Synthesis, spectroscopic characterization, antineoplastic, in vitro-cytotoxic, and antibacterial activities of mononuclear ruthenium(II) complexes

Article abstract of DOI:10.1080/00958972.2012.663082

The synthesis, antineoplastic, cytotoxic, and antibacterial activities of Ru(II) complexes derived from quinazoline and thiosemicarbazone ligands are reported. These complexes have been prepared and characterized by UV-Vis, IR, ¹H-NMR, FAB-mass

Full text of DOI:10.1080/00958972.2012.663082

This article was downloaded by: [Istanbul Universitesi Kutuphane ve Dok]
On: 31 May 2013, At: 18:50
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Coordination Chemistry
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/gcoo20
Synthesis, spectroscopic
characterization, antineoplastic,
in vitro-cytotoxic, and antibacterial
activities of mononuclear ruthenium(II)
complexes
Sreekanth Thota a , Mohammad Imran a , Manasa Udugula a ,
Subhas Somalingappa Karki b , Narasimha Kanjarla a , Rajeshwar
Yerra a , Jan Balzarini c & Erik De Clercq c
a Department of Pharmaceutical Chemistry & Toxicology,
SR 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 Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Published online: 21 Feb 2012.
To cite this article: Sreekanth Thota , Mohammad Imran , Manasa Udugula , Subhas Somalingappa
Karki , Narasimha Kanjarla , Rajeshwar Yerra , Jan Balzarini & Erik De Clercq (2012): Synthesis,
spectroscopic characterization, antineoplastic, in vitro-cytotoxic, and antibacterial activities of
mononuclear ruthenium(II) complexes, Journal of Coordination Chemistry, 65:5, 823-839
To link to this article: http://dx.doi.org/10.1080/00958972.2012.663082
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-
conditions
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation
that the contents will be complete or accurate or up to date. The accuracy of any

instructions, formulae, and drug doses should be independently verified with primary
sources. The publisher shall not be liable for any loss, actions, claims, proceedings,
demand, or costs or damages whatsoever or howsoever caused arising directly or
indirectly in connection with or arising out of the use of this material.

Journal of Coordination Chemistry
Vol. 65, No. 5, 10 March 2012, 823–839
Synthesis, spectroscopic characterization, antineoplastic,
in vitro-cytotoxic, and antibacterial activities of mononuclear
ruthenium(II) complexes
SREEKANTH THOTA*y, MOHAMMAD IMRANy, MANASA UDUGULAy,
SUBHAS SOMALINGAPPA KARKIz, NARASIMHA KANJARLAy,
RAJESHWAR YERRAy, JAN BALZARINIx and ERIK DE CLERCQx
yDepartment of Pharmaceutical Chemistry & Toxicology, SR 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
xRega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium
(Received 21 September 2011; in final form 2 January 2012)
The synthesis, antineoplastic, cytotoxic, and antibacterial activities of Ru(II) complexes derived
from quinazoline and thiosemicarbazone ligands are reported. These complexes have been
prepared and characterized by UV-Vis, IR, H-NMR, FAB-mass spectroscopy, and elemental
1
analysis. The ligands and resulting complexes were subjected to in vivo antineoplastic activity
against a transplantable murine tumor cell line Ehrlich ascites carcinoma (EAC) and in vitro
cytotoxic activity against human cancer cell line Molt 4/C8, CEM, and murine tumor cell line L
1210. The ruthenium complexes show promising biological activity especially in decreasing
tumor volume and viable ascitic cell counts. These complexes prolonged the life span of mice
bearing EAC tumors by 10–52%. In vitro evaluation of these ruthenium complexes revealed
cytotoxic activity from 0.29 to 2.9 mmol Lꢀ1 against Molt 4/C8, 0.22 to 2.1 mmol Lꢀ1 against
CEM and 0.42 to 4.7 mmol Lꢀ1 against L1210 cell proliferation, depending on the nature of the
compound. The metal complexes are more active than the parent ligand and exhibit mild to
moderate antibacterial activity.
Keywords: Antineoplastic; Quinazoline; Proliferation; Ru(II) complexes
1. Introduction
A large number of transition metal complexes with heterocyclic ligands containing
nitrogen, oxygen, and sulfur have pharmacological importance. Antineoplastic is said
of a drug that inhibits or prevents maturation and proliferation of neoplasms that may
become malignant, by targeting the DNA; most chemotherapy drugs are antineoplastic.
In searching for antineoplastic active metal complexes several ruthenium compounds
have been reported to be promising as anticancer drugs, including series of
*Corresponding author. Email: stsreekanththota@gmail.com
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2012.663082

824
S. Thota et al.
mononuclear Ru(II) complexes [Ru[M]2[U]]2þ, where M ¼ 2,20-bipyridine/1,10-phenan-
throline and U ¼ tpl, 4-Cl-tpl, 4-CH3-tpl, 4-OCH3-tpl, 4-NO2-tpl, pai [1].
The objectives of the present investigation were to develop analogs of [Ru[T]2[S]]2þ
,
where T ¼ 2,20-bipyridine/1,10-phenanthroline and S ¼ 4-NO2-PQZ, 4-CH3-PQZ,
3,4-di-OCH3-PQZ, 3-OCH3, 4-OH-PQZ, 4-OH-PQZ, 4 -N(CH3)2-PQZ, 4-NO2-PTSZ,
3,4,5-tri-OCH3-PTSZ as candidate cytotoxins. The discovery of anticancer activity of
cisplatin in 1965 marked the development of metallopharmaceuticals in cancer
chemotherapy [2]. 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 as potential antineoplastic agents [3].
The Erst systematic investigation of ruthenium compounds and their antineoplas-
tic properties was done in the early 1980s with fac-[RuCl3(NH3)3] and cis-
[RuCl2(NH3)4]Cl [4]. Several ruthenium complexes have been investigated for potential
antitumor activity such as trans-[RuCl4(Im)(DMSO)] ImH(NAMI-A) [5], (H2ind)
[trans-RuCl4(Hind)2] (Hind ¼ 1 H-indazole) [6], imidazolium[trans-tetrachloro (1 H-
imidazole) (S-dimethyl sulfoxide) ruthenate(III)] (NAMI-A) [7], [ImH] trans-
[RuCl4(Im)(dmso-s)] (NAMI-A, Im ¼ imidazole) [8], tethering Ru-arene drugs to
macromolecules [9], [Ru(ꢀ6-p-arene)Cl2 (pta)] [10], [IndH] trans-[RuCl4(Ind)2]
(KP1019) [11].
Ruthenium(II) arene complexes show remarkable cytotoxic properties in vitro as well
as in vivo [12, 13]. In comparison with Ru(III) complexes, Ru(II) complexes are
kinetically more reactive [14]. We have reported that Ru(II) compounds bearing
thiosemicarbazides and 8-hydroxy quinolines have in vivo anticancer and in vitro
antibacterial activities [15, 16]. In this work, we describe the synthesis and character-
ization of some ruthenium complexes, their in vitro cytotoxic activity against human
cancer cell line CEM, Molt 4/C8, and murine tumor cell line L 1210, their in vivo
anticancer activity, and in vitro antibacterial activity against transplantable murine
tumor cell line Ehrlich ascites carcinoma (EAC). Our research has focused on
complexes of general formula [Ru[T]2[S]]Cl2, where T ¼ 2,20-bipyridine/1,10-phenan-
throline and S ¼ 4-NO2-PQZ, 4-CH3-PQZ, 3,4-di-OCH3-PQZ, 3-OCH3, 4-OH-PQZ,
4-OH-PQZ, 4 -N(CH3)2-PQZ [17], 4-NO2-PTSZ, 3,4,5-tri-OCH3-PTSZ [18, 19].
2. Experimental
2.1. General methods
AR grade solvents were obtained from E. Merck, Mumbai, and SD Fine Chem. All
solvents were distilled prior to use. Fluka and E. Merck supplied the reagents (puriss
grade). Anthranilic acid was purchased from Merck (Germany) and used as received.
Hydrated ruthenium trichloride was purchased from Loba Chemie, Mumbai, and used
as received. Silica gel plates were used for the TLC analysis by using CHCl3 : CH3OH as
mobile phase. UV-Vis spectra were recorded on a Jasco spectrophotometer. IR spectra
(in KBr pellets) were recorded on the Vertex 70 Bruker apparatus. NMR spectra were
recorded in CDCl3 and DMSO-d6 on a Bruker Ultraspec 500 MHz/AMX 400 MHz/
300 MHz spectrometer using TMS as internal standard. The content of C, H, and N
were done with an ECS-40-10-Costech micro-dosimeter after drying the complexes.

Ruthenium(II) complexes
825
FAB mass spectra were recorded on a JEOL JMS600 spectrometer with an mNBA
matrix.
2.2. General procedure for synthesis of 3 -(aryldeneamino)-2-phenylquinazolin-
4(3 H)-ones [17]
Step 1. Synthesis of 2-phenyl-3,1-benzoxazin-4-one: To a solution of anthranilic acid
(0.1 mol) dissolved in pyridine (60 mL), benzoyl chloride (0.2 mol) was added. The
mixture was stirred for 2 h followed by treatment with 5% NaHCO3 (15 mL). The solid
obtained was crystallized from ethanol.
Step 2. Synthesis of 3-amino-2-phenyl quinazolin-4(3 H)-one: A mixture of 2-phenyl-3-
benzoxazin-4-one (0.05 mol) and hydrazine hydrate (0.05 mol) in ethanol was refluxed
for 3 h and cooled. The separated solid was recrystallized from ethanol.
Step 3. Synthesis of 3 -(arylideneamino)-2-phenyl-quinazolin-4(3 H)-one: A mixture of
3-amino-2-phenyl quinazolin-4(3 H)-one (0.01 mol), the appropriate aromatic aldehyde
(0.01 mol), and ethanol (20 mL) was refluxed for 4–6 h. The resulting mixture was
cooled and poured into ice water. The separated solid was filtered, washed with water,
and recrystallized from ethanol.
2.2.1. 4-NO2-PQZ. Yield 82%, m.p. 241–242ꢁC. IR (KBr) cmꢀ1: 3144 (C–H), 2924
(C–H), 1679 (C¼O). Calcd for C21H14O3N4 (%): C, 68.11; H, 3.78; N, 15.13. Found
(%): C, 67.96; H, 3.69; N, 15.04. ꢁmax nm (MeOH): 232, 245, 355. 1H-NMR (DMSO-d6):
ꢂ ¼ 8.88 (1-H, s), 8.04 (2-H, d), 7.91 (2-H, d), 7.58 (5-H, m), 7.72 (2-H, d, J ¼ 8.6 Hz),
7.31 (2-H, d).
2.2.2. 4-CH3-PQZ. Yield 69%, m.p. 210–211ꢁC. IR (KBr) cmꢀ1: 3157 (C–H), 2983
(C–H), 1669 (C¼O). Calcd for C22H17ON3 (%): C, 77.87; H, 5.01; N, 12.38. Found
(%): C, 77.48; H, 4.99; N, 12.28. ꢁmax nm (MeOH): 236, 295, 374. 1H-NMR (DMSO-d6):
ꢂ ¼ 9.02 (1-H, s), 8.24 (2-H, d), 8.11 (1-H, s), 8.08 (2-H, d), 7.86 (4-H, m), 7.64 (2-H, d,
J ¼ 8.6 Hz), 7.59 (2-H, d), 1.58 (3-H, s).
2.2.3. 3,4-di-OCH3-PQZ. Yield 84%, m.p. 232–233ꢁC. IR (KBr) cmꢀ1: 3132 (C–H),
2912 (C–H), 1680 (C¼O). Calcd for C23H19O3N3 (%): C, 71.68; H, 4.94; N, 10.91.
1
Found (%): C, 71.66; H, 4.89; N, 10.84. ꢁmax nm (MeOH): 217, 303, 371. H-NMR
(DMSO-d6): ꢂ ¼ 8.94 (1-H, s), 8.36 (2-H, d), 8.21 (2-H, d), 8.04 (5-H, m), 7.98 (1-H, s),
7.67 (2-H, d, J ¼ 8.6 Hz), 3.71 (3-H, m), 3.65 (3-H, m).
2.2.4. 3-OCH3,4-OH-PQZ. Yield 74%, m.p. 154–155ꢁC. IR (KBr) cmꢀ1: 3531
(O–H), 3153 (C–H), 2924 (C–H), 1680 (C¼O). Calcd for C22H17O3N3 (%): C, 71.16;
H, 4.58; N, 11.32. Found (%): C, 71.06; H, 4.52; N, 11.24. ꢁmax nm (MeOH): 240, 292,
367. 1H-NMR (DMSO-d6): ꢂ ¼ 10.01 (1-H, s), 9.12 (1-H, s), 8.44 (2-H, d), 8.28 (2-H, d),
8.01 (1-H, s), 7.98 (5-H, m), 7.76 (2-H, d, J ¼ 8.6 Hz), 3.74 (3-H, m).

826
S. Thota et al.
2.2.5. 4-OH-PQZ. Yield 76%, m.p. 166–167ꢁC. IR (KBr) cmꢀ1: 3528 (O–H), 3042
(C–H), 2904 (C–H), 1682 (C¼O). Calcd for C21H15O2N3 (%): C, 73.90; H, 4.39; N,
12.32. Found (%): C, 73.72; H, 4.32; N, 12.29. ꢁmax nm (MeOH): 263, 305, 356.
1H-NMR (DMSO-d6): ꢂ ¼ 10.28 (1-H, s), 9.04 (1-H, s), 8.21 (2-H, d), 8.10 (5-H, m),
7.98 (2-H, d), 7.74 (2-H, d, J ¼ 8.6 Hz), 7.53 (2-H, d).
2.2.6. 4 -N-(CH3)2-PQZ. Yield 70%, m.p. 177–178ꢁC. IR (KBr) cmꢀ1: 3148 (C–H),
2929 (C–H), 1680 (C¼O). Calcd for C23H20ON4 (%): C, 75.00; H, 5.43; N, 15.22.
1
Found (%): C, 74.88; H, 5.39; N, 15.18. ꢁmax nm (MeOH): 252, 295, 369. H-NMR
(DMSO-d6): ꢂ ¼ 8.89 (1-H, s), 8.34 (2-H, d), 8.14 (5-H, m), 7.92 (2-H, d), 7.68 (2-H, d,
J ¼ 8.6 Hz), 7.41 (2-H, d), 2.84 (3-H, m), 2.78 (3-H, m).
2.3. General procedure for synthesis of aryl substituted thiosemicarbazones
Aryl substituted thiosemicarbazones [18, 19] were prepared according to the literature
method.
2.3.1. 4-NO2-PTSZ. Yield 84%, m.p. 242–243ꢁC (lit., 242ꢁC). IR (KBr) cmꢀ1: 3417–
3380 (NH2 and NH), 3136 (C–H), 2901 (C–H), 1370 (C¼S). Calcd for C8H8O2N4S (%):
C, 42.85; H, 3.57; N, 25.00. Found (%): C, 42.69; H, 3.52; N, 24.94. ꢁmax nm (MeOH):
230, 307, 375. 1H NMR (DMSO-d6): ꢂ ¼ 10.36 (1-H, s), 9.02 (1-H, s), 8.04 (2-H, d), 7.83
(2-H, d), 7.24 (2-H, d, J ¼ 8.6 Hz).
2.3.2. 3,4,5-tri-OCH3-PTSZ. Yield 85%, m.p. 187–188ꢁC (lit., 187.5ꢁC). IR
(KBr) cmꢀ1: 3528 (O–H), 3042 (C–H), 2904 (C–H), 1682 (C¼O). Calcd for
C11H15O3N3 (%): C, 49.08; H, 5.57; N, 15.62. Found (%): C, 49.02; H, 5.48; N,
1
15.59. ꢁmax nm (MeOH): 240, 285, 372. H NMR (DMSO-d6): ꢂ ¼ 10.14 (1-H, s), 9.16
(1-H, s), 8.43 (2-H, d), 7.44 (2-H, d, J ¼ 8.6 Hz), 3.78 (3-H, m), 3.72 (3-H, m), 3.64
(3-H, m).
2.4. Preparation of cis-[bis(T)dichlororuthenium(II)] cis-[Ru(T)2Cl2] [20](where
T ^ 2,20-bipyridine/1,10-phenanthroline)
A mixture of ligand T (5 mmol) and RuCl3 ꢂ XH2O 1.15 g (2.5 mmol), DMF (50 mL) was
heated under reflux for 3 h under nitrogen atmosphere. After the reaction was
completed the reddish-brown solution slowly turned purple and the product precip-
itated. The solution was kept overnight in the refrigerator at 0ꢁC and then the
crystalline mass was filtered off. The residue was repeatedly washed with 30% LiCl
solution and finally recrystallized from ethanol. The product was dried in a vacuum
desiccator over P2O5 for further use (yield 75%).

Ruthenium(II) complexes
827
2.5. General procedure for preparing [Ru(T)2(S)Cl2] (where T ^ 1,10-phenanthro-
line (Ru1)/2,20-bipyridine (Ru2) and S ^ 4-NO2-PQZ, 4-CH3-PQZ, 3,4-di-
OCH3-PQZ, 3-OCH3,4-OH-PQZ, 4-OH-PQZ, 4 -N(CH3)2-PQZ, 4-NO2-
PTSZ, 3,4,5-tri-OCH3-PTSZ)
A mixture of excess ligand (2.5 mmol), black microcrystalline cis-Ru(T)2Cl2 (2 mmol),
and ethanol (100 mL) was heated under reflux for 5 h under nitrogen. The initial colored
solution slowly changed to brownish orange at the end of the reaction and verified by
TLC on silica plates. Then the excess ethanol was distilled off and silica gel (60–120
mesh) added to this solution. The residue was purified by column chromatography;
orange red band was collected using silica gel as stationary phase and chloroform-
methanol (8 : 2 ratio) as mobile phase.
2.5.1. [Ru[phen]2[4-NO2-PQZ]Cl2. 45%, black crystals, IR (KBr) cmꢀ1: 3083 (C–H),
2931 (C–H), 1681 (C¼O). Calcd for C45H30Cl2N8O3Ru1 (%): C, 59.80; H, 3.32; N,
1
12.41. Found (%): C, 59.74; H, 3.29; N, 12.38. H NMR (DMSO-d6): ꢂ ppm: 8.91
(s, 1-H), 8.64 (s, 1-H), 8.58 (2-H, t), 8.44 (d, 2-H), 8.28 (t, 3-H), 8.12 (d, J ¼ 8.4 Hz, 2-H),
7.93 (d, 2-H), 7.87 (m, 3-H),7.76 (m, 3-H), 7.68 (m, 5-H), 7.52 (d, 2-H), 7.24 (m, 4-H),
FAB-MS (mNBA): 902 [Ru(phen)2(4-NO2-PQZ)]2þ(Cl2)ꢀ; 831 [Ru(phen)2(4-NO2-
PQZ)]2þ; 651 [Ru(phen)(4-NO2-PQZ)]2þ; 462 [Ru(phen)2].
2.5.2. [Ru[bpy]2[4-NO2-PQZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3102 (C–H),
2987 (C–H), 1679 (C¼O). Calcd for C41H30Cl2N8O3Ru1 (%): C, 57.61; H, 3.51; N,
1
13.11. Found (%): C, 57.54; H, 3.49; N, 13.09. H NMR (DMSO-d6): ꢂ ppm: 9.18
(s, 8.66 (2-H, d), 8.46 (d, J ¼ 4.9 Hz, 2-H), 8.38 (m, J ¼ 8.4-Hz, 4-H), 8.26 (d, 2-H), 8.14
(d, 2-H), 8.04 (d, J ¼ 5.0 Hz, 2-H), 7.92 (m, 4-H), 7.84 (m, 5-H), 7.54 (d, 2-H), 7.62
(m, 4-H). FAB-MS (mNBA): 854 [Ru(bpy)2(4-NO2-PQZ)]2þ(Cl2)ꢀ; 783 [Ru(bpy)2
(4-NO2-PQZ)]2þ; 627 [Ru(bpy)(4-NO2-PQZ)]2þ; 413 [Ru(bpy)2].
2.5.3. [Ru[phen]2[4-CH3-PQZ]Cl2. 45%, black crystals, IR (KBr) cmꢀ1: 3134 (C–H),
2986 (C–H), 1681 (C¼O). Calcd for C46H33Cl2N7O1Ru1 (%): C, 63.37; H, 3.78; N,
1
11.25. Found (%): C, 63.28; H, 3.69; N, 11.22. H NMR (DMSO-d6): ꢂ ppm: 10.01
(s, 1-H), 8.86 (s, 1-H), 8.52 (d, 2-H), 8.46 (d, 2-H), 8.34 (3-H, m), 8.22 (d, 2-H), 8.01
(d, J ¼ 8.4 Hz, 2-H), 7.96 (t, 3-H), 7.82 (s, 1-H), 7.64 (m, 5-H), 7.54 (m, 4-H), 6.49
(m, 4-H), 1.56 (s, 3-H). FAB-MS (mNBA): 871 [Ru(phen)2(4-CH3-PQZ)]2þ(Cl2)ꢀ; 800
[Ru(phen)2(4-CH3-PQZ)]2þ; 620 [Ru(phen)(4-CH3-PQZ)]2þ; 462 [Ru(phen)2].
2.5.4. [Ru[bpy]2[4-CH3-PQZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3139 (C–H),
2928 (C–H), 1683 (C¼O). Calcd for C42H33Cl2N7O1Ru1 (%): C, 61.23; H, 4.01; N,
1
11.91. Found (%): C, 61.14; H, 3.99; N, 11.87. H NMR (DMSO-d6): ꢂ ppm: 10.45
(s, 1-H), 8.72 (1-H, s), 8.44 (m, J ¼ 4.9 Hz, 4-H), 8.26 (d, J ¼ 8.4 Hz, 2-H), 8.18 (d, 2-H),
8.02 (d, 2-H), 7.96 (d, J ¼ 5.0 Hz, 2-H), 7.78 (m, 4-H), 7.68 (m, 4-H), 7.54 (m, 4-H), 6.37
(m, 4-H), 1.58 (m, 3-H). FAB-MS (mNBA): 823 [Ru(bpy)2(4-CH3-PQZ)]2þ(Cl2)ꢀ; 752
[Ru(bpy)2(4-CH3-PQZ)]2þ; 596 [Ru(bpy)(4-CH3-PQZ)]2þ; 413 [Ru(bpy)2].

828
S. Thota et al.
2.5.5. [Ru[phen]2[3,4-di-OCH3-PQZ]Cl2. 45%, black crystals, IR (KBr) cmꢀ1: 3121
(C–H), 2990 (C–H), 1668 (C¼O). Calcd for C47H35Cl2N7O3Ru1 (%): C, 61.51; H, 3.82;
1
N, 10.68. Found (%): C, 61.46; H, 3.79; N, 10.64. H NMR (DMSO-d6): ꢂ ppm: 10.12
(s, 1-H), 8.58 (s, 1-H), 8.52 (1-H, s), 8.38 (d, 2-H), 8.34 (d, 2-H), 8.24 (d, 2-H), 8.16
(t, 3-H), 8.01 (m, 5-H), 7.96 (m, 5-H), 7.78 (d,2-H), 7.67 (m, 3-H), 6.46 (d, 2-H), 3.81
(m, 3-H), 3.76 (m, 3-H). FAB-MS (mNBA): 917 [Ru(phen)2(3,4-di-OCH3-
PQZ)]2þ(Cl2)ꢀ; 846 [Ru(phen)2(3,4-di-OCH3-PQZ)]2þ; 666 [Ru(phen)(3,4-di-OCH3-
PQZ)]2þ; 462 [Ru(phen)2].
2.5.6. [Ru[bpy]2[3,4-di-OCH3-PQZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3109
(C–H), 2916 (C–H), 1678 (C¼O). Calcd for C43H35Cl2N7O3Ru1 (%): C, 59.37; H, 4.03;
1
N, 11.27. Found (%): C, 59.34; H, 3.99; N, 11.21. H NMR (DMSO-d6): ꢂ ppm: 9.92
(s, 1-H), 8.62 (4-H, m), 8.54 (s, 1-H), 8.44 (d, J ¼ 4.9 Hz, 2-H), 8.36 (d, J ¼ 8.4 Hz, 2-H),
8.28 (m, 4-H), 8.14 (m, 5-H), 8.01 (d, J ¼ 5.0 Hz, 2-H), 7.86 (m, 4-H), 7.64 (d, 2-H), 6.23
(d, 2-H), 3.78 (m, 3-H), 3.65 (m, 3-H). FAB-MS (mNBA): 869 [Ru(bpy)2(3,4-di-OCH3-
PQZ)]2þ(Cl2)ꢀ; 798 [Ru(bpy)2(3,4-di-OCH3-PQZ)]2þ
PQZ)]2þ; 413 [Ru(bpy)2].
; 642 [Ru(bpy)(3,4-di-OCH3-
2.5.7. [Ru[phen]2[3-OCH3,4-OH-PQZ]Cl2. 45%, black crystals, IR (KBr) cmꢀ1: 3490
(O–H), 3086 (C–H), 2913 (C–H), 1681 (C¼O). Calcd for C46H33Cl2N7O3Ru1 (%): C,
61.13; H, 3.65; N, 10.85. Found (%): C, 61.06; H, 3.61; N, 10.74. 1H NMR (DMSO-d6):
ꢂ ppm: 10.84 (s, 1-H), 10.16 (s, 1-H), 8.66 (d, 2-H), 8.52 (s, 1-H), 8.41 (d, 2-H), 8.34
(d, 2-H), 8.22 (t, 3-H), 8.01 (d, 2-H), 7.94 (m, 3-H), 7.82 (m, 5-H), 7.74 (m, 3-H), 7.58
(t, 3-H), 6.93 (d, 2-H), 3.65 (m, 3-H). FAB-MS (mNBA): 903 [Ru(phen)2(3-OCH3,
4-OH-PQZ)]2þ(Cl2)ꢀ; 832 [Ru(phen)2(3-OCH3,4-OH-PQZ)]2þ; 652 [Ru(phen)(3-OCH3,
4-OH-PQZ)]2þ; 462 [Ru(phen)2].
2.5.8. [Ru[bpy]2[3-OCH3,4-OH-PQZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3511
(O–H), 3099 (C–H), 2936 (C–H), 1666 (C¼O). Calcd for C42H33Cl2N7O3Ru1 (%): C,
58.95; H, 3.86; N, 11.46. Found (%): C, 58.89; H, 3.85; N, 11.41. 1H NMR (DMSO-d6):
ꢂ ppm: 10.68 (s, 1-H), 9.87 (s, 1-H), 8.62 (d, 1-H), 8.42 (d, 2-H), 8.38 (m, 4-H), 8.24
(d, J ¼ 8.4 Hz, 2-H), 8.06 (m, 4-H), 7.92 (d, J ¼ 5.0 Hz, 2-H), 7.88 (d, 2-H), 7.74
(m, 5-H), 7.54 (m, 4-H), 6.55 (d, 2-H), 3.62 (m, 3-H). FAB-MS (mNBA): 855
[Ru(bpy)2(3-OCH3,4-OH-PQZ)]2þ(Cl2)ꢀ; 784 [Ru(bpy)2(3-OCH3,4-OH-PQZ)]2þ; 628
[Ru(bpy)(3-OCH3,4-OH-PQZ)]2þ; 413 [Ru(bpy)2].
2.5.9. [Ru[phen]2[4-OH-PQZ]Cl2. 45%, black crystals, IR (KBr) cmꢀ1: 3520 (O–H),
3128 (C–H), 2901 (C–H), 1678 (C¼O). Calcd for C45H31Cl2N7O2Ru1 (%): C, 61.85; H,
1
3.55; N, 11.23. Found (%): C, 61.78; H, 3.49; N, 11.20. H NMR (DMSO-d6): ꢂ ppm:
11.03 (s, 1-H), 9.45 (s, 1-H), 8.59 (d, 2-H), 8.36 (3-H, m), 8.21 (t, 3-H), 8.09
(d, J ¼ 8.4 Hz, 2-H), 8.01 (d, 2-H), 7.92 (m, 5-H), 7.86 (t, 3-H), 7.78 (d, 2-H), 7.64
(m, 3-H), 7.54 (d, 2-H), 6.87 (d, 2-H). FAB-MS (mNBA): 873 [Ru(phen)2(4-OH-
PQZ)]2þ(Cl2)ꢀ; 802 [Ru(phen)2(4-OH-PQZ)]2þ; 622 [Ru(phen)(4-OH-PQZ)]2þ; 462
[Ru(phen)2].

Ruthenium(II) complexes
829
2.5.10. [Ru[bpy]2[4-OH-PQZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3513 (O–H),
3094 (C–H), 2915 (C–H), 1680 (C¼O). Calcd for C41H31Cl2N7O2Ru1 (%): C, 59.88; H,
1
3.76; N, 11.88. Found (%): C, 59.84; H, 3.70; N, 11.79. H NMR (DMSO-d6): ꢂ ppm:
10.91 (s, 1-H), 9.24 (s, 1-H), 8.76 (4-H, m), 8.12 (d, J ¼ 4.9 Hz, 2-H), 8.04 (d, J ¼ 8.4 Hz,
2-H), 7.98 (m, 5-H), 7.88 (m, 4-H), 7.82 (d, 2-H), 7.64 (m, 2-H), 7.54 (m, 4-H), 7.52
(d, 2-H), 6.37 (d, 2-H). FAB-MS (mNBA): 825 [Ru(bpy)2(4-OH-PQZ)]2þ(Cl2)ꢀ; 754
[Ru(bpy)2(4-OH-PQZ)]2þ; 598 [Ru(bpy)(4-OH-PQZ)]2þ; 413 [Ru(bpy)2].
2.5.11. [Ru[phen]2[4-N-(CH3)2-PQZ]Cl2. 45%, black crystals, IR (KBr) cmꢀ1: 3136
(C–H), 2943 (C–H), 1679 (C¼O). Calcd for C47H36Cl2N8O1Ru1 (%): C, 62.66; H, 4.00;
1
N, 12.45. Found (%): C, 62.58; H, 3.91; N, 12.39. H NMR (DMSO-d6): ꢂ ppm: 10.04
(s, 1-H), 8.56 (d, 2-H), 8.48 (d, 2-H), 8.28 (5-H, m), 8.16 (m, 3-H), 8.10 (d, J ¼ 8.4 Hz,
2-H), 8.04 (d, 2-H), 7.98 (m, 3-H), 7.84 (t, 3-H), 7.56 (m, 3-H), 6.86 (d, 2-H), 6.45
(d, 2-H), 2.89 (s, 3-H), 2.82 (s, 3-H). FAB-MS (mNBA): 900 [Ru(phen)2(4 -N-(CH3)2-
PQZ)]2þ(Cl2)ꢀ; 829 [Ru(phen)2(4 -N-(CH3)2-PQZ)]2þ; 649 [Ru(phen)(4 -N-(CH3)2-
PQZ)]2þ; 462 [Ru(phen)2].
2.5.12. [Ru[bpy]2[4-N-(CH3)2-PQZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3133
(C–H), 2908 (C–H), 1661 (C¼O). Calcd for C43H36Cl2N8O1Ru1 (%): C, 60.56; H, 4.23;
1
N, 13.15. Found (%): C, 60.45; H, 4.19; N, 13.09. H NMR (DMSO-d6): ꢂ ppm: 10.11
(s, 1-H), 8.58 (2-H, d), 8.52 (d, J ¼ 4.9 Hz, 2-H), 8.32 (m, J ¼ 8.4 Hz, 4-H), 8.22 (m, 5-H),
8.04 (d, 2-H), 7.94 (m, 4-H), 7.62 (d, 2-H), 7.50 (m, 4-H), 6.96 (d, 2-H), 6.46 (d, 2-H), 2.81
(s, 3-H), 2.78 (s, 3-H). FAB-MS (mNBA): 852 [Ru(bpy)2(4 -N-(CH3)2-PQZ]2þ(Cl2)ꢀ;
781 [Ru(bpy)2(4 -N-(CH3)2-PQZ)]2þ
[Ru(bpy)2].
;
625 [Ru(bpy)(4 -N-(CH3)2-PQZ)]2þ
;
413
2.5.13. [Ru[phen]2[4-NO2-PTSZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3417–3380
(NH2&N–H), 3136 (C–H), 2958 (C–H), 1370 (C¼S). Calcd for C32H24Cl2N8O2Ru1S1
1
(%): C, 50.79; H, 3.17; N, 14.81. Found (%): C, 50.65; H, 3.15; N, 14.73. H NMR
(DMSO-d6): ꢂ ppm: 12.49 (s, 1-H), 9.04 (s, 1-H), 8.58 (3-H, t), 8.36 (d, J ¼ 4.9 Hz, 2-H),
8.26 (d, J ¼ 8.4 Hz, 2-H), 8.06 (t, 3-H), 8.01 (d, J ¼ 5.0 Hz, 2-H), 7.96 (m, 3-H), 7.74
(t, 3-H), 7.62 (d, 2-H, NH2), 6.23 (d, 2-H). FAB-MS (mNBA): 756 [Ru(phen)2(4-NO2-
PTSZ)]2þ(Cl2)ꢀ; 685 [Ru(phen)2(4-NO2-PTSZ)]2þ; 505 [Ru(phen)(4-NO2-PTSZ)]2þ
462 [Ru(phen)2].
;
2.5.14. [Ru[bpy]2[4-NO2-PTSZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3424–3347
(NH2&N–H), 3175 (C–H), 2983 (C–H), 1328 (C¼S). Calcd for C28H24Cl2N8O2Ru1S1
1
(%): C, 47.46; H, 3.39; N, 15.82. Found (%): C, 47.38; H, 3.34; N, 15.76. H NMR
(DMSO-d6): ꢂ ppm: 11.24 (s, 1-H), 9.26 (1-H, s), 8.46 (m, 4-H), 8.34 (d, J ¼ 4.9 Hz,
2-H), 8.14 (m, 4-H), 8.01 (d, 2-H), 7.87 (m, 4-H), 7.78 (s, NH2, 2-H), 7.69 (d, 2-H), 6.43
(d, 2-H). FAB-MS (mNBA): 708 [Ru(bpy)2(4-NO2-PTSZ)]2þ(Cl2)ꢀ; 637 [Ru(bpy)2
(4-NO2-PTSZ)]2þ; 481 [Ru(bpy)(4-NO2-PTSZ)]2þ; 413 [Ru(bpy)2].
2.5.15. [Ru[phen]2[3,4,5-tri-OCH3-PTSZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1
3426–3247 (NH2&N–H), 3155 (C–H), 2987 (C–H), 1332 (C¼S). Calcd for
:

830
S. Thota et al.
C35H31Cl2N7O3Ru1S1 (%): C, 52.43; H, 3.87; N, 12.24. Found (%): C, 52.38; H, 3.84;
N, 12.21. 1H NMR (DMSO-d6): ꢂ ppm: 12.24 (s, 1-H), 8.79 (2-H, d), 8.54 (d, 4-H), 8.12
(d, 2-H), 8.04 (d, J ¼ 5.0 Hz, 2-H), 7.96 (d, 2-H), 7.73 (d, 2-H), 7.31 (d, 2-H, NH2), 7.24
(m, 3-H), 6.81 (d, 2-H), 3.78 (s, 3-H), 3.66 (s, 3-H), 3.63 (s, 3-H). FAB-MS (mNBA): 801
[Ru(phen)2(3,4,5-tri-OCH3-PTSZ)]2þ(Cl2)ꢀ; 730 [Ru(phen)2(3,4,5-tri-OCH3-PTSZ)]2þ
550 [Ru(phen)(3,4,5-tri-OCH3-PTSZ)]2þ; 462 [Ru(phen)2].
;
2.5.16. [Ru[bpy]2[3,4,5-tri-OCH3-PTSZ]Cl2. 44%, black crystals, IR (KBr) cmꢀ1
:
3444–3245 (NH2&N–H), 3056 (C–H), 2931 (C–H), 1384 (C¼S). Calcd for
C31H31Cl2N7O3Ru1S1 (%): C, 49.41; H, 4.12; N, 13.02. Found (%): C, 49.38; H,
4.08; N, 12.98. 1H NMR (DMSO-d6): ꢂ ppm: 12.32 (s, 1-H), 8.62 (2-H, d), 8.48
(d, J ¼ 8.4 Hz, 2-H), 8.39–8.26 (m, 4-H), 8.18 (s, 1-H), 8.07 (d, J ¼ 5.0 Hz, 2-H), 7.99
(d, 2-H), 7.56 (d, 2-H), 7.19 (d, 2-H, NH2), 6.93 (d, J ¼ 14.6 Hz, 2-H), 6.13 (d, 2-H) 3.78
(s, 3-H), 3.66 (s, 3-H), 3.63 (s, 3-H). FAB-MS (mNBA): 753 [Ru(bpy)2(3,4,5-tri-OCH3-
PTSZ)]2þ(Cl2)ꢀ; 682 [Ru(bpy)2(3,4,5-tri-OCH3-PTSZ)]2þ
OCH3-PTSZ)]2þ; 413 [Ru(bpy)2].
; 526 [Ru(bpy)(3,4,5-tri-
2.6. Antineoplastic activity
All the mice (Albino Swiss mice) body weight of 18–20 g were maintained in identical
laboratory conditions and given standard food pellets (Hindustan Lever Ltd, Mumbai,
India) and water ad libitum. LD50 value of the synthesized complexes was determined
according to the literature [21]. The animals were divided into 19 groups each
containing 10 mice group I was vehicle controls (5 mLkgꢀ1 body weight ip) and group II
was EAC control (2 ꢃ 106 EAC cells/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–XIX, respectively. All the ruthenium
complexes and cisplatin were treated daily for 9 days starting 24 h after tumor
transplantation. Six animals from each group were sacrificed 18 h after the last dose.
The ascetic cell count parameters and ascetic fluid volume were noted. Mean survival
time (MST) for the remaining six mice of each group was noted. Tumor volume and
viable count ascites volume was noted according to the literature method.
2.7. Biological assays
The cytotoxic assays on inhibition of tumor cell proliferation in exponentially growing
tumor cell cultures.
2.8. Cytotoxic evaluation [22]
Murine leukemia L1210 and human lymphocyte Molt4/C8 and CEM cells were seeded
in 96-well microtiter plates at 50,000 (L1210) or 75,000 (CEM, Molt4/C8) cells per
200 mL-well in the presence of different concentrations of the test compounds. After
2 (L1210) or 3 (CEM, Molt4/C8) days, the viable cell number was counted using
a Coulter counter apparatus. The 50% cytotoxic concentration (CC50) was defined as
the compound concentration required to inhibit tumor cell proliferation by 50%.

Ruthenium(II) complexes
831
2.9. Antibacterial activity [20]
A stock solution of ruthenium complexes of 200 mg mLꢀ1 was made in sterile containing
5% DMF under aseptic conditions and further dilutions were made with the same
solvent in a similar manner. All the dilutions and stock solutions were sterilized by
membrane filtration. Solid agar and liquid broth culture media No. 1 were used for all
the test organisms and the pH was adjusted to 7.2. Antimicrobial activity of the
ruthenium complexes against different strains of bacteria was determined by the cup–
plate method and activity was expressed in terms of diameters in mm zones of
inhibition. Inoculum was prepared by washing a fresh 5 mL medium slant of test
organism with 5 mL sterile water and further diluting the 1 mL washing to 10 mL. The
suspension was added to 15 mL melted medium at a temperature 45–50ꢁC and plates
were prepared. Holes were dug into the agar plates with a sterile borer and filled with
the drug. The plates were incubated at 35ꢁC for 24 h. The results were compared with
that of standard chloramphenicol.
3. Results and discussion
3.1. Chemistry
The crude ruthenium complexes were purified by column chromatography with the
orange-red band collected by using silica gel as stationary phase and CHCl3 : CH3OH
(8 : 2) ratio as mobile phase.
The arylidene amino-2-phenyl quinazoline-4(3 H)-ones were prepared by reacting
3-amino-2-phenyl quinazolin-4(3 H)-one with appropriate substituted aromatic alde-
hyde in alcohol at 1 : 1 molar ratio and substituted aromatic thiosemicarbazones were
prepared by reacting substituted benzaldehyde with thiosemicarbazide in alcohol at 1 : 1
molar ratio (schemes 1 and 2). Structures of the ligands and complexes (schemes 3
and 4) were established on the basis of UV-Vis, FT-IR, 1H–NMR, 13C-NMR, and mass
spectral analysis (Supplementary figures S1–S9). In IR spectra, phenyl quinazoline
ligands showed absorptions at 3150–3000 cmꢀ1 for C–H aromatic stretching, 2980–
2850 cmꢀ1 for C–H aliphatic stretching, and 1685–1680 cmꢀ1 for C¼O stretching. The
thiosemicarbazones showed absorptions at 3410–3200 cmꢀ1 for NH2 and NH stretch,
from 3150 to 3000 cmꢀ1 for C–H aromatic stretch, and from 1370 to 1320 cmꢀ1 for C¼S
stretch. Rf values of all ligands were determined.
A comparison of IR spectra of ruthenium complexes with PQZ ligands confirms
coordination to ruthenium by oxygen and nitrogen (Supplementary figures S1–S9).
Thiosemicarbazones coordinate to ruthenium by sulfur and imino nitrogen, confirmed
by the spectra. These compounds do not possess any C2-axis of symmetry. Such loss of
C2-axis of symmetry was seen for [Ru(L)2(R)] (where L ¼ 2,20-bipyridine/1,10-
phenanthroline and R ¼ acetazolamide, 7-iodo-8-hydroxy-quinoline, etc.). All com-
pounds 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.
In UV spectra the ruthenium complexes showed broad and intense visible bands
between 320 and 530 nm due to metal-to-ligand charge transfer (MLCT) transition.
In the UV region, bands at 270 and 300 nm were assigned to 2,20-bipyridine/
1,10-phenanthroline ꢃ–ꢃ* charge transfer transitions. The same transition was found in

832
S. Thota et al.
O
N
COOH
a
b
O
c
NH2
Anthranilic acid
R
O
N
O
N
NH2
N
N
d
N
R = 4-NO2, 4-CH3, 3,4-di-OCH3, 3-OCH34-OH, 4-OH, 4-N-(CH3)2
Reagents: a = C6H5COCl; b = C5H5N; c = NH2NH2H2O; d = R-CHO
Scheme 1. Preparation of ligands (4-NO2-PQZ, 4-CH3-PQZ, 3,4-di-OCH3-PQZ, 3-OCH3, 4-OH-PQZ,
4-OH-PQZ, 4 -N-(CH3)2-PQZ), (PQZ ¼ phenyl quinazolines).
O
H
N
NH2
a
N
H
S
b
R
R
R = 4-NO2, 3,4,5-tri-OCH3
Reagents: a = NH2NHCSNH2; b = C2H5OH, CH3COOH
Scheme 2. Preparation
thiosemicarbazones).
of
ligands
(4-NO2-PTSZ,
3,4,5-tri-OCH3-PTSZ),
(PTSZ ¼ phenyl
T
N
N
N2 DMF
"T" Ligand
T
N
Cl
Ru
N
RuCl3.xH2O
Cl
cis-[Ru(T)2Cl2]
'-bipyridine/1,10-phenanthroline
where T = 2,2
Scheme 3. Preparation of cis-[Ru(T)2Cl2].
free 2,20-bipyridine/1,10-phenanthroline at 270 nm, so that coordination of the ligand
resulted in a red shift. There were also two shoulders at 380 and 500 nm, which were
tentatively attributed to MLCT transitions involving 2,20-bipyridine, 1,10-phenanthro-
line, and the third ligand.

Ruthenium(II) complexes
833
2+
T
T
N
N
N
N
Reflux in Ethanol
Cl2
Ru N
O
S = 4-Nitro-PQZ, 4-Methyl-PQZ,
3,4-di-methoxy-PQZ,
3-methoxy4-hydroxy-PQZ,
4-N-dimethyl-PQZ,
S
4-hydroxy-PQZ
T
N
N
T
N
Cl
Ru
N
2+
Cl
T
cis-[Ru(T)2Cl2]
T
N
Cl2
Reflux in Ethanol
N
N
N
Ru N
S
S = 4-niro-PTSZ ,
3,4,5-tri-methoxy-PTSZ
S
Scheme 4. Preparation of tris chelates from cis-[Ru(T)2Cl2].
1
In H-NMR spectra of the complexes, there were resonances at ꢂ 12.49 (s, br, NH).
For [Ru(phen)(4-NO2[Ru[phen]2[4-NO2-PTSZ]Cl2 there were 24 resonances (ꢂ 12.49–
6.23) and 24 well-resolved peaks (ꢂ 11.24–6.43) for [Ru[bpy]2[4-NO2-PTSZ]]Cl2. There
were also resonances at ꢂ 8.91. Thus for [Ru[phen]2[4-NO2-PQZ]Cl2 there were 30
resonances (ꢂ 8.1–7.24) and 30 peaks (ꢂ 9.18–7.54) also for [Ru[bpy]2[4-NO2-PQZ]]Cl2.
The mass spectra of the complexes confirmed the suggested formula by their
molecular ion peaks. The spectrum showed numerous peaks representing successive
degradation of the molecules. FAB mass spectroscopic data in figure 1 clearly suggest
that mononuclear complexes had formed in each case, the first fragment being due to
[Ru(T)2(S)]2þ Cl2ꢀ ion pair. The complex also showed a peak due to the complex cation
[Ru(T)2(S)]2þ and others due to [Ru(T)2(S)]2þ, [Ru(T)2]2þ, respectively (where T ¼ 1,10-
phenanthroline/2,20-bipyridine and S ¼ 4-NO2-PQZ, 4-CH3-PQZ, 3,4-di-OCH3-PQZ,
3-OCH3,4-OH-PQZ, 4-OH-PQZ, 4 -N(CH3)2-PQZ, 4-NO2-PTSZ, 3,4,5-tri-OCH3-
PTSZ). This type of fragmentation was reported for [Ru(phen)2(tpl)], where
tpl ¼ thiopicolinanilide. In all the cases, loss of chloride was detected [1]. Thus, based
on the above UV, IR, 1H-NMR, 13C-NMR, and mass spectral data, the Ru(II)
complexes show an octahedral geometry.
3.2. Biological activity and discussion
All the ruthenium complexes were tested for their antineoplastic activity in EAC
bearing mice. Results are shown in table 1 and the pharmacological data were analyzed
statistically by analysis of variance (ANOVA). The statistical significance was
considered only when p 5 0.05 and F 4 Fcritical. This study clearly demonstrates
tumor inhibitory activity of the ruthenium complexes against the transplantable murine

834
S. Thota et al.
Figure 1. Mass spectrum of [Ru(phen)2(3,4,5-tri-OCH3-ptsz)]2þCl2 shows intense peak at 799,
[Ru(phen)2(3,4,5-tri-OCH3-ptsz)]2þ shows intense peak at 731, [Ru(phen)(Cl-bitsz)]2þ shows intense peak
at 550, [Ru(phen)2] shows intense peak at 461 by the loss of Cl2.
ꢀ
Table 1. Antineoplastic activity of ruthenium complexes against EAC bearing mice.
Total body
weight (g)
MST
(days)
ILS
(%)
Tumor
volume (mL)
Viable cells in
ascitic fluid (%)
Parameters
Group I
Group II
Group III
Group IV
Group V
22.4 ꢄ 0.4
26.8 ꢄ 0.7
20.2 ꢄ 0.4
23.4 ꢄ 0.4
21.8 ꢄ 0.8
24.6 ꢄ 0.6
22.6 ꢄ 0.5
23.2 ꢄ 0.8
22.8 ꢄ 1.2
28.3 ꢄ 0.3
20.8 ꢄ 0.4
22.1 ꢄ 0.5
25.2 ꢄ 0.9
23.6 ꢄ 0.6
22.2 ꢄ 0.6
24.2 ꢄ 0.8
25.6 ꢄ 0.6
21.4 ꢄ 0.8
20.8 ꢄ 0.4
–
–
–
5
–
–
21
22
25
24
29
23
24
29
31
24
25
32
23
31
30
24
32
30
3.8 ꢄ 0.2
95.4 ꢄ 4.1
–
–
19
14
38
10
14
38
48
14
19
52
10
48
43
14
52
43
1.6 ꢄ 0.06
1.4 ꢄ 0.02
0.8 ꢄ 0.08
1.8 ꢄ 0.04
1.3 ꢄ 0.03
0.9 ꢄ 0.06
0.6 ꢄ 0.05
1.4 ꢄ 0.04
1.7 ꢄ 0.08
0.5 ꢄ 0.03
1.8 ꢄ 0.06
0.7 ꢄ 0.04
0.8 ꢄ 0.02
1.3 ꢄ 0.06
0.6 ꢄ 0.02
0.8 ꢄ 0.06
46.4 ꢄ 1.2
47.8 ꢄ 1.1
41.2 ꢄ 1.6
48.0 ꢄ 1.2
47.2 ꢄ 1.4
40.8 ꢄ 1.8
34.6 ꢄ 1.2
46.6 ꢄ 1.2
47.1 ꢄ 1.4
31.4 ꢄ 1.2
48.6 ꢄ 1.5
33.2 ꢄ 1.4
36.8 ꢄ 1.2
47.6 ꢄ 1.2
30.8 ꢄ 1.6
36.4 ꢄ 1.4
Group VI
Group VII
Group VIII
Group IX
Group X
Group XI
Group XII
Group XIII
Group XIV
Group XV
Group XVI
Group XVII
Group XVIII
Group XIX
Values are means ꢄ 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 XIX, ruthenium complexes (2 mg kgꢀ1) þ EAC.

Ruthenium(II) complexes
Table 2. Cytotoxic studies of ligands.
835
IC50 (mmol Lꢀ1
)
a
Compound
L1210
Molt 4/C8
CEM
4-NO2-PQZ
4-CH3-PQZ
3,4-di-OCH3-PQZ
3-OCH3,4-OH-PQZ
4-OH-PQZ
4 -N-(CH3)2-PQZ
4-NO2-PTSZ
3,4,5-tri-OCH3-PTSZ
164 ꢄ 28
78 ꢄ 13
86 ꢄ 16
102 ꢄ 18
188 ꢄ 16
63 ꢄ 25
58 ꢄ 14
48 ꢄ 12
116 ꢄ 14
98 ꢄ 16
74 ꢄ 14
116 ꢄ 16
94 ꢄ 21
76 ꢄ 14
84 ꢄ 12
66 ꢄ 14
122 ꢄ 16
86 ꢄ 12
104 ꢄ 18
128 ꢄ 12
98 ꢄ 18
84 ꢄ 16
78 ꢄ 12
48 ꢄ 14
a50% inhibitory concentration, required to inhibit tumor cell proliferation by 50%.
Table 3. Cytotoxic studies of ruthenium compounds.
IC50 (mmol Lꢀ1
)
a
Compound
L1210
Molt 4/C8
CEM
Ru[phen]2[4-NO2-PQZ]Cl2
Ru[bpy]2[4-NO2-PQZ]Cl2
1.1 ꢄ 0.4
0.92 ꢄ 0.06
1.9 ꢄ 0.8
0.98 ꢄ 0.03
1.6 ꢄ 0.5
0.67 ꢄ 0.2
0.82 ꢄ 0.04
1.2 ꢄ 0.06
0.49 ꢄ 0.02
1.1 ꢄ 0.08
0.84 ꢄ 0.06
0.22 ꢄ 0.02
0.28 ꢄ 0.03
1.1 ꢄ 0.07
0.33 ꢄ 0.04
2.1 ꢄ 0.5
Ru[phen]2[4-CH3-PQZ]Cl2
Ru[bpy]2[4-CH3-PQZ]Cl2
1.2 ꢄ 0.05
0.84 ꢄ 0.4
1.4 ꢄ 0.2
0.88 ꢄ 0.02
2.3 ꢄ 0.07
1.5 ꢄ 0.8
Ru[phen]2[3,4-di-OCH3-PQZ]Cl2
Ru[bpy]2[3,4-di-OCH3-PQZ]Cl2
Ru[phen]2[3-OCH3,4-OH-PQZ]Cl2
Ru[bpy]2[3-OCH3,4-OH-PQZ]Cl2
Ru[phen]2[4-OH-PQZ]Cl2
0.95 ꢄ 0.04
0.29 ꢄ 0.05
0.38 ꢄ 0.04
1.3 ꢄ 0.09
0.42 ꢄ 0.06
2.2 ꢄ 0.3
0.42 ꢄ 0.04
0.89 ꢄ 0.06
2.5 ꢄ 0.6
Ru[bpy]2[4-OH-PQZ]Cl2
0.54 ꢄ 0.05
3.4 ꢄ 0.5
Ru[phen]2[4-N-(CH3)2-PQZ]Cl2
Ru[bpy]2[4-N-(CH3)2-PQZ]Cl2
Ru[phen]2[4-NO2-PTSZ]Cl2
Ru[bpy]2[4-NO2-PTSZ]Cl2
Ru[phen]2[3,4,5-tri-OCH3-PTSZ]Cl2
Ru[bpy]2[3,4,5-tri-OCH3-PTSZ]Cl2
Cisplatin
1.8 ꢄ 0.7
1.4 ꢄ 0.4
1.0 ꢄ 0.08
0.46 ꢄ 0.04
0.78 ꢄ 0.5
0.36 ꢄ 0.04
0.48 ꢄ 0.04
0.26 ꢄ 0.02
0.58 ꢄ 0.2
0.82 ꢄ 0.05
0.47 ꢄ 0.03
0.79 ꢄ 0.2
0.28 ꢄ 0.2
0.63 ꢄ 0.05
0.94 ꢄ 0.04
0.52 ꢄ 0.6
0.82 ꢄ 0.04
0.34 ꢄ 0.04
a50% inhibitory concentration, required to inhibit tumor cell proliferation.
tumor cell line. Ru(bpy)2(4-OH-pqz)Cl2 and Ru(phen)2(3,4,5-tri-OCH3-ptsz)Cl2
increase life span of the tumor hosts by 52%; the remaining ruthenium complexes
increase life span in the tumor hosts by 14–48%.
The in vitro cytotoxic activity was evaluated for the ligands and ruthenium
complexes against human 4/C8, CEM, T-lymphocytes as well as murine L1210
cells. The relative potencies between ligands and their ruthenium complexes
revealed the importance of ruthenium metal using the 4/C8, CEM, and murine
L1210 assays. These determinations showed that in comparison to ligands, the
ruthenium complexes were more potent.

836
S. Thota et al.
Table 4. Antibacterial activity of ruthenium compounds.
Compound
S.A 6571
S.A 8530
V.C 865
S.F
Ru[phen]2[4-NO2-PQZ]Cl2
Ru[bpy]2[4-NO2-PQZ]Cl2
16
18
09
12
16
10
14
12
08
11
12
09
14
12
16
09
26
14
12
08
10
14
09
15
12
10
10
14
08
12
08
15
08
26
20
14
10
15
18
09
16
14
09
12
12
10
14
10
18
10
30
12
14
09
12
10
10
15
14
10
11
12
08
12
09
15
08
20
Ru[phen]2[4-CH3-PQZ]Cl2
Ru[bpy]2[4-CH3-PQZ]Cl2
Ru[phen]2[3,4-di-OCH3-PQZ]Cl2
Ru[bpy]2[3,4-di-OCH3-PQZ]Cl2
Ru[phen]2[3-OCH3,4-OH-PQZ]Cl2
Ru[bpy]2[3-OCH3,4-OH-PQZ]Cl2
Ru[phen]2[4-OH-PQZ]Cl2
Ru[bpy]2[4-OH-PQZ]Cl2
Ru[phen]2[4-N-(CH3)2-PQZ]Cl2
Ru[bpy]2[4-N-(CH3)2-PQZ]Cl2
Ru[phen]2[4-NO2-PTSZ]Cl2
Ru[bpy]2[4-NO2-PTSZ]Cl2
Ru[phen]2[3,4,5-tri-OCH3-PTSZ]Cl2
Ru[bpy]2[3,4,5-tri-OCH3-PTSZ]Cl2
Chloramphenicol
S.A ¼ Staphylococcus. aureus, V.C ¼ Vibrio cholera, S.F ¼ Shigella flexneri.
Zone of inhibition in mm (including bore size of 6 mm).
The cytotoxicity data in tables 2 and 3 reveal that ruthenium complexes have
significant cytotoxic potencies (IC50 figures at 0.29–1.6 mmol Lꢀ1 for Molt 4/C8, 0.42–
3.4 mmol Lꢀ1 for L1210, and 0.22–2.1 mmol Lꢀ1 for CEM), while for ligands the IC50
values were larger (66–116 mmol Lꢀ1 against Molt 4/C8, 48–188 mmol Lꢀ1 for L1210,
and 48–128 mmol Lꢀ1 for CEM). Ru(phen)2(3-OCH3,4-OH-pqz)Cl2 showed cytotoxic-
ity against all three cell lines in the range of 0.29, 0.42, and 0.22 mmol Lꢀ1 for Molt 4/C8,
CEM, and L1210, respectively. Ru(bpy)2(4-OH-pqz)Cl2 showed cytotoxicity against
cell lines tested 0.42 mmol Lꢀ1 for Molt 4/C8, 0.33 for CEM, and 0.54 for L1210;
Ru(phen)2(3,4,5-tri-OCH3-ptsz)Cl2 0.52 mmol Lꢀ1 for Molt 4/C8, 0.47 for CEM, and
0.36 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 concentration.
Several ruthenium complexes exhibit marked inhibitory effect on the proliferation of
tumor cells with IC50 as low as 0.29 mmol Lꢀ1 for Molt 4/C8, 0.22 mmol Lꢀ1 for CEM,
and 0.42 mmol Lꢀ1 for L1210, inhibiting tumor growth at submicromolar concentration.
The ligands were not antitumorally active. The mechanism of action of the ruthenium
complexes is not known but ruthenium antitumor agents contain an electron deficient
metal that acts as a magnet for electron-rich DNA nucleophiles.
The synthesized complexes were also evaluated for their antibacterial activity
(table 4) by the cup–plate method. Moderate antibacterial activity was observed
for Ru(phen)2(4-NO2-PQZ), Ru(phen)2(3,4,5-tri-OCH3-PTSZ) against microorgan-
isms such as Staphylococcus aureus, Vibrio cholera, and Shigella flexneri.
However some of the complexes show mild antibacterial activity against tested
organism. All the results of the complexes were compared with that of the
standard chloramphenicol.

Ruthenium(II) complexes
837
2
+
NO
2
+
2
CH
3
N
N
N
N
Ru
O
Ru
O
N
N
N
N
N
N
Cl
Cl
2
N
2
N
N
N
Ru[phen]
Ru[phen]2 [4-NO2-pqz]Cl2
2 [4-CH3-pqz]Cl2
2
+
OMe
OMe
2
+
OH
OMe
N
N
N
N
Ru
O
Ru
N
N
N
N
N
N
O
Cl
2
Cl
2
N
N
N
N
Ru[phen]2 [3,4-diOCH3-pqz]Cl2
Ru[phen]2 [3-OCH3 4-OH-pqz]Cl2
2
+
2
+
OH
N(CH )
3 2
N
N
N
N
Ru
O
Ru
O
N
N
N
N
N
N
Cl
Cl
2
2
N
N
N
N
Ru[phen]2 [4-OH-pqz]Cl2
Ru[phen]2 [4-N-(CH3)2-pqz]Cl2
2
+
2
+
CH
3
NO
2
N
N
N
N
Ru
O
Ru
O
N
N
N
N
Cl
Cl
2
2
N
N
N
N
N
N
Ru[bpy]2 [4-NO2-pqz]Cl2
Ru[bpy]2 [4-CH3-pqz]Cl2
Figure 2. Schematic presentation of the 16 complexes.
4. Conclusion
Sixteen ruthenium complexes (figure 2) bearing 2,20-bipyridine and 1,10-phenanthroline
with r-pqz or r-ptsz were synthesized in ethanol in the presence of nitrogen atmosphere.
The coordination involved for Ru is bidentate. Several ruthenium complexes exhibited
marked inhibitory effect on the proliferation of tumor cells with IC50 as low as

838
S. Thota et al.
2
+
2
+
OH
OMe
OMe
OMe
N
N
N
N
Ru
O
Ru
O
N
N
N
N
Cl
2
Cl
2
N
N
N
N
N
N
Ru[bpy]2 [3,4-diOCH3-pqz]Cl2
Ru[bpy]2 [3-OCH3 4-OH-pqz]Cl2
2
2
+
+
N(CH )
3 2
OH
N
N
N
N
Ru
O
Ru
O
N
N
N
N
Cl
Cl
2
2
N
N
N
N
N
N
Ru[bpy]2 [4-OH-pqz]Cl2
Ru[bpy]2 [4-N-(CH3)2-pqz]Cl2
2
+
2
+
N
N
N
N
S
Ru S
NH
Ru
N
2
NH
N
N
2
N
N
N
N
H
N
H
Cl
2
Cl
2
CH
CH
MeO
OMe
NO
OMe
2
Ru[phen]2 [4-NO2-ptsz]Cl2
Ru[phen]2 [3,4,5-tri-OCH3-ptsz]Cl2
2
+
2
+
N
N
N
N
Ru
S
N
N
Ru
S
NH
N
N
2
NH
2
N
N
H
Cl
2
N
N
H
Cl
2
CH
CH
MeO
OMe
NO
2
OMe
Ru[bpy]2 [4-NO2-ptsz]Cl2
Figure 2. Continued.
Ru[bpy]2 [3,4,5-tri-OCH3-ptsz]Cl2
0.29 mmol Lꢀ1 for Molt 4/C8, 0.42 mmol Lꢀ1 for L1210, and 0.22 mmol Lꢀ1 for CEM,
inhibiting tumor growth at submicromolar concentration. The ligands were not
antitumorally active. Ruthenium complexes have significant in vitro antibacterial
activity. In comparison to previously reported ruthenium complexes most of the newly
synthesized Ru(II)complexes show significant antineoplastic activity and antibacterial
activity [23–27].

Ruthenium(II) complexes
839
Acknowledgments
The authors are grateful to the Correspondent A. Madhukar Reddy, S.R. College of
Pharmacy, Hanamkonda for providing the chemicals for carrying out this research.
Appreciation is extended to the Belgian Geconcerteerde Onderzoeksacties which
supported the cytotoxic activity assays.
References
[1] U.K. Mazumder, M. Gupta, S.S. Karki, S. Bhattacharya, S. Rathinasamy, T. Sivakumar. Bioorg. Med.
Chem., 13, 5766 (2005).
[2] B. Rosenberg, L.V. Camp, T. Krigas. Nature, 205, 698 (1965).
[3] M.J. Clarke, F. Zhu, D.R. Frasca. Chem. Rev., 99, 2511 (1999).
[4] M.J. Clarke. Metal Ions in Biol. Syst., 42, 425 (2004).
[5] L. Ronconi, P.J. Sadler. Coord. Chem. Rev., 251, 1633 (2007).
[6] P. Schluga, C.G. Hartinger, A. Egger, E. Reisner, M. Galanski, M.A. Jakupec, B.K. Keppler. Dalton
Trans., 1796 (2006).
[7] M. Groensl, E. Reisner, C.G. Hartinger, R. Eichinger, O. Semenova, M.A. Jakupec, V.B. Arion,
B.K. Keppler. J. Med. Chem., 50, 2185 (2007).
[8] L. Bratsos, S. Jedner, T. Gianferara, E. Alessio. Chimia, 61, 692 (2007).
[9] W.H. Ang, E. Daldini, P.J. Dyson. Inorg. Chem., 46, 9048 (2007).
[10] W.H. Ang, A. Casini, G. Sava, P.J. Dyson. J. Org. Met. Chem., 696, 989 (2011).
[11] C.G. Hartinger, P.J. Dyson. Chem. Soc. Rev., 38, 391 (2009).
[12] C.S. Allardyce, A. Dorcier, C. Scolaro, P.J. Dyson. Appl. Organomet. Chem., 19, 1 (2005).
[13] A. Vessieres, S. Top, W. Beck, E. Hillard, G. Jaouen. Dalton Trans., 529 (2006).
[14] S.A. Clairs, J.D. Paul. Platinum Met. Rev., 45, 62 (2001).
[15] U.K. Mazumder, M. Gupta, A. Bera, S. Bhattacharya, S.S. Karki, L. Manikadan, S. Patra. Indian J.
Chem., 42A, 313 (2003).
[16] R. Suresh, S.S. Karki, S. Bhattacharya, L. Manikadan, S.G. Prabhakaran, U.K. Mazumder, M. Gupta.
J. Enzyme Inhib. Med. Chem., 21, 501 (2006).
[17] A. Nanda, S. Ganguli, K. Chakraborty. Molecules, 12, 2413 (2007).
[18] S.N. Pandeya, P. Yogeshwari, J.P. Stables. J. Med. Chem., 35, 879 (2000).
[19] A.B. Tomchin, I.L. Zhmykhova, M.M. Ponomareva, L.G. Pastushenkov, A.G. Gromova. Pharm. Chem.
J., 20, 619 (1986).
[20] U.K. Mazumder, M. Gupta, S.S. Karki, S. Bhattacharya, S. Rathinasamy, T. Sivakumar. J. Enzyme
Inhib. Med. Chem., 19, 185 (2004).
[21] J.T. Litchfield Jr, F. Wilcoxon. J. Pharmacol. Exp. Ther., 96, 99 (1949).
[22] J. Balzarini, E.D. Clercq, M.P. Mertes, D. Shugar, P.F. Torrence. Biochem. Pharmacol., 31, 3673 (1982).
[23] S. Thota, S.S. Karki, K.N. Jayaveera, J. Balzarini, E. De Clercq. J. Coord. Chem., 63, 4332 (2010).
[24] S. Arunachalam, N. Padma Priya, C. Saravanakumar, C. Jayabalakrishnan, V. Chinnusamy. J. Coord.
Chem., 63, 1795 (2010).
[25] N. Padmapriya, S. Arunachalam, N. Sathya, C. Jayabalakrishnan. J. Coord. Chem., 63, 1440 (2010).
[26] N. Muthukumar, P. Vishwanathamurthi. J. Coord. Chem., 63, 1263 (2010).
[27] N. Thilagavathi, A. Manimaran, C. Jayabalakrishnan. J. Coord. Chem., 63, 1252 (2010).