Synthesis and pharmacological activities of some mononuclear Ru(II) complexes

Article abstract of DOI:10.1016/j.bmc.2005.05.047

A series of mononuclear Ru(II) complexes of the type [Ru(M) ₂(U)]²⁺, where M=2,2′-bipyridine/1,10-phenanthroline and U=tpl (Ru1), 4-Cl-tpl (Ru2), 4-CH₃-tpl (Ru3), 4-CH ₃O-tpl (Ru4), and 4-NO₂-tpl (Ru5), -pai (Ru6), where tpl = thiopicolinanilide and pai = 2-phenyl-azo-imidazole, have been prepared and characterized by IR, UV-Vis, ¹H NMR, ¹³C-NMR, FAB-Mass spectrophotometer, and elemental analysis. The complexes display metal-ligand charge transfer (MLCT) transitions in the visible region. The title complexes were subjected to in vivo anticancer activity tests against a transplantable murine tumor cell line, Ehrlich's ascitic carcinoma (EAC) and in vitro antibacterial activity against Gram positive and Gram negative microorganisms. Ru1-Ru6 were found to increase the life span of the tumor hosts by 19-52%, and decreased tumor volume and viable ascitic cell count. The results of the present study clearly demonstrated the tumor inhibitory activity of the ruthenium chelates against transplantable murine tumor cell line. The treatment with ruthenium complexes could be secondary to tumor regression or due to the action of the compounds itself. The significant antibacterial activity was observed for Ru1-Ru4 against microorganisms like Vibrio cholera 865, Staphylococcus aureus 6571, and Shigella flexneri as compared to that of standard drug chloramphenical. Ru5 showed moderate activity against S. aureus 8530. However, all the complexes fail to show significant antibacterial activity against V. cholera 14033 and Shigella sonnai.

Full text of DOI:10.1016/j.bmc.2005.05.047

Bioorganic & Medicinal Chemistry 13 (2005) 5766–5773
Synthesis and pharmacological activities of some
mononuclear Ru(II) complexes
Upal K. Mazumder,a,* Malaya Gupta,a Subhas S. Karki,b Shiladitya Bhattacharya,a
Suresh Rathinasamya and Thangavel Sivakumara
aDivision of Pharmaceutical Chemistry, Department of Pharmaceutical Technology, Jadavpur University,
Jadavpur, Kolkata 700 032, India
bDepartment of Pharmaceutical Chemistry, KLES College of Pharmacy, P.O. Box 1062, 2nd Block,
Rajajinagar, Bangalore 560 010, India
Received 2 April 2005; revised 28 May 2005; accepted 28 May 2005
Available online 27 June 2005
Abstract—A series of mononuclear Ru(II) complexes of the type [Ru(M)2(U)]2+, where M=2,20-bipyridine/1,10-phenanthroline and
U=tpl (Ru1), 4-Cl-tpl (Ru2), 4-CH3-tpl (Ru3), 4-CH3O-tpl (Ru4), and 4-NO2-tpl (Ru5), -pai (Ru6), where tpl = thiopicolinanilide
and pai = 2-phenyl-azo-imidazole, have been prepared and characterized by IR, UV–Vis, 1H NMR, 13C-NMR, FAB-Mass spectro-
photometer, and elemental analysis. The complexes display metal–ligand charge transfer (MLCT) transitions in the visible region.
The title complexes were subjected to in vivo anticancer activity tests against a transplantable murine tumor cell line, EhrlichÕs ascitic
carcinoma (EAC) and in vitro antibacterial activity against Gram positive and Gram negative microorganisms. Ru1–Ru6 were
found to increase the life span of the tumor hosts by 19–52%, and decreased tumor volume and viable ascitic cell count. The results
of the present study clearly demonstrated the tumor inhibitory activity of the ruthenium chelates against transplantable murine
tumor cell line. The treatment with ruthenium complexes could be secondary to tumor regression or due to the action of the com-
pounds itself. The significant antibacterial activity was observed for Ru1–Ru4 against microorganisms like Vibrio cholera 865,
Staphylococcus aureus 6571, and Shigella flexneri as compared to that of standard drug chloramphenical. Ru5 showed moderate
activity against S. aureus 8530. However, all the complexes fail to show significant antibacterial activity against V. cholera 14033
and Shigella sonnai.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
es well suited for medicinal applications: (i) rate of li-
gand exchange, (ii) the range of accessible oxidation
states, and (iii) the ability of ruthenium to mimic iron
in binding to certain biological molecules. There has
been considerable interest in ruthenium complexes, in
recent years, because of their redox stability, excited
state reactivities, and excited state lifetime.4 Owing to
the octahedral structure of Ru(II) and Ru(III) complex-
es as opposed to the square–planar geometry of Pt(II),
ruthenium antitumor complexes probably function in a
manner differently than cisplatin, which appears to bind
DNA by cross-linking adjacent guanine, thereby causing
a class of DNA-binding proteins to adhere to the site.5–7
Activation by reduction hypothesis, Ru(III) complexes
may serve as prodrugs that are activated by reduction
in vivo to coordinate more rapidly to biomolecules.3,8,9
Because tumors rapidly utilize oxygen and other nutri-
ents, and as the development of blood vessels often fails
to keep pace with tumor growth, there is usually a lower
oxygen content (hypoxia) in tumor cell.10–14
Cisplatin is the most widely used antitumor drug, espe-
cially for the treatment of testicular and ovarian can-
cers.1,2 However, cisplatin has some major drawbacks:
severe toxic side effects, a limited applicability to a rela-
tively small range of tumors, and often occurring resis-
tance. This resistance is either developed or intrinsic.2
The second-generation platinum drug carboplatin also
has wide clinical applications. It has less toxic side effects
than cisplatin.2 In search for other antitumor active met-
al complexes, several ruthenium complexes have been
reported to be promising as anticancer drugs.3 There
are three main properties that make ruthenium complex-
Keywords: Ruthenium complexes; Thiopicolinanalide; Anti-cancer;
Anti-bacterial.
*
Corresponding author. Tel.: +91 33 24404123; fax: +91 80
23425373; e-mail: subhasskarki@hotmail.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.047

U. K. Mazumder et al. / Bioorg. Med. Chem. 13 (2005) 5766–5773
5767
Since then, complexes such as cis-[Ru(dmso)4Cl2]15,16
(dmso = dimethyl sulfoxide), trans-[(IndH)Ru(ind)2Cl4]
(Ind = indazole), mer-[Ru(terpy)]Cl3 (terpy = 2,20-ter-
pyridine), and Ru(ChdH2)Cl2 (Chd = 1,2-cyclohexane
diamine tetraacetate) have been reported to be highly
active.17–19 Arene complexes like [Ru(dmso)4(arene)]Cl2
(where dmso = dimethyl sulfoxide and arene=C6H6)
were shown to inhibit topoisomerase II (DNA gyrase)
activity.20 Other arene complexes, such as X(g6-arene)
(en)Ru(II)+ (where arene = C6H6 substituted C6H6,
en = ethylene diamine, and X = halide), were found to
inhibit the growth of the human ovarian cancer cell line
A 2780.21
Table 2. Antibacterial activity of ruthenium complexes at 20 lg/0.1 ml
Complex V C 865 V C 14033 S A 6571 S A 8530 S F S S
Ru 1
Ru 2
Ru 3
Ru 4
Ru 5
STD
24
24
25
20
NA
30
NA
NA
NA
16
17
16
14
18
NA
26
20
20
25
18
12
26
14 NA
15 NA
15 NA
16 NA
NA NA
20 19
NA
20
STD = chloramphenicol 10 lg/ml. Zone inhibitions in mm (including
bore size 6 mm). V C 865 = V. cholera 865; V C 14033 = V. cholera
14033; S A 6571 = S. aureus 6571; S A 8530 = S. aureus 8530; S F = S.
flexneri; S S = S. sonnai. Ru1–Ru5 = ruthenium complexes.
Ruthenium complexes bearing 2-hydroxy-1-naphthal-
dehyde thiosemicarbazone as a ligand have been studied
for their antimicrobial and antifungal activities.22 Sulu
et al.23 have evaluated the ruthenium complexes for in
vitro antifungal activity with a range of l values, be-
tween 16 and 250 lg/ml. Recently, Reedijk et al.24 have
reported water-soluble complexes of the type a-[Ru(az-
py)2(L)] (azpy = 2-phenyl azo pyridine and L = 1,1-cyc-
lobutane dicarboxylic acid, oxalic acid, and malonic
acid) as cytotoxic against A 2780 human ovarian carci-
noma and A 2780 cis-R, the corresponding cisplatin-re-
sistant cell line. A previous investigation by our group
has dealt with the antitumor and antibacterial activities
of the type [Ru(R)2(L)]2+ (R = 1,10-phenanthroline/2,20-
bipyridine and L = 5,7-disubstituted-8-hydroxy-quino-
line, picolinic acid, 3-hydroxy coumarin, N-methyl-isat-
in-3-thiosemicarbazone, acetazolamide, 4-substituted
thiosemicarbazides, etc.).25,26 Taking into account the
pharmacological potential of these class of compounds,
we have synthesized a novel range of Ru(II) complexes
of the type [Ru(M)2(U)]2+ (M = 1,10-phenanthroline/
2,20-bipyridine and U = tpl, 4-Cl-tpl, 4-CH3O-tpl,
4-CH3-tpl, 4-NO2-tpl, pai, where tpl = thiopicolinanilide
and pai = 2-phenyl azo-imidazole) in order to evaluate
their anticancer and antibacterial activities.
Scheme 1. Preparation of cis-Ru(phen)2Cl2 and cis-Ru(bpy)2Cl2.
for the synthesis of these homoleptic complexes. Ruthe-
nium trichloride undergoes reduction in a number
of organic solvents. The starting material for the synthesis
of the complexes was cis-bis(1,10-phenanthroline)dichlo-
roruthenium(II)/cis-bis(2,20-bipyridine)dichlororuthe-
nium(II). Ruthenium trichloride was refluxed in DMF in
the presence of 1,10-phenanthroline/2,20-bipyridine and
in excess of the stoichiometric amount, which afforded
the final product cis-bis(1,10-phenanthroline)dichlo-
roruthenium(II)/cis-bis(2,20-bipyridine)dichlororutheni-
um(II)10 (Scheme 1). The third ligand was introduced
in the presence of alcohol (Scheme 2).
In order to obtain products of high purity, it was neces-
sary to use column chromatography. TLC was attempt-
ed in order to determine the efficient supports, eluent
compositions, and reaction endpoints. It was carried
out using a solvent mixture such as CHCl3–CH3OH/
CH2Cl2–isopropanol. The solvent mixture provided
sharp spots of the main complex and one or two second-
ary spots, which had convenient Rf values. Column
2. Results and discussion
2.1. Chemistry
Results are summarized in Tables 1 and 2, and Schemes 1
and 2 show the details of the synthetic strategy adopted
Table 1. Antineoplastic activity of ruthenium complexes against EAC bearing mice
Parameters
Total body weight (g)
Mean survival time (days)
ILS (%)
Tumor volume (ml)
Viable cells in ascitic fluid (%)
Group I
22.2 0.5
28.3 0.6
18.2 0.7
22.9 1.2
22.8 1.0
23.1 0.5
23.2 0.8
25.0 0.9
24.5 0.9
—
21
22
29
32
30
28
25
26
—
—
5
—
3.4 0.3
—
—
95.2 3.5
—
Group II
Group III
Group IV
Group V
Group VI
Group VII
Group VIII
Group IX
38
52
43
33
19
24
0.9 0.05
0.7 0.04
0.8 0.07
1.1 0.04
1.8 0.05
1.5 0.06
42.1 1.1
39.2 1.3
40.5 2.3
42.9 2.6
69.1 2.9
63.2 1.8
Values are means SEM. Group I: Vehicle (5 ml/kg); Group II: EAC (2 · 106 cells/mouse); Group III: cisplatin (2 mg/kg) + EAC; Group IV–Group
VIII: ruthenium complexes (2 mg/kg) + EAC.

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U. K. Mazumder et al. / Bioorg. Med. Chem. 13 (2005) 5766–5773
Scheme 2. Preparation of tris chelates from cis-Ru(phen)2Cl2 and cis-Ru(bpy)2Cl2.
chromatography was performed using silica gel (230–
400 mesh) or neutral alumina as the support and
CHCl3–CH3OH/CH2Cl2–isopropanol as the eluate.
(ddd, 1H, py), and 7.49–7.46 (dddd, 1H, py), and
7.41–7.40 (d, 2H, Ar) d ppm.
We attempted to prepare 2-(4-carboxy)thiopicolinani-
lide by the same method; unfortunately; the product
was unsubstituted thiopicolinanilide, which was con-
firmed from its FTIR, NMR, and EI–MS spectra.
The ligands 4-R-tpl27 (where R = H, Cl, CH3, OCH3,
and NO2) were prepared by heating a-picoline, sulfur,
and their respective aromatic amines at 160 ꢁC for
12 h with slight modifications (Scheme 3A). 4-Cl-tpl
and 4-NO2-tpl are new ligands, and they were confirmed
In the IR spectra of the ligand pai, the band observed at
3220 cmꢀ1 corresponds to N–H stretching. On complex-
ation, there was no change in the vibration frequency of
N–H. Therefore, the coordination involved was with
both the ring imine nitrogens. In the IR spectra of the
complexes, [Ru(phen)2(R-tpl)]2+, there were changes in
the vibration mode for C @ S to 1429. Therefore, the
coordination of ruthenium metal with the ligand was
via C @ S and endocyclic pyridine ring nitrogen but
not with the exocyclic N–H, because there is no change
in vibration frequency of N–H.
1
for their purity by their mp, IR H NMR, EI–MS, and
elemental analyses. The other ligand pai28 (2-phenyl azo
imidazole) was prepared according to the literature with
little modification (Scheme 3B). All the ligands were
confirmed for their purity by their mp, FTIR,
1H NMR, and elemental analyses. Structural models
of these ligands are shown in Figure 1. These models
are capable of exhibiting bidentate behavior.
There are bands at 3225–3186 cmꢀ1 for N–H, at 3100–
2900 cmꢀ1 for C–H, and at 1375 cmꢀ1 (C @ S). In
4-Cl-tpl ligand, there are well-resolved resonance peaks
at low field at 12.05 (s, br, NH) 8.54–8.53 (d, 1H, py),
8.78–8.76 (d, 1H, py), 8.06–8.04 (d, 2H, Ar) 7.90–7.86
The coordination of 4-Cl-thiopicolinanilide (4-Cl-tpl)
with Ru(phen)2Cl2 resulted in a compound, {Ru(phen)2
(4-Cl-tpl)}(ClO4)2, that no longer showed a C2 axis,
Scheme 3. (A) Preparation of 2-(4-substituted)-thiopicolinanilides (4-R-tpl). (B) Preparation of 2-phenyl azo imidazole (pai).

U. K. Mazumder et al. / Bioorg. Med. Chem. 13 (2005) 5766–5773
5769
Figure 1. Structure of 4-substituted thiopicolinanalide and 2-phenyl
azo imidazole.
resulting in nonequivalent ligands. Such loss of C2 axis
resulting in nonequivalancy of ligands was observed
for [Ru(phen)2(nmit)]Cl2 and [Ru(bpy)2(ihqs)]Cl2,25,26
where phen = 1,10-phenanthroline, nmit = N-methyl
isatin thiosemicarbazone, bpy = 2,20-bipyridine, and
Figure 2. Structure of various ruthenium complexes as chlorate salts.
ihqs = 7-iodo-8-hydroxy
quinoline-5-sulfonic
acid.
Therefore, such NMR spectra will become more compli-
cated. In the aromatic region, 22 well-resolved reso-
nance peaks were obsereved: (9.85–7.10, 24H,
aromatic) d ppm. The complexes bearing R-tpl ligand
did not show NH protons in their proton NMR spectra.
It might be merged with the baseline, which shows that,
there are six different aromatic ring protons of two 1,10-
phenanthroline and one 4-Cl-tpl.
Thus, based on the above observations, the proposed
structures for the complexes are shown in Figure 2.
2.2. Biological activity and discussion
The Results are summarized in Tables 1 and 2. The
pharmacological data were analyzed statistically by
analysis of variance followed by DunnettÕs test of signif-
icance. The statistical significance were considered only
when p < 0.05 and F > Fcritical. All the complexes were
tested for their anti-cancer activity against EAC bearing
mice. Ru1–Ru6 were found to increase the life span of
the tumor hosts by 19–52%. The results of the present
study clearly demonstrated the tumor inhibitory activity
of the ruthenium chelates against transplantable murine
tumor cell lines (Table 1). The treatment with ruthenium
complexes could be secondary to tumor regression or
could be due to the action of the compounds itself. In
the EAC bearing mice, the cells were present in the per-
itoneal cavity and the compounds were administered
directly into the peritoneum. Thus, tumor inhibition
might be due to the direct effect of these compounds
on DNA synthesis or due to some unknown effect but
certain structurally related tris-chelates of ruthenium
were reported to have DNA-binding property in vitro.29
Thus, the action of these synthesized complexes could
also be mediated via its effect, if any, on the DNA.
In the case of 13C NMR, in aromatic region, 32 well-
resolved resonance peaks were observed: (177.304–
123.038) d ppm, which also showed that all the ligands
that coordinated with ruthenium were nonequivalent.
The coordination of ligand 2-phenyl azo imidazole (pai)
with Ru(bpy)2Cl2 resulted in a compound, [Ru(bpy)2
(pai)]Cl2, that no longer showed a C2 axis, resulting in
nonequivalent ligands. In the aromatic region, 14 well-
resolved resonance peaks were observed: (8.76–6.53,
24H, aromatic) d ppm.
These complexes showed broad and intense visible
bands between 350 and 450 nm due to metal to ligand
charge transfer transition.9 In the UV region, the bands
at 290 and 310 nm were assigned to the phenanthroline
ligand. The same transition was found in free phenan-
throline at 280 nm, so that coordination of the ligand re-
sults in a red shift in the transition energy. There were
also two shoulders at 390 and 500 nm, which are tenta-
tively attributed to a metal to ligand charge transfer
transitions involving phenanthroline ligand.
Myelosupression is a frequent and major complication
of cancer chemotherapy. Cisplatin was known to cause
bonemarrow depression and acute nephrotoxicity. Thus,
the severe depression of the bonemarrow coupled with
acute nephrotoxicity30–33 probably caused a negative
nitrogen balance in the cisplatin-treated animals, which
resulted in a loss of body weight. The nephrotoxicity of
cisplatin was due to the capability of the compound to
generate free radicals and reactive oxygen species within
the liver and kidney.34–41
The FAB-MS of the prepared complexes showed frag-
mentation in the following manner. The first fragment
was due to [Ru(M)2(U)]2+(ClO4)ꢀ ion pair. The complex
also showed a peak due to the complex cation
[Ru(M)2(U)]2+ and others due to [Ru(M)(U)]2+
[Ru(M)2]2+. The FAB-MS thus confirms the authenticity
of the complex. This type of fragmentaion was reported
for [Ru(phen)2(nmit)]Cl2 and [Ru(bpy)2(ihqs)]Cl2,25,26
where phen = 1,10-phenanthroline, nmit = N-methyl
isatin thiosemicarbazone, bpy = 2,20-bipyridine, and
ihqs = 7-iodo-8-hydroxy quinoline-5-sulfonic acid.
The complexes were also evaluated for their antibacterial
activity by cup–plate method.42 The significant antibac-
terial activity was observed for Ru1–Ru4 against micro-
organisms such as Vibrio cholera 865, Staphylococcus

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U. K. Mazumder et al. / Bioorg. Med. Chem. 13 (2005) 5766–5773
aureus 6571, and Shigella flexneri as compared to that of
the standard drug chloramphenical. Ru5 showed moder-
ate activity against S. aureus 8530, but no activity was
found for the remaining microorganisms. However, all
the complexes failed to show significant antibacterial
activity against V. cholera 14033 and Shigella Sonnai.
4.2.1. tpl. Silica gel as stationary phase, petroleum ether
and benzene as mobile phase. 60%, yellow, FTIR (KBr)
cmꢀ1: 3220 (N–H), 3054 (C–H), 1535 (C @ C), 1375
(C @ S). 1H NMR (CDCl3):
d
12.06 (1H, s,
S–H), 8.82 (1H, d, J = 8.03 Hz), 8.57 (1H, d,
J = 5.29 Hz), 8.09 (2H, d, J = 7.75 Hz), 7.92–7.87
(1H, m), 7.50–7.40 (3H, m), and 7.32–7.26 (1H, m).
Mp 45 ꢁC (46.5 ꢁC). Anal. calcd for C12H10N2S: C,
67.26; H, 4.70; N, 13.07. Found C, 67.02; H, 4.67; N,
12.55.
3. Conclusions
In conclusion, five (Ru1–Ru5) complexes, bearing 1,10-
phenanrhroline with 4-R-tpl (where R = H, Cl, CH3,
OCH3, and NO2), were synthesized; and one (Ru6) com-
plex, bearing 2,20-bipyridine with pai (where pai = 2-
phenyl azo imidazole) was synthesized in alcohol in
nitrogen atmosphere. The coordination involved for
ruthenium complexes (Ru1–Ru5) is via C @ S and endo-
cyclic pyridine ring nitrogen.
4.2.2. 4-CH3-tpl. Silica gel as stationary phase, petro-
leum ether and benzene as mobile phase. 45%, orange,
FTIR (KBr) cmꢀ1: 3225 (N–H), 3039 (C–H), 1540
(C @ C), 1380 (C @ S). Mp = 96 ꢁC (98.5 ꢁC). Anal.
calcd for C13H12N2OS: C, 63.91; H, 4.95; N, 11.47.
Found C, 63.70; H, 5.01; N, 11.05.
4.2.3. 4-Cl-tpl. Silica gel as stationary phase, and petro-
leum ether as mobile phase. 58%, yellow, FTIR (KBr)
cmꢀ1: 3186 (N–H), 3050 (C–H), 1531 (C @ C), 1370
The results of the present study clearly demonstrated the
tumor inhibitory activity of the ruthenium complexes
against transplantable murine tumor cell line, EAC.
The ruthenium complex bearing 4-Cl-tpl (Ru2) showed
increase in the life span of tumor host by 52% as com-
pared to cisplatin (5%).
1
(C @ S). H NMR (CDCl3): d 12.06 (1H, s, S–H), 8.77
(1H, d, J = 4.80 Hz), 8.55 (1H, d, J = 2.50 Hz), 8.05
(2H, d, J = 5.26 Hz), 7.90–7.87 (1H, m), 7.49–7.47 (1H,
m), and 7.41 (2H, d, J = 5.27 Hz). Mp = 73–75 ꢁC. Anal.
calcd for C12H9Cl N2S: C, 57.95; H, 3.65; N, 11.26. Found
C, 57.31; H, 3.41; N, 10.82.
The study of in vitro antibacterial activity reveals the
significant activity of Ru1–Ru4 against microorganisms
such as V. cholera 865, S. aureus 6571, and S. flexneri as
compared to the standard drug chloramphenicol.
4.2.4. 4-CH3O-tpl. Silica gel as stationary phase, and
benzene as mobile phase. 54%, orange, FTIR (KBr)
cmꢀ1: 3200 (N–H), 3055 (C–H), 1529 (C @ C), 1378
(C @ S). Mp = 98.5 ꢁC (100.5 ꢁC). Anal. calcd for
C12H9Cl N2S: C, 57.95; H, 3.65; N, 11.26. Found C,
57.32; H, 3.26; N, 10.24.
4. Experimental
4.1. General methods
4.2.5. 4-NO2-tpl. Silica gel as stationary phase, petro-
leum ether and benzene as mobile phase. 32%, yellowish
orange, FTIR (KBr) cmꢀ1: 3180 (N–H), 3029 (C–H),
1520 (C @ C), 1372 (C @ S). Mp = 178–184 ꢁC. Anal.
calcd for C12H9ClN2S: C, 57.95; H, 3.65; N, 11.26.
Found C, 57.99; H, 3.18; N, 10.34.
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. UV–Vis
spectra were run on a Beckmann DU 64 spectrophoto-
meter. FTIR spectra were recorded in KBr discs on a
Jasco V410/Schimadzu IR spectrometer. 1H NMR spec-
tra were measured in CDCl3 and d6-DMSO on a Bruker
Ultraspec 500 MHz/AMX400 MHz/300 MHz spectro-
meter. The reported chemical shifts were against that
of TMS. FAB mass spectra were recorded on a JEOL
JMS600 spectrum with mNBA matrix.
4.2.6. General procedure for preparing 2-phenyl azo
imidazole (pai).28 To an ice-cold solution of imidaz-
ole, sodium bicarbonate, and sodium nitrite dil.
HCl was added with stirring. The orange colored
azo product precipitated almost instantaneously. Stir-
ring was continued for 3 h after all the acid had been
added. The product was kept at 0 ꢁC overnight and
filtered off. It was recrystallized from hot aqueous
ethanol.
4.2. General procedure for preparing 4-substituted
thiopicolinanilide27
65%, red, FTIR (KBr) cmꢀ1: 3220 (N–H), 3005 (C–H),
1520 (C @ C). Mp = 187 ꢁC. Anal. calcd for C9H8N4: C,
62.78; H, 4.68; N, 32.54. Found C, 62.01; H, 4.11; N,
31.05.
A suspension of 4.7 g (0.15 g of atom) of sulfur, 5.2 ml
(0.05 mol) of a-picoline, and amine (0.055 mol) were
heated in an oil bath, maintained at 160 ꢁC for 12 h;
unreacted a-picoline and amine were then removed by
distillation in vacuo. The residue in the still pot was then
extracted by heating ethanol to remove sulfur complete-
ly. Distillation of the ethanol extract produced 2.5–3.8 g
of crude thiopicolinanilides. The crude product was then
separated by column chromatography on efficient sup-
ports then separately choosen for each ligand.
4.3. General procedure for preparing cis-[Ru(phen)2 (4-R-
tpl)](ClO4)2
Where phen = 1,10-phenanthroline; R = H (Ru 1), Cl
(Ru 2), CH3 (Ru 3), OCH3 (Ru 4), and NO2 (Ru 5);
and tpl = thiopicolinanilide.

U. K. Mazumder et al. / Bioorg. Med. Chem. 13 (2005) 5766–5773
5771
To the black microcrystalline cis-bis(phen)dichlororu-
thenium(II) cis-Ru(phen)2Cl2/ (106 mg, 2 mmol), excess
of 4-substituted thiopicolinanilide (4-R-tpl) (2.5 mmol)
was added and refluxed in ethanol under nitrogen
atmosphere. The initial colored solution slowly chan-
ged to a brownish orange at the end of the reaction,
which was verified by TLC on silica plates. Then, the
excess of ethanol was distilled off, the saturated
aqueous solution of sodium perchlorate was added to
the mixture, and the resultant solution was cooled at
0 ꢁC overnight. A microcrystalline precipitate was
obtained. The crystals were filtered and the solid was
washed with very little amount of water and dried over
CaCl2 in vacuum (yield 45–50%). Finally, they were
purified on column chromatography by using neutral
alumina as stationary phase and dichloromethane–
isopropanol as eluent.
4.3.4. Ru 4. 48%, black crystals, FTIR (KBr) cmꢀ1: 3210
(N–H), 3041 (C–H), 1532 (C @ C), 1438 (C @ S). kmax
nm (methanol): 246, 268, 365, and 490. Anal. calcd for
RuC37H28N6O9Cl2S: C, 49.12; H, 3.10; N, 9.29. Found
C, 49.24; H, 2.89; N, 9.35.
FAB-MS m/z: 805 [Ru(phen)2(MeO-tpl)]2+(ClO4)ꢀ; 705
[Ru(phen)2(MeO-tpl)]2+; 525 [Ru(phen)(MeO-tpl)]2+
.
4.3.5. Ru 5. 40%, black crystals, FTIR (KBr) cmꢀ1: 3195
(N–H), 3025 (C @ H), 1525 (C @ C), 1425 (C @ S). kmax
nm (methanol): 255, 275, 380, and 498. Anal. calcd for
RuC37H25N7O10Cl2S: C, 47.00; H, 2.72; N, 10.66.
Found C, 46.29; H, 2.22; N, 11.01.
FAB-MS m/z: 811 [Ru(phen)2(NO2-tpl)]2+(ClO4)ꢀ; 711
2+
[Ru(phen)2(NO2-tpl)]2+; 531 [Ru(phen)(NO2-tpl)]
.
4.3.1. Ru 1. 48%, black crystals, FTIR (KBr) cmꢀ1: 3215
(N–H), 3058 (C–H), 1533 (C @ C), 1429 (C @ S). kmax
nm (methanol): 245, 260, 355, and 490. Anal. calcd for
RuC36H26N6Cl2O8S: C, 49.43; H, 2.97; N, 9.61. Found
C, 49.12; H, 2.15; N, 9.99.
4.3.6. General procedure for preparing cis-[Ru(bpy)2
(pai)]Cl2 Ru 6. Where
bpy = 2,20-bipyridine,
pai = 2-phenyl azo imidazole.
The complex was prepared in a similar manner to Ru 1
with microcrystalline cis-bis(bpy)dichlororuthenium(II)
{cis-Ru(bpy)2Cl2} and ligand (pai). Finally, they were
purified on column chromatography by using silica gel
as stationary phase and chloroform–methanol as eluent.
FAB-MS m/z: 775 [Ru(phen)2(tpl)]2+(ClO4)ꢀ; 675
[Ru(phen)2(tpl)]2+; 495 [Ru(phen)(tpl)]2+
.
4.3.2. Ru 2. 45%, Black crystals, FTIR (KBr) cmꢀ1: 3190
(N–H), 3058 (C–H), 1523 (C @ C), 1426 (C @ S). kmax
nm (methanol) : 240, 265, 340, and 485. Anal. calcd
for RuC36H25N6O8Cl3S: C, 47.52; H, 2.75; N, 9.24.
Found C, 47.45; H, 2.66; N, 9.41.
41%, black crystals, FTIR (KBr) cmꢀ1: 3205 (N–H), 1520
(C @ C). Anal. calcd for RuC29H24N8Cl2: C, 53.05; H,
3.66; N, 17.07. Found C, 52.29; H, 3.17; N, 17.29.
1H NMR (DMSO-d6) d ppm: 8.78–8.75 (2H, m), 8.57
(1H, d, J = 8.07 Hz), 8.50 (1H, d, J = 8.03 Hz), 8.29
(1H, d, J = 5.27 Hz), 8.07–7.99 (3H, tt, J = 15.25 Hz,
15.38 Hz), 7.91–7.88 (1H, t, J = 15.31 Hz), 7.72 (1H,
br, NH), 7.70 (1H, d, J = 5.26 Hz), 7.60 (1H, d,
J = 5.26 Hz), 7.44 (1H, d, J = 5.15 Hz), 7.41–7.34 (4H,
m), 7.12 (1H, m), 7.01–6.98 (3H, m), and 6.90 (2H, d,
J = 7.61 Hz).
1H NMR (DMSO-d6): d 9.85 (1H, d, J = 4.34 Hz), 8.82
(2H, dd, J = 11.47, 8.54 Hz), 8.70 (1H, d, J = 8.14 Hz),
8.50 (1H, d, J = 8.09 Hz), 8.40–8.32 (5H, m), 8.25 (1H,
d, J = 8.91 Hz), 8.21 (1H, dd, J = 8.17, 5.31 Hz), 8.06
(1H, dd, J = 8.17, 5.60 Hz), 8.00 (1H, d, J = 5.11 Hz),
7.89 (1H, t, J = 14.23 Hz), 7.76 (1H, d, J = 4.49 Hz),
7.72 (1H, dd, J = 8.12, 5.23 Hz), 7.51 (1H, t,
J = 11.55 Hz), 7.47 (1H, d, J = 5.35 Hz), 7.31 (2H, d,
J = 8.62 Hz), 7.22 (1H, t, J = 11.67 Hz), and 7.13 (2H,
d, J = 8.61 Hz).
FAB-MS m/z: 669 [Ru(phen)2(pai)]2+Cl2; 633 [Ru
(phen)2(pai)]2+; 453 [Ru(phen)(pai)]2+
.
13C NMR (DMSO-d6): d 177.3 (s), 162.5 (s), 154.3 (s),
151.8 (s), 151.6 (s), 151.5 (s), 151.3 (s), 150.9 (s), 148.2
(s), 147.7 (s), 147.4 (s), 147.1 (s), 136.4 (s), 136.1 (s),
135.7 (s), 135.0 (s), 134.9 (s), 130.4 (s), 130.2 (s), 129.7
(s), 128.4 (s), 128.1 (s), 127.8 (d), 127.6 (s), 127.0 (s),
126.8 (s), 126.3 (s), 126.0 (s), 125.8 (s), 125.2 (s), 123.8
(s), and 123.0 (s).
5. Evaluation of therapeutic effect in vivo
Albino Swiss mice (18–20 g body weight) were main-
tained in identical laboratory conditions and given
standard food pellets (Hindustan Lever, Bombay, In-
dia) and water ad libitum. LD50 values of the synthe-
sized complexes were obtained from the literature.44
The animals were divided into nine groups each con-
taining 12 mice. Group I acted as a vehicle control
(5 ml/kg bodyweight, i.p.) and group II as Ehrlich asci-
tes Carcinoma control (EAC; 2 · 106 EAC cells/mouse,
i.p.). Group III was treated with the standard drug cis-
platin (2 mg/kg body weight). All the complexes were
administered (i.p.) at a dose of 2 mg/kg body weight
in groups IV–IX. All the complexes (Ru1–Ru6) and
cisplatin were administered daily for nine days starting
24 h after tumor transplantation. Six animals from
each group were sacrificed 24 h after the last dose
FAB-MS m/z: 809 [Ru(phen)2(Cl- tpl)]2+(ClO4)ꢀ; 709
[Ru(phen)2(Cl-tpl)]2+; 529[Ru(phen)(Cl-tpl)]2+
.
4.3.3. Ru 3. 45%, black crystals, FTIR (KBr) cmꢀ1: 3215
(N–H), 3052 (C–H), 1539 (C @ C), 1435 (C @ S). kmax
nm (methanol): 245, 270, 360, and 485. Anal. calcd for
RuC37H28N6O9Cl2S: C, 50.00; H, 3.15; N, 9.46. Found
C, 49.05; H, 2.89; N, 9.35.
FAB-MS m/z: 789 [Ru(phen)2(Me-tpl)]2+(ClO4)ꢀ; 689
[Ru(phen)2(Me-tpl)]2+; 509 [Ru(phen)(Me-tpl)]2+
.

5772
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Acknowledgments
The authors are thankful to All India Council of Tech-
nical Education (AICTE), New Delhi, for providing the
funds for carrying out research. Dr. Subhas S. Karki
expresses his thanks to Dr. R. Mukharjee and Dr. V.S.
Giri, Indian Institute of Chemical Biology, Kolkata,
India, for their valuable help in performing spectral
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