Synthesis, spectroscopic characterization, in vitro cytotoxic and structure activity relationships of some mononuclear Ru(II) complexes

Article abstract of DOI:10.1080/00958972.2013.773318

New mononuclear Ru(II) complexes [Ru(A)₂(B)}₂+, where A= 2,2'-bipyridine/1,10-phenanthroline and B= 3,4,5-tri-OCH3-DPC, 4-CH3-DPC, 4-N(CH3)₂-DPC, 4-NO₂-DPC, N-BITSZ, PTSZ and PINH, were prepared and characterized by spectroscopic methods. The in vitro cytotoxic activities of the complexes and their corresponding ligands were investigated against the human cancer Tly^MPhocyte cell lines molt 4/c8 and CEM and the murine tumor leukemia cell line L¹210, human promyelocytic leukemia cells (HL-60) and Bel-7402 liver cancer cells by MTT assay. The complexes [Ru(A)₂(B)}₂+ (A= 1,10-phenanthroline, B= 3,4,5-tri-OCH3-DPC) exerts rather more potent activities against all of these cell lines, especially for CEM and L¹210. Ru complexes and structure-activity relationships and anticancer mechanisms are also discussed.

Full text of DOI:10.1080/00958972.2013.773318

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Synthesis, spectroscopic
characterization, in vitro cytotoxic and
structure activity relationships of some
mononuclear Ru(II) complexes
Sreekanth Thota a , Srujana Vallala a , Mohammad Imran a
,
Sravani Mekala a , Shyam Sunder Anchuri a , Subhas Somalingappa
Karki b , Rajeshwar Yerra a , Jan Balzarini c & Erik De Clercq c
a Department of Pharmaceutical Chemistry & Toxicology, SR
College of Pharmacy, Warangal, India
b Department of Pharmaceutical Chemistry, KLE University’s
College of Pharmacy, Bangalore, India
c Rega Institute for Medical Research, Katholieke Universiteit
Leuven, Leuven, Belgium
Accepted author version posted online: 04 Feb 2013.Published
online: 25 Mar 2013.
To cite this article: Sreekanth Thota , Srujana Vallala , Mohammad Imran , Sravani Mekala ,
Shyam Sunder Anchuri , Subhas Somalingappa Karki , Rajeshwar Yerra , Jan Balzarini & Erik De
Clercq (2013): Synthesis, spectroscopic characterization, in vitro cytotoxic and structure activity
relationships of some mononuclear Ru(II) complexes, Journal of Coordination Chemistry, 66:6,
1031-1045
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Journal of Coordination Chemistry
Vol. 66, No. 6, 20 March 2013, 1031–1045
Synthesis, spectroscopic characterization, in vitro cytotoxic
and structure activity relationships of some mononuclear
Ru(II) complexes
SREEKANTH THOTA*†, SRUJANA VALLALA†, MOHAMMAD IMRAN†, SRAVANI
MEKALA†, SHYAM SUNDER ANCHURI†, SUBHAS SOMALINGAPPA KARKI‡,
RAJESHWAR YERRA†, JAN BALZARINI§ and ERIK DE CLERCQ§
†Department of Pharmaceutical Chemistry & Toxicology, SR College of Pharmacy,
Warangal, India
‡Department of Pharmaceutical Chemistry, KLE University’s College of Pharmacy,
Bangalore, India
§Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium
(Received 28 December 2011; in final form 29 November 2012)
New mononuclear Ru(II) complexes [Ru(A)2(B)]2+, where A= 2,2′-bipyridine/1,10-phenanthroline
and B = 3,4,5-tri-OCH3-DPC, 4-CH3-DPC, 4-N(CH3)2-DPC, 4-NO2-DPC, N-BITSZ, PTSZ and
PINH, were prepared and characterized by spectroscopic methods. The in vitro cytotoxic activities
of the complexes and their corresponding ligands were investigated against the human cancer T-
lymphocyte cell lines molt 4/c8 and CEM and the murine tumor leukemia cell line L1210, human
promyelocytic leukemia cells (HL-60) and Bel-7402 liver cancer cells by MTT assay. The com-
plexes [Ru(A)2(B)]2+ (A= 1,10-phenanthroline, B = 3,4,5-tri-OCH3-DPC) exerts rather more potent
activities against all of these cell lines, especially for CEM and L1210. Ru complexes and struc-
ture–activity relationships and anticancer mechanisms are also discussed.
Keywords: Ruthenium complexes; MTT; Structure-activity relationship
1. Introduction
Ruthenium complexes are widely used in bioinorganic chemistry, organometallic chemistry
[1–8] and in medicine [9, 10]. The properties of ruthenium complexes can be altered by
choice of ligand for multiple applications [11–14]. Ruthenium complexes have many bio-
logical and pharmacological activities, including anticancer [9], antioxidant [15, 16], anti-
bacterial [17–20], antifungal [19, 20], antiviral [6], antimalarial [21], and cytotoxicity [22].
Ruthenium complexes having the general formula [Ru(S)2(U)], where S = 2,2′-bipyri-
dine/1,10-phenanthroline and U = tpl, 4-Cl-tpl, 4-CH3-tpl, 4-OCH3-tpl, 4-NO2-tpl and pai
provide examples [4]. The most prominent examples, Na(trans-RuCl4(DMSO)(imid)]
NAMI and its more stable imidazolium complexes [Indit][trans-RuCl4(ind)2], KP1019
*Corresponding author. Email: srph_office@yahoo.co.in
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online Ó 2013 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2013.773318

2
S. Thota et al.
show high selectivity for tumor metastasis [23, 24] and low toxicity at pharmacologically
active doses [25–27], successfully completing phase-I clinical trials [28].
Ruthenium(II) arene complexes show cytotoxic properties in vitro as well as in vivo
[29, 30]. A series of complexes [Ru(n6-arene)Cl(en)](PF6), en = ethylene diamine,
arene = benzene, p-cymene, tetrahydroanthracene, etc. have been studied for their in vitro
anticancer activity [31]. We recently reported certain Ru(II) complexes that are sufficiently
stable under physiological conditions and possess excellent antitumor cytotoxic activity
towards human cell lines [32].
Our research has focused on [Ru(A)2(B)]2+, where A= 2,2′-bipyridne/1,10-phenanthro-
line, B = aryl substituted phenyl hydrazones, and their anticancer activities against transplan-
table murine tumor cell line, Ehrlich ascitic carcinoma. The in vitro cytotoxic activities of
these complexes and their corresponding Nitrogen, Sulphur, and Oxygen containing ligands
were investigated against human cancer T-lymphocyte cell lines molt 4/C8 and CEM, the
murine tumor leukemia cell line L1210, human oral epidermoid carcinoma KB cells, human
promyelocytic leukemia cells (HL-60), and Bel-7402 liver cancer cells by MTT assay.
2. Experimental
2.1. General chemicals
AR grade solvents and reagents were purchased commercially from E.Merck, Mumbai and
S.D. Fine Chem., Mumbai. Hydrated ruthenium trichloride was purchased commercially
from Loba Chemie, Mumbai and used without purification. UV–Visible (UV–vis) absorp-
tion spectra were recorded on a Shimadzu UVPC-3000 spectrophotometer. FTIR spectra
were recorded in KBr powder on a Jasco V410 FTIR spectrophotometer by diffuse reflec-
1
tance technique. H-NMR and 13C-NMR spectra were measured in DMSO-d6 and CDCl3
on a Bruker Ultraspec 500 MHz/AMX 400/300 MHz spectrophotometer. The chemical
shifts are reported against that of TMS. FAB-mass spectra were recorded on a JEOL
JMS600 spectrometer with 3-nitro benzyl alcohol as matrix. Microanalyses were carried
out on an Elementar Vario elemental analyser. Pyrazoline derivatives were prepared
according to the literature method [33, 34].
2.2. Synthesis of 3-phenyl-5-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide
(PCT)
2.2.1. Preparation of chalcones (benzylidene acetophenone or 1,3-diphenyl prop-2-
en-1-one). A solution of 22 g of sodium hydroxide in 200 mL of water and 125 mL
ethanol is placed in a 500 mL bolt head flask provided with a mechanical stirrer [33]. The
flask is immersed in an ice bath, 52 g (0.43 M) of freshly distilled acetophenone is added,
the stirrer started, and then, 46 g (44 mL, 0.43 M) of pure benzaldehyde added. Keeping
the temperature at 25 °C (15–30 °C), stir vigorously until the mixture is so thick that
stirring is no longer effective (2–3 h). The stirrer is removed and the reaction mixture left
in an ice-chest or refrigerator overnight. The product is collected by filtration, washed with
cold water until the washings are neutral to litmus and then with 20 mL of ice-cold
ethanol. The crude chalcone (88 g), after drying in air, melts at 50–54 °C. The crude
product was recrystallized from ethanol at 50 °C (about 5 mL/g).

Ru(II) Complexes
3
2.2.2. Synthesis of 3-phenyl-5-(1H-pyrrol-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothio-
amide. To solution of chalcone derivatives (0.01 M) and thiosemicarbazide (0.012 M) in
25 mL of ethanol, a solution of sodium hydroxide (0.025 M) in 5 mL of water was added
and refluxed for 8 h [34]. The products were poured into crushed ice and the solid mass
which separated was filtered, dried, and recrystallized from appropriate solvents.
2.3. Preparation of cis-[bis (A) dichlororuthenium(II)] cis-[Ru(A)2Cl2] (where A = 2,2′-bi-
pyridine/1,10-phenanthroline)
In a round bottom flask fitted with a reflux condenser RuCl3·H2O, 1 g (2.5 mM) and ligand
A (5 mM) were added [22]. The mixture was refluxed in DMF (50 mL) for 3–4 h under
nitrogen. The reddish brown color of the solution slowly turned purple and precipitation of
the product occurs. The solution was cooled to room temperature and left overnight at 0 °
C. A fine microcrystalline mass was filtered off, repeatedly washed with 30% LiCl solution
and dried. The crude solid was purified by recrystallization from anhydrous alcohol to give
crystals.
2.4. General procedure for preparing [Ru(A)2(B)Cl2] (where A = 2,2-bipyridine/1,10-phen-
anthroline; B = 3,4,5-tri-OCH3-DPC, 4-CH3-DPC, 4-N-(CH3)2-DPC, 4-NO2-DPC, N-BITSZ,
PTSZ and PINH)
To the black microcrystalline cis-Ru(A)2Cl2 (2 mM), excess of ligand B (2.5 mM) was
added and refluxed in anhydrous 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. Excess ethanol was distilled off and silica gel (60–120 mesh) added to this
solution. The final complex was purified by column chromatography using silica gel as
stationary phase and chloroform–methanol as mobile phase.
2.4.1. [Ru(phen)2(3,4,5-tri-OCH3-DPC)]Cl2. 44%, black crystals, IR (KBr) cmꢀ1
:
3483-NH2, 3040 (C–H), 2946 (C–H), 1610 (C=C), 1324 (C==S). Calcd for
C43H37Cl2N7O3Ru1S1: C, 57.14; H, 4.13; N, 10.85. Found: C, 57.02; H, 4.09; N, 10.69%.
1H NMR (DMSO-d6): δ ppm: 8.84 (s, 1H), 8.72 (d, 2H), 8.49 (d, J = 4.9 Hz, 2H),
8.42–8.31 (d, J = 5.0 Hz, 2H), 8.23 (s, 1H), 8.21 (s, 1H), 8.04–7.94 (d, 4H), 7.83 (s, 1H),
7.76 (d, J = 14.6 Hz, 2H), 7.68–7.58 (m, 4H), 7.57–7.54 (m, 2H), 6.94 (d, 2H), 6.48
(d, 2H), 3.83 (OCH3, 3H), 3.78 (OCH3, 3H), 3.76 (OCH3, 3H), 3.21 (d, 2H). 13C-NMR
(d6-DMSO) 159.96–43.82 (43 C). FAB-MS (mNBA): 903 [Ru(phen)2(3,4,5-tri-
OCH3-DPC)]2+(Cl2)ꢀ; 832 [Ru(phen)2(3,4,5-tri-OCH3-DPC)]2+; 461 [Ru(phen)2]; 371
[3,4,5-tri-OCH3-DPC].
2.4.2. [Ru(bpy)2(3,4,5-tri-OCH3-DPC)]Cl2. 42%, black crystals, IR (KBr) cmꢀ1: 3510-
NH2, 3050 (C–H), 2958 (C–H), 1639 (C=O), 1320 (C==S). Calcd for
C39H37Cl2N7O3Ru1S1: C, 54.73; H, 4.36; N, 11.46. Found: C, 54.64; H, 4.29; N, 11.42%.
1H NMR (DMSO-d6): δ ppm: 8.26 (s, 1H), 8.15 (d, 2H). 8.02–7.96 (d, J = 4.9 Hz, 2H),
7.94–7.92 (d, J = 5.0 Hz, 2H), 7.86–7.80 (d, 2H), 7.74–7.52 (d, 2H), 7.46–7.45 (d,
2H), 7.44 (d, 2H), 7.37 (s, J = 14.6 Hz, 1H), 7.22–7.00 (m, 4H), 6.75 (d, 2H), 6.4–5.90

4
S. Thota et al.
(d, 2H), 3.91–3.88 (OCH3, 3H), 3.84–3.82 (OCH3, 3H), 3.81–3.80 (OCH3, 3H), 3.16–3.14
(d, 2H), 3.07 (s, 1H), 2.32 (s, 1H). 13C-NMR (d6-DMSO) 174.22–54.82 (39 C). FAB-MS
(mNBA): 855 [Ru(bpy)2(3,4,5-tri-OCH3-DPC)]2+(Cl2)ꢀ; 784 [Ru(bpy)2(3,4,5-tri-OCH3-
DPC)]2+; 413 [Ru(bpy)2]; 371 [3,4,5-tri-OCH3-DPC].
2.4.3. [Ru(phen)2(4-CH3-DPC)]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3460 (NH2),
3068 (C–H), 2946 (C–H), 1640 (C=O), 1347 (C==S). Calcd for C41H33Cl2N7Ru1S1: C,
1
59.49; H, 4.02; N, 11.84. Found: C, 59.36; H, 4.00; N, 11.78%. H NMR (DMSO-d6): δ
ppm: 8.86 (s, 1H), 8.83 (s, 1H), 8.80 (s, J = 4.9 Hz, 1H), 8.56 (d, J = 8.4 Hz, 2H), 8.38 (d,
2H), 8.06 (d, 2H), 7.94 (d, J = 5.0 Hz, 2H), 7.86 (d, 2H), 7.58 (d, 2H), 7.46 (d, 3H), 7.32
(d, 2H), 7.28 (d, J = 14.6 Hz, 2H), 7.25 (d, 2H), 7.18 (d, 2H), 6.25 (d, 2H), 3.86 (s, 1H),
3.82 (s, 1H), 2.35 (s, 3H). 13C-NMR (d6-DMSO) 168.48–22.14 (41 C). FAB-MS (mNBA):
827 [Ru(phen)2(4-CH3-DPC)]2+(Cl2)ꢀ; 756 [Ru(phen)2(4-CH3-DPC)]2+; 461 [Ru(phen)2];
295 [4-CH3-DPC].
2.4.4. [Ru(bpy)2(4-CH3-DPC)]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3480 (NH2),
3100 (C–H), 2978 (C–H), 1680 (C=O), 1326 (C==S). Calcd for C37H33Cl2N7Ru1S1: C,
1
56.99; H, 4.27; N, 12.57. Found: C, 56.81; H, 4.22; N, 12.38%. H-NMR (DMSO-d6): δ
ppm: 8.93 (s, 1H), 8.59 (s, 1H), 8.48 (s, J = 4.9 Hz, 1H), 8.26 (d, J = 8.4 Hz, 2H), 8.14 (s,
1H), 7.98 (d, 2H), 7.60 (d, J = 5.0 Hz, 2H), 7.24 (d, 2H), 7.18 (d, 2H), 7.14 (d, 2H), 6.94
(d, 2H), 6.80 (d, J = 14.6 Hz, 2H), 6.74 (d, 2H), 6.28 (d, 2H) 6.18 (d, 2H), 3.90 (s, 1H),
3.83 (s, 1H), 2.56 (s, 2H), 2.34 (s, 3H). 13C-NMR (d6-DMSO) 172.6–24.28 (37 C). FAB-
MS (mNBA): 779 [Ru(bpy)2(4-CH3-DPC)]2+(Cl2)ꢀ; 708 [Ru(bpy)2(4-CH3-DPC)]2+; 413
[Ru(bpy)2]; 295 [4-CH3-DPC].
2.4.5. [Ru(phen)2(4-N(CH3)2-DPC)]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3421
(NH2), 3155 (C–H), 2985 (C–H), 1680 (C=O), 1336 (C=S). Calcd for C42H36Cl2N8Ru1S1:
1
C, 58.87; H, 4.23; N, 13.08. Found: C, 58.72; H, 4.19; N, 13.04%. H NMR (DMSO-d6):
δ ppm: 9.01 (s, 1H), 8.98 (s, 1H), 8.84 (s, J = 4.9 Hz, 1H), 8.76 (d, J = 8.4 Hz, 2H), 8.44
(d, 2H), 8.36 (d, 2H), 8.24 (d, J = 5.0 Hz, 2H), 8.16 (d, 2H), 8.04 (d, 2H), 7.98 (d, 3H),
7.82 (d, 2H), 7.83 (d, J = 14.6 Hz, 2H), 7.64 (d, 2H), 7.42 (d, 2H), 6.43 (d, 2H), 3.98 (s,
1H), 3.88 (s, 1H), 2.54–2.34 (d, 6H). 13C-NMR (d6-DMSO) 178.04–42.24 (42 C).
FAB-MS (mNBA): 856 [Ru(phen)2(4-N(CH3)2-DPC)]2+(Cl2)ꢀ; 785 [Ru(phen)2(4-N(CH3)2-
DPC)]2+; 461 [Ru(phen)2]; 324 [4-N(CH3)2-DPC].
2.4.6. [Ru(bpy)2(4-N(CH3)2-DPC)]Cl2. 44%, black crystals, IR (KBr) cmꢀ1: 3467
(NH2), 3044 (C–H), 2929 (C–H), 1682 (C=O), 1331 (C=S). Calcd for C38H36Cl2N8Ru1S1:
1
C, 56.43; H, 4.49; N, 13.85. Found: C, 56.38; H, 4.37; N, 13.72%. H NMR (DMSO-d6):
δ ppm: 8.98 (s, 1H), 8.64 (s, 1H), 8.40 (d, J = 4.9 Hz, 2H), 8.28 (d, J = 8.4 Hz, 2H), 8.08
(s, 1H), 7.85 (d, 2H), 7.58 (d, J = 5.0 Hz, 2H), 7.36 (m, 3H), 7.22 (d, 2H), 7.16 (d, 2H),
6.98 (d, 2H), 6.72 (d, J = 14.6 Hz, 2H), 6.64 (d, 2H), 6.36 (d, 2H), 6.28 (d, 2H), 4.01 (s,
1H), 3.96 (s, 1H), 2.68–2.32 (d, 6H). 13C-NMR (d6-DMSO) 177.84–41.44 (38 C).
FAB-MS (m-NBA): 808 [Ru(bpy)2(4-N(CH3)2-DPC)]2+(Cl2)ꢀ; 727 [Ru(bpy)2(4-N(CH3)2-
DPC)]2+; 413 [Ru(bpy)2]; 324 [4-N(CH3)2-DPC].

Ru(II) Complexes
5
2.4.7. [Ru(phen)2(4-NO2–DPC)]Cl2. 46%, black crystals, IR (KBr) cmꢀ1: 3426 (NH2),
3038 (C–H), 1339 (C=S). Calcd for C40H30Cl2N8O2Ru1S1: C, 55.94; H, 3.52; N, 13.05.
1
Found: C, 55.88; H, 3.48; N, 13.02%. H NMR (DMSO-d6): δ ppm: 8.86 (s, 1H), 8.83
(s, 1H), 8.80 (s, J = 4.9 Hz, 1H), 8.56 (d, J = 8.4 Hz, 2H), 8.38 (d, 2H), 8.06 (d, 2H), 7.94
(d, J = 5.0 Hz, 2H), 7.86 (d, 2H), 7.58 (d, 2H), 7.46 (d, 3H), 7.32 (d, 2H), 7.28 (d,
J = 14.6 Hz, 2H), 7.25 (d, 2H), 7.18 (d, 2H), 6.25 (d, 2H), 3.86 (s, 1H), 3.82 (s, 1H).
13C-NMR (d6-DMSO) 174.26–116.28 (40 C). FAB-MS (mNBA): 858 [Ru(phen)2(4-NO2-
DPC)]2+(Cl2)ꢀ; 787 [Ru(phen)2(4-NO2-DPC)]2+; 461 [Ru(phen)2]; 326 [4-NO2-DPC].
2.4.8. [Ru(bpy)2(4-NO2–DPC)]Cl2. 43%, black crystals, IR (KBr) cmꢀ1: 3452 (NH2),
3065 (C–H), 1328 (C=S). Calcd for C36H30Cl2N8O2Ru1S1: C, 53.33; H, 3.73; N, 13.82.
1
Found: C, 53.29; H, 3.66; N, 13.78%. H NMR (DMSO-d6): δ ppm: 8.76 (s, 1H), 8.72
(s, 1H), 8.62 (s, J = 4.9 Hz, 1H), 8.44 (d, J = 8.4 Hz, 2H), 8.26 (s, 1H), 8.24–8.18 (d, 2H),
8.04 (d, J = 5.0 Hz, 2H), 7.84 (d, 2H), 7.61 (d, 2H), 7.36 (d, 2H), 7.08 (d, 2H), 6.92 (d,
J = 14.6 Hz, 2H), 6.88 (m, 4H), 6.56 (d, 2H), 6.17 (d, 2H), 3.23 (s, 1H), 3.12 (s, 1H). 13C-
NMR (d6-DMSO) 169.42–121.64 (36 C). FAB-MS (mNBA): 810 [Ru(bpy)2(4-NO2-
DPC)]2+(Cl2)ꢀ; 729 [Ru(bpy)2(4-NO2-DPC)]2+; 413 [Ru(bpy)2]; 326 [4-NO2-DPC].
2.4.9. [Ru(phen)2(N-BITSZ)]Cl2. 46%, black crystals, IR (KBr) cmꢀ1: 3445–3282
(NH2 & N–H), 3062 (C–H), 2957 (C–H), 1682 (C=O), 1381 (C==S). Calcd for
C40H30Cl2N8O1Ru1S1: C, 57.01; H, 3.59; N, 13.30. Found: C, 56.98; H, 3.42; N, 13.22%.
1H NMR (DMSO-d6): δ ppm: 10.13 (d, J = 5.2 Hz, 1H), 9.11 (s, 1H, CH=N), 8.84 (d,
J = 8.8 Hz, 2H), 8.66 (m, J = 8.6 Hz, 4H), 8.54 (d, J = 8.6 Hz, 2H), 8.47–8.21 (m, 6H),
8.10–8.01 (m, 2H), 7.81 (d, J = 5.0 Hz, 2H), 7.62–7.44 (m, 4H), 7.39 (m, 4H), 6.84 (s, 2H,
br, NH2). 13C-NMR (d6-DMSO) 180.48–46.84 (40 C). FAB-MS (mNBA): 842
[Ru(phen)2(N-BITSZ)]2+(Cl2)ꢀ; 772 [Ru(phen)2(N-BITSZ)]2+; 461 [Ru(phen)2]; 381
[N-BITSZ].
2.4.10. [Ru(bpy)2(N-BITSZ)]Cl2. 43%, black crystals, IR (KBr) cmꢀ1: 3488–3385
(NH2 & N–H), 3056 (C–H), 2968 (C–H), 1682 (C=O), 1361 (C==S). Calcd for
C36H30Cl2N8O1Ru1S1: C, 54.41; H, 3.80; N, 14.10. Found: C, 54.34; H, 3.79; N, 14.04%.
1H NMR (DMSO-d6): δ ppm: 10.05 (d, J = 5.2 Hz, 1H), 9.18 (s, 1H, CH=N), 8.76 (d,
J = 8.8 Hz, 2H), 8.62 (d, J = 8.6 Hz, 2H), 8.51 (d, J = 8.6 Hz, 2H), 8.39–8.22 (m, 6H), 8.20–
8.03 (m, 2H), 7.86 (d, J = 5.0 Hz, 2H), 7.64–7.45 (m, 4H), 7.28 (m, 4H), 6.54 (s, 2H, br,
NH2), 6.34 (d, 2H). 13C-NMR (d6-DMSO) 182.22–44.84 (36 C). FAB-MS (mNBA): 794
[Ru(bpy)2(N-BITSZ)]2+(Cl2)ꢀ; 723 [Ru(bpy)2(N-BITSZ)]2+; 413 [Ru(bpy)2]; 381 [N-
BITSZ].
2.4.11. [Ru(phen)2(PTSZ)]Cl2. 43%, black crystals, IR (KBr) cmꢀ1: 3450–3292 (NH2
&
N–H), 3060 (C–H), 2965 (C–H), 1630 (NH), 1328 (C==S). Calcd for
C30H24Cl2N8Ru1S1: C, 51.43; H, 3.45; N, 15.99. Found: C, 51.38; H, 3.39; N, 15.92%.
1H NMR (DMSO-d6): δ ppm: 8.71 (s, J = 5.2 Hz, 1H), 8.62 (s, 1H, CH = N), 8.57
(d, J = 8.8 Hz, 2H), 8.34 (d, J = 8.6 Hz, 1H), 8.26 (d, J = 8.6 Hz, 1H), 7.95 (m, 3H), 7.86
(m, 2H), 7.63 (d, J = 5.0 Hz, 2H), 7.58 (d, 2H), 7.46 (d, 2H), 7.31 (s, 2H, br, NH2), 6.86

6
S. Thota et al.
(d, 2H), 3.81 (d, 2H), 2.21 (s, 1H). 13C-NMR (d6-DMSO) 184.86–106.62 (30 C). FAB-
MS (mNBA): 700 [Ru(phen)2(PTSZ)]2+(Cl2)ꢀ; 629 [Ru(phen)2(PTSZ)]2+; 461 [Ru
(phen)2]; 168 [PTSZ].
2.4.12. [Ru(bpy)2(PTSZ)]Cl2. 43%, black crystals, IR (KBr) cmꢀ1: 3441–3278 (NH2 &
N–H), 3148 (C–H), 2980 (C–H), 1670 (NH), 1349 (C=S). Calcd for C26H24Cl2N8Ru1S1:
1
C, 47.85; H, 3.71; N, 17.17. Found: C, 47.74; H, 3.69; N, 17.12%. H NMR (DMSO-d6):
δ ppm: 8.75 (s, J = 5.2 Hz, 1H), 8.64 (s, 1H, CH=N), 8.61 (d, J = 8.8 Hz, 2H), 8.56 (d,
J = 8.6 Hz, 1H), 8.47 (d, J = 8.6 Hz, 1H), 8.36 (m, 3H), 8.24 (s, 1H), 7.98 (d, J = 5.0 Hz,
2H), 7.88 (d, 2H), 7.62 (d, 1H), 7.34 (s, 2H, br, NH2), 7.22 (d, 2H), 6.75 (d, 2H), 3.98 (d,
2H), 2.06 (s, 1H). 13C-NMR (d6-DMSO) 181.28–112.40 (26 C). FAB-MS (mNBA): 652
[Ru(bpy)2(PTSZ)]2+(Cl2)ꢀ; 581 [Ru(bpy)2(PTSZ)]2+; 413 [Ru(bpy)2]; 168 [PTSZ].
2.4.13. [Ru(phen)2(PINH)]Cl2. 43%, black crystals, IR (KBr) cmꢀ1: 3320 (N–H), 3120
(C–H), 2943 (C–H), 1648 (C==O). Calcd for C35H26Cl2N8O1Ru1S1: C, 56.30; H, 3.51; N,
15.01. Found: C, 56.18; H, 3.49; N, 14.96%. 1H NMR (DMSO-d6): δ ppm: 8.82 (d,
J = 5.2 Hz, 1H), 8.80 (s, 1H, CH=N), 8.64 (d, J = 8.8 Hz, 2H), 8.51 (d, J = 8.6 Hz, 1H),
8.41 (d, J = 8.6 Hz, 1H), 8.23 (s, 1H), 7.93 (m, 2H), 7.81 (d, J = 5.0 Hz, 2H), 7.74–7.58
(m, 4H), 7.49 (d, 2H), 7.02 (s, 1H), 6.80 (s, 2H, br, NH2), 6.65 (d, 2H), 5.68 (s, 1H), 5.65
(d, 2H), 2.08 (s, 1H). 13C-NMR (d6-DMSO) 166.04–104.26 (35 C). FAB-MS (mNBA):
746 [Ru(phen)2(PINH)]2+(Cl2)ꢀ; 675 [Ru(phen)2(PINH)]2+; 461 [Ru(phen)2]; 214 [PINH].
2.4.14. [Ru(bpy)2(PINH)]Cl2. 43%, black crystals, IR (KBr) cmꢀ1: 3242 (N–H), 3164
(C–H), 2980 (C–H), 1652 (C==O). Calcd for C31H26Cl2N8O1Ru1S1: C, 53.30; H, 3.75; N,
16.04. Found: C, 53.28; H, 3.69; N, 15.99%. 1H NMR (DMSO-d6): δ ppm: 8.93 (d,
J = 5.2 Hz, 2H), 8.64 (s, 1H, CH=N), 8.58 (s, J = 8.8 Hz, 1H), 8.21 (d, J = 8.6 Hz, 1H),
7.70–7.62 (m, J = 8.6 Hz, 3H), 7.42–7.31 (m, 4H), 6.78 (m, 2H), 6.61 (d, J = 5.0 Hz, 2H),
6.58–6.28 (m, 4H), 6.18 (d, 2H), 5.61 (s, 2H), 5.54 (s, 1H) 2.01 (s, 1H). 13C-NMR (d6-
DMSO) 162.46–108.28 (31 C). FAB-MS (mNBA): 698 [Ru(bpy)2(PINH)]2+(Cl2)ꢀ; 627
[Ru(bpy)2(PINH)]2+; 413 [Ru(bpy)2]; 214 [PINH].
3. Results and discussion
3.1. Chemistry
The ligands like 3-phenyl-5-(3,4,5,-tri-OCH3-phenyl)-4,5-dihydro 1H-pyrazole-1-carbo-
thioamide were prepared by reacting 3,4,5-tri-methoxy benzylidene chalcone with
appropriate thiosemicarbazide in alcohol at 1 : 1 M ratio, ligands like 3-phenyl-5-(p-tolyl)-
4,5-dihydro 1H-pyrazole-1-carbothioamide were prepared by reacting 4-methyl benzylidene
chalcone with appropriate thiosemicarbazide in alcohol at 1 : 1 M ratio. Ligands like PTSZ
and PINH were prepared by reacting pyrrole-2-carbaldehyde with thiosemicarbazide and
isoniazide in alcohol at 1 : 1 M ratio. N-BITSZ was prepared by reacting N-benzyl isatin
with thiosemicarbazide in alcohol at 1 : 1 M ratio (schemes 1–3). Structures of the

Ru(II) Complexes
7
synthesized ligands and complexes were established by UV–vis, FT-IR, 1H-NMR,
13C-NMR, elemental analysis and mass spectral analysis (Supplementary material).
3.1.1. UV spectra. The ruthenium complexes showed broad and intense visible bands
320–530 nm due to metal to ligand charge transfer transition (MLCT). In the UV region,
bands at 270 and 300 nm were assigned to 2,2′-bipyridine/1,10-phenanthroline π–π⁄ transi-
tions, found in free 2,2′-bipyridine/1,10-phenanthroline at 270 nm, so that coordination
resulted in a red shift in the transition energy. There were also two shoulders at 380 and
500 nm, which were, tentatively, attributed to MLCTs involving 2,2′-bipyridine, 1,10-phe-
nanthroline, and the third ligand (schemes 4 and 5).
3.1.2. IR spectra. In the phenyl pyrazoline ligands, absorptions from 3500–3400 for NH2,
3150–3000 cmꢀ1 for C–H aromatic stretching, 2980–2850 cmꢀ1 for C–H aliphatic stretching,
and 1585–1500 cmꢀ1 for C=N stretching, 1380–1320 cmꢀ1 for C=S stretching were
observed. Pyrrole thiosemicarbazone ligands showed absorptions from 3410–3200 cmꢀ1 for
NH2 and NH stretching, from 3150–3000 cmꢀ1 for C–H aromatic stretching, and from 1370–
1320 cmꢀ1 for C=S stretching. In pyrrole isonicotinylhydrazones, absorptions from 3300–
3200 for NH stretching, 3150–3000 cmꢀ1 for C–H aromatic stretching, and 1680–1620 cmꢀ1
for C=O stretching were observed. Rf value of all the ligands were determined.
Ru(phen)2(3,4,5-tri-OCH3-DPC)]Cl2 has bands at 3483 for NH2, 3040 for CH and 1324
for C=S, whereas 3,4,5-tri-OCH3-DPC they are at 3480 for NH2, 3042 for C–H and 1346
for C=S. By comparison of IR spectra of complexes with pyrazolines coordination occurs
R
R
R
R
R
R
NaOH
CH
3
stirring 3-4 hrs
H
O
O
2a-2d
O
Ref lux
8hrs
Thiosemicarbazide,
Ethanol
R
R
R
N
N
NH
2
S
3a-3d
Scheme 1. Preparation of pyrazole ligands. 2a, R = OCH3, R′ = OCH3, R″ = OCH3; 2b, R = H, R′ = CH3, R″ = H;
2c, R = H, R′ = N(CH3)2, R″ = H; 2d, R = H, R′ = NO2, R″ = H. 3a, R = OCH3, R′ = OCH3, R″ = OCH3; 3b, R = H,
R′ = CH3, R″ = H; 3c, R = H, R′ = N(CH3)2, R″ = H; 3d, R = H, R′ = NO2, R″ = H.

8
S. Thota et al.
O
NOH
a
b
c
O
NH2
N
H
O
N
H
Isatin
Aniline
Isonitrosoacetanilide
d
e
H2N
S
HN
N
O
N
O
O
N
f
CH2
CH2
g
N-Benzyl isatin
BITSZ
Scheme 2. Preparation of N-benzyl isatin thiosemicarbazone [35, 36]. Reagents (a) Cl3CCH(OH)2; (b) NH2OH.
HCl; (c) H2SO4; (d) DMF/K2CO3; (e) C6H5–CH2Cl; (f) NH2NHCSNH2; (g) Alcohol, CH3COOH.
by sulfur and nitrogen (Supplementary figures 1–18). In the complexes with pyrrole thio-
semicarbazones, spectra confirm coordination by sulfur and imino nitrogen. In pyrrole
isonicotinyl hydrazone complexes, coordination by oxygen and imino nitrogen was con-
firmed by the spectra. These compounds do not possess C2 axes of symmetry. Such a loss
of C2 axis of symmetry was seen for [Ru(L)2(R)] [37] (where L= 2,2′-bipyridine/1,10-phe-
nanthroline and R = acetazolamide, 7-iodo-8-hydroxy-quinoline, etc.). All compounds had
well-resolved resonances, which correspond to four different aromatic ring protons of the
two 2,2′-bipyridine/1,10-phenanthroline ligands and third ligand.
1
3.1.3. 1H-NMR spectra. In H-NMR spectra of the 3-phenyl-5-(3,4,5-tri-OCH3-phenyl)-
4,5-dihydro 1H-pyrazole-1-carbothioamide, there were 12 resolved resonances at
δ 8.05–5.86 and nine resolved resonances at δ 3.86–3.79 due to 3 OCH3 groups.
1
In H-NMR spectra of [Ru(bpy)2(3,4,5-tri-OCH3-DPC)]Cl2, there were 28 resonances (δ
1
8.26–5.9) and nine resolved resonances at δ 3.91–3.80 due to 3 OCH3 groups. H-NMR
of the complexes having more protons confirmed the authenticity of complex.
1
In H-NMR spectra of the complexes, there were resolved resonances at δ 10.13. Thus,
for [Ru(phen)2(N-BITSZ)]Cl2, there were 30 resonances (δ 10.13–6.84) and 30 well-
resolved peaks (δ 10.05–6.54) for [Ru(bpy)2(N-BITSZ)]Cl2.

Ru(II) Complexes
9
H2N
S
H2N
S
HN
HN
N
NH2
glacial aceticacid, Alcohol
N
H
H
ptsz
O
H
N
N
O
NH2
H
O
N
H
HN
N
N
glacial aceticacid, Alcohol
N
H
H
pinh
Scheme 3. Preparation of ptsz and pinh [38].
A
N
N
A
N2
DMF
Ru
N
N
RuCl3.xH2O
Cl
cis-[Ru(A)2Cl2]
Where A =2,2'-bipyridine/ 1,10-phenanthroline
Cl
"A" Ligand
Scheme 4. Preparation of cis-[Ru(A)2Cl2].
3.1.4. Mass spectra. Spectra showed numerous peaks representing successive degrada-
tion of the molecules. FAB mass spectroscopic data in Supplementary Figure 15 clearly
suggest that monoꢀnuclear complexes formed in each case, the first fragment being due to
[Ru(A)2(B)]2+ Cl2 ion pair. The complex also showed a peak due to the complex cation
[Ru(A)2(B)]2+ and others due to [Ru(A)(B)]2+, [Ru(A)2]2+, respectively (where A= 1,10-
phenanthroline/2,2′-bipyridine and B = 3,4,5-tri-OCH3-DPC, 4-CH3-DPC, 4-N-(CH3)2-
DPC, 4-NO2-DPC, N-BITSZ, PTSZ and PINH]. This type of fragmentation was reported
for [Ru(phen)2(nmit)]Cl2 and [Ru(bpy)2(ihqs)]Cl2, where nmit = N-methyl isatin thiosemi-
carbazone and ihqs = 7-iodo-8-hydroxy quinoline-5-sulfonic acid [45]. Loss of chloride
was detected where A= 2,2′-bipyridine/1,10-phenanthroline and B = r-DPC, PTSZ, PINH.
Thus, based on the above observations, it is tentatively suggested that Ru(II) complexes
have pseudooctahedral geometry.
3.2. Biological activity and discussion
The in vitro cytotoxic activity was evaluated for ligands and complexes against human 4/
C8, CEM, T-lymphocytes as well murine L1210 cells, HL-60, and BEL-7402; results are

10
S. Thota et al.
2+
A
A
N
N
N
N
Reflux inAlcohol
Cl2
N
Ru
S
B = 3,4,5-tri-methoxy-DPC,
4-Methyl-DPC,
4-N-dimethyl-DPC,
4-Nitro-DPC,
N-BITSZ, PTSZ
B
A
N
N
Cl
N
A
Ru
N
2+
Cl
cis-[Ru(A)2Cl2]
A
A
N
Cl2
Reflux inAlcohol
B = PINH
N
N
N
Ru
N
O
B
Scheme 5. Preparation of tris chelates from cis-[Ru(A)2Cl2].
Table 1. Cytotoxic studies of ligands.
a
IC50 (μM)
Compound
CEM
L1210
Molt 4/C8
HL60
BEL7402
3,4,5-tri-OCH3-DPC
4-CH3-DPC
N-(CH3)2-DPC
4-NO2-DPC
N-BITSZ
54 08
86 14
48 06
106 04
128 16
84 22
158 18
28 04
98 12
44 08
116 12
122 14
96 14
184 06
88 16
42 08
104 18
134 12
68 16
48 14
78 12
58 02
82 08
54 06
231 08
118 04
42 08
144 14
66 04
50 02
92 14
225 12
104 08
96 06
100 14
PTSZ
PINH
summarized in tables 1 and 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 ruthenium complexes were more
potent.
Results are summarized in table 2 for 14 complexes tested for in vitro cytotoxic activity
against human cancer cell lines CEM, L1210, Molt 4/C8, HL60, Molt 4/C8, and murine
tumor cell line, BEL7402. Cells were seeded in 200 l-micro titer plate wells in RPMI-
1640 culture medium (supplemented with fetal calf serum (10%), 2 Mm L-glutamine and
0.075% NaHCO3) in the absence or presence of test compounds (200, 40, 8, 1.6 and
0.32 μg/mL). The cell number was counted after two (L1210) or three (CEM, Molt4) days
using a Coulter counter.
In vitro evaluation of these ruthenium complexes revealed cytotoxic activities from 0.24
to >2 μM against CEM, 0.28 to >2 μM against L1210, 0.36 to >2 μM against Molt4/C8,
0.24 to >2 μM against HL60 and 0.25 to >2 μM against BEL7402. Of the tested ruthenium

Ru(II) Complexes
Table 2. Cytotoxic studies of ruthenium complexes.
11
a
IC50 (μM)
Comp. code
CEM
L1210
Molt 4/C8
HL60
BEL7402
R-1
R-2
R-3
R-4
R-5
R-6
R-7
R-8
0.75 0.06
1.1 0.4
0.28 0.04
0.99 0.2
0.35 0.02
0.49 0.03
0.59 0.04
1.5 0.3
0.95 0.3
1.7 0.5
0.24 0.02
0.58 0.04
0.34 0.04
0.63 0.07
0.28 0.04
0.45 0.05
0.94 0.06
0.58 0.04
0.44 0.04
0.30 0.02
1.0 0.4
0.58 0.06
0.36 0.04
0.84 0.06
0.92 0.08
0.44 0.06
0.25 0.03
1.2 0.4
0.64 0.06
0.46 0.02
0.51 0.03
1.6 0.06
1.2 0.6
0.24 0.03
0.71 0.05
0.54 0.05
0.97 0.2
1.4 0.4
0.88 0.1
0.47 0.04
0.68 0.05
1.2 0.07
2.4 0.08
1.6 0.04
3.4 0.08
0.51 0.1
0.57 0.05
0.44 0.04
0.86 0.06
0.94 0.3
1.9 0.7
0.62 0.05
0.36 0.04
0.77 0.03
0.98 0.04
2.9 0.5
1.2 0.2
0.42 0.03
0.98 0.3
0.46 0.04
1.8 0.2
1.4 0.04
0.92 0.2
2.2 0.02
1.2 0.02
R-9
R-10
R-11
R-12
R-13
R-14
STD
3.2 0.06
1.4 0.06
1.9 0.05
0.98 0.02
1.1 003
2.8 0.06
0.87 0.06
2.4 0.04
1.8 0.02
0.78 0.04
a50% inhibitory concentration, required to inhibit tumor cell proliferation by 50%.
complexes, R-3 shows potent cytotoxic activity against the entire five cell lines tested,
0.24, 0.35, 0.57, 0.34, and 0.84 μM for CEM, L1210, Molt4/C8, HL60, BEL7402, respec-
tively. R-5 shows significant cytotoxic activity against the entire five cell lines of 0.54,
0.59, 0.86, 0.28, 0.44 μM for CEM, L1210, Molt4/C8, HL60, BEL7402, respectively.
The remaining ruthenium complexes showed low μM values for Molt 4, HL60, and
CEM and higher μM values for L1210 and BEL7402. Some ruthenium complexes showed
low μM values for L1210, BEL7402, and CEM and higher μM values for Molt 4 and
HL60.
In comparison with ruthenium compounds, the ligands displayed cytotoxicities at higher
concentration. Thus, the ruthenium compounds proved inhibitory to tumor growth at sub-
micromolar concentration where the ligands were not antitumorally active.
All of these Ru(II) complexes can bind to DNA through intercalation with high
DNA-binding affinities and also possess DNA-cleaving capacity. At 50 and 100 μM,
almost complete DNA unwinding can be observed.
3.3. DNA-binding and DNA-unwinding properties
The electronic spectra of all these Ru(II) compounds in water (0.1% DMSO) are character-
ized by an intense MLCT in the visible region [2]. Visible bands around 450 nm arise from
metal to ligand (π) charge transfer transitions. UV–Visible spectra showed that all of these
ruthenium complexes exhibit hypochromism accompanied by bathochromic shifts upon
addition of CT-DNA.
3.3.1. Electronic absorption titration. Absorption titrations of Ru(II) complexes in buf-
fer A were performed by using a fixed Ru(II) concentration to which increments of DNA
stock solutions were added. Initially, 3000 μL solutions of the blank buffer solution and
the Ru(II) complex were placed in the reference and sample cuvettes (1 cm path length),
respectively, and then, the first spectrum was recorded from 200 to 640 nm. During the

12
S. Thota et al.
titration, an aliquot (3–10 μL) of DNA stock solution was added to each cuvette to elimi-
nate the absorbance of the DNA itself, and the solutions were mixed by repeated inver-
sion. The solutions were allowed to incubate for 5 min before absorption spectra were
recorded. The titration processes were repeated until there was no change in the spectra for
at least 4 titrations, indicating binding saturation. The changes in Ru(II) complex concen-
tration due to dilution at the end of each titration were negligible. The intrinsic binding
constants of both complexes, Kb to DNA were obtained by monitoring the changes of the
MLCT absorbance for both complexes according to equations (1) and (2) [39–42]
1=2
½ðea ꢀ ef Þ=ðeb ꢀ ef Þ ¼ ðb ꢀ ðb2 ꢀ 2Kb2Ct½DNAꢁ=SÞ ꢁ2KbCt
ð1Þ
ð2Þ
b ¼ 1 þ KbCt þ Kb½DNAꢁ=2s
where ɛa is the extinction coefficient (Aabs/[M]) observed for the MLCT absorption band at
a given DNA concentration, ɛf and ɛb the extinction coefficient for the free Ru(II) complex
and the extinction coefficient for the Ru(II) complex in the fully bound form, respectively,
the concentration of DNA in nucleotides, Ct the total Ru(II) complex concentration, Kb the
equilibrium binding constant (in M L )
ꢀ1 ꢀ1, and s the binding site size.
3.4. Structure–activity relationships and antitumor mechanism
DNA is the main target of antitumor activity in most antitumor compounds [39]. The
heavy metal complexes can be designed to target particular sequences or structural features
of the DNA double helix with binding greatly influenced by the structure of DNA [40].
Many antiviral, antitumor and antibacterial agents take action through binding to DNA.
The binding of these drugs can be attributed to electrostatic forces, hydrophobic interac-
tion, hydrogen bonds, etc. The DNA binding constants (Kb) are rather high (0.31–
1.49 ꢂ 105 mꢀ1). These complexes also possess good DNA-cleaving ability. The most
important phenomena for complexes showing antitumor activity is interaction between the
complexes and DNA by intercalation.
In cancer chemotherapy, DNA-targeting agents play an important role [41], such as cis-
platin, [cis-(PtCl2(NH3)2], carboplatin ([Pt(CBDCA)-(NH3)2], CBDCA= 1,1-cyclobutane
dicarboxylato), and oxoplatin ([Pt(dach)(oxaloto)] dach = R,R-1,2-diamino cyclohexane).
All these structures have the general structure of cis-[PtX2(amine)2], where X is a leaving
group such as chloride or dicarboxylate. All of the Ru(II) complexes are different from cis-
platin, coordination-saturated with no leaving group. The most important structural feature
is they contain aromatic and heterocyclic (pyrazole) moieties and all of them can bind to
DNA. Most of the Ru(II) complexes show strong DNA-binding affinities and they can eas-
ily interact with the DNA of tumor cells.
In our research, it is found that all these synthesized complexes possess excellent antitu-
mor activities, whereas their corresponding ligands do not, which is closely correlated with
the structural (N,S,O) characteristics (including the electronic structure) of these Ru(II)
bipyridyl and phenanthroline complexes. The mononuclear Ru(II) complexes (Ru
(phen)2L)2+ adopt pseudooctahedral geometry with an N-, S-, and O-containing heterocy-
clic ligand as an intercalative ligand (L) of the complex. Free ligands have the same pla-
narity and can intercalate between DNA-base pairs. Energies of the lowest unoccupied
molecular orbitals (LUMO and LUMO + x), which usually accept the electron offered from

Ru(II) Complexes
13
DNA-base pairs, are different [42, 43]. The Ru+2 introduces high-positive charge making
the energies of the frontier molecular orbital (LUMO) reduced. The comparisons of some
frontier molecular orbital energies between the complexes and corresponding ligands are
given in Supplementary material.
Some of the complexes possess the most potent anticancer activity, although their DNA-
binding affinities are not strongest in this series of complexes. The energy difference
(ΔEL ꢀ H) and the hydrophobic parameters usually expressed as logP of the complexes
may be important. Quantitative–structure–activity relationships for pharmaceutical com-
pounds have shown that the anticancer activity (PIc50) of ruthenium complexes is closely
related to logp [44]. In our research, the dipole moments of the complexes can be obtained
with DFT calculations and used to quantitatively analyze the trend in the hydrophobic val-
ues (logP), although logP data cannot be experimentally measured. The complexes have
poor water solubility; according to the similarity theorem, it means that they have rather
good fat solubility. Since most antimetastatic agents perform their activity in organic sol-
vents, this may be one reason why the complexes possess higher anticancer activity. From
the table, the energy differences (ΔEL ꢀ H) of the ruthenium complexes 5 and 6 are greater
than can be reasonably explained.
In some of the heterocyclic ligands, substitution of the p-site on the intercalative ligand
seems to be more advantageous than that of the o-site. It may be related to atomic net
charge population on the intercalative ligand. Substitution of the p-site provides more bal-
anced polarity alteration of the charge populations on the intercalative ligand than that of
the o-site; research on QSAR is underway.
4. Conclusion
The in vitro anticancer activities of a series of Ru(II) polypyridine complexes
[Ru(A)2(B)]2+, where A= 2,2′-bipyridine/1,10-phenanthroline and B = 3,4,5-tri-OCH3-DPC,
4-CH3-DPC, 4-N(CH3)2-DPC, 4-NO2-DPC, N-BITSZ, PTSZ, PINH and their correspond-
ing ligands have been measured against human cancer T-lymphocyte cell lines molt 4/c8
and CEM, murine tumor leukemia cell line L1210, human promyelocytic leukemia cells
(HL-60) and Bel-7402 liver cancer cells by MTT assay. These ruthenium complexes
possess excellent in vitro cytotoxic activities; the corresponding ligands are not as
cytotoxic. The relationship between anticancer potency and the DNA-binding affinity was
analyzed to get possible QSAR, but the anticancer efficiency is not in line with DNA-bind-
ing affinity. The complexes show good binding to calf thymus DNA and most exhibit effi-
cient DNA cleavage upon irradiation via a mechanistic pathway involving formation of
singlet oxygen as the reactive species. These results suggest that both ancillary ligand and
intercalative ligand influence the binding of these complexes to DNA. In comparison with
previously reported ruthenium complexes these new complexes show significant cytotoxic
activities and DNA-binding constants [45–52].
Acknowledgement
One of the authors Mrs. Srujana Vallala is thanking the Department of Science &
Technology (DST), New Delhi, India, for providing the funds for carrying research (SR/
WOS-A/LS-562/2011) dated 27/03/2012.

14
S. Thota et al.
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