and cisplatin 2 C H ClNRuS: C, 54.62; H, 3.72; N, 13.52. Found C, 53.26; daily for 9 days starting 24 h after tumor transplantation. Six 33 27 2 7 H, 3.72, N, 14.47%. 1H NMR (DMSO-d): δ ppm: 10.15–10.04 animals from each group were sacrificed 18 h after the last 6 41/81/60 no lavaL fo ytisrevinU yb moc.erachtlaehamrofni morf dedaolnwoD yrtsimehC lanicideM dna noitibihnI emyznE fo lanruoJ .ylno esu lanosrep roF 516 Sreekanth Thota et al. dose. Ascitic fluid volume and Ascitic cell count parameters reflectance technique, and are reported in their respective were noted. Mean survival time (MST) for the remaining six titles by tentative assignments. The r-btsz ligands showed mice of each group was noted. vibrational frequency from 3400 to 3200 cm−1 for NH and 2 N-H stretching, and from 1335 to 1325 cm−1for C=S stretching. Tumor volume and viable cell count The r-binh ligands showed vibrational frequency from 3320 Ascites volume was noted by taking it in a graduated centri- to 3200 cm−1 for N-H stretching and from 1690 to 1670 cm−1 fuge tube, and packed cell volume determined by centrifug- for C=O stretching. ing at 1000g for 5 min. The viability of ascitic cells was checked A comparison of IR spectra of r-btsz ligands and ruthenium by Trypan blue (0.4% in normal saline) dye exclusion test complexes confirmed coordination to the metal center by the and the count was taken in a Neubauer counting chamber. sulfur atom and imine nitrogen. Comparing the IR spectra of The effect of the ruthenium complexes on tumor growth was r-binh ligands and ruthenium compounds confirmed coordi- monitored by recording the mortality daily, and percentage nation to the metal center by an oxygen atom and imine nitro- increase in life span (ILS%) was calculated by the following gen. In complexes such as Ru 1–Ru 2, Ru 5–Ru 6, Ru 9–Ru 10, formula: coordination occurred via the sulfur and imine nitrogen but not with the terminal amine group; this was confirmed by the ILS (%) = [(mean survival of treated group)/(mean survival of spectra, which indicated no change in vibrational frequency control group) – 1] × 100 of the NH group between 3400 and 3300 cm−1. 2 Coordination of ligands (K = r-binh, r-btsz) to ruthenium Cytotoxic evaluation resulted in compounds such as [Ru(S)(K)]2+Cl(Ru 1–Ru 12), 2 2 The compounds prepared in the laboratory were evalu- respectively. These compounds did not possess any C2 axes ated against Molt 4/C, CEM, and L1210 cells by a literature of symmetry. Such a loss of C2 axis of symmetry was seen 8 procedure39. for [Ru(L)(R)]33–35 (where L = 2,2’-bipyridine/1,10-phenan- 2 throline and R = acetazolamide, 7-iodo-8-hydroxy-quinoline, Results and discussion 4-substituted thiopicolinanalide, etc.). All compounds had well-resolved resonance peaks, which corresponded to four Chemistry Ligands type r-binh (r-binh = substituted benzyl isonico- tinyl hydrazones) were prepared by reacting substituted O H N S benzaldehydes with isoniazid in alcohol at 1:1 molar ratio 2 (Scheme 1), and r-btsz (r-btsz = substituted benzyl thiosemi- H carbazones) were prepared by reacting substituted benzal- H 2 N S R HN N dehydes with thiosemicarbazide in alcohol at 1:1 molar ratio H (Scheme 1). All ligands were confirmed for their purity by HN Reflux in Alcohol, 3Hrs R NH their melting point, elemental analysis, and other spectral 2 studies. Details of the strategy adopted for the synthesis of thiosemicarbazide r-btsz these ruthenium homoleptic compounds are as follows. R = 4-OH, 4-CH , 3,4-di-OCH 3 3 The starting material for synthesis of the compounds was cis-bis(1,10- phenanthroline) dichlororuthenium(II)/cis- O N H H bis(2,2’-bipyridine) dichlororuthenium(II). Ruthenium O N N trichloride was refluxed in DMF in the presence of 1,10- NH 2 H N R phenanthroline/2,2’-bipyridine and in excess of the O stoichiometric amount, which afforded the final product R Reflux in cis-bis(1,10-phenanthroline) dichlororuthenium(II)/cis- N Alcohol, 3Hrs bis(2,2’-bipyridine)dichlororuthenium (II)37 (Scheme 2). The isoniazide r-binh third ligand was introduced in alcohol in the presence of a nitrogen atmosphere (Scheme 3). R = 4-OH, 4-CH 3 , 3,4-di-OCH 3 The structures of the ligands, especially r-inh and r-btsz, Scheme 1. Preparation of ligands (r-btsz and r-binh). were capable of exhibiting bidentate behavior. There are very few cases in which the thiosemicarbazide acts as a monoden- tate ligand, binding to the metal center through the sulfur S atom40,41. In the case of r-btsz ligands the chelating mode was N N via the sulfur atom and imine nitrogen by a coordination RuCl 3 ·xH 2 O " N S 2 " L i g D a M n F d N Ru N S covalent bond. In the case of r-binh ligands a covalent bond Cl Cl was formed between the metal ion and oxygen atom and a cis-[Ru(S) 2 Cl 2 ] coordinate covalent bond with the imine nitrogen. Where S = 2,2'-bipyridine/ 1,10-phenanthroline The infrared spectra of all ligands and their ruthenium(II) compounds were recorded in KBr powder by the diffuse Scheme 2. Preparation of cis-[Ru(S)Cl]. 2 2 41/81/60 no lavaL fo ytisrevinU yb moc.erachtlaehamrofni morf dedaolnwoD yrtsimehC lanicideM dna noitibihnI emyznE fo lanruoJ .ylno esu lanosrep roF Activities of mononuclear Ru(II) complexes 517 2+ S S N N Reflux in Alcohol N Ru N Cl 2 Ligand r-btsz, N 2 atm N S K S N N Ru1-Ru2, Ru5-Ru6, Ru9-Ru10 S N Ru N Cl Cl 2+ cis-[Ru(S) Cl ] S 2 2 S N N Reflux in Alcohol N Ru N Cl 2 Ligand r-binh, N 2 atm N O K Ru3-Ru4, Ru7-Ru8, Ru11-Ru12 Scheme 3. Preparation of tris chelates from cis-[Ru(S)Cl]. 2 2 different aromatic ring protons of the two 2,2’-bipyridine/1,10- phenanthroline ligands and the third ligand. 2+ 2+ These compounds showed broad and intense visible bands between 340 and 510 nm due to a metal–ligand charge trans- N N N N N Ru S N Ru O fer transition (MLCT). In the UV region the bands at 280 and N NH 2 Cl 2 N N Cl 2 310 nm were assigned to 2,2’-bipyridine/1,10-phenanthroline N N N N ligand p–p* charge transfer transitions. The same transition H H was found in free 2,2’-bipyridine/1,10-phenanthroline at 270 nm, so that coordination of the ligand resulted in a red shift in the transition energy. There were also two shoulders at R R 380 and 500 nm, which were, tentatively, attributed to metal– Ru 1, Ru 2, Ru 5, Ru 3, Ru 4, Ru 7, ligand charge transfer transitions involving 2,2’-bipyridine, Ru 6, Ru 9, Ru 10 Ru 8, Ru 11, Ru 12 1,10-phenanthroline, and the third ligand. In the 1H-NMR spectra of the complexes, there were Figure 1. Structures of the ruthenium(II) complexes, where N = 1,10- phenanthroline/2,2’-bipyridine, R= 4-OH, 4-CH, 3,4-di-OCH. resolved resonance peaks at low field at δ 10.02 (s, br, NH), 3 3 7.68 (s, 1H, O-H). Thus, in the case of Ru 1, there were 25 Biological activity and discussion resonance peaks (δ 10.03–6.13), and 25 well-resolved peaks Results are summarized in Tables 1 and 2 and the pharma- (δ 10.00–6.34) for Ru 2. cological data were analyzed statistically by ANOVA (analysis The mass spectra of the complexes confirmed the formulae of variance). Statistical significance was considered only suggested by their molecular ion peaks. The spectrum showed when p < 0.05 and F > F . All the complexes were tested for critical numerous peaks representing successive degradation of the their anticancer activity in mice bearing EAC tumors. Ru 6 molecule. FAB mass spectroscopic data clearly suggested was found to increase the life span of the tumor hosts by that mononuclear complexes had been formed in each case, 52%, while the remaining ruthenium complexes were able to the first fragment being due to the [Ru(S)(K)]2+–Cl− ion pair. increase the life span in the tumor hosts by 10–38% only. The 2 2 The complex also showed a peak due to the complex cation results of the present study clearly demonstrated the tumor [Ru(S)(K)]2+ and others due to [Ru(S)(K)]2+ and [Ru(S)]2+ inhibitory activity of the ruthenium complexes against the 2 2 respectively (where S = 1,10-phenanthroline/2,2’-bipyridine transplantable murine tumor cell line (Table 1). and K = r-binh, r-btsz). This type of fragmentation has been The in vitro cytotoxic activity was evaluated for all the reported for [Ru(phen)(nmit)]Cl and [Ru(bpy)(ihqs)]Cl synthesized ligands and the ruthenium complexes against 2 2 2 2 (where phen = 1,10-phenanthroline, bpy = 2,2’-bipyridine, human Molt 4/C and CEM T-lymphocytes as well as murine 8 nmit = N-methyl isatin thiosemicarbazone, ihqs = 7-iodo-8- L1210 cells, and the results are summarized in Table 2. The hydroxyquinoline-5-sulfonicacid)33. In all cases, the loss of relative potencies between ligands and their ruthenium com- chlorine ions was detected where S = 2,2’-bipyridine/1,10- plexes revealed the importance of ruthenium metal using phenanthroline and K = r-binh, r-btsz. Thus, based on the the 4/C and CEM assays and murine L1210 assay. These 8 above observations, it is tentatively suggested that Ru(II) determinations showed that in comparison to the ligands, complexes show an octahedral geometry (Figure 1). the ruthenium complexes were more potent. 41/81/60 no lavaL fo ytisrevinU yb moc.erachtlaehamrofni morf dedaolnwoD yrtsimehC lanicideM dna noitibihnI emyznE fo lanruoJ .ylno esu lanosrep roF 518 Sreekanth Thota et al. Table 1. Antineoplastic activity of ruthenium complexes against EAC bearing mice. Parameter Total body weight (g) Mean survival time (days) ILS% Tumor volume (mL) Viable cells in ascitic fluid (%) Group I 24.2 ± 0.5 — — — — Group II 27.8 ± 0.6 21 — 3.4 ± 0.3 94.8 ± 3.8 Group III 19.6 ± 0.5 22 5 — — Group IV 22.4 ± 0.4 29 38 0.9 ± 0.07 36.2 ± 1.1 Group V 23.2 ± 0.7 26 24 1.1 ± 0.03 43.5 ± 1.4 Group VI 23.7 ± 0.8 25 19 1.4 ± 0.04 45.6 ± 1.2 Group VII 28.4 ± 0.6 28 33 1.0 ± 0.04 38.8 ± 1.7 Group VIII 25.3 ± 0.3 24 14 1.2 ± 0.03 46.9 ± 1.4 Group IX 26.8 ± 0.2 32 52 0.7 ± 0.03 28.4 ± 1.6 Group X 26.4 ± 0.5 26 24 1.1 ± 0.02 43.4 ± 1.3 Group XI 24.2 ± 0.5 25 19 1.4 ± 0.06 45.2 ± 1.4 Group XII 22.9 ± 0.4 28 33 1.0 ± 0.02 38.6 ± 1.8 Group XIII 24.8 ± 0.6 26 24 1.1 ± 0.04 43.8 ± 1.2 Group XIV 22.6 ± 0.8 23 10 1.3 ± 0.06 47.9 ± 1.5 GroupXV 23.8 ± 0.2 25 19 1.9 ± 0.04 45.1 ± 1.3 Note. Values are mean ± SEM. ILS% = [(mean survival of treated group)/(mean survival of control group) – 1] × 100. Group I, vehicle (5 mL/kg); Group II, EAC (2 × 106 cells/mouse); Group III, cisplatin (2 mg/kg) + EAC; Group IV, Ru 1; Group IV–Group XV, ruthenium complexes (2 mg/kg) + EAC. Table 2. Cytotoxic studies of ligands and ruthenium compounds. IC a (µM) 50 Compound L1210 Molt 4/C CEM 8 4-OH-btsz 244 ± 8 328 ± 12 223 ± 4 4-CH-btsz 186 ± 21 126 ± 34 136 ± 22 3 3,4-di-OCH-btsz 72 ± 4 88 ± 12 84 ± 33 3 4-OH-binh 232 ± 12 180 ± 24 163 ± 26 4-CH-binh 94 ± 22 227 ± 13 128 ± 42 3 3,4-di-OCH-binh 64 ± 32 96 ± 28 202 ± 64 3 Ru 1 18 ± 4 3.1 ± 1.8 2.9 ± 0.8 Ru 2 32 ± 12 24 ± 0.6 19 ± 5 Ru 3 0.78 ± 0.6 0.21 ± 0.02 0.24 ± 0.21 Ru 4 8.7 ± 0.3 0.65 ± 0.11 0.96 ± 0.53 Ru 5 0.82 ± 0.04 0.39 ± 0.03 0.48 ± 0.16 Ru 6 1.8 ± 0.2 1.2 ± 0.4 0.19 ± 0.14 Ru 7 0.75 ± 0.06 0.29 ± 0.07 0.16 ± 0.09 Ru 8 5.9 ± 1.3 1.4 ± 0.1 2.1 ± 0.2 Ru 9 0.91 ± 0.08 0.26 ± 0.03 0.22 ± 0.02 Ru 10 3.9 ± 1.5 0.92 ± 0.24 2.3 ± 0.5 Ru 11 1.5 ± 0.3 0.36 ± 0.04 1.6 ± 0.4 Ru 12 12 ± 1.4 18 ± 12 10 ± 06 a50% inhibitory concentration, required to inhibit tumor cell proliferation by 50%. The cytotoxicity data in Table 2 revealed that most ruthe- for L1210. In comparison with the ruthenium complexes, the nium complexes had significant cytotoxic potencies (IC ligands displayed cytotoxicty at higher-µM concentration. 50 values in the range 0.21–3.1 for Molt 4/C, and 0.75–5.9 µM From the results presented in Table 2, it is clear that several 8 for L1210). On the other hand, for the ligands, the IC values ruthenium complexes exhibited a marked inhibitory effect on 50 were in excess (84–223 µM against CEM, 96–328 µM for Molt the proliferation of tumor cells, with IC values from as low 50 4/C, and 64–244 µM for L1210). Of the tested ligands and as 0.21 µM for Molt 4/C, 0.16 µM for CEM, and 0.75 µM for 8 8 ruthenium complexes, Ru 3 showed cytotoxicity against all L1210. Thus, the ruthenium complexes proved inhibitory to three cell lines tested in the region of 0.21, 0.24, and 0.78 µM tumor growth at submicromolar concentration. Their ligands, for Molt 4/C, CEM, and L1210, respectively. Another com- however, were not antitumorally active. 8 plex, Ru 5, showed cytotoxicity against the cell lines tested at 0.39 µM for Molt 4/C, 0.48 for CEM, and 0.82 for L1210. 8 Acknowledgement Yet another complex, Ru 7, showed cytotoxicity against the cell lines tested at 0.29 µM for Molt 4/C, 0.16 for CEM, and The authors are thankful to the Principal, S.R. College of 8 0.75 for L1210. The remaining ruthenium complexes showed Pharmacy, Hanamkonda, for providing the chemicals for low-µM values for Molt 4/C and CEM and higher-µM values carrying out this research. 8 41/81/60 no lavaL fo ytisrevinU yb moc.erachtlaehamrofni morf dedaolnwoD yrtsimehC lanicideM dna noitibihnI emyznE fo lanruoJ .ylno esu lanosrep roF Activities of mononuclear Ru(II) complexes 519 Declaration of interest 20. Gagliardi R, Sava G, Pacor S, Mestroni G, Alessio E. Antimetastatic action and toxicity on healthy tissues of Na [trans-RuCl (DMSO) Im] in the 4 The authors declare no conflict of interest. The authors alone mouse. Clin Exp Metastasis 1994;12:93–100. are responsible for the writing and content of this paper. 21. Maganarin M, Bergamo A, Carotenuto ME, Zorzet S, Sava G. Increase of tumor infiltrating lymphocytes in mice treated with antimetastatic doses of NAMI-A. Anticancer Res 2000;20:2939–44. References 22. Cocchietto M, Sava G. Blood concentration and toxicity of the antime- tastasis agent NAMI-A following repeated intravenous treatment in mice. 1. Clarke MJ, Zhu F, Frasca DR. Non-platinum chemotherapeutic metallo Pharmacol Toxicol 2000;87:193–7. pharmaceuticals. Chem Rev 1999;99:2511–34. 23. Zorzet S, Sorc A, Casarsa C, Cocchietto M, Sava G. Pharmacological effects 2. Kureshy RI, Khan NH. Mononuclear chiral ruthenium(II) Schiff base com- of the ruthenium complex NAMI-A given orally to CBA mice with MCa plexes; synthesis, physicochemical studies and reactivity with π-acceptor mammary carcinoma. Met Based Drugs 2001;8:1–7. ligands. Polyhedron 1999;12:195–201. 24. Sava G, Clerici K, Capozzi I, Cocchietto M, Gagliardi R, Alessio. E, et al. 3. Chakravarty J, Bhattacharya S. Ruthenium phenolates, synthesis, Reduction of lung metastasis by ImH (trans-RuCl (DMSO) Im: mecha- 4 characterization and electron-transfer properties of some salylaldimi- nism of the selective action investigated on mouse tumors. Anticancer nato and 2-(arylazo) phenolates complexes of ruthenium. Polyhedron Drugs 1999;10:129–38. 1996;15:1047–55. 25. Sava G, Gagliardi R, Cocchietto M, Clerici K, Capozzi I, Marella M, et al. 4. Baitalik S, Adhikary B. Heterochelates of ruthenium(II): electrochem- Comparison of the effects of the antimetastatic compound ImH (trans- istry, absorption spectra, and luminescence properties. Polyhedron RuCl (DMSO) (Im)] (NAMI-A) on the arthritic rat and on MCa mammary 4 1997;16:4073–80. carcinoma in mice. Pathol Oncol Res 1998;4:30–6. 5. Clarke MJ. Ruthenium metallo pharmaceuticals. Coord Chem Rev 26. Keppler BK, Henn M, Juhl UM, Berger MR, Niebel R, Wagner FE. New 2003;236:209–33. ruthenium complexes for the treatment of cancer. Prog Clin Biochem Med 6. Clarke MJ. Oncological implications of the chemistry of ruthenium. Met 1989;10:41–69. Ions Biol Syst 1980;11:231–83. 27. Kreuser ED, Keppler BK, Berdel WE, Piest A, Thiel E. Synergistic antitumor 7. Rudolph R. Electron microscopy demonstration of sulphurated acid interactions between newly synthesized ruthenium complexes and cytokines monopolysaccarides in canine mast cell tumours, using barium chloride in human colon carcinoma cell lines. Semin Oncol 1992;19:73–81. and ruthenium red, together with comments on the classification and dif- 28. Sava G, Pacor S, Zorzet S, Alessio E, Mestroni G. Antitumor properties of ferentiation of tumor cells. Arch Exp Veterinarmed 1971;25:925–35. dimethyl sulphoxide ruthenium (II) complexes in the Lewis lung carci- 8. Anghileri LJ, Krebsforsch Z. The in vivo inhibition of tumor growth noma system. Pharmacol Res 1989;21:617–28. by ruthenium red: its relationship with the metabolism of cal- 29. Zorzet S, Bergamo A, Cocchietto M, Sorc A, Gava B, Alessio E, et al. Lack cium in the tumor. Z Krebsforsch Klin Onkol Cancer Res Clin Oncol of in vitro cytotoxicity, associated to increased G-M cell fraction and inhi- 2 1975;83:213–17. bition of matrigel invasion, may predict in vivo selective antimetastasis 9. Keppler BK, Henn M, Juhl UM, Berger MR, Niebi R, Wagner FE. New activity of ruthenium complexes. Pharmacol Exp Ther 2000;295:927–33. ruthenium complexes for the treatment of cancer. Prog Clin Biochem Med 30. Clarke MJ, Galang RD, Roudriguez VM, Kumar R, Pell S, Bryan DM. 1989;10:41–69. Chemical considerations in the design of ruthenium anticancer agents. 10. Novakova O, Kasparkova J, Vrana O, Van Vilet PM, Reedijk J, Brabec V. In: Nicolini M, ed. Platinum and Other Metal Coordination Compounds Correlation between cytotoxicty and DNA binding polypyridyl ruthenium in Cancer Chemotherapy. Boston, MA: Martinus Nijhoff, 1988:582–601. complexes. Biochemistry 1995;34:12369–78. 31. Tysoe SA, Morgan RJ, Baker D. Spectroscopic investigation of differential 11. Vilaplana RA, Gonazalez-Vichez F, Gutierrez-Puebla E, Ruiz-Valero C. binding modes of Δ- and λ- Ru (bpy) (ppz)2+ with calf thymus DNA. J Phys 2 The first isolated antineoplatic Ru(IV) complex; synthesis and structure Chem 1993;97:1707–11. of [Cl [1,2-cyclohexane diaminotetra acetate) Ru] 2HO. Inorg Chim Acta 32. Clairs SA, Paul JD. Ruthenium in medicine: current clinical uses and 2 2 1994;224:15–18. future prospects. Platinum Met Rev 2001;45:62–9. 12. Sava G, Bergamo A, Zorzet S, Gava B, Casarsa C. Influence of chemi- 33. Mazumder UK, Gupta M., Bera A, Bhattacharya S, Karki S, Manikadan cal stability on the activity of the antimetastasis ruthenium compound L, et al. Synthesis, antitumor and antibacterial activity of some NAMI-A. Eur J Cancer 2002;38:427–35. Ru(bpy)2+/4-substituted thiosemicarbazide complexes. Indian J Chem 2 13. Keppler BK, Wehe D, Enders H, Rupp W. Syntheisis, antitumor activity, 2003;42A:313–17. and X-ray structure of bis (imidazolium) imidazole-pentachloro ruthenate 34. Mazumder UK, Gupta M, Karki S, Bhattacharya S, Suresh R, Sivakumar (III), (ImH) (RuImCl). Inorg Chem 1987;26:844–6. T. Synthesis, and pharmacological activities of some mononuclear Ru(II) 2 5 14. Keppler BK, Rupp W, Juhl UM, Endres H, Nieu R, Blazer WS. Synthesis, complexes. Bioorg Med Chem 2005;13:5766–73. molecular structure and tumor-inhibiting property of imidazolium-trans- 35. Suresh R, Karki SS, Bhattacharya S, Manikadan L, Prabhakaran SG, bis(imidazole) tetra chlororuthenate (III) and its methyl substituted deriv- Mazumder UK, et al. Synthesis, and anticancer activity of certain mono- atives. Inorg Chem 1987;26:4366–70. nuclear Ru(II) complexes. J Enzyme Inhib Med Chem 2006;21:501–7. 15. Sava G, Gangliyardi R, Bergamo A, Alessio E, Mestroni G. Treatment 36. Karki SS, Sreekanth T, Balzarini J, Clercq DE. Synthesis, anticancer and of metastases of solid mouse tumors by NAMI-A; comparison with cytotoxic activities of some mononuclear Ru(II) compounds. Bioorg Med cisplatin, cyclophosphamide and decarbazine. Anticancer Res Chem 2007;15:6632–41. 1999;19:969–72. 37. Giordano PJ, Bock CR, Wrighton MS. Excited state proton transfer 16. Velders AH, Pazderski L, Ugozzoli F, Biagini-Cingi M, Reedijk J. Synthesis, of ruthenium(II) complexes of 4, 7-dihydroxy-1,10-phenanthroline. characterization and crystal structure of trans-aquatrichlorobis (5,7- Increased acidity in the excited state. J Am Chem Soc 1978;100:6960–6. dimethyl 1,2,4 triazolo 1,5-a pyrimidine-N) ruthenium (III) monohydrate. 38. Litchfield JT, Wilcoxon F. A simplified method of evaluating dose-effect 3 Inorg Chim Acta 1998;273:259–65. experiments. J Pharmacol Exp Ther 1949;96:99–113. 17. Velders AH, Ugozzoli F, Biagini-Cingi M, Reedijk JJ. A unique fourfold 39. Balzarini J, De Clercq E, Mertes MP, Shugar D, Torrence PF. 5-Substituted intramolecular hydrogen bonding stabilizes the structure of trans-bis 2’-deoxy uridines: correlation between inhibition of tumor cell growth (2-amino-5,7-dimethyl [1,2,4] triazolo [1,5-a] pyrimidine-N) aquatrichloro and inhibition of thymidine kinase and thymidylate synthetase. Biochem 3 ruthenium (III) monohydrate. Eur J Inorg Chem 1999;273:213–15. Pharmacol 1982;31:3673–82. 18. Sava G, Bergamo A. Ruthenium-based compounds and tumor growth 40. Nardelli M, Gasparri GF, Battislini GG, Musatti A. Configuration of thi- control. Int J Oncol 2000;17:353–65. osemicarbazide molecules in monochloro monothiosemicarbazide silver. 19. Rademaker-Lakhai JM, Bongard DV, Pluim D, Beijnen JH. A phase I and Chem Commun 1965:187–188. pharmacological study with imidazolium-trans-DMSO-imidazole-tetra- 41. Gasparri GF, Mangia A, Musatti A, Nardelli M. The crystal and molecular chlororuthenate, a novel ruthenium anticancer agent. Clin Cancer Res structure of monothiosemicarbazide silver(I) chloride. Acta Crystallogr 2004;10:3717–27. 1968;24:367–74. 41/81/60 no lavaL fo ytisrevinU yb moc.erachtlaehamrofni morf dedaolnwoD yrtsimehC lanicideM dna noitibihnI emyznE fo lanruoJ .ylno esu lanosrep roF