Design, synthesis, characterization, cytotoxic and structure activity relationships of novel Ru(II) complexes

Article abstract of DOI:10.1016/j.cclet.2015.03.011

Abstract Platinum containing compounds have shown antineoplastic potential, but their clinical applications have been limited by high toxicity. Ruthenium containing complexes have long been known to be well suited for biological applications, and have lon

Full text of DOI:10.1016/j.cclet.2015.03.011

Accepted Manuscript
Title: Design, synthesis, characterization, cytotoxic and
structure activity relationships of novel Ru(II) complexes
Author: Sreekanth Thota Srujana Vallala Rajeshwar Yerra
Eliezer J. Barreiro
PII:
S1001-8417(15)00094-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2015.03.011
CCLET 3240
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
12-12-2014
11-2-2015
27-2-2015
Please cite this article as: S. Thota, S. Vallala, R. Yerra, E.J. Barreiro,
Design, synthesis, characterization, cytotoxic and structure activity
relationships of novel Ru(II) complexes, Chinese Chemical Letters (2015),
http://dx.doi.org/10.1016/j.cclet.2015.03.011
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Graphical Abstract
Design, synthesis, characterization, cytotoxic and structure activity relationships
of novel Ru(II) complexes
Sreekanth Thotaa, c*, Srujana Vallalab, Rajeshwar Yerrab, Eliezer J. Barreiroc
a National Institute for Science and Technology on Innovation on Neglected Diseases (INCT/IDN), Center for Technological
Development in Health (CDTS), Fundação Oswaldo Cruz - Ministério da Saúde, Av. Brasil 4036 - Prédio da Expansão, 8º
Andar - Sala 814, Manguinhos, 21040-361 - Rio de Janeiro - RJ – Brazil
bS. R. College of Pharmacy, Department of Pharmaceutical Chemistry & Toxicology, Ananthasagar, Warangal, Telangana-
506371, India
cPrograma de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio
de Janeiro, RJ, Brazil
Ruthenium containing complexes have long been known to be well suited for biological applications, and have long been
utilized as replacements to popular platinum based-drugs.
Page 1 of 9

Original article
Design, synthesis, characterization, cytotoxic and structure activity relationships of novel Ru(II) complexes
Sreekanth Thota a, c∗, Srujana Vallalab, ∗, Rajeshwar Yerrab, Eliezer J. Barreiroc
a National Institute for Science and Technology on Innovation on Neglected Diseases (INCT/IDN), Center for Technological Development in Health (CDTS),
Fundação Oswaldo Cruz - Ministério da Saúde, Av. Brasil 4036 - Prédio da Expansão, 8º Andar - Sala 814, Manguinhos, 21040-361 - Rio de Janeiro - RJ –
Brazil
bS.R.College of Pharmacy, Department of Pharmaceutical Chemistry & Toxicology, Ananthasagar, Warangal, Telangana-506371, India
cPrograma de Desenvolvimento de Fármacos, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil
ARTICLE INFO
ABSTRACT
Article history:
Platinum containing compounds have shown antineoplastic potential, but their clinical applications
have been limited by high toxicity. Ruthenium containing complexes have long been known to be well
suited for biological applications, and have long been utilized as replacements to popular platinum
Received 12 December 2014
Received in revised form 11 February 2015
Accepted 27 February 2015
Available online
based-drugs. Here, we report
a novel series of ruthenium(II) arene compounds bearing
thiosemicarbazone and isonicotinylhydrazone ligands with potent anticancer activity their structure
activity relationships and apoptosis was studied. The cytototoxic activity of the new ruthenium(II)
arene compounds has been evaluated in several cell lines (Molt 4/C8, L1210, CEM, HL60 and
BEL7402). Among them, ten complexes were found to be excellent in vitro growth inhibitory activity
against various cellines with IC50 in the sub-micromolar range.
Keywords:
Cytotoxic
Ligand
Ruthenium compounds
Structure activity relationships
1. Introduction
Metal coordination chemistry, in recent years, has provided a significant contribution to the development of efficient diagnostic and
therapeutic agents for biomedical applications [1]. Metal coordination complexes offer biological and chemical diversity that is distinct
from that of organic drugs. This diversity arises from not only the choice of the metal itself and its oxidation state, but from the types
and numbers of coordinated ligands and the coordination geometry of the complex [2]. Platinum-based drugs are widely used to treat
cancer, but their therapeutic use can be altered by intrinsic or acquired resistance and the occurrence of numerous side effects including
neurotoxicity, nephrotoxicity, neuropathy, myelosuppression, thrombocytopenia and neutropenia [3]. Ruthenium, a second row
transition metal, continues to attract much attention in the world of scientific research, as it possesses a wide array of applicable
properties. Ruthenium-containing complexes have long been known to be well suited for biological applications, and have long been
utilized as replacements to popular platinum based-drugs. In particular, ruthenium complexes have attracted significant attention with
two complexes, namely NAMI-A3 and KP1019, advancing through clinical trials [4]. The organometallic Ru(II) arene complexes, with
the half-sandwich type structure, have demonstrated their potential increasingly [5]. Their coordination sites can be filled with various
ligands, which offer numerous possibilities to modulate biological and pharmacological properties by proper ligand selection [6].
Thiosemicarbazones (TSCs) have attracted considerable attention by chemists and biologists because of their wide range of
pharmacological effects, and their metal complexes have shown marked antibacterial, antiviral, antifungal and particularly antitumor
activity [7]. The complexes consisted of transition metals and TSC ligands usually possess more potent pharmacological effects than
the thiosemicarbazone ligands alone [8]. The biological properties of the TSC ligands can be modified and improved by the linkage to
transition metal ions [9]. Ruthenium complexes of diimine ligands such as 2,2'-bipyridine (bpy) and 1,10-phenanthroline (phen) are
widely used in bioinorganic chemistry as anticancer agents [10-14] and a few studies have been carried out on the anticancer activity of
ruthenium(II) polypyridyl complexes [15-19], but many more studies need to be undertaken to develop a novel and interesting group of
antitumor transition metal complexes. The design, synthesis and reactivity of novel ruthenium complexes have become the central
focus of research in several laboratories, including ours. In this manuscript we report on a study of a family of mixed ligand ruthenium
complexes of the type [Ru(phen)2(TSZ)]2+Cl2, [Ru(phen)2(INH)]2+Cl2, [Ru(bpy)2(TSZ)]2+Cl2, [Ru(bpy)2(INH)]2+Cl2, where TSZ is a
chelating thiosemicarbazone ligand and INH is isonicotinyl hydrazone ligands derived from the various benzaldhyde derivatives. We
report the synthesis and characterization of the complexes and their cytotoxicity towards a various human cancer celllines, and
structure activity relationships.
2. Experimental
The solvents AR grades were obtained from Sd Fine Chem., Mumbai, and E.Merck, Mumbai. The reagents (puriss grade) were
obtained from Fluka and E.Merck. Hydrated ruthenium trichloride was purchased from Loba Chemie, Mumbai, and used as received.
———
All the melting points were determined in open capillary and are uncorrected. UV-visible spectra were on a Jasco spectrophotometer.
∗ Corresponding author.
E-mail address: sthota@cdts.fiocruz.br
Page 2 of 9

1
FTIR spectra were recorded in KBr powder on a Jasco V410 FTIR spectrometer by diffuse reflectance technique. H-NMR spectra
were measured in CDCl3 and DMSO-d6 on a Bruker Ultraspec 500 MHz/AMX 400 MHz/300 MHz spectrometer. The reported
chemical shifts were against that of TMS. Mass spectra were recorded on a JEOL JMS600 spectrum with m-NBA matrix.
Synthetic procedure for the preparation of r-btsz and r-binh: A mixture of substituted benzaldehyde (1 mmol) and
thiosemicarbazide/Isoniazid (1mmol) in 100 mL of ethanol was refluxed for 3h and left overnight. The crude solid was purified by
recrystallization from alcohol to give crystals.
Preparation of cis-[bis (S)dichlororuthenium(II)] cis-[Ru(S)2Cl2] (where S = 2,2'-bipyridine/1,10-phenanthroline): [21]
General procedure for preparing-[Ru(S)2(L)Cl2] (where S = 1,10-phenanthroline/2,2'-bipyridine; [21] where L = 2-OMe-btsz, 2-Me-
btsz, 4-F-btsz, 2,4-dinitro-btsz, 2-OMe-binh, 2-Me-binh, 4-F-binh, 2,4-dinitro-binh:
2-MeO-btsz: Yield 85%. FTIR (KBr, cm-1): 3414–3328 (NH2 and NH), 3136 (C–H), 2989 (C–H), 1609 (N–H), 1332 (C=S). λmax nm
(MeOH): 226, 312 and 395. Mp 189–190 °C. Anal. Calcd for C9H11N3OS: C, 51.65; H, 5.30; N, 20.08. Found: C, 51.26; H, 5.24; N,
20.05.1H-NMR (DMSO-d6): δ 11.02 (s, 1H), 8.22 (s, 1H), 7.96 (s, 1H), 7.78 (s, 1H), 7.64 (d, 2H, J = 8.4 Hz), 6.88 (d, 2H, J = 8.4 Hz),
3.72 (s, 3H, OCH3). ESI-MS: m/z 209 (100).
2-CH3-btsz: Yield 82%. FTIR (KBr, cm−1): 3448–3298 (NH2 and NH), 3142 (C-H), 1614 (N-H), 1328 (C=S). λmax nm (MeOH): 242,
1
326, 398. Mp 174–176°C. Calcd. for C9H11N3S: C, 55.93; H, 5.74; N,21.74. Found C, 55.89; H, 5.70; N, 21.68%. H-NMR (DMSO-
d6): δ 11.28 (s, 1H), 8.18 (s, 1H), 8.02 (s, 1H), 7.94 (s, 1H), 7.72 (d, 2H, J = 8.7 Hz), 6.84 (d, 2H, J = 8.7 Hz), 1.62 (s, 3H, CH3). ESI-
MS: m/z 193 (100).
4-F-btsz: Yield 88%. FTIR (KBr, cm-1): 3408–3234 (NH2 and N–H), 3085 (C-H), 1602 (N–H), 1325 (C=S). λmax nm (MeOH): 222,
346. Mp 191–192 °C. Anal. Calcd. for C8H8FN3S: C, 48.72; H, 4.09; N, 21.30. Found: C, 48.56; H, 4.04; N, 21.18. 1H-NMR (DMSO-
d6): δ 10.98 (s, 1H), 8.04(s, 1H), 7.96 (s, 1H), 7.82 (s, 1H), 7.64 (d, 2H, J = 8.6 Hz), 6.88 (d, 2H, J = 8.6 Hz), ESI-MS: m/z 197 (100).
2,4-di-NO2-btsz: Yield 72%. FTIR (KBr, cm-1): 3398–3249 (NH2 and N–H), 3018 (C-H), 1664 (NO2), 1610 (N–H), 1334 (C=S).
λmax nm (MeOH): 216, 264 and 386. Mp 183–185 °C. Anal. Calcd. for C8H7N5O4S: C, 35.69; H, 2.62; N, 26.01. Found: C, 35.64; H,
2.58; N, 25.96. 1H-NMR (DMSO-d6): δ 10.82 (s, 1H), 8.94 (s, 1H), 8.04 (s, 1H), 7.98 (s, 1H), 7.62 (d, 2H, J = 8.6 Hz), 6.88 (1H, d),
ESI-MS: m/z 269 (100).
2-OCH3-binh: Yield 85%, FTIR (KBr, cm−1): 3328(NH), 3142 (C-H), 1682 (C=O), 1608 (N-H). λmax nm (MeOH): 246, 322, 394.
Mp. 257–258 °C. Anal. Calcd. for C14H13N3O2: C, 65.87; H, 5.13; N,16.46. Found C, 65.77; H, 5.10; N, 16.42%. 1H-NMR (DMSO-d6):
δ 10.26 (s, 1H), 9.02 (s, 1H, CH=N), 8.03-7.78 (m, 4H, Ar-H), 7.69 (d, 2H, J = 8.7 Hz), 7.56 (d, 2H, J = 8.4 Hz), 3.78 (s, 3H, OCH3).
ESI-MS: m/z 255 (100).
2-CH3-binh: Yield 74%, FTIR (KBr, cm−1): 3375 (NH), 3094 (C-H), 1680 (C=O), 1602 (N-H). λmax nm (MeOH): 224, 328, 375. Mp
1
184–186 °C. Anal. Calcd. for C14H13N3O: C, 70.28; H, 5.48; N,17.56. Found C, 70.22; H, 5.43; N, 17.52%. H-NMR (DMSO-d6): δ
10.06 (s, 1H), 9.02 (s, 1H, CH=N), 8.21-7.98 (m, 4H, Ar-H), 7.83 (dd, 2H,), 7.48 (d, 2H, J = 8.4 Hz), 1.58 (s, 3H, CH3). ESI-MS: m/z
239 (100).
4-F-binh: Yield 88%, FTIR (KBr, cm−1): 3402 (NH), 3125 (C-H), 1682 (C=O), 1615 (N-H). λmax nm (MeOH): 228, 306, 388. Mp.
232–233 °C. Anal. Calcd. for C13H10N3O2F: C, 64.19; H, 4.14; N,17.28. Found C, 64.17; H, 4.08; N, 17.22%. 1H-NMR (DMSO-d6): δ
9.94 (s, 1H,), 8.98 (s, 1H,), 8.12-7.84 (m, 4H), 7.72 (d, 2H, J = 8.7 Hz), 7.48 (dd, 2H, J = 8.4 Hz). ESI-MS: m/z 243 (100).
2,4-di-NO2-binh: Yield 92%. FTIR (KBr, cm-1): 3402 (N–H), 3128 (C-H), 1681 (C=O), 1664 (NO2), 1614 (N–H). λmax nm (MeOH):
1
212, 284 and 392. Mp 201–202 °C Anal. Calcd. for C13H9N5O5: C, 49.53; H, 2.88; N, 22.22. Found: C, 49.48; H, 2.82; N, 22.16. H-
NMR (DMSO-d6): δ 10.02 (s, 1H), 8.84 (s, 1H), 8.06 (d, 2H), 7.86 (s, 1H), 7.58 (d, 2H, J = 8.7 Hz), 7.24 (d, 1H), 7.06 (s, 1H) ESI-MS:
m/z 315 (100).
Ru(Phen)2(2-OMe-btsz)]2+Cl2 (Ru-1): Yield 48%. FTIR (KBr, cm-1): 3453–3284 (NH2 and N–H), 3014 (C-H) 1612 (N–H), 1318
(C=S). λmax nm (MeOH): 215, 274, 326, 398 and 444. Anal. Calcd. for C33H27N7ORuS: C, 59.09; H, 4.06; N, 14.62. Found: C, 59.02;
H, 4.02; N, 14.58. 1H-NMR (DMSO-d6): δ 10.04 (s, 1H), 8.96 (d, 1H, J = 4.9 Hz ), 8.88 (t, 2H, J = 5.0 Hz), 8.74 (s, 1H,), 8.52 (d, 1H, J
= 8.7 Hz), 8.44–8.12 (m, 3H), 8.05–7.98 (m, 2H), 7.94 (d, 1H, J = 8.4 Hz), 7.84 (d, 1H, J = 8.4 Hz) 7.76–7.68 (m, 2H) 7.62 (d, 1H, J =
5.0 Hz), 7.48–7.45 (m, 3H), 6.91 (s, br, 2H), 6.74 (d, 2H, J = 8.7 Hz), 6.14 (s, 1H), 3.69 (s, 3H, OCH3). MS (ESI) (35 eV) m/z%: 670
(100) for [Ru(phen)2(2-MeO-btsz)].
Ru(bpy)2(2-OMe-btsz)]2+Cl2 (Ru-2): Yield 43%. FTIR (KBr, cm-1): 3426–3318 (NH2 and N–H), 3125 (C-H) 1610 (N–H), 1325
(C=S). λmax nm (MeOH): 202, 248, 298, 316, 385 and 453. Anal. Calcd. for C29H27N7ORuS: C, 55.93; H, 4.37; N, 15.75. Found: C,
1
55.78; H, 4.29; N, 15.68. H-NMR (DMSO-d6): δ 10.16 (s, 1H), 9.02 (s, 1H), 8.94 (s, 1H), 8.32–8.18 (m, 4H), 8.03–7.97 (m, 4H),
7.84–7.62 (dd, 2H, J = 8.4 Hz), 7.48–7.32 (t, 2H, J = 8.6 Hz), 7.28–7.26 (s, 1H), 7.18–6.98 (m, 2H), 6.93–6.71 (m, 2H), 6.60 (s, 2H),
6.35–6.12 (m, 2H), 3.72 (s, 3H, OCH3). MS (ESI) (35 eV) m/z%: 622 (100) for [Ru(bpy)2(2-MeO-btsz)].
Page 3 of 9

Ru(Phen)2(2-Me-btsz)]2+Cl2 (Ru 3): Yield 46%, black crystals, FTIR (KBr, cm−1): 3398–3264 (NH2 and N-H), 3045 (C-H), 1632 (N-
1
H), 1328 (C=S). Calcd. for C33H27N7RuS: C, 60.54; H, 4.16; N, 14.97. Found C, 60.38; H, 4.12, N, 14.88%. H-NMR (DMSO-d6): δ
9.98 (s, 1H), 9.48 (s, 1H,), 8.94 (s, 1H), 8.83 (d, 1H, J = 5.0 Hz), 8.74 (s, 1H), 8.46–8.21–8.12 (m, 3H), 8.01 (s, 2H), 7.94–7.82 (m.
3H), 7.69–7.54 (dd, 1H, J = 8.1 Hz), 7.42–7.28 (m, 2H), 7.22 (dd, 2H, J = 8.7 Hz), 7.14 (d, 2H, J = 8.3 Hz), 6.95 (d, 1H, J = 8.5 Hz),
6.98 (d, 2H), 6.13 (s, 1H), 1.52 (s, 3H, CH3). MS (ESI) (35 eV) m/z%: 654 (100) for [Ru(phen)2(2-Me-btsz)].
Ru(bpy)2(2-Me-btsz)]2+Cl2 (Ru 4): Yield 52%, black crystals, FTIR (KBr, cm−1): 3409–3219 (NH2 and N-H), 3035 (C-H), 1615 (N-
1
H), 1327 (C=S). Calcd. for C29H27N7RuS: C, 57.41; H, 4.49; N, 16.16. Found C, 57.38; H, 4.32; N, 16.12%. H-NMR (DMSO-d6): δ
10.12 (s, 1H), 8.94 (s, 1H), 8.84 (s, 1H), 8.75 (d, 1H, J = 5.6 Hz), 8.68 (d, 1H, J = 8.0 Hz), 8.42 (d, 1H, J = 8.0 Hz), 8.02–7.88 (m, 4H)
7.76–7.58 (m, 4H), 7.62–7.58 (dd, 2H, J = 8.7 Hz), 7.42 (d, 1H, J = 5.6 Hz), 7.26–7.20 (m, 3H), 6.97 (d, 2H, J = 12.0 Hz), 6.22 (s, 2H),
1.61 (s, 3H, -CH3). MS (ESI) (35 eV) m/z%: 606 (100) for [Ru(bpy)2(2-Me-btsz)].
Ru(Phen)2(4-F-btsz)]2+Cl2 (Ru-5): Yield 42%, black crystals, FTIR (KBr, cm-1): 3397–3311 (NH2 and N–H), 1611 (N–H), 1308
(C=S). λmax nm (MeOH): 211, 244, 294 340, 400 and 485. Anal. Calcd. for C32H24FN7RuS: C, 58.35; H, 3.67; N, 14.88. Found: C,
1
58.26; H, 3.64; N, 14.82. H-NMR (DMSO-d6): δ 10.24 (s, 1H), 9.06 (s, 1H), 8.84 (dd, 2H, J = 5.0 Hz), 8.78 (s, 1H), 8.56 (s, 1H),
8.20–8.02 (m, 3H), 7.88 (s, 1H), 7.62 (d, 1H, J = 8.4 Hz) 7.54–7.48 (m, 3H) 7.36 (d, 1H), 7.28–7.15 (m, 3H), 6.96 (m, 3H), 6.78 (d,
2H), 6.64 (s, 1H,). MS (ESI) (35 eV) m/z%: 658 (100) for [Ru(phen)2(4-F-btsz)].
Ru(bpy)2(4-F-btsz)]2+Cl2 (Ru-6): Yield 38%, black crystals, FTIR (KBr, cm-1): 3396–3318 (NH2 and N–H), 1609 (N–H), 1307
(C=S). λmax nm (MeOH): 223, 265, 402 and 455. Anal. Calcd. for C28H24FN7RuS: C, 55.07; H, 3.46; N, 16.06. Found: C, 55.02; H,
1
3.42; N, 15.98. H-NMR (DMSO-d6): δ 9.98 (s, 1H), 9.02 (s, 1H), 8.92 (m, 2H), 8.84 (s, 1H), 8.62 (s, 1H), 8.14–8.04 (m, 4H), 7.96
(dd, 2H, J = 8.7 Hz), 7.84–7.68 (m, 4H) 7.44 (d, 1H, J = 8.5 Hz), 7.38–7.22 (dd, 2H, J = 8.7 Hz), 7.04 (m, 2H), 6.92 (dd, 2H, J = 9.2
Hz), 6.88 (s, 1H). MS (ESI) (35 eV) m/z%: 610 (100) for [Ru(bpy)2(4-F-btsz)].
Ru(Phen)2(2,4-di-NO2-btsz)]2+Cl2 (Ru-7): Yield 44%, black crystals, IR (KBr, cm−1): 3446-3218 (NH2 and N-H), 3041 (C-H), 1662
1
(NO2), 1328 (C=S). Calcd. for C32H23N9O4RuS: C, 52.60; H, 3.17; N, 17.25. Found C, 52.56; H, 3.14; N, 17.20%. H-NMR (DMSO-
d6): δ 10.16 (d, 1H, J = 5.2 Hz), 9.08 (s, 1H), 8.98 (d, 1H, J = 5.6 Hz), 8.76 (d, 1H, J = 8.4 Hz), 8.34 (d, 1H, J = 8.6 Hz), 8.36–8.24 (m,
3H), 8.22–8.07 (m, 3H), 7.95 (d, 2H, J = 5.1 Hz), 7.86–7.79 (m, 2H), 7.62–7.58 (s, 1H), 7.44–7.40 (m, 2H), 7.36–7.32 (m, 2H), 6.93 (s,
2H), 6.77 (d, 2H, J = 15.4 Hz), 6.11 (s, 1H). MS (ESI) (35 eV) m/z%: 730 (100) for [Ru(phen)2(2,4-di-NO2-btsz)].
Ru(bpy)2(2,4-di-NO2-btsz)]2+Cl2 (Ru-8): Yield 44%, black crystals, IR (KBr, cm−1): 3389-3264 (NH2 and N-H), 3115 (C-H), 1668
1
(NO2), 1612 (N-H), 1332 (C=S). Calcd. for C28H23N9O4RuS: C, 49.26; H, 3.40; N, 18.47. Found C, 49.18; H, 3.38; N, 18.38%. H-
NMR (DMSO-d6): δ 10.09 (d, 1H, J = 5.1 Hz), 9.02 (s, 1H,), 8.87 (d, 1H, J = 5.6 Hz), 8.64 (d, 1H, J = 8.3 Hz), 8.46 (d, 1H, J = 8.6
Hz), 8.37–8.19 (m, 4H), 8.13–8.07 (m, 2H), 7.93 (d, 2H, J = 5.1 Hz), 7.84–7.78 (m, 2H), 7.64–7.60 (s, 1H), 7.46–7.43 (m, 2H), 7.38–
7.32 (m, 2H), 7.04 (s, 2H), 6.98 (s, 1H). MS (ESI) (35 eV) m/z%: 682 (100) for [Ru(bpy)2(2,4-di-NO2-btsz)].
Ru(Phen)2(2-OMe-binh)]2+Cl2 (Ru-9): Yield 44%, black crystals, IR (KBr, cm−1): 3362 (N-H), 3098 (C-H), 1676 (C=O). Calcd. for
C38H29N7O2Ru: C, 63.68; H, 4.08; N, 13.68. Found C, 63.59; H, 4.02; N, 13.58%. 1H-NMR (DMSO-d6): δ 10.24. (d, 1H, J = 4.9 Hz),
9.09 (s, 1H), 8.98 (d, 1H, J = 5.3 Hz), 8.34–8.22 (m, 3H), 8.14–7.98 (m, 3H), 7.89–7.66 (m, 2H), 7.62–7.48 (m, 3H), 7.44–7.32 (dd,
2H, J = 8.7 Hz), 7.38-7.24 (m, 3H), 7.22–7.14 (s, 1H), 7.11–7.02 (m, 2H), 6.63 (s, 2H), 6.36–6.15 (m, 2H) 3.64 (s, 3H, OMe). MS
(ESI) (35 eV) m/z%: 716 (100) for [Ru(phen)2(2-OMe-binh)].
Ru(bpy)2(2-OMe-binh)]2+Cl2 (Ru-10): Yield 48%, black crystals, IR (KBr, cm−1): 3298 (N-H), 3066 (C-H), 1680 (C=O). Calcd. for
C34H29N7O2Ru: C, 61.07; H, 4.37; N, 14.66. Found C, 61.02; H, 4.32; N, 14.58%. 1H-NMR (DMSO-d6): δ 10.18 (1H, s), 9.14 (s, 1H),
8.97 (d, 1H, J = 5.4 Hz), 8.78 (s, 1H), 8.56 (d, 1H, J = 8.6 Hz), 8.44–8.23 (m, 4H), 8.21–8.08 (m, 3H), 7.99 (dd, 2H, J = 5.1 Hz), 7.86–
7.74 (m, 4H), 7.62 (s, 1H), 7.48–7.36 (m, 3H), 7.28 (m, 2H), 7.01 (dd, 2H, J = 8.4 Hz), 3.78 (s, 3H, OMe). MS (ESI) (35 eV) m/z%:
668 (100) for [Ru(bpy)2(2-OMe-binh)].
Ru(Phen)2(2-Me-binh)]2+Cl2 (Ru 11): Yield 52%, black crystals, IR (KBr, cm−1): 3302 (N-H), 3076 (C-H), 1682 (C=O). Calcd. for
C38H29N7ORu: C, 65.13; H, 4.17; N, 13.99. Found C, 65.10; H, 4.12, N, 13.86%. 1H-NMR (DMSO-d6): δ 10.12–10.03 (d, 1H, J = 4.9
Hz), 9.56 (s, 1H,), 8.99 (s, 1H), 8.72 (s, 1H), 8.68 (s, 1H), 8.54–8.46 (dd, 2H, J = 8.4 Hz), 8.22–8.14 (m, 3H), 8.11–8.04 (m, 3H), 7.98
(dd, 2H, J = 8.7 Hz), 7.96–7.84 (m, 3H), 7.58–7.36 (dd, 2H, J = 8.4 Hz), 7.34–7.28 (m, 3H), 7.23 (d, 2H, J = 9.8 Hz), 6.98 (s, 1H), 1.64
(s, 3H, CH3). MS (ESI) (35 eV) m/z%: 700 (100) for [Ru(phen)2(2-Me-binh)].
Ru(bpy)2(2-Me-binh)]2+Cl2 (Ru 12): Yield 44%, black crystals, IR (KBr, cm−1): 3269 (N-H), 3008 (C-H), 1678 (C=O). Calcd. for
1
C34H29N7ORu: C, 62.56; H, 4.48; N, 15.02. Found C, 62.48; H, 4.42; N, 14.98%. H-NMR (DMSO-d6): δ 9.76 (s, 1H), 8.95 (s, 1H),
8.88 (d, 1H, J = 5.6 Hz),8.74-8.69 (dd, 2H, J = 8.7 Hz), 8.64 (d, 1H, J = 8.2 Hz), 8.52 (d, 1H, J = 8.4 Hz), 8.16–8.02 (m, 4H), 7.89–
7.75 (m, 4H), 7.68–7.54 (dd, 2H, J = 8.7 Hz), 7.48 (d, 1H, J = 5.6 Hz), 7.34–7.25 (m, 3H), 7.16–7.04 (m, 3H), 6.86 (d, 2H, J = 12.0
Hz), 1.64(s, 3H, -CH3). MS (ESI) (35 eV) m/z%: 652 (100) for [Ru(bpy)2(2-Me-binh)].
Ru(Phen)2(4-F-binh)]2+Cl2 (Ru-13): Yield 42%, FTIR (KBr, cm-1): 3397–3311 (N–H), 1611 (N–H), 1308 (C=S). λmax nm (MeOH):
211, 244, 294 340, 400 and 485. Anal. Calcd. for C37H26FN7ORu: C, 63.06; H, 3.72; N, 13.91. Found: C, 63.01; H, 3.66; N, 13.88. 1H-
NMR (DMSO-d6): δ 10.16 (s, 1H), 9.16 (s, 1H), 8.92 (s, 1H), 8.88 (s, 1H), 8.76 (s, 1H), 8.34–8.16 (m, 3H), 8.04 (dd, 2H, J = 8.7 Hz),
Page 4 of 9

7.98–7.78 (m, 3H), 7.66–7.52 (s, 1H), 7.46–7.32 (m, 3H), 7.24 (s, 1H), 7.18 (dd, 2H, J = 8.4 Hz), 6.98 (d, 1H, J = 8.5 Hz), 6.84 (d, 2H,
J = 4.9 Hz) 6.75-6.72 (t, 2H, J = 8.4 Hz), 6.52 (s, 1H) . MS (ESI) (35 eV) m/z%: 704 (100) for [Ru(phen)2(4-F-binh)].
Ru(bpy)2(4-F-binh)]2+Cl2 (Ru-14): Yield 38%, black crystals, FTIR (KBr, cm-1): 3366 (N–H), 3126 (C-H), 1685 (C=O). λmax nm
(MeOH): 223, 265, 402 and 455. Anal. Calcd. for C33H26FN7ORu: C, 60.36; H, 3.99; N, 14.93. Found: C, 60.28; H, 3.92; N, 14.88. 1H-
NMR (DMSO-d6): δ 9.92 (s, 1H), 8.98 (s, 1H), 8.86 (m, 2H), 8.78 (s, 1H), 8.55 (s, 1H), 8.24–8.06 (m, 4H), 8.01 (dd, 2H, J = 8.4 Hz),
7.98 (d, 2H, J = 8.6 Hz) 7.89–7.66 (m, 4H) 7.42 (s,1H), 7.35–7.21 (dd, 2H, J = 5.0 Hz), 7.14 (dd, 2H, J = 8.4 Hz), 6.94 (t, 2H, J = 5.0
Hz), 6.72 (s, 1H). MS (ESI) (35 eV) m/z%: 656 (100) for [Ru(bpy)2(4-F-binh)].
Ru(Phen)2(2,4-dinitro-binh)]2+Cl2 (Ru-15): Yield 42%, black crystals, FTIR (KBr, cm−1): 3326 (N-H), 3041 (C-H), 1681 (C=O), 1662
(NO2). Calcd. for C37H25N9O5Ru: C, 57.21; H, 3.24; N, 16.23. Found C, 57.16; H, 3.19; N, 16.20%. 1H-NMR (DMSO-d6): δ 10.28 (s,
1H), 9.14 (s, 1H), 8.98 (d, 1H, J = 5.6 Hz), 8.84 (d, 1H, J = 8.3 Hz), 8.54 (d, 1H, J = 8.7 Hz), 8.45–8.22 (m, 3H), 8.20–8.08 (m, 3H),
7.95 (dd, 2H, J = 9.4 Hz), 7.86–7.72 (m, 3H), 7.62 (s, 1H), 7.48–7.42 (m, 3H), 6.93 (s, 2H), 6.82 (d, 2H, J = 15.2 Hz), 6.24 (s, 1H). MS
(ESI) (35 eV) m/z%: 776 (100) for [Ru(phen)2(2,4-di-NO2-binh)].
Ru(bpy)2(2,4-dinitro-binh)]2+Cl2 (Ru-16): Yield 44%, black crystals, IR (KBr, cm−1): 3395 (N-H), 3088 (C-H), 1675 (C=O). Calcd.
for C33H25N9O5Ru: C, 54.39; H, 3.46; N, 17.30. Found C, 54.28; H, 3.42; N, 17.28%. 1H-NMR (DMSO-d6): δ 9.82(s, 1H), 9.06 (s, 1H),
8.92 (s, 1H), 8.84 (s, 1H), 8.32–8.20 (m, 4H), 8.04–7.87 (m, 4H), 7.75 (d, 2H, J = 5.1 Hz), 7.68–7.46 (m, 3H), 7.25–7.21 (dd, 2H, J =
8.4 Hz), 7.14–7.02 (m, 2H), 6.96 (s, 2H), 6.84 (s, 1H), 6.58 (s, 1H). MS (ESI) (35 eV) m/z%: 728 (100) for [Ru(bpy)2(2,4-di-NO2-
btsz)].
3. Results and discussion
3.1 Chemistry
The ligands like r-btsz (r-btsz = substituted benzyl thiosemicarbazones) were prepared by reacting substituted benzaldehydes with
thiosemicarbazide in alcohol at 1:1 molar ratio (Scheme 1). r-binh (r-btsz = substituted benzyl isonicotinylhydrazones) were prepared
by reacting substituted benzaldehydes with thiosemicarbazide in alcohol at 1:1 molar ratio (Scheme 1). The details of the synthetic
strategy adopted for the synthesis of these ruthenium homoleptic compounds was as follows. Ruthenium trichloride was refluxed in
DMF in the presence of 1,10-phenanthroline/2,2'-bipyridine and in excess of the stoichiometric amount, which afforded the final
product cis-bis(1,10-phenanthroline)dichlororuthenium(II)/cis-bis(2,2'-bipyridine) dichlororuthenium (II) [20]. The third ligand was
introduced in alcohol in the presence of nitrogen atmosphere (Scheme 2). The structures of the ligands especially r-binh, and r-btsz
were capable of exhibiting bidentate behavior. There were very few cases in which the thiosemicarbazide acts as monodentate ligand
binding to the metal center through the sulfur atom [21]. In case of ligands (r-btsz) the chelating mode was via sulfur atom and imine
nitrogen by coordination covalent bond. In case of ligands (r-binh) the covalent bond formed between metal ion and oxygen atom and
coordinate covalent bond with imine nitrogen.
The infrared spectra of all the ligands and their ruthenium(II) compounds were recorded in KBr powder by diffuse reflectance
technique and are reported in their respective titles by tentative assignments. Vibrational frequencies of the r-btsz ligands shown 3450
cm-1 to 3230 cm-1 for NH2 and N–H stretching and from 1330 cm-1 to 1310 cm-1 for C=S stretching. In r-binh ligands showed vibrational
frequency from 3420 cm-1 to 3210 cm-1 for N–H stretching and from 1690-1670 cm-1 for C=O stretching. Vibrational frequencies of the
Ruthenium complexes bearing r-btsz ligands shown 3460 cm-1 to 3200 cm-1 for NH2 and N–H stretching and from 1335 cm-1 to 1305
cm-1 for C=S stretching. In Ruthenium complexes bearing r-binh ligands showed vibrational frequency from 3400 cm-1 to 3250 cm-1 for
N–H stretching and from 1685-1675 cm-1 for C=O stretching.
A comparison of IR spectra of ligands r-btsz with ruthenium complexes indicates this was coordinated to the metal center by sulfur
atom and imine nitrogen, which was confirmed by the IR spectra. In case of IR spectra of ligands r-binh with ruthenium compound
indicates this was coordinated to the metal center by oxygen atom and imine nitrogen. In the complexes such as Ru-1 to Ru-8 the
coordination had occurred via sulfur and imine nitrogen but not with terminal amine group, which was confirmed by the spectra, which
indicates no change in vibrational frequency of NH2 group between 3400 and 3300 cm-1. A comparison of IR spectra of ligand r-binh
with ruthenium complexes indicates this was coordinated to the metal center by oxygen atom and imine nitrogen, which was confirmed
by the IR spectra. Coordination of ligands (L = r-binh, r-btsz,) to ruthenium results in compounds such as [Ru(S)2(L)]2+Cl2(Ru1–Ru16)
(Fig. 1) , respectively. All these compounds do not possess any C2 axes of symmetry.
In the 1H-NMR spectra of the complexes, Ru-1 there were 24 resonance peaks (δ 10.04-6.14), and 24 well resolved peaks (δ 10.16-
6.12) for Ru-2. The mass spectra of the complexes confirmed the suggested formula by their molecular ion peak. ESI mass
spectroscopic data clearly suggested that authentification of ruthenium complexes. In all the cases, the loss of chloride ions was
detected where S = 2,2'-bipyridine/1,10-phenanthroline and L = r-binh, r-btsz. Thus, based on the above observations, it is tentatively
suggested that Ru(II) complexes showed an octahedral geometry.
Page 5 of 9

3.2 Biological activity
Results are summarized in Table 1 and Table 2. The in vitro cytotoxic activity was evaluated for all the synthesized ligands and its
ruthenium complexes against human Molt 4/C8, CEM, HL60, BEL7402, and L1210. The relative potencies between ligands and their
ruthenium complexes revealed the importance of ruthenium metal using the Molt4/C8, CEM, HL60, BEL 7402 assays and murine
L1210 assays. The cytotoxicity data in table revealed that most ruthenium complexes have significant cytotoxic potencies (IC50 figures
in the 0.24-2.4 for Molt 4/C8, 0.26-1.82 µmol/L for L1210, 0.37-4.6 µmol/L for CEM, 0.22-3.5 µmol/L and 0.32-4.2 µmol/L for
BEL7402). While for ligands, the IC50 values were in excess (100-228 µmol/L against Molt4/C8, 86-198 µmol/L for CEM, 99-248
µmol/L for L1210, 66-218 µmol/L against HL60, 99-221 µmol/L for BEL7402). Of the tested ligands and ruthenium complexes
Ru(phen)2 (2-(OMe)-btsz)Cl2 showed cytotoxicity against all five cell lines tested in range of 0.34, 0.26, 0.38, 0.22, and 0.48 µmol/L
for Molt 4/C8, L1210, CEM, HL60, BEL7402 respectively. Whereas another complex Ru(bpy)2 (2-(OMe)-btsz)Cl2 did show
cytotoxicity against cell lines tested 0.62 µmol/L for Molt 4/C8, 0.46 for L1210, 0.44 for CEM, 0.51 for HL60 and 0.32 for BEL7402.
Whereas another complex Ru(bpy)2 (2-(OMe)-binh)Cl2 did show cytotoxicity against cell lines tested 1.1 µM for Molt 4/C8, 0.92 for
L1210, 1.0 for CEM, 0.86 for HL60 and 0.94 for BEL7402. Comparison of structure activity relationships (Fig. 2) with ruthenium
complexes bearing thiosemicarbazone ligands (Ru-1 to Ru-8) displayed the better cytotoxicity comparison with the ruthenium
complexes bearing isonicotinyl hydrazone ligands (Ru-9 to Ru-16). On comparison to ruthenium complexes the ligands displayed the
cytotoxicty at higher µmol/L concentration. On comparison with other ruthenium complexes (e.g. NAMI-A & KP1019) these reported
ruthenium complexes displayed the cytotoxicity at lower µmol/L concentration.
3.3 Apoptosis activity [22]
Most of the cytotoxic drugs in current use have been shown to induce apoptosis in susceptible cells. To further address the death
pattern, BEL-7402 cells were stained with acridine orange (AO) and ethidium bromide (EB). The AO/EB staining is sensitive to DNA
and was used to access changes in nuclear morphology. In the absence of complex Ru-1, the living cells were stained bright green in
spots (Fig. 3A). After treatment of BEL-7402 cells with complex (Ru-1) for 48 h, the green apoptotic cells with apoptotic features such
as nuclear shrinkage, chromatin condensation, as well as red necrotic cells, were observed (Fig. 3B). Similar results for complexes (Ru-
1 to Ru-16) were also observed.
3.4 Cytotoxic evaluation
The compounds prepared in laboratory were evaluated against various celllines by a literature procedure [23].
4. Conclusion
A series of 16 arene Ru(II) complexes (Ru-1 to Ru-16) bearing thiosemicarbazone and ligands were prepared. The cytototoxic
activity of the new ruthenium(II) arene compounds has been evaluated in several cell lines (Molt4/C8, L1210, CEM, HL60, BEL7402)
in order to establish structure−activity relationships. From the cytotoxic data indicate that ruthenium complexes bearing
thiosemicarbzone lignads more active than the ruthenium complexes bearing isonicotinyl hydrazone ligands. It was found that the
ruthenium complexes were stronger anticancer activity than the free ligands, it could be due to more efficient uptake of the
mononuclear ruthenium complexes. The present study may stimulate the development of novel anticancer molecules, with the
increasing competition and reducing development time.
Acknowledgment
This work was supported by National Council for Scientific and Technological Development (CNPq), Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Oswaldo Cruz Foundation (Fiocruz) and Department of Science and
Technology, New Delhi, India [No. SR/WOS-A/LS-562/2011) dated 27/03/2012]. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
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[8] D.R. Richardson, D.S. Kalinowski, V. Richardson, et al., 2-Acetylpyridine thiosemicarbazones are potent iron chelators and antiproliferative agents:
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substituted thiosemicarbazones and their Cu(II) complexes, J. Med. Chem. 54 (2011) 2391-2398.
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NH2
S
NH
N
O
S
H2N
N
NH2
H
R
R
Ethanol, Reflux, 3-4 hrs
R = 2-OCH3, 2-CH3, 4-F, 2,4-di-NO2
Scheme 1: Synthesis of ligands r-bitsz and r-binh.
Scheme 2: Synthesis of [Ru(S)2(L)]Cl2.
Page 7 of 9

2
+
2
+
N
N
N
N
Ru
N
S
Ru
N
O
NH2
N
N
N
NH
NH
Cl2
Cl2
CH
CH
N
N
R
R
Figure 1. Chemical structures of [Ru(phen)2(btsz)]2+ and [Ru(phen)2(binh)]2+.
Figure 2. Structure activity relationship of [Ru(phen)2(btsz)]2+Cl2
Figure 3. BEL-7402 cell without treatment (A) and in the presence of Ru complex 2 (B) incubated at 37 °C and 5% CO2 for 48 h. Cells in a, b
and c are living, apoptotic and necrotic cells, respectively.
Table 1. Cytotoxic studies of ligands
Compound
IC50a (µmol/L)
CEM
Molt4/C8
118 ± 04
144 ± 14
100 ± 12
112 ± 06
228 ± 16
204 ± 08
185 ± 12
L1210
HL60
BEL7402
134 ± 12
99 ± 02
2-OCH3-BTSZ
2-CH3-BTSZ
4-F-BTSZ
2,4-dinitro-BTSZ
2-OCH3-BINH
2-CH3-BINH
4-F-BINH
158 ± 18
198 ± 12
134 ± 08
86 ± 12
106 ± 04
104 ± 08
142 ± 18
94 ± 12
128 ± 16
110 ± 08
66 ± 06
114 ± 08
218 ± 12
184 ± 06
162 ± 08
94 ± 06
92 ± 12
125 ± 04
166 ± 08
221 ± 09
148 ± 12
200 ± 16
182 ± 14
198 ± 12
196 ± 14
184 ± 06
168 ± 16
216 ± 04
248 ± 04
202 ± 12
2,4-Dinitro-BINH 175 ± 08
a50% inhibitory concentration, required to inhibit tumor cell proliferation by 50%
Table 2. Cytotoxic studies of Ruthenium complexes
Comp. code
IC50a (µmol/L)
CEM
Molt4/C8
L1210
HL60
BEL7402
Page 8 of 9

0.34 ± 0.06
0.62 ± 0.05
0.24 ± 0.06
0.68 ± 0.05
0.84 ± 0.4
0.51 ± 0.06
0.98 ± 0.4
1.1 ± 0.04
1.4 ± 0.08
0.98 ± 0.6
1.2 ± 0.4
1.4 ± 0.08
2.1 ± 0.06
1.8 ± 0.2
2.4 ± 0.06
1.9 ± 0.5
0.26 ± 0.02
0.46 ± 0.04
0.55 ± 0.5
0.79 ± 0.03
0.95 ± 0.05
0.56 ± 0.4
1.0 ± 0.02
0.92 ± 0.06
0.99 ± 0.6
1.4 ± 0.04
1.8 ± 0.02
0.98 ± 0.06
1.82 ± 0.4
1.2 ± 0.6
0.38 ± 0.4
0.44 ± 0.02
0.37 ± 0.04
0.54 ± 0.08
0.66 ± 0.4
0.78 ± 0.03
0.86 ± 0.4
1.0 ± 0.05
2.3 ± 0.4
1.2 ± 0.03
0.98 ± 0.02
2.2 ± 0.5
1.4 ± 0.4
2.6 ± 0.04
4.6 ± 0.8
0.22 ± 0.04
0.51 ± 0.05
0.64 ± 0.04
0.28 ± 0.6
0.86 ± 0.04
0.68 ± 0.2
0.88 ± 0.02
0.86 ± 0.04
0.94 ± 0.8
0.90 ± 0.06
1.2 ± 0.42
1.8 ± 0.06
2.4 ± 0.04
3.5 ± 0.05
2.5 ± 0.3
0.48 ± 0.02
0.32 ± 0.04
0.52 ± 0.06
0.68 ± 0.02
0.64 ± 0.06
0.85 ± 0.3
0.92 ± 0.4
0.94 ± 0.06
1.2 ± 0.2
1.51 ± 0.04
1.6 ± 0.6
1.8 ± 0.04
2.2 ± 0.24
2.8 ± 0.02
3.6 ± 0.4
Ru-1
Ru-2
Ru-3
Ru-4
Ru-5
Ru-6
Ru-7
Ru-8
Ru-9
Ru-10
Ru-11
Ru-12
Ru-13
Ru-14
Ru-15
Ru-16
STD
1.5 ± 0.5
2.2 ± 0.08
1.2 ± 0.02
3.6 ± 0.6
0.87 ± 0.06
2.8 ± 0.22
0.98 ± 0.02
4.2 ± 0.6
0.78 ± 0.04
0.51± 0.1
a50% inhibitory concentration, required to inhibit tumor cell proliferation by 50%
STD = Cisplatin
Page 9 of 9