Synthesis, characterization, antitumor, and cytotoxic activity of mononuclear Ru(II) complexes

Article abstract of DOI:10.1080/00958972.2010.534140

In the search for antitumor active metal complexes several ruthenium complexes have been reported to be promising. A series of mononuclear Ru(II) complexes, [Ru(T)₂(S)]₂+, where T = 2,2'-bipyridine/1,10- phenanthroline and S = CH₃-bitsz, Cl-bitsz, Br-bitsz, tmtsz, dmtsz, have been prepared and characterized by UV-Vis, IR, ¹H-NMR, FAB-mass spectroscopy, and elemental analysis. The complexes were subjected to invivo anticancer activity against a transplantable murine tumor cell line Ehrlich's ascitic carcinoma (EAC) and invitro cytotoxic activity against human cancer cell line Molt 4/C₈, CEM, and murine tumor cell line L1210. Ruthenium complexes showed promising biological activity especially in decreasing tumor volume and viable ascitic cell counts. Treatment with these complexes prolonged the life span of EAC-tumor-bearing mice by 10-48%. Invitro evaluation of these ruthenium complexes revealed cytotoxic activity from 0.21 to 24 mol L ^-1 against Molt 4/C₈, 0.16-19 μmol L^-1 against CEM, and 0.75-32 μmol L^-1 against L1210 cell proliferation, depending on the nature of the compound.

Full text of DOI:10.1080/00958972.2010.534140

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Synthesis, characterization, antitumor,
and cytotoxic activity of mononuclear
Ru(II) complexes
Sreekanth Thota a , Subhas Somalingappa Karki b , K.N. Jayaveera
c , Jan Balzarini d & Erik De Clercq d
a Department of Pharmaceutical Chemistry, S.R. College of
Pharmacy, Ananthasagar, Warangal – 506371, Andhrapradesh, India
b Department of Pharmaceutical Chemistry, KLE Academy of
Higher Education and Research, College of Pharmacy, 2nd Block,
Rajajinagar, Bangalore – 560010, Karnataka, India
c Department of Chemistry, JNTU Oil Technological Research
Institute, Anantapur – 515001, Andhrapradesh, India
d Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Published online: 19 Nov 2010.
To cite this article: Sreekanth Thota , Subhas Somalingappa Karki , K.N. Jayaveera , Jan Balzarini
& Erik De Clercq (2010): Synthesis, characterization, antitumor, and cytotoxic activity of
mononuclear Ru(II) complexes, Journal of Coordination Chemistry, 63:24, 4332-4346
To link to this article: http://dx.doi.org/10.1080/00958972.2010.534140
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Journal of Coordination Chemistry
Vol. 63, No. 24, 20 December 2010, 4332–4346
Synthesis, characterization, antitumor, and cytotoxic activity of
mononuclear Ru(II) complexes
SREEKANTH THOTA*y, SUBHAS SOMALINGAPPA KARKIz,
K.N. JAYAVEERAx, JAN BALZARINI{ and ERIK DE CLERCQ{
yDepartment of Pharmaceutical Chemistry, S.R. College of Pharmacy, Ananthasagar,
Warangal – 506371, Andhrapradesh, India
zDepartment of Pharmaceutical Chemistry, KLE Academy of Higher Education and
Research, College of Pharmacy, 2nd Block, Rajajinagar, Bangalore – 560010,
Karnataka, India
xDepartment of Chemistry, JNTU Oil Technological Research Institute,
Anantapur – 515001, Andhrapradesh, India
{Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
(Received 16 July 2010; in final form 6 October 2010)
In the search for antitumor active metal complexes several ruthenium complexes have been
reported to be promising. A series of mononuclear Ru(II) complexes, [Ru(T)2(S)]2þ, where
T ¼ 2,20-bipyridine/1,10-phenanthroline and S ¼ CH3-bitsz, Cl-bitsz, Br-bitsz, tmtsz, dmtsz,
have been prepared and characterized by UV-Vis, IR, 1H-NMR, FAB-mass spectroscopy, and
elemental analysis. The complexes were subjected to in vivo anticancer activity against a
transplantable murine tumor cell line Ehrlich’s ascitic carcinoma (EAC) and in vitro cytotoxic
activity against human cancer cell line Molt 4/C8, CEM, and murine tumor cell line L1210.
Ruthenium complexes showed promising biological activity especially in decreasing tumor
volume and viable ascitic cell counts. Treatment with these complexes prolonged the life span of
EAC-tumor-bearing mice by 10–48%. In vitro evaluation of these ruthenium complexes
revealed cytotoxic activity from 0.21 to 24 mmol Lꢀ1 against Molt 4/C8, 0.16–19 mmol Lꢀ1
against CEM, and 0.75–32 mmol Lꢀ1 against L1210 cell proliferation, depending on the nature
of the compound.
Keywords: Ru(II) complexes; Antitumor; EAC; Cytotoxicity
1. Introduction
Metal ions show good effect on cellular process, not only natural processes, such as cell
division and gene expression, but also toxicity, carcinogenicity, and antitumor
chemistry. In the search for antitumor metal complexes several ruthenium complexes
have shown promise as anticancer drugs. A series of mononuclear Ru(II) complexes
of the type [Ru[M]2[U]]2þ, where M ¼ 2,20-bipyridine/1,10-phenanthroline and U ¼ tpl,
4-Cl-tpl, 4-CH3-tpl, 4-OCH3-tpl, 4-NO2-tpl, and pai [1].
*Corresponding author. Email: sreekanth_237@yahoo.in
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2010 Taylor & Francis
DOI: 10.1080/00958972.2010.534140

Mononuclear Ru(II)
4333
The objectives of the present investigations were to develop analogs of [Ru[T]2[S]]
principally as candidate cytotoxins. The discovery of antitumor properties of cisplatin
in 1965 marked the development of metallo-pharmaceuticals in cancer chemotherapy
[2]. Platinum drugs induce cytotoxicity by cross-linking DNA, causing changes to the
DNA structure resulting in the inhibition of replication and protein synthesis. However,
the application of platinum drugs suffers from their high general toxicity leading to
severe toxic side effects. In comparison, ruthenium complexes have attracted attention
in the last 20 years as potential antitumor agents. Some exhibit very encouraging
pharmacological profiles [3].
Ruthenium compounds are regarded as promising alternatives to platinum com-
pounds and offer many approaches to metallo-pharmaceuticals; the compounds are
known to be stable and to have predictable structures both in the solid state and in
solution with the tuning of ligand affinities and steadily increasing knowledge of the
biological effects of ruthenium compounds [4]. The first systematic investigation of
ruthenium compounds and their antitumor properties was done in the beginning of the
1980s with fac-[RuCl3(NH3)3] and cis-[RuCl2(NH3)4]Cl [5] preceded by the discovery
that ruthenium red possesses antitumor properties in the 1970s [6, 7]. Since then
compounds, such as trans-(IndH)[Ru(ind)2Cl4] (Ind ¼ indazole), mer-[Ru(terpy)Cl3
(terpy ¼ 2,20-terpyridine) [8–10], [Ru(dmso)4Cl2] [11], ImH[Ru(im)Cl5] [12],
ImH[Ru(im)2-Cl4] [13], and ImH[Ru(im)(dmso)Cl4] [14] NAMI-A (im ¼ imidazole)
are also well-known antitumor agents.
Ruthenium(II) arene complexes show remarkable cytotoxic properties in vitro as well
as in vivo [15, 16]. A series of complexes with general formula [Ru(ꢀ6-arene)Cl(en)][PF6]
(en ¼ ethylene diamine, arene ¼ benzene, p-cymene, tetrahydroanthracene, etc.) have
been studied for their in vitro anticancer activity [17].
Ruthenium compounds with bidentate ligands show intercalation properties with
DNA [18]. Ru(II) compounds are kinetically more reactive than Ru(III) [19]. So, we
have reported that Ru(II) compounds bearing thiosemicarbazides (TSC), 8-hydroxy
quinolines, have in vivo anticancer and in vitro antibacterial activity [20, 21]. Recently
we reported Ru(II) compounds bearing isatin thiosemicarbazones, chloro-fluoro-
phenyl imino methyl phenol have in vivo anticancer and in vitro cytotoxic activity [22].
In this work, we describe the synthesis and characterization of some ruthenium
complexes, their in vitro cytotoxic activity against human cancer cell line Molt 4/C8,
CEM, and murine tumor cell line L1210, and their in vivo anticancer activity against
transplantable murine tumor cell line Ehrlich’s ascitic carcinoma (EAC). Our research
has focused on the complexes of the general formula [Ru[T]2[S]]Cl2, where
T ¼ 2,20-bipyridine/1,10-phenanthroline and S ¼ CH3-bitsz, Cl-bitsz, Br-bitsz, tmtsz,
and dmtsz.
2. Experimental
2.1. General methods
AR grade solvents 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

4334
S. Thota et al.
as received. UV-Vis spectra were on a Jasco spectrophotometer. FTIR spectra were
recorded in KBr powder on a Jasco V410 FTIR spectrometer by diffuse reflectance
1
technique. H/13C NMR spectra were measured in CDCl3 and DMSO-d6 on a Bruker
Ultraspec 500 MHz/AMX 400/300 MHz spectrometer. The reported chemical shifts
were against that of TMS. FAB-mass spectra were recorded on a JEOL JMS600
spectrum with mNBA matrix. Substituted N-benzyl isatin thiosemicarbazones [23, 24]
were prepared according to the literature method.
1
1
R
R
1
O
c
a
b
NOH
O
R
NH2
N
N
O
H
H
Aniline
Isonitrosoacetanilide
Isatin
d
e
H2N
HN
S
1
N
1
R
O
R
N
O
N
f
O
CH2
CH2
g
F
F
r-bitsz
R1
CH , Cl, Br
=
3
a = CCl3CHOH2
b = NH2OHHCl
c = H2SO4
d = DMF/ K2CO3
e = F-C6 H4-CH2Cl
f = NH2NHCSNH2
g = C2H5OH, CH3COOH
2.2. General procedure for preparing thiophene-2-methylene thiosemicarbazone
In a round bottom flask fitted with a reflux condenser thiophene-2-carboxyaldehyde
(0.01 mol) and TSC (0.01 mol) were added. The mixture was refluxed in anhydrous
alcohol for 4–5 h and left overnight. The solid that separated was filtered and dried. The
crude solid was purified by recrystallization from alcohol to give white crystals.

Mononuclear Ru(II)
4335
2.2.1. CH3-bitsz. Yield 74%, m.p. 259–260ꢁC (lit., 260ꢁC). IR (KBr) cmꢀ1: 3416–3262
(NH2 and NH), 3031 (C–H), 2924 (C–H), 1693 (C¼O), 1341 (C¼S). Calcd for
C17H15O1N4S1F1 (%): C, 59.64; H, 4.42; N, 16.36. Found (%): C, 59.20; H, 4.35; N,
16.28. ꢁmax nm (MeOH): 270, 366. 1H NMR (DMSO-d6): ꢂ ¼ 10.64 (1H, s), 8.07 (1H, s),
7.99 (2H, d), 7.87 (2H, s), 7.73 (2H, d, J ¼ 8.6 Hz), 7.62 (2H, d, NH2), 6.45 (2H, d,
J ¼ 8.6 Hz), 1.56 (3H, s, CH3).
2.2.2. Cl-bitsz. Yield 69%, m.p. 243–245ꢁC (lit., 245ꢁC). IR (KBr) cmꢀ1: 3427–3272
(NH2 and NH), 3069 (C–H), 2922 (C–H), 1687 (C¼O), 1335 (C¼S). Calcd for
C16H12O1N4S1F1Cll (%): C, 52.97; H, 3.33; N, 15.44. Found (%): C, 52.76; H, 3.31; N,
1
15.29. ꢁmax nm (MeOH): 250, 287, 369. H NMR (DMSO-d6): ꢂ ¼ 10.29 (1H, s), 8.04
(2H, d), 7.98 (2H, s), 7.87 (2H, d, J ¼ 8.6 Hz), 7.80 (1H, s), 7.74 (2H, d, NH2), 6.58
(2H, d, J ¼ 8.6 Hz).
2.2.3. Br-bitsz. Yield 85%, m.p. 241–242ꢁC (lit., 242ꢁC). IR (KBr) cmꢀ1: 3422–3238
(NH2 and NH), 3143 (C–H), 1682 (C¼O), 1346 (C¼S). Calcd for C16H12O1N4S1F1Br1
(%): C, 47.19; H, 2.97; N, 13.76. Found (%): C, 47.16; H, 2.94; N, 13.69. ꢁmax nm
1
(MeOH): 242, 277, 358. H NMR (DMSO-d6): ꢂ ¼ 10.01 (1H, s), 8.12 (2H, d), 8.00
(2H, s), 7.89 (2H, d, J ¼ 8.6 Hz), 7.72 (1H, s), 7.56 (2H, d, NH2), 6.39 (2H, d, J ¼ 8.6 Hz).
2.2.4. tmtsz. Yield 78%, m.p. 182–184ꢁC. IR (KBr) cmꢀ1: 3447–3263 (NH2 and NH),
3021 (C–H), 1326 (C¼S). Calcd for C6H7N3S2 (%): C, 38.90; H, 3.81; N, 22.68. Found
1
(%): C, 38.86; H, 3.76; N, 22.49. ꢁmax nm (MeOH): 267, 369. H NMR (DMSO-d6):
ꢂ ¼ 10.45 (1H, s), 8.89 (1H, s, CH¼N), 7.89 (2H, d, NH2, J ¼ 8.6 Hz), 7.72–7.43 (3H, m).
2.3. General procedure for preparing dichloro phenyl methylene thiosemicarbazone
In a round bottom flask fitted with a reflux condenser 2,4-dichlorobenzaldehyde
(0.01 mol) and TSC (0.01 mol) were added. The mixture was refluxed in anhydrous
alcohol for 4–5 h and left overnight. The solid that separated was filtered and dried. The
crude solid was purified by recrystallization from alcohol to give off white crystals.
2.3.1. dmtsz. Yield 80%, m.p. 234–235ꢁC. IR (KBr) cmꢀ1: 3414–3238 (NH2 and NH),
3016 (C–H), 2894 (C–H), 1339 (C¼S). Calcd for C8H7N3Cl2S1 (%): C, 38.72; H, 2.84;
N, 16.93. Found (%): C, 38.66; H, 2.78; N, 16.89. ꢁmax nm (MeOH): 238, 366. 1H NMR
(DMSO-d6): ꢂ ¼ 10.08 (1H, s), 8.98 (1H, s, CH¼N), 8.01 (2H, d), 7.62 (2H, d, NH2,
J ¼ 8.6 Hz), 7.34 (1H, s).
2.4. Preparation of cis-[bis(S)dichlororuthenium(II)] cis-[Ru(T)2Cl2] (where
T ¼ 2,20-bipyridine/1,10-phenanthroline) [21]
RuCl3 ꢂ H2O, 1 g (2.5 mmol) and ligand T (5 mmol) was refluxed in 50 mL DMF for 3 h
under nitrogen. The reddish-brown solution slowly turned purple and the product
precipitated. The solution was cooled overnight at 0ꢁC. A fine microcrystalline mass
was filtered off, repeatedly washed with 30% LiCl solution, and finally recrystallized
from the same. The product was dried and stored in a vacuum desiccator over P2O5 for
further use (yield 75%).

4336
S. Thota et al.
2.5. General procedure for preparing [Ru(T)2(S)Cl2] (where
T ¼ 1,10-phenanthroline (Ru1)/2,20-bipyridine (Ru2); S ¼ CH3-bitsz,
Cl-bitsz, Br-bitsz, tmtsz, dmtsz)
To the black microcrystalline cis-bis(T)dichloro ruthenium(II){cis-Ru(T)2Cl2} (2 mmol)
excess of ligand (2.5 mmol) was added and refluxed in ethanol under nitrogen.
The initial colored solution slowly changed to brownish orange at the end of the
reaction, which was verified by TLC on silica plates. Then excess ethanol distilled off
and silica gel (60–120 mesh) added to this solution. The product was purified by column
chromatography by using silica gel as stationary phase and chloroform–methanol as
mobile phase.
2.5.1. [Ru[phen]2[CH3-bitsz]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3438–3254
(NH2 and N–H), 3098 (C–H), 2946 (C–H), 1682 (C¼O), 1334 (C¼S). Calcd for
C41H31Cl2N8O1Ru1S1F1 (%): C, 56.29; H, 3.54; N, 12.82. Found (%): C, 56.24; H, 3.49;
1
N, 12.79. H NMR (DMSO-d6): ꢂ ppm: 10.42 (s, 1H), 8.86 (2H, t), 8.65 (d, J ¼ 4.9 Hz,
1H), 8.51 (d, J ¼ 8.4 Hz, 1H), 8.31–8.19 (m, 6H), 8.09 (s, 1H), 8.00 (d, J ¼ 5.0 Hz, 2H),
7.82 (d, 2H), 7.76 (d, 2H), 7.22 (d, 2H, NH2), 7.10 (d, 2H), 7.01 (d, J ¼ 14.6 Hz, 2H),
6.73 (d, 2H), 6.47 (d, 2H), 1.58 (3H, s, CH3). FAB-MS (mNBA): 874
[Ru(phen)2(CH3-bitsz)]2þ(Cl2)ꢀ; 803 [Ru(phen)2(CH3-bitsz)]2þ
(CH3-bitsz)]2þ; 461 [Ru(phen)2].
;
623 [Ru(phen)
2.5.2. [Ru[bpy]2[CH3-bitsz]Cl2. 42%, black crystals, IR (KBr) cmꢀ1: 3426–3267 (NH2
and N–H), 3124 (C–H), 2958 (C–H), 1686 (C¼O), 1342 (C¼S). Calcd for
C37H31Cl2N8O1Ru1S1F1 (%): C, 53.75; H, 3.75; N, 13.56. Found (%): C, 53.64;
1
H, 3.69; N, 13.42. H NMR (DMSO-d6): ꢂ ppm: 10.29 (s, 1H), 8.71 (2H, d), 8.59
(d, J ¼ 4.9 Hz, 2H), 8.47 (d, J ¼ 8.4 Hz, 2H), 8.36–8.22 (m, 4H), 8.10 (s, 1H), 8.02
(d, J ¼ 5.0 Hz, 2H), 7.87 (d, 2H), 7.64 (d, 2H), 7.12 (d, 2H, NH2), 7.09 (d, 2H), 7.00
(d, J ¼ 14.6 Hz, 2H), 6.68 (d, 2H), 6.39 (d, 2H), 1.62 (3H, s, CH3). FAB-MS (mNBA):
826 [Ru(bpy)2(CH3-bitsz)]2þ(Cl2)ꢀ; 755 [Ru(bpy)2(CH3-bitsz)]2þ
(CH3-bitsz)]2þ; 413 [Ru(bpy)2].
; 599 [Ru(bpy)
2.5.3. [Ru[phen]2[Cl-bitsz]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3417–3238 (NH2
and N–H), 3079 (C–H), 2958 (C–H), 1680 (C¼O), 1328 (C¼S). Calcd for
C40H28Cl3N8O1Ru1S1F1 (%): C, 53.69; H, 3.13; N, 12.52. Found (%): C, 53.56;
1
H, 3.10; N, 12.44. H NMR (DMSO-d6): ꢂ ppm: 10.18 (s, 1H), 8.73 (2H, t), 8.59
(d, J ¼ 4.9 Hz, 1H), 8.47 (d, J ¼ 8.4 Hz, 1H), 8.26–8.18 (m, 6H), 8.14 (s, 1H), 8.06
(d, J ¼ 5.0 Hz, 2H), 7.96 (d, 2H), 7.79 (d, 2H), 7.02 (d, 2H, NH2), 7.00 (d, 2H), 6.89
(d, J ¼ 14.6 Hz, 2H), 6.72 (d, 2H), 6.38 (d, 2H). FAB-MS (mNBA): 894
[Ru(phen)2(Cl-bitsz)]2þ(Cl2)ꢀ; 823 [Ru(phen)2(Cl-bitsz)]2þ; 643 [Ru(phen)(Cl-bitsz)]2þ
461 [Ru(phen)2].
;
2.5.4. [Ru[bpy]2[Cl-bitsz]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3451–3242 (NH2
and N–H), 3082 (C–H), 2983 (C–H), 1688 (C¼O), 1344 (C¼S). Calcd for
C36H28Cl3N8O1Ru1S1F1 (%): C, 51.06; H, 3.31; N, 13.24. Found (%): C, 51.01;
1
H, 3.28; N, 13.19. H NMR (DMSO-d6): ꢂ ppm: 10.27 (s, 1H), 8.87 (2H, d), 8.69
(d, J ¼ 4.9 Hz, 2H), 8.58 (d, J ¼ 8.4 Hz, 2H), 8.42–8.17 (m, 4H), 8.06 (s, 1H), 8.01

Mononuclear Ru(II)
4337
(d, J ¼ 5.0 Hz, 2H), 7.91 (d, 2H), 7.68 (d, 2H), 7.15 (d, 2H, NH2), 7.09 (d, 2H), 7.00
(d, J ¼ 14.6 Hz, 2H), 6.61 (d, 2H), 6.37 (d, 2H). FAB-MS (mNBA): 846
[Ru(bpy)2(Cl-bitsz)]2þ(Cl2)ꢀ; 775 [Ru(bpy)2(Cl-bitsz)]2þ; 619 [Ru(bpy)(Cl-bitsz)]2þ
413 [Ru(bpy)2].
;
2.5.5. [Ru[phen]2[Br-bitsz]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3411–3303 (NH2
and N–H), 3125 (C–H), 2995 (C–H), 1681 (C¼O), 1324 (C¼S). Calcd for
C40H28Cl2N8O1Ru1S1F1Br1 (%): C, 51.17; H, 2.98; N, 11.94. Found (%): C, 51.12;
1
H, 2.92; N, 11.90%. H NMR (DMSO-d6): ꢂ ppm: 10.07 (s, 1H), 8.79 (2H, t), 8.68
(d, J ¼ 4.9 Hz, 1H), 8.54 (d, J ¼ 8.4 Hz, 1H), 8.39–8.21 (m, 6H), 8.12 (s, 1H), 8.04
(d, J ¼ 5.0 Hz, 2H), 7.96 (d, 2H), 7.73 (d, 2H), 7.31 (d, 2H, NH2), 7.24 (d, 2H), 7.11
(d, J ¼ 14.6 Hz, 2H), 6.81 (d, 2H), 6.35 (d, 2H). FAB-MS (mNBA): 938
[Ru(phen)2(Br-btsz)]2þ(Cl2)ꢀ; 867 [Ru(phen)2(Br-btsz)]2þ; 687 [Ru(phen)(Br-btsz)]2þ
461 [Ru(phen)2].
;
2.5.6. [Ru[bpy]2[Br-bitsz]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3467–3232 (NH2
and N–H), 3058 (C–H), 2927 (C–H), 1682 (C¼O), 1341 (C¼S). Calcd for
C36H28Cl2N8O1Ru1S1F1Br1 (%): C, 48.53; H, 3.14; N, 12.58. Found (%): C, 48.46;
1
H, 3.11; N, 12.52%. H NMR (DMSO-d6): ꢂ ppm: 10.00 (s, 1H), 8.62 (2H, d), 8.59
(d, J ¼ 4.9 Hz, 2H), 8.48 (d, J ¼ 8.4 Hz, 2H), 8.39–8.26 (m, 4H), 8.18 (s, 1H), 8.07
(d, J ¼ 5.0 Hz, 2H), 7.99 (d, 2H), 7.56 (d, 2H), 7.19 (d, 2H, NH2), 7.08 (d, 2H), 6.93
(d, J ¼ 14.6 Hz, 2H), 6.67 (d, 2H), 6.37 (d, 2H). FAB-MS (mNBA): 890
[Ru(bpy)2(Br-bitsz)]2þ(Cl2)ꢀ; 819 [Ru(bpy)2(Br-bitsz)]2þ; 663 [Ru(bpy)(Br-bitsz)]2þ
413 [Ru(bpy)2].
;
2.5.7. [Ru[phen]2[tmtsz]Cl2. 46%, black crystals, IR (KBr) cmꢀ1: 3418–3226 (NH2
N–H), 3035 (C–H), 1339 (C¼S). Calcd for C30H23Cl2N7Ru1S2 (%): C, 50.21; H, 3.21;
1
N, 13.67. Found (%): C, 50.11; H, 3.18; N, 13.62. H NMR (DMSO-d6): ꢂ ppm: 10.43
(d, J ¼ 5.2 Hz, 1H), 8.98 (s, 1H, CH¼N), 8.80 (d, J ¼ 8.8 Hz, 2H), 8.64 (d, J ¼ 8.6 Hz,
1H), 8.51 (d, J ¼ 8.6 Hz, 1H), 8.40–8.20 (m, 6H), 8.11–8.03 (m, 2H), 7.89 (d, J ¼ 5.0 Hz,
2H), 7.67–7.48 (m, 3H), 7.42 (d, 2H), 6.98 (s, 2H, br, NH2). FAB-MS (mNBA): 717
[Ru(phen)2(tmtsz)]2þ(Cl2)ꢀ; 646 [Ru(phen)2(tmtsz)]2þ; 466 [Ru(phen)(tmtsz)]2þ; 461
[Ru(phen)2].
2.5.8. [Ru[bpy]2[tmtsz]Cl2. 43%, black crystals, IR (KBr) cmꢀ1: 3472–3284 (NH2
N–H), 3050 (C–H), 1342 (C¼S). Calcd for C26H23Cl2N7Ru1S2 (%): C, 46.64; H, 3.43;
1
N, 14.65. Found (%): C, 46.57; H, 3.41; N, 14.42. H NMR (DMSO-d6): ꢂ ppm: 10.52
(d, J ¼ 5.2 Hz, 1H), 9.03 (s, 1H, CH¼N), 8.83 (d, J ¼ 8.8 Hz, 2H), 8.59 (d, J ¼ 8.6 Hz,
1H), 8.52 (d, J ¼ 8.6 Hz, 1H), 8.39–8.26 (m, 6H), 8.14–8.01 (m, 2H), 7.73 (d, J ¼ 5.0 Hz,
2H), 7.54–7.43 (m, 3H), 7.32 (d, 2H), 6.87 (s, 2H, br, NH2). FAB-MS (mNBA): 669
[Ru(bpy)2(tmtsz)]2þ(Cl2)ꢀ; 598 [Ru(bpy)2(tmtsz)]2þ; 442 [Ru(bpy)(tmtsz)]2þ; 413
[Ru(bpy)2].
2.5.9. [Ru[phen]2[dmtsz]Cl2. 46%, black crystals, IR (KBr) cmꢀ1: 3482–3220 (NH2
and N–H), 3072 (C–H), 2957 (C–H), 1329 (C¼S). Calcd for C32H23Cl4N7Ru1S1 (%): C,
49.29; H, 2.95; N, 12.58. Found (%): C, 49.23; H, 2.88; N, 12.52. 1H NMR (DMSO-d6):

4338
S. Thota et al.
ꢂ ppm: 10.13 (d, J ¼ 5.2 Hz, 1H), 9.11 (s, 1H, CH¼N), 8.84 (d, J ¼ 8.8 Hz, 2H), 8.66 (d,
J ¼ 8.6 Hz, 1H), 8.54 (d, J ¼ 8.6 Hz, 1H), 8.47–8.21 (m, 6H), 8.10–8.01 (m, 2H), 7.81 (d,
J ¼ 5.0 Hz, 2H), 7.62–7.44 (m, 4H), 7.39 (d, 1H), 6.84 (s, 2H, br, NH2). FAB-MS
(mNBA): 779 [Ru(phen)2(dmtsz)]2þ(Cl2)ꢀ; 708 [Ru(phen)2(dmtsz)]2þ
[Ru(phen)(dmtsz)]2þ; 461 [Ru(phen)2].
;
528
2.5.10. [Ru[bpy]2[dmtsz]Cl2. 43%, black crystals, IR (KBr) cmꢀ1: 3450–3263 (NH2
and N–H), 3061 (C–H), 2980 (C–H), 1338 (C¼S). Calcd for C28H23Cl4N7Ru1S1 (%): C,
45.96; H, 3.14; N, 13.41. Found (%): C, 45.88; H, 3.09; N, 13.39. 1H NMR (DMSO-d6):
ꢂ ppm: 10.05 (d, J ¼ 5.2 Hz, 1H), 9.18 (s, 1H, CH¼N), 8.76 (d, J ¼ 8.8 Hz, 2H), 8.62
(d, J ¼ 8.6 Hz, 1H), 8.51 (d, J ¼ 8.6 Hz, 1H), 8.39–8.22 (m, 6H), 8.20–8.03 (m, 2H), 7.86
(d, J ¼ 5.0 Hz, 2H), 7.64–7.45 (m, 4H), 7.28 (d, 1H), 6.54 (s, 2H, br, NH2). FAB-MS
(mNBA):
731
[Ru(bpy)2(dmtsz)]2þ(Cl2)ꢀ;
660
[Ru(bpy)2(dmtsz)]2þ
;
494
[Ru(bpy)(dmtsz)]2þ; 413 [Ru(bpy)2].
2.6. Antineoplastic activity
Albino swiss mice (18–20 g body weight) were maintained in identical laboratory
conditions, given standard food pellets (Hindustan Lever Ltd, Bombay, India) and
water ad libitum. LD50 value of the synthesized compound was determined according to
the literature [25]. All compounds were dissolved in 10% DMSO solution. The animals
were divided into 15 groups each containing 12 mice. Group I was vehicle controls
(5 mL kgꢀ1 body weight ip) and group II as EAC control (2 ꢃ 106 EAC cells per mouse
ip). Group III was treated with standard drug cisplatin (2 mg kgꢀ1 body weight). All the
compounds were administered (ip) at a dose of 2 mg kgꢀ1 body weight in groups
IV–XIII, respectively. All the compounds and cisplatin treatments were provided daily
for 9 days, starting at 24 h after tumor transplantation. Six animals from each group
were sacrificed 18 h after the last dose. The ascitic fluid volume and ascitic cell count
parameters were noted. Mean survival time (MST) for remaining six mice of each group
was noted.
2.6.1. Tumor volume and viable count. Ascitic volume was noted by taking it in a
graduated centrifuge tube and packed cell volume determined by centrifuging at
1000ꢃ g for 5 min. The viability of ascitic cells was checked by Trypan blue (0.4% in
normal saline) dye exclusion test and the count was taken in Neubauer counting
chamber. The effect of the ruthenium complexes on tumor growth was monitored by
recording the mortality daily and percentage increase in life span (ILS%) was calculated
by the following formula:
ILS ð %Þ ¼ ½ðmean survival of treated groupÞ=ðmean survival of control groupÞꢀ1ꢄꢃ100:
2.6.2. Biological assays. The antiviral assays were based on the inhibition of
virus-induced cytopathicity in confluent cell cultures and the cytostatic assays on the
inhibition of tumor cell proliferation in exponentially growing tumor cell cultures.

Mononuclear Ru(II)
4339
T
N
N
N2 DMF
¥T¥ Ligand
T
N
Cl
Ru
N
.
RuCl3 xH2O
Cl
cis-[Ru(T)2Cl2]
where T = 2,2„-bipyridine/1,10-phenanthroline
Scheme 1. Preparation of cis-[Ru(T)2Cl2].
2.6.3. Cytotoxic evaluation. The compounds prepared in laboratory were evaluated
against Molt 4/C8, CEM, and L1210 cells by a literature procedure [26].
3. Results and discussion
3.1. Chemistry
Ruthenium trichloride undergoes reduction when refluxed in DMF; when refluxed
along with two-fold molar ratios of the bidentate ligand, namely (1,10-phenanthroline/
2,20-bipyridine),
a
homoleptic complex is formed. The product cis-bis(1,10-
phenanthroline/2,20-bipyridine)dichlororuthenate(II) is the starting material for the
synthesis of complexes. The product cis-bis(1,10-phenanthroline/
2,20-bipyridine)dichlororuthenate(II) was then refluxed in ethanol in the presence of
nitrogen with various ligands to yield the final octahedral ruthenium complexes.
In this homoleptic chelate the first two ligands to enter the complex in a stepwise
assembly were 1,10-phenanthroline/2,20-bipyridine, respectively. Since both ligands
are the same, a single step method was adopted for its synthesis. Ruthenium trichloride
was refluxed in DMF in the presence of 1,10-phenanthroline/2,20-bipyridine, in excess
of the stoichiometric amount, which afforded the final product cis-bis(1,10-
phenanthroline)dichlororuthenium(II)/cis-bis(2,20-bipyridine)dichlororuthenium(II)
(scheme 1). The introduction of the third ligand was carried out in the presence of
alcohol. The final chelate formed had ionic chloride in the molecule.
Ligands like r-bitsz (r-bitsz ¼ N-benzyl isatin thiosemicarbazones) were prepared by
reacting N-benzyl isatin with TSC in alcohol at 1 : 1 molar ratio (scheme 2). Tmtsz
(tmtsz ¼ thiophene methylene thiosemicarbazones) and dmtsz (dichloro methylene
thiosemicarbazones) were prepared by reacting thiophene carboxyaldehyde, dichlor-
obenzaldehyde with TSC in alcohol at 1 : 1 molar ratio (scheme 3). All ligands were
confirmed for their purity by their melting points, elemental analyses, and other spectral
studies.
The structures of the ligands especially r-bitsz, tmtsz, and dmtsz are capable of
exhibiting bidentate behavior. There are very few cases in which the TSC is
monodentate binding to the metal through sulfur [27, 28]. In the case of r-bitsz, the
chelating mode was via sulfur and imine nitrogen, not with amide carbonyl oxygen. For
tmtsz and dmtsz, the chelating mode was via sulfur and imine nitrogen by coordination
covalent bonds.

4340
S. Thota et al.
2
+
2
+
N
N
N
N
Ru
S
N
N
Ru
S
N
N
NH2
CH2
Cl2
NH2
CH2
N
N
H
Cl2
N
N
O
H
O
N
N
H3C
H3C
F
F
2+
2
+
N
N
N
N
Ru
S
N
N
NH2
Cl2
Ru
S
N
N
N
N
H
NH2
Cl2
O
N
N
H
O
H2
C
N
CH2
N
Cl
Cl
F
F
2
+
2
+
N
N
N
N
Ru
S
N
N
Ru
N
S
N
N
NH2
Cl2
NH2
CH2
N
N
H
Cl2
N
H
O
O
N
CH2
N
Br
Br
F
F

Mononuclear Ru(II)
4341
2
+
2+
N
N
N
N
Ru
S
N
N
Ru
S
N
N
NH2
Cl2
NH2
Cl2
N
N
N
N
H
H
CH
CH
S
S
2
+
2+
N
N
N
N
Ru
S
N
Ru
S
N
N
NH2
Cl2
NH2
Cl2
N
N
H
N
N
H
N
CH
CH
Cl
Cl
Cl
Cl
Infrared spectra of all the ligands and their ruthenium(II) compounds were recorded
in KBr powder by diffuse reflectance technique and are reported with tentative
assignments. The r-bitsz, tmtsz and dmtsz showed vibrational frequency from 3400 to
3200 cmꢀ1 for NH2 and N–H stretching, from 1690 to 1670 cmꢀ1 for C¼O stretching,
and from 1345 to 1320 cmꢀ1 for C¼S stretching.
A comparison of IR spectra of r-bitsz with ruthenium complexes indicates
coordination to the metal center by sulfur and imine nitrogen. In [Ru[phen]2
[CH3-bitsz]Cl2,
[Ru[bpy]2[CH3-bitsz]Cl2,
[Ru[phen]2[Cl-bitsz]Cl2,
[Ru[bpy]2
[Cl-bitsz]Cl2, [Ru[phen]2[Br-bitsz]Cl2, [Ru[bpy]2[Br-bitsz]Cl2, [Ru[phen]2[tmtsz]Cl2,
[Ru[bpy]2[tmtsz]Cl2, [Ru[phen]2[dmtsz]Cl2, and [Ru[bpy]2[dmtsz]Cl2 the coordination
occurred via sulfur and imine nitrogen but not with terminal amine group, which was
conErmed by no change in vibrational frequency of NH2 between 3400 and 3200 cmꢀ1
.
The coordination of ligands (S ¼ r-bitsz, tmtsz, dmtsz) to ruthenium results in
[Ru(T)2(S)]2þCl2 (Ru1–Ru10), respectively. These compounds do not possess any C2
axes of symmetry. Such loss of C2 axis of symmetry was seen for [Ru(L)2(R)] [22, 23]
(where L ¼ 2,20-bipyridine/1,10-phenanthroline and R ¼ acetazolamide, 7-iodo-
8-hydroxy-quinoline, etc.). All compounds had well-resolved resonances, which
correspond to four different aromatic ring protons of the two 2,20-bipyridine/
1,10-phenanthroline ligands and the third ligand.

4342
S. Thota et al.
H2N
S
O
HN
N
H
S
S
Alcohol
H
tmtsz
H2N
HN
S
H2N
H
S
NH2
HN
Thiosemicarbazide
N
Alcohol
Cl
H
O
Cl
Cl
dmtsz
Cl
Scheme 2. Preparation of ligands (r-bitsz, tmtsz, dmtsz).
2+
T
T
T
Alcohol, Ligand r-bitsz, tmtsz, dmtsz
N2 atm
N
N
N
Cl
N
N
T
Cl2
Ru
N
N
Ru N
S
N
Cl
cis-[Ru(T)2Cl2]
S
Ru1-Ru10
Scheme 3. Preparation of tris chelates from cis-[Ru(T)2Cl2].
These compounds showed broad and intense visible bands between 330 and 520 nm
due to metal-to-ligand charge-transfer (MLCT) transition. In the UV region tꢅhe bands
at 270 and 300 nm were assigned to 2,20-bipyridine/1,10-phenanthroline p–p charge-
transfer transitions. The same transition was found in free 2,20-bipyridine/
1,10-phenanthroline at 270 nm, so that the coordination of the ligand resulted in a
red shift in the transition energy. There were also shoulders at 380 and 500 nm, which
were, tentatively, attributed to MLCT transitions involving 2,20-bipyridine,
1,10-phenanthroline, and the third ligand.
1
In H-NMR spectra of the complexes, there were resolved resonances at low field at
ꢂ 10.42 (s, br, NH), 1.58 (s, 3H, CH3). Thus in the case of [Ru[phen]2[CH3-bitsz]Cl2
there were 31 resonances (ꢂ 10.42–1.58) and 31 well-resolved peaks (ꢂ 10.29–1.62) for
[Ru[bpy]2[CH3-bitsz]]Cl2.
The mass spectra of the complexes confirmed the suggested formula by their
molecular ion peaks. The spectrum showed numerous peaks representing the successive

Mononuclear Ru(II)
4343
Figure 1. Mass spectrum of [Ru(phen)2(Cl-bitz)]2þ shows intense peak at 824, [Ru(phen)(Cl-bitsz)]2þ shows
intense peak at 643, and [Ru(phen)2] shows intense peak at 460 by loss of Cl2.
degradation of the molecule. FAB mass spectroscopic data in figure 1 clearly suggested
that mononuclear complexes had been formed in each case, the first fragment being due
to [Ru(T)2(S)]2þ Clꢀ2 ion pair. The complex also showed a peak due to the complex
cation [Ru(T)2(S)]2þ and others due to [Ru(T)(S)]2þ, [Ru(T)2]2þ, respectively [where
T ¼ 1,10-phenanthroline/2,20-bipyridine and S ¼ r-bitsz, tmtsz, dmtsz]. This type of
fragmentation reported for [Ru(phen)2(nmit)]Cl2 and [Ru(bpy)2(ihqs)]Cl2, where
phen ¼ 1,10-phenanthroline, bpy ¼ 2,20-bipyridine, nmit ¼ N-methyl isatin thio semi-
carbazone, ihqs ¼ 7-iodo-8-hydroxy quinoline-5-sulfonic acid. In all cases, the loss of
chlorine ions was detected, where T ¼ 2,20-bipyridine/1,10-phenanthroline and
S ¼ r-bitsz, tmtsz, and dmtsz. Thus, based on the above observations, it is suggested
that Ru(II) complexes have octahedral geometry.
3.2. Biological activity and discussion
Results are summarized in tables 1 and 2 and the pharmacological data were analyzed
statistically by ANOVA. The statistical significance was considered only when p 5 0.05
and F 4 Fcritical. All the complexes were tested for their anticancer activity in
EAC-bearing mice. Ru(phen)2(tmtsz)Cl2 was found to increase life span of the tumor
hosts by 48%; remaining ruthenium complexes increased life span in the tumor hosts by
38% only. The results of this study clearly demonstrated the tumor-inhibitory activity

4344
S. Thota et al.
Table 1. Antitumor activity of ruthenium complexes against EAC-bearing mice.
Viable cells in
Total body weight (g) MST (days) ILS (%) Tumor volume (mL) ascitic fluid (%)
Parameters
Group I
21.6 ꢆ 0.3
27.2 ꢆ 0.6
19.8 ꢆ 0.5
22.2 ꢆ 0.9
23.1 ꢆ 0.5
23.8 ꢆ 0.5
24.3 ꢆ 0.6
24.8 ꢆ 0.5
24.1 ꢆ 0.5
23.6 ꢆ 0.4
22.9 ꢆ 0.8
24.5 ꢆ 0.8
23.1 ꢆ 0.6
–
–
–
5
–
–
Group II
Group III
Group IV
Group V
Group VI
Group VII
Group VIII
Group IX
Group X
Group XI
Group XII
Group XIII
21
22
24
25
23
29
26
25
31
25
24
29
3.3 ꢆ 0.3
94.8 ꢆ 3.8
–
–
14
19
10
38
24
19
48
19
14
38
1.5 ꢆ 0.01
1.7 ꢆ 0.05
1.8 ꢆ 0.03
0.9 ꢆ 0.05
1.2 ꢆ 0.05
1.3 ꢆ 0.05
0.7 ꢆ 0.03
1.4 ꢆ 0.04
1.3 ꢆ 0.06
0.9 ꢆ 0.04
46.3 ꢆ 1.5
44.9 ꢆ 1.6
47.9 ꢆ 1.4
40.1 ꢆ 1.8
43.2 ꢆ 1.2
44.8 ꢆ 1.5
33.8 ꢆ 1.7
45.1 ꢆ 1.6
46.5 ꢆ 1.5
40.7 ꢆ 1.8
Values are means ꢆ SEM; ILS (%) ¼ [(mean survival of treated group)/(mean survival of control group) ꢀ 1] ꢃ 100. Group I,
vehicle (5 mL kgꢀ1); group II, EAC (2 ꢃ 106 cells/mouse); group III, cisplatin (2 mg kgꢀ1) þ EAC; group IV, (Ru1); group
IV–group XIII, ruthenium complexes (2 mg kgꢀ1) þ EAC.
Table 2. Cytotoxic studies of ligands and ruthenium compounds.
IC50 (mmol Lꢀ1
)
a
Compound
L1210
Molt 4/C8
CEM
CH3-bitsz
Cl-bitsz
Br-bitsz
tmtsz
dmtsz
Ru[phen]2[CH3-bitsz]Cl2
Ru[bpy]2[CH3-bitsz]Cl2
Ru[phen]2[Cl-bitsz]Cl2
Ru[bpy]2[Cl-bitsz]Cl2
Ru[phen]2[Br-bitsz]Cl2
Ru[bpy]2[Br-bitsz]Cl2
Ru[phen]2[tmtsz]Cl2
Ru[bpy]2[tmtsz]Cl2
Ru[phen]2[dmtsz]Cl2
Ru[bpy]2[dmtsz]Cl2
241 ꢆ 45
98 ꢆ 29
178 ꢆ 6
63 ꢆ 31
144 ꢆ 21
49 ꢆ 5
74 ꢆ 8
84 ꢆ 7
58 ꢆ 13
102 ꢆ 9
93 ꢆ 12
76 ꢆ 05
204 ꢆ 19
15 ꢆ 1
186 ꢆ 39
17 ꢆ 8
111 ꢆ 16
9.1 ꢆ 0.5
2.8 ꢆ 0.4
0.89 ꢆ 0.05
1.4 ꢆ 0.3
0.34 ꢆ 0.04
1.8 ꢆ 0.6
12 ꢆ 0.9
1.4 ꢆ 0.3
0.98 ꢆ 0.06
2.2 ꢆ 0.3
0.86 ꢆ 0.08
2.5 ꢆ 0.4
1.6 ꢆ 0.4
0.89 ꢆ 0.06
8.7 ꢆ 0.9
1.2 ꢆ 0.3
9.6 ꢆ 0.9
0.71 ꢆ 0.05
1.9 ꢆ 0.6
0.45 ꢆ 0.03
1.1 ꢆ 0.4
0.98 ꢆ 0.05
0.38 ꢆ 0.04
2.4 ꢆ 0.4
1.8 ꢆ 0.5
0.28 ꢆ 0.02
1.6 ꢆ 0.2
0.94 ꢆ 0.06
a50% Inhibitory concentration, required to inhibit tumor cell proliferation by 50%.
of the ruthenium complexes against transplantable murine tumor cell line. The in vitro
cytotoxic activity was evaluated for all the synthesized ligands and ruthenium
complexes against human Molt 4/C8, CEM, T-lymphocytes as well as murine L1210
cells, and its results are summarized in table 2. The relative potencies between ligands
and their ruthenium complexes revealed the importance of ruthenium using the Molt
4/C8, CEM assays, and murine L1210 assays. These determinations showed that in
comparison to ligands, the ruthenium complexes were more potent.
The cytotoxicity data in table 2 reveal that most ruthenium complexes have
significant cytotoxic potencies (IC50 figures in the 0.38-17 for Molt 4/C8,

Mononuclear Ru(II)
4345
0.86–15 mmol Lꢀ1 for L1210 and 0.34–9.1 mmol Lꢀ1 for CEM). For ligands, the IC50
values were in excess (49–144 mmol Lꢀ1 against CEM, 63–186 mmol Lꢀ1 for Molt 4/C8,
and 74–241 mmol Lꢀ1 for L1210). Of the tested ligands and ruthenium complexes,
Ru(phen)2(Br-bitsz)Cl2 showed cytotoxicity against all three cell lines tested in range of
0.45, 0.34, and 0.86 mmol Lꢀ1 for Molt 4/C8, CEM, and L1210, respectively.
Ru(bpy)2(tmtsz)Cl2 shows cytotoxicity against cell lines tested, 0.38 mmol Lꢀ1 for
Molt 4/C8, 0.28 mmol Lꢀ1 for CEM, and 0.89 mmol Lꢀ1 for L1210; Ru(phen)2
(Cl-bitsz)Cl2 shows cytotoxicity against cell lines tested, 0.71 mmol Lꢀ1 for Molt 4/C8,
0.89 mmol Lꢀ1 for CEM, and 0.98 mmol Lꢀ1 for L1210; remaining ruthenium complexes
for Molt 4/C8 and CEM (low) mmol Lꢀ1 and L1210 (higher) mmol Lꢀ1. On comparison
to ruthenium complexes the ligands displayed the cytotoxicity at higher mmol Lꢀ1
concentration.
From the results presented in table 2, it is clear that several ruthenium complexes
exhibit a marked inhibitory effect on the proliferation of tumor cells: IC50 from as low
as 0.38 mmol Lꢀ1 for Molt 4/C8, 0.28 mmol Lꢀ1 CEM, and 0.86 mmol Lꢀ1 for L1210.
Thus, the ruthenium complexes proved inhibitory to tumor growth at submicromolar
concentrations. Their ligands however were not antitumorally active.
The mechanism of action of the ruthenium complexes is not known but
organoruthenium antitumor agents contain an electron-deficient metal atom that acts
as a magnet for electron-rich DNA nucleophiles. Organoruthenium complexes are
bifunctional and can accept electrons from two DNA nucleophiles.
Acknowledgments
The authors are grateful to the Principal Dr Y. Rajeshwar, S.R. College of Pharmacy,
Hanamkoda for providing the chemicals for carrying out this research. Appreciation is
extended to the Belgian Geconcerteerde Onderzoeksacties which supported the
cytotoxic activity assays.
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