Potential inhibitors of DNA topoisomerase II: Ruthenium(II) poly-pyridyl and pyridyl-azine complexes

Article abstract of DOI:10.1016/j.jorganchem.2004.03.044

In search of new DNA probes a series of new mono and binuclear cationic complexes [RuH(CO)(PPh₃)₂(L)] ⁺ and [RuH(CO)(PPh₃)₂(-μ-L)RuH(CO) (PPh₃)₂]²⁺ [L=pyridine-2-carbaldehyde azine (paa), p -phenylene-bis(picoline)aldimine (pbp) and p -biphenylene-bis(picoline)aldimine (bbp)] have been synthesized. The reaction products were characterized by microanalyses, spectral (IR, UV-Vis, NMR and ESMS and FAB-MS) and electrochemical studies. Structure of the representative mononuclear complex [RuH(CO)(PPh₃) ₂(paa)]BF₄ was crystallographically determined. The crystal packing in the complex [RuH(CO)(PPh₃) ₂(paa)]BF₄ is stabilized by intermolecular π-π stacking resulting into a spiral network. Topoisomerase II inhibitory activity of the complexes and a few other related complexes [RuH(CO)(PPh₃)₂(L)]⁺ {L=2,4,6-tris(2-pyridyl)-1,3,5-triazine (tptz) and 2,3-bis(2-pyridyl) -pyrazine (bppz)} have been examined against filarial parasite Setaria cervi. Absorption titration experiments provided good support for DNA interaction and binding constants have also been calculated which were found in the range 1.2 × 10³-4.01 × 10⁴ M^-1.

Full text of DOI:10.1016/j.jorganchem.2004.03.044

Journal of Organometallic Chemistry 689 (2004) 2256–2267
www.elsevier.com/locate/jorganchem
Potential inhibitors of DNA topoisomerase II: ruthenium(II)
poly-pyridyl and pyridyl-azine complexes
a
a
a,
b
b
Manish Chandra , A.N. Sahay , D.S. Pandey *, R.P. Tripathi , J.K. Saxena ,
b
V.J.M. Reddy , M. Carmen Puerta , Pedro Valerga
c
c
a
Department of Chemistry, Awadhesh Pratap Singh University, Rewa 486 003 (M.P.), India
Medicinal and Biochemistry Division, Central Drug Research Institute, P.B. No.173, Chattar Manzil, Lucknow (U.P.), India
b
c
Departmento de Ciencia de los Materiales e Ingenieria Metallurgica y Quimica Inorganica, Facultad de Ciencia, Universidad de Cadiz,
Apartado – 40, Puerto Real 11510, Spain
Received 24 January 2004; accepted 26 March 2004
Abstract
In search of new DNA probes a series of new mono and binuclear cationic complexes [RuH(CO)(PPh3)2(L)]þ and
[RuH(CO)(PPh3)2(-l-L)RuH(CO)(PPh3)2]2þ [L ¼ pyridine-2-carbaldehyde azine (paa), p-phenylene-bis(picoline)aldimine (pbp) and
p-biphenylene-bis(picoline)aldimine (bbp)] have been synthesized. The reaction products were characterized by microanalyses,
spectral (IR, UV–Vis, NMR and ESMS and FAB-MS) and electrochemical studies. Structure of the representative mono-
nuclear complex [RuH(CO)(PPh3)2(paa)]BF4 was crystallographically determined. The crystal packing in the complex [RuH(CO)-
(PPh3)2(paa)]BF4 is stabilized by intermolecular p–p stacking resulting into a spiral network. Topoisomerase II inhibitory activity of
the complexes and a few other related complexes [RuH(CO)(PPh3)2(L)]þ {L ¼ 2,4,6-tris(2-pyridyl)-1,3,5-triazine (tptz) and 2,3-
bis(2-pyridyl)-pyrazine (bppz)} have been examined against filarial parasite Setaria cervi. Absorption titration experiments provided
good support for DNA interaction and binding constants have also been calculated which were found in the range 1.2 ꢀ 103–
4.01 ꢀ 104 Mꢁ1
.
Ó 2004 Elsevier B.V. All rights reserved.
Keywords: Ruthenium; Poly-pyridyl and pyridyl-azine ligands; Hydride; DNA; Topoisomerase II; p–p interaction
1. Introduction
polynuclear, homo/hetero-metallic complexes possessing
interesting spectroscopic, photo-physical and photo-
chemical properties [2a–2g]. Although, closely related
pyridyl azine ligands like pyridine-2-carbaldehyde azine
(paa), p-phenylene-bis(picoline)aldimine (pbp) or p-bi-
phenylene-bis(picoline)aldimine (bbp) have long been
known (Fig. 1), there are only a few reports dealing with
their complexes with the metal ions. Further, most of the
reports are mainly on the complexes of the first transi-
tion series metal ions and molybdenum and iridium
[3a–3g].
At the same time, synthesis and characterization of
metallo-intercalators based on Ru(II) poly-pyridyl
complexes that can probe and target nucleic acids have
drawn much attention [4a–4e]. A detailed study of the
mode of interaction of the complexes with DNA will
Ruthenium(II) poly-pyridyl complexes have received
considerable recent attention, because of their interest-
ing photo-physical and photo-chemical properties, pos-
sible use in photo-chemical molecular devices and as
light sensitive probes in biological systems [1a–1d]. In
this regard, poly-pyridyl bridging ligands viz. 2,4,6-
tris(2-pyridyl)-1,3,5-triazine (tptz) and 2,3-bis(2-pyr-
idyl)-pyrazine (bppz) and other related diimines have
drawn special attention. Poly-pyridyl ligands have ex-
tensively been used in the synthesis of mono and bi/
* Corresponding author. Tel./fax: +91-766230684.
E-mail address: dsprewa@yahoo.com (D.S. Pandey).
0022-328X/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2004.03.044

M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
2257
H
2. Experimental
H
C
C
N p
Np
Na
N p
Np
Na
C
Na Na
2.1. General
C
H
H
pyridine-2-carbaldehyde azine (paa)
p-phenylene-bis(picoline)aldimine (pbp)
All the reactions were carried out under nitrogen at-
mosphere and in de-aerated solvents. The solvents were
of AR grade and were purified rigorously by standard
procedures prior to their use [8]. Triply distilled and
deionized water was used for the preparation of various
buffers. 2,3-Bis(2-pyridyl)-pyrazine (bppz); 2,4,6-tris(2-
pyridyl)-1,3,5-triazine (tptz), ammonium tetrafluorobo-
rate, ammonium hexafluorophosphate, ruthenium(III)
chloride hydrate (all Aldrich) and tetrabutylammonium
perchlorate (Fluka) were used as received without fur-
ther purification. Calf thymus (CT) DNA, Supercoiled
pBR322 DNA, bovine serum albumin (BSA), ATP, di-
thiothreitol (DTT), agarose, ethidium bromide, sodium
dodecyl sulphate (SDS) and proteinage were procured
from Sigma Chemical Co., St. Louis. Topo II from fi-
larial parasite S. cervi was partially purified according to
the method by Pandeya et al. [7a]. The ligands pyridine-
2-carbaldehyde azine (paa), p-phenylene-bis(pico-
line)aldimine (pbp) and p-biphenylene-bis(picoline)
aldimine (bbp) and the precursor complex [RuH-
(CO)Cl(PPh3)3] were prepared and purified following
the literature procedures [9a–9c].
H
C
N p
Na
Np
Na
C
H
p-biphenylene-bis(picoline)-aldimine (bbp)
Fig. 1. Diagrammatical representation of ligands used in the synthesis
of complexes (paa, pbp, bbp).
lead, not only to new novel chemotherapeutics, but to
highly sensitive diagnostic agents too [5a–5l]. It is well
documented that, human lymphatic filariasis caused by
helminths, Wuchereria bancrofti (90% cases) and Burgia
malayi (10% cases), affects approximately 120 million
people, and one billion people are considered to be at
the risk of being affected [6]. During past few years, we
are working towards identification of novel therapeutic
targets. Only recently, DNA topoisomerase II (topo II)
of the filarial parasite S. cervi have been identified as a
target for the development of anti-filarial compounds
[7a,7b].
Because of our interests in this area, we have initi-
ated work on designing of metallo-intercalators con-
taining functionalized poly-pyridyl ligands to achieve
site-specific DNA recognition. During studies on Ru(II)
azine/poly-pyridyl complexes, it was felt that the
complexes containing pyridine-2-carbaldehyde (paa),
p-phenylene-bis(picoline)aldimine (pbp), p-biphenylene-
bis(picoline)aldimine (bbp), 2,4,6-tris(2-pyridyl)-1,3,5-
triazine (tptz) and 2,3-bis(2-pyridyl)-pyrazine (bppz)
ligands, may not only accentuate DNA-binding of the
ensuing complexes, but facilitate the study of other
interesting associated functional aspects too. Although,
a number of reports dealing with DNA cleavage and
inhibitory activity of ruthenium complexes are available
in the literature, the application of hydride complexes
in this regard is unusual. In this direction, mono and
binuclear Ru(II) hydrido carbonyl complexes [RuH-
(CO)(PPh3)2(paa)]BF4 (1), [RuH(CO)(PPh3)2(paa)]PF6 (2),
[RuH(CO)(PPh3)2(pbp)]PF6 (3), [RuH(CO)(PPh3)2(-l-
pbp)RuH(CO)(PPh3)2](BF4)2 (4), [RuH(CO)(PPh3)2(bbp)
RuH(CO)(PPh3)2](BF4)2 (5) were synthesized. Further,
anti-filarial activity of the complexes 1–5 and their efficacy
as anti-filarial drugs and that of some other related
complexes [RuH(CO)(PPh3)2(g2-tptz)] BF4 (6), [RuH(CO)-
(PPh3)2(g2-bppz)]BF4 (7), [RuH (CO)(PPh3)2(g2-bppz)]-
PF6 (8) have been examined [12b]. In this paper, we report
the studies on the synthesis, spectral, electrochemical and
structural characterization and topo II activity of some
Ru(II) poly-pyridyl hydrido carbonyl complexes.
2.2. Physical measurements
Microanalyses of the complexes were obtained from
micro-analytical section of the Sophisticated Analytical
Instrumentation Facility, Central Drug Research Insti-
tute, Lucknow. IR and electronic spectra using quartz
cuvette with a path length of 1 cm were recorded on a
Shimadzu-8201PC and Shimadzu-UV-1601 spectro-
photometers, respectively. H, H–1H COSY, 13C and
31P NMR spectra were recorded on a Bruker DRX-300
NMR instrument. Electrospray mass spectra were re-
corded on a Micromass-Quattro-II triple quadrupole
mass spectrometer. Samples were introduced into the
ESI source through a syringe pump at 0.4 ml hꢁ1. The
ESI capillary was 3.5 kV and cone-voltage 25–50 V.
Spectra were collected in 4s scans and printouts were
averaged spectra of 5–10 scans. FAB mass spectra were
recorded on a JEOL SX 102/DA 6000 mass spectrom-
eter using xenon (6 kV and 10 mA) as the FAB gas. The
accelerating voltage was 10 kV and the spectra were
recorded at room temperature with m-nitrobenzyl al-
cohol as the matrix. Thermal denaturation analyses were
conducted on a DU-640 spectrophotometer. Electro-
chemical data were acquired on a PAR model 273A
electrochemistry system at a scan rate of 50 mV sꢁ1. The
sample solutions (10ꢁ3 M) were prepared in purified
acetonitrile containing Etþ4 ClO4ꢁ (0.1 M) as a supporting
electrolyte. Solution was deoxygenated by bubbling
dinitrogen for about 20 min for each experiment.
1
1

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M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
Platinum wire working and auxiliary electrodes and an
aqueous saturated calomel reference electrode were used
in a three-electrode configuration.
2.3.3. Preparation of [RuH(CO)(PPh3)2(pbp)]PF6 (3)
Complex 3 was prepared by the same method as de-
scribed for complex
1
by the reaction of
[RuH(CO)Cl(PPh3)3] (477 mg and 0.5 mmol) and p-
phenylene-bis(picoline)aldimine (pbp) (286 mg and 1
mmol). It was separated as yellow microcrystalline
product. Yield: 72% (391.32 mg); Anal. Calc. for
C55F6H45N4OP3Ru: C, 60.17; H, 4.13; N, 5.15; Found:
C, 59.36; H, 4.22; N, 5.46%. FAB MS (m=z): 939 (Calc.
for [RuH(CO)(PPh3)2(pbp)]þ) (940). IR (cmꢁ1, nujol):
2002 m(Ru–H), 1946 m(CO), 1627, 1479, 1431, 1396,
1313, 1163, 1159, 1091, 999, 840, 746, 696, 555 (char-
acteristic bands due to pbp, PPh3, and counter anion
2.3. Preparation of the complexes
2.3.1. Preparation of [RuH(CO)(PPh3)2(paa)]BF4 (1)
A suspension of [RuH(CO)Cl(PPh3)3] (955 mg and 1
mmol) in methanol (60 mL) was treated with pyridine-2-
carbaldehyde azine (paa) (420 mg and 2 mmol) and the
resulting solution was heated under reflux for about 20
h. The precursor complex [RuH(CO)Cl(PPh3)3] slowly
dissolved and gave a bright orange solution. It was
cooled to room temperature and filtered through celite
to remove any solid residue. Saturated solution of
NH4BF4 in methanol (25 mL) was added to the filtrate,
the solution was reduced in volume using a rotatory
evaporator to about 25 mL and was left in refrigerator
for slow crystallization. Crystalline product appeared in
a couple of days. The crystals (complex 1) were sepa-
rated by filtration, washed with methanol, diethylether
and dried in vaccuo. Yield: 80% (762.4 mg); Anal. Calc.
for BC49F4H41N4OP2Ru: C, 61.69; H, 4.30; N, 5.87,
Found: C, 60.13; H, 4.13; N, 5.67%. ESMS (m=z): 865.1
Calc. for [RuH(CO)(PPh3)2(paa)]þ (864). FAB MS m=z
1
PFꢁ6 ); H NMR (ppm, CDCl3, 300 MHz, 25 °C): 8.92
(d), 8. 67 (s), 8.60 (d, 4.4 Hz), 7.89 (m), 7.62 (t, 4.3 Hz),
7.03 (m) )10.89 (t, 19 Hz Ru–H); 13C{1H}: d 206 (car-
bonyl carbon), 166.4, 162.3, 150.3, 150.1, 138.0, 133.8,
130.08, 127.9, 117 ppm (carbons of the pbp ligand);
31P{1H}: 43.21 (s) UV–Vis{CH2Cl2; kmax, nmðeÞ}:
465(6.1 ꢀ 104), 310(1.6 ꢀ 104), 295(2.35 ꢀ 104).
2.3.4. Preparation of [RuH(CO)(PPh3)2(-l-pbp)-
RuH(CO)(PPh3)2](BF4)2 (4)
Complex 4 was prepared following the above proce-
dure by starting with [RuH(CO)Cl(PPh3)3] (477 mg and
0.5 mmol) and p-phenylene-bis(picoline)aldimine (pbp)
(118 mg, 0.5 mmol). Yellow brown crystals separated
out. Yield 68% (599.40 mg). Anal. Calc. for
B2C92F8H76N4O2P4Ru2: C, 62.30; H, 4.28; N, 3.16;
Found: C, 61.67; H, 4.40; N, 3.44%. IR (cmꢁ1, nujol):
2000 m(Ru–H), 1951 m(CO), 1618, 1581, 1479, 1433,
1389, 1311, 1085, 1029, 750, 840, 746, 698 (characteristic
obs. (Calc.): 864 (864), 600 (602), 572 (572); IR (cmꢁ1
,
nujol): 2005 m(Ru–H), 1954 m(CO), 1625, 1593, 1475,
1434, 1394, 1087, 1055, 950.8, 848, 746, 696 (bands due
to paa, PPh3, and counter anion BFꢁ4 ); 1H NMR (d
ppm, acetone-d6, 300 MHz, 25 °C): 9.30 (d, 4.8 Hz), 8.94
(t, 2.4 Hz), 8.76 (s), 8.65 (d, 6.8 Hz), 7.99 (d, 3.6 Hz),
7.46 (m, 1H), )10.71 (t, 19.4 Hz); 31P{1H}: 43.28 (s);
UV–Vis{CH2Cl2; kmax, nmðeÞ}: 605(5.0 ꢀ 103), 475
(1.98 ꢀ 104), 428(3.8 ꢀ 104), 390(3.5 ꢀ 104), 311(1.68 ꢀ
104).
1
bands due to pbp, PPh3, and counter anion BFꢁ4 ); H
NMR (d ppm, CDCl3, 300 MHz, 25 °C): 9.01 (d, 5.4
Hz), 8.70 (s), 8.60 (d, 4.5 Hz), 7.91 (t, 5.8 Hz), 7.62 (t, 6.0
Hz), 7.03 (m) )11.8 (m), Ru–H; 13C{1H}: d 206 (car-
bonyl carbon), 164.4, 162.3, 150.3, 149.1, 138.0, 133.8,
130.08, 128.9, 113 ppm (carbons of the pbp ligand);
31P{1H}: d 47.6 ppm (s); UV–Vis{CH2Cl2; kmax, nmðeÞ}:
540(8.0 ꢀ 103), 410(2.2 ꢀ 104), 394(1.1 ꢀ 104), 241(1.2 ꢀ
104).
2.3.2. Preparation of [RuH(CO)(PPh3)2(paa)]PF6 (2)
Complex 2 was prepared by the same procedure as
described for complex
1
by the reaction of
[RuH(CO)Cl(PPh3)3] (477 mg and 0.5 mmol) in
methanol (30 mL) with pyridine-2-carbaldehyde azine
(paa) (210 mg and 1 mmol). The complex separated as
bright yellow needles. Yield: 64% (324 mg); Anal.
Calc. for C49F6H41N4OP3Ru: C, 58.16; H, 4.05; N,
5.53; Found: C, 58.01; H, 3.92; N, 5.20%. FAB MS
m=z obs. (calc.): 865 (864), 600 (602), 572 (572). IR
(cmꢁ1, nujol): 2003 m(Ru–H), 1953.8 m(CO), 1625,
1591, 1477, 1434, 1305, 1091, 997, 840, 775, 746, 696,
516 (bands due to paa, PPh3 and counter anion PFꢁ6 );
1H NMR (d ppm, Acetone-d6, 300 MHz, 25 °C):
9.338 (d, 4.9 Hz), 8.96 (t, 2.3 Hz), 8.791 (s), 8.586 (d,
6.7 Hz), 7.983 (d, 3.5 Hz), 7.45 (m, 1H), )10.769 (t,
2.3.5. Preparation of [RuH(CO)(PPh3)2(-l-bbp)RuH-
(CO)(PPh3)2](BF4)2 (5)
Complex 5 was prepared by the same method as de-
scribed for complex 1 by treating [RuH(CO)Cl(PPh3)3]
(477 mg, 0.5 mmol) in methanol (30 mL) with bbp (181
mg, 0.5 mmol). The complex separated as yellowish red
granules. Yield: 60% (554.4 mg). Anal. Calc. for
B2C98F8H80N4O2P4Ru2: C, 63.63; H, 4.32; N, 3.03;
Found: C, 62.51; H, 4.45; N, 3.22%. FAB m=z obs.
(Calc.): 1756 (1757), 1668 (1670), 1410 (1408), 1144
(1146), 1017 (1017), 753 (755), 625 (625), 363(362). IR
(cmꢁ1, nujol): 2003 m(Ru–H), 1953.8 m(CO), 1618, 1580,
1481, 1433, 1421, 1389, 1311, 1085, 1029, 750, 840, 746,
698 (characteristic bands due to bbp, PPh3, and counter
19 Hz); 31P{1H}: 44.213 (s); UV–Vis{CH2Cl2; kmax
,
nmðeÞ}: 591(7.0 ꢀ 103), 466(2.2 ꢀ 104), 387(3.1 ꢀ 104),
290(1.2 ꢀ 104).

M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
2259
1
anion BFꢁ4 ). H NMR (d ppm, Acetone-d6, 300 MHz,
25 °C): 9.10 (d, 5.5 Hz), 8.76 (s), 8.63 (d, 6.5 Hz), 8.43 (t,
5.8 Hz), 7.64 (t, 4.5 Hz), 6.57 (d, 9.0 Hz), )10.78 (m);
31P{1H}: 44.213 (s); UV–Vis{CH2Cl2; kmax, nmðeÞ}:
570(1.1 ꢀ 104), 415(2.02 ꢀ 104), 399(1.6 ꢀ 104), 267(9.5 ꢀ
103).
2.5. DNA binding and cleavage studies
The concentration of CT DNA was confirmed by
using its extinction coefficient at 260 nm (6600
M
DNA in buffer gave a ratio of UV absorbance at A260
ꢁ1 cmꢁ1) [11a]. After sonication, the solutions of CT
/
A280 of ca. 1.87 indicating that the DNA was sufficiently
free of protein [11b]. Chloride salts were prepared and
fully characterized for binding, cleavage and inhibition
2.4. X-ray structure determination of [RuH(CO)-
(PPh3)2(paa)]BF4 (1)
1
studies. H NMR and UV–Vis spectral analyses were
Suitable crystals for X-ray crystallographic studies
for the complex 1 were obtained from CH2Cl2/petro-
leum ether (60–80) at room temperature over a period
of three days. All the pertinent crystallographic data
are recorded in Table 1. Intensity data were collected
at 293(2)° on Enraf Nonius MACH3 diffractometer
using graphite monochromatized Mo Ka radiation
used to check the stability of the complexes towards the
solvent (water and DMSO) and no change was ob-
served. Buffer A (5 mM Tris–HCl, fs 7.1, 50 mM NaCl)
was used for absorption titration and buffer B (50 mM
Tris–HCl, pH 7.5, 50 mM KCl) was used for inhibition
studies.
Values obtained from absorption titration were used
to calculate the intrinsic binding constant Kb [5i] by
following equation:
ꢀ
(k ¼ 0:7093 A) from plate like dark orange crystal with
the dimensions 0.4 ꢀ 0.4 ꢀ 0.1 mm in the x–2h scan
mode in the range from 1.62° to 24.91°. Intensities of
these reflections were measured periodically to monitor
crystal decay.
½DNAꢂ=ðea ꢁ ef Þ ¼ ½DNAꢂ=ðeb ꢁ ef Þ þ 1=Kbðeb ꢁ ef Þ;
where ea is the extinction coefficient observed for the
absorption band at a given DNA concentration, ef is the
extinction coefficient of the free complex in solution, eb
is the extinction coefficient of the complex fully bound to
the DNA (it is assumed that further DNA addition will
not change the absorbance), Kb is the intrinsic binding
constant and [DNA] is the DNA concentration in the
nucleotides. A plot of [DNA]/ðea ꢁ ef Þ vs. [DNA] gave a
slope of 1/ðeb ꢁ ef Þ and a y intercept equal to 1/Kb
ðeb ꢁ ef Þ; Kb is the ratio of slope to y intercept.
The structure was solved by direct methods and re-
fined by full-matrix least squares on F2
(SHELX-97)
[10a,10b,10c]. In the final cycles of refinement all the
non-H atoms were treated anisotropically. The contri-
bution due to H-atoms attached to carbon atoms was
included as fixed contribution. Final value of
R ¼ 0:0423; wR2 ¼ 0:1092; GOF ¼ 1.068.
2.6. DNA topo II estimation
Table 1
Crystal data for the complex 1
The reaction catalyzed by DNA topo II was mea-
sured as reported by Pandya et al. [7a]. Relaxation of the
super coiled DNA pBR322 and separation of the to-
pological isomers of DNA were monitored by gel elec-
trophoresis. For activity measurement, reaction was
carried out (10 ll) in a reaction mixture containing
buffer B, 10 mM MgCl2; 1 mM ATP; 0.1 mM EDTA;
0.5 mM DTT; 30 lg mlꢁ1 BSA and enzyme protein. The
reaction was started by incubation at 37 °C for 30 min
and stopped by adding 5 ll loading dye. The sample
components were separated by electrophoresis on 1%
agarose gel in Tris–acetate buffer for 18 h at 20 V. Gels
were stained with ethidium bromide (0.5 lg mlꢁ1) visu-
alized and photographed on UVP GDS 7500 UV–
transilluminator. The percent relaxation was measured
by microdensitometry of gel by Gel base/Gel blot Pro
Gel analysis software programme.
Empirical formula
Formula weight
Crystal size (mm)
Temperature
C49H41B F4N4O P2Ru
951.68
0.4 ꢀ 0.4 ꢀ 0.1
293(2) K
ꢀ
0.70930 A
Wavelength
Crystal system, space group
Monoclinic, P
14.2461(13)
19.313(2)
16.574(2)
90.000(9)
92.035(9)
90.000(8)
4557.2(8)
4
ꢀ
a (A)
ꢀ
b (A)
ꢀ
c (A)
a (°)
b (°)
c (°)
3
ꢀ
Volume (A )
Z
Density (mg mꢁ3
)
1.387
0.472
Absorption coefficient (mmꢁ1
)
F ð000Þ
1944
6915
Reflections collected
Unique reflections
Parameters
6915
571
GOF (on F 2)
1.068
0.0423
R1 (on F )a
2.7. Cleavage studies
wR2
Largest difference peak and hole (e A
0.1092
0.494 and )0.718
ꢁ3
ꢀ
)
The cleavage experiments were carried out with a
little modification to the method reported by Mark and
P
P
P
P
2
1=2
a R1 ¼ ½jFoj ꢁ jFcjꢂ= ½jFojꢂ, wR2 ¼ f ½wðFo2 ꢁ Fc2Þ ꢂ= ½ðFo2Þꢂg
.

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M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
co-workers [7c]. The complexes were mixed with the
enzyme and incubated for 10 min at 37 °C after that
pBR322 was added and again incubated at 37 °C for 1/2
an hour. It was followed by addition of 2 ll 10% SDS, 2
ll (1 mg mlꢁ1) proteinase K and again incubated for one
hour at 37 °C. Reaction was stopped by addition 2 ll of
loading dye and loaded into 1% agarose gel. Electro-
phoresis was carried out as described in DNA topo II
estimation.
complexes has also been obtained from ESMS and FAB
mass spectrometry. Presence of the different peaks and
overall fragmentation pattern (recorded in Section 2) in
the FAB mass spectra of the respective complexes con-
formed well and strongly supported formulation of the
complexes.
Infrared spectra of the complexes exhibited charac-
teristic bands due to coordinated ligands paa, pbp and
bbp, PPh3, CO, hydride group and counter anions in the
respective complexes. The mCꢁN bands of the pyridyl
ring in the complexes shifted towards lower wave
numbers and appeared around 1595 cmꢁ1 as compared
to that in the free ligand (ꢃ1640 cmꢁ1). The band as-
sociated with pyridyl ring breathing mode appeared at
about 1020 cmꢁ1. The shift in the position of mCꢁN and
pyridine ring breathing mode suggested co-ordination of
the ligands through pyridyl and diazine nitrogen with
the metal ion. Interestingly, the position of m(CBO) and
m(Ru–H) bands shifted toward higher and lower fre-
quencies, respectively, indicating a decrease in the metal
to carbonyl carbon interaction. Broad bands in the re-
gion 1145 and 845 cmꢁ1 have been assigned to counter
anions BFꢁ4 and PFꢁ6 , respectively.
2.8. Tm determination
Tm analyses were conducted in a DU-640 spectro-
photometer by using Tm analysis programme and high
performance temperature controller for regulating the
temperature of the micro-cuvettes. The temperature
range was 40–90 °C and data were recorded at 260 nm.
The DNA was prepared in 0.1ꢀ SCC buffer, pH 7.2, and
metal coordinate compounds were incubated with DNA
in a total volume of 300 ll. The change in Tm (DTm)
following interaction of DNA with added compounds
was calculated by:
DTm ¼ Tm0 ðDNA þ CompoundÞ ꢁ TmðDNAÞ:
1
The H NMR spectra of the complexes 1 and 2 (re-
corded in Section 2) displayed distinct signals at d 9.30
(d, 4.8 Hz), 8.94 (t, 2.4 Hz), 8.76 (s), 8.65 (d, 6.8 Hz),
7.99 (d, 3.6 Hz), 7.466 (m, 1H) ppm which can be as-
signed to pyridyl ring and N@CH protons of the ligand
paa [12]. The aromatic protons of PPh3 resonated
around d 7.27–7.33 ppm (as a broad multiplet) and the
hydride proton as a triplet in the high field side at
)10.713 (t, 19.4 Hz). The protons associated with the
bonded ligand paa exhibited downfield shift with respect
to those in the free ligand upon coordination with the
metal center. 13C{1H} NMR spectra of the complex 1
followed the same trends observed in the 1H NMR
spectra. The paa carbons resonated at d 208.00 (CBO),
167.8 (N@CH) 157.1; 140.8; 133.3; 131.0–128.24 (pyr-
3. Results and discussion
The cationic mono and binuclear complexes with the
general formulation [RuH(CO)(PPh3)2(L)]þ and
[RuH(CO)(PPh3)2(l-L)RuH(CO)(PPh3)2]þ2 (L ¼ paa,
pbp or bbp) could be easily prepared in quantitative
yield by reaction of [RuH(CO)Cl(PPh3)3] with the
bridging ligands in methanol in 1:1/1:2 molar ratio
(Scheme 1).
The mono and binuclear complexes [RuH(CO)-
(PPh3)2(paa)]BF4 (1), [RuH(CO)(PPh3)2-(paa)] PF6 (2),
[RuH(CO)(PPh3)2(pbp)]PF6 (3), [RuH(CO)(PPh3)2(l-
pbp)RuH(CO)(PPh3)2](BF4)2 (4), [RuH(CO)(PPh3)2-
(l-bbp)RuH(CO)(PPh3)2](BF4)2 (5) are bright yellow to
golden orange, air-stable, non-hygroscopic shiny crys-
talline solids. These are sparingly soluble in dichlorom-
ethane, chloroform, methanol and ethanol, highly
soluble in acetone, acetonitrile, dimethylsulfoxide, dim-
ethylformamide and insoluble in benzene, petroleum
ether and diethylether.
1
idyl carbons and aromatic carbons of PPh3). H NMR
spectra of the complex 3 also exhibited signals in the
same region as that observed for the complex 1 with the
additional resonances at 6.4 ppm assigned to C6H4
protons of the pbp ligand. The spectrum of the binuclear
complex [RuH(CO)(PPh3)2(l-pbp)RuH(CO)(PPh3)2]-
(BF4)2 (4), displayed signals at d 9.01 (d, 5.4 Hz), 8.70
(s), 8.60 (d, 4.5 Hz), 7.91 (t, 5.8 Hz), 7.62 (t, 6.0 Hz)
corresponding to pyridyl protons, d 8.36 ppm corre-
sponding to N@CH proton and 7.03 ppm (m) corre-
sponding to C6H4 protons of the pbp. The pbp protons
exhibited a downfield shift with respect to those in the
free ligand. Presence of only five signals in the 1H NMR
corresponding to pbp ligand strongly suggested coordi-
nation of both the pyridyl and diazine moieties of the
ligand bis-chelating two [RuH(CO)(PPh3)2]. It is in
good agreement for a symmetrically bridged complex in
which two Ru(II) centers are bridged by the ligand pbp.
Carbon, hydrogen and nitrogen analyses of all the
complexes conformed well to their respective formula-
tions. Further information about composition of the
1:2
[RuH(CO)(PPh3)2(µ-L)]BF4
Cl
Stirring
Reflux
/
PPh3
PPh3
Ph3P
H
Ru
+
L
MeOH
CO
1:1
[RuH(CO)(PPh3)2(µ-L)RuH(CO)(PPh3)2](BF4)2
Scheme 1.

M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
2261
Table 2
The aromatic protons of PPh3 ligand resonated in its
ꢀ
Selected bond lengths (A) bond angles (°) and torsion angles (°)
characteristic region at ꢃ7.91–7.62 ppm with the pyridyl
protons of the ligand pbp. The 13C{1H} spectrum of the
complex 4 in CDCl3 exhibited resonance d 206 (carbonyl
carbon), 164.4, 162.3, 150.3, 149.1, 138.0, 133.8, 130.08,
128.9, 113 ppm (carbons of the pbp ligand). It is in good
Ru(1)–P(1)
Ru(1)–P(2)
Ru(1)–N(1)
Ru(1)–N(2)
Ru(1)–C(51)
Ru(1)–H(5)
2.3619(11)
2.3643(10)
2.178(3)
2.169(3)
1.848(4)
1.317
1
agreement with the conclusions drawn from H NMR
1
spectral studies. The H NMR spectra of the complex 5
P(1)–Ru(1)–P(2)
P(1)–Ru(1)–N(1)
P(1)–Ru(1)–N(2)
P(1)–Ru(1)–H(5)
P(2)–Ru(1)–H(5)
P(1)–Ru(1)–C(51)
P(2)–Ru(1)–C(51)
P(2)–Ru(1)–N(1)
P(2)–Ru(1)–N(2)
N(1)–Ru(1)–N(2)
N(1)–Ru(1)–C(51)
N(2)–Ru(1)–C(51)
N(1)–Ru(1)–H(51)
N(2)–Ru(1)–H(51)
C(51)–Ru(1)–H(5)
164.55(4)
94.22(9)
90.69(9)
83.50
showed resonances corresponding to pyridyl and aro-
matic protons of the ligand bbp and PPh3 at d 9.10 (d),
8.76 (s), 8.63 (d), 8.43 (t), 7.64 (t), 6.57 (d) ppm. The
position and integrated intensity of the different signals
suggested binuclear nature of the complex.
81.22
88.54(13)
88.71(13)
101.23(9)
92.89(9)
75.00(12)
101.81(15)
176.66(15)
169.93
1
The metal bound hydride protons in the H NMR
spectra of complexes 1–5 resonated in the high field side
around )10 ppm (cf. Section 2). The presence of a triplet
or a multiplet in complexes 4 and 5 corresponding to
metal bound hydride proton suggested hydride group to
be coupled with two equivalent 31P nuclei [13]. The
31P{1H} NMR spectra of all the complexes (1–5) dis-
played sharp signals around d 43.5 (recorded in Section
2) indicating equivalence of both the triphenylphosphine
ligands in the trans position.
95.18
87.96
C(6)–N(2)–N(3)–C(7)
N(1)–C(5)–C(6)–N(2)
N(3)–C(7)–C(8)–N(4)
C(5)–C(6)–N(2)–N(3)
C(8)–C(7)–N(3)–N(2)
174.8(4)
)2.4(6)
5.7(7)
)177.7(3)
)177.1(3)
Interaction of the filled orbitals of proper symmetry
on d6 Ru(II), with the low lying pꢄ orbital of the ligands
(tptz, bppz or bptz) should show MLCT transition
ðt2g ! pꢄÞ in the electronic spectra of these complexes,
with the transition energy varying with nature of the
ligands acting as a p acceptor. Our assignments of the
bands are based on the same trends as observed in the
similar Ru(II) polyazine complexes [13b]. These display
p ! pꢄ ligand transition bands in the UV region fol-
lowed by MLCT band in the visible region. MLCT
bands of the complex 1 appeared at about 591, 369, 311
nm, assigned on the basis of intensity and position of the
lowest energy absorption [14]. Though, the band at 369
nm may be of MLCT character, but the possibility of r
bond-to-ligand charge transfer (SBLCT) can also not be
ruled out [15]. The high-energy band has been assigned
to intra-ligand p ! pꢄ transition or p (phenyl) to pꢄ
(phenyl) transitions [16]. The mononuclear ruthenium
complex 3 displayed bands at 465, 310 and 295 nm. The
low energy band at 465 nm has been assigned to Ru pbp
CT transition while the one at 310 nm to intra-ligand
p ! pꢄ transition. It was further observed that, in the
electronic spectra of the binuclear complex 4, the
Ru ! pbp CT transition band appeared at 540 nm while
the band associated with intra-ligand transitions are
observable at 394 nm. The red shift in the position of
Ru ! pbp CT transition towards lower energy in the
binuclear complex 4, may be due to stabilization of pbp
pꢄ orbital upon coordination of the second metal center.
In general, bridging results in stabilization of the pꢄ level
of the bridging ligand leading to enhanced p–pꢄ overlap
[17]. This effect lowers the HOMO–LUMO energy gap
which, in turn, results in a shift of MLCT bands in the
binuclear complexes towards lower energy. The stabil-
ization of the pbp pꢄ also lead to a red shift of the ligand
based p ! pꢄ transitions. These observations are con-
sistent with other reports [2f].
Cyclic voltammogram of the complexes 1, 3 and 4,
were recorded in previously purified acetonitrile and the
data is summarized in (Table 4). On the anodic potential
window (0 to +2 V vs. SCE) mononuclear complexes 1
and 3 (Fig. 2) exhibited single irreversible and reversible
peaks attributed to metal-based oxidation Ru(II/III) at
potential 1.38 and 1.17 V, respectively, whereas on the
cathodic potential window (0 to )2 V vs. SCE) these
complexes exhibited two and three ligand-based reduc-
tion couple. Reduction potential of mononuclear
complexes RuIIH(CO)(PPh3)2(paa)]þ and RuIIH(CO)-
(PPh3)2(pbp)]þ move anodically in order of L ¼ paa <
pbp this reflects the lowering of LUMO energy of the
ligand. Ligand-based reduction peaks in the complexes
were shifted 0.5–0.7 V more positive with respect to
those of free ligands.
In the binuclear complex 4, the metal-centered oxi-
dation occurs at two distinct potentials. The first peak
appears at almost the same potential 1.21 V with a small
shift with respect to mononuclear complexes whereas
the second oxidation process appears at slightly more
positive potential 1.32 V. The latter is ascribed to the
second Ru ion oxidation. The splitting of the metal-
centered oxidation with positive shift compared to that
in the mononuclear complexes is a common feature for

2262
M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
Table 3
C–Hꢅ ꢅ ꢅF and p–p stacking interaction distances
D–Hꢅ ꢅ ꢅA
d(D–H) d(Hꢅ ꢅ ꢅA) d(Dꢅ ꢅ ꢅA) \(DHA)
C(1)–H(1)ꢅ ꢅ ꢅF(2)b
C(11)–H(11)ꢅ ꢅ ꢅF(4)c
C(29)–H(29)ꢅ ꢅ ꢅF(4)a
0.93
0.93
0.93
2.48
2.53
2.48
3.400(6)
3.366(7)
3.399(8)
170.2
149.8
170.1
a x; y; z.
b x ꢁ 1=2; ꢁy þ 1=2; z þ 1=2.
c x ꢁ 1=2; ꢁy þ 1=2; z ꢁ 1=2.
Fig. 2. Cyclic voltammogramm for complex 3.
all the binuclear complexes and also supports metal–
metal interaction through the bridging ligands
[18a–18b].
Fig. 3. (a) ORTEP view of complex cation for complex 1. (b) I. Helical
motif obtained by p–p stacking intermolecular interaction. (Hydrogen
atom have been omitted for clarity.) and II. Space fill diagram.
3.1. Single crystal X-ray structure of the complex
[RuH(CO)(PPh3)2(paa)]BF4
The ligand paa is almost planar and the C(6)–N(2)–
Structure of the complex 1 has been confirmed by
single crystal X-ray diffraction analysis. Details about
data collection, reduction and structure solution are
recorded in Table 1. An ORTEP [10d] view of the
complex cation along with the atom numbering is shown
in Fig. 3. Selected bond lengths, bond angles and torsion
angles are recorded in Table 2. Coordination geometry
about the Ru(II) center is distorted octahedron formed
by the atoms N(1), N(2), C(51) and H(5) occupying the
equatorial plane and the P donor atoms of the phos-
phine ligands lie in axial positions. The N(1)–Ru(1)–
N(2) angle of 75.00(12)° suggested inward bending of
the coordinated pyridyl group. The smaller value of
N(1)–Ru(1)–N(2) bite angle 75.00(12)° as compared to
the ideal value of 90° is probably the source of observed
distortion. The triphenylphosphine ligands lie in trans
position as indicated by the P(1)–Ru(1)–P(2) angle of
164.55(4)°. The Ru(1)–P(1) and Ru(1)–P(2) distances
N(3)–C(7) torsion angle is 174.8(4)°. The Ru(1) to pyr-
idyl N(1) bond length is 2.178(3) A and it is slightly
larger than the Ru(1) to coordinated azine nitrogen N(2)
ꢀ
ꢀ
[2.169(3) A]. These are comparable to those observed in
other Ru(II) amine complexes, poly-pyridyl complexes
and closely related hydrido carbonyl complex
[RuH(CO)(PPh3)2(g2-tptz)]BF4 [22a–22f]. The N(2)–
ꢀ
N(3) bond distance is 1.413(4) A, it can be defined as
single bond and it is comparable with N–N bond length
ꢀ
in hydrazine (1.47) A. The C@N bond lengths C(6)–
N(2) and C(7)–N(3) are essentially equal and are
ꢀ
1.278(5) and 1.257(5) A, respectively, and can be con-
sidered to have double bond character.
Weak interaction studies were made by computa-
tional programme PLUTON [10e]. The crystal packing in
the complex 1 revealed intermolecular C–Hꢅ ꢅ ꢅF and
intermolecular and intramolecular p–p interaction.
Relevant interaction distances are summarized in Table
3. The importance of p–p stacking interactions between
aromatic rings has been widely recognized in intercala-
tion of drugs into DNA [23]. Similar aromatic interac-
tions were observed in biological systems, which lie
ꢀ
are 2.3619(11) and 2.3643(10) A, respectively. These are
essentially equivalent and comparable to those in the
related complexes [19]. The Ru(1)–C(51) bond length is
ꢀ
1.848(4) A, which is normal for Ru(II) carbonyls
[20a,20b] The Ru(1)–H(5) distance in the complex cat-
ꢀ
ꢀ
within 3.4–3.5 A distance [23c]. In complex 1, both inter
and intramolecular (C1–C46) type of p–p interactions
has been found with the interaction distances of 3.491
ion is 1.317 A. It is shorter than those found in
[RuH(H2O)(CO)2(PPh3)2]þ (1.7 A), [RuHCl(PPh3)3]
ꢀ
ꢀ
(1.7 A), and in other related systems [21a–21c].

M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
2263
Table 4
Electrochemical data for mono and binuclear Ru(II) complexes in acetonitrile solution at (rt), scan rate 50 mV sꢁ1
S. No. Complex
E
ð1=2Þ
Oxidation
Reduction
I
II
I
II
III
1.
2.
3.
[RuH(CO)(PPh3)2(paa)]BF4 (1)
[RuH(CO)(PPh3)2(pbp)]PF6 (3)
1.38 (irr)
1.17 (60)
1.21 (72)
–
)0.97 (70) )1.61 (90)
)1.03 (80) )1.27 (40)
)1.04 (80) )1.19 (30)
–
–
1.32 (irr)
)1.55 (90)
)1.63 (irr)
[RuH(CO)(PPh3)2(-l-pbp)RuH(CO)(PPh3)2](BF4)2 (4)
Eð1=2Þ ¼ 0.5(Epa + Epc), where Epa and Epc are the anodic and cathodic potential, respectively, the value of DEp in (mV is given in parentheses),
DEp ¼ Epa ) Epc, irr ¼ irreversible.
ꢀ
and 3.211 A, respectively. Intermolecular p–p stacking
ligand in mononuclear complexes with the DNA major
groove [25d]. The interaction results in an ordered
stacking of the bound species between base pairs. The
intercalating surface is sandwiched tightly between the
aromatic heterocyclic base pairs and stabilized elec-
tronically in the helix by p-stacking and dipole–dipole
interactions.
Ru(II) complexes used in the present study are octa-
hedral in which the coordination geometry is completed
by the carbonyl group, hydride group, pyridyl and azine
nitrogen from the ligands (paa, pbp, bbp, bppz, tptz)
alongwith the two PPh3 groups lying trans to each other.
If we examine the single crystal X-ray structure of the
complex 1, it is evident that planar portion may get
stacked between the base pairs of the DNA major
groove. However, bulky PPh3 groups that are trans
disposed to each other may interact with the sugar. Such
interaction is not possible in the binuclear complexes 4
or 5 due to their non-planar structures. Further due to
an increase in the number of bulky PPh3 groups in these
complexes, the possibilities of intercalative stacking by
the complexes have been ruled out. It therefore favours
the interaction of polynucleotide through a surface
bound interaction in the minor groove.
Unwinding of the DNA helix is accompanied by a
decrease in the intensity of the absorbance [24a–24c].
Binding of these complexes have successfully been
monitored by absorption titration and summarized in
preceding section. The ruthenium(II) complexes exhib-
ited the presence of isobestic points, hypochromicity and
red shift in the position of absorption maxima with in-
creasing DNA concentration. Hypochromic shifts of 6%
and bathochromic shift of upto 4.5 nm were observed.
The representative UV–Vis spectra of complexes 3 and 6
are shown in Figs. 4(a) and (b).Complexes under study
exhibited hypochromic and bathochromic shift in the
position of bands upon addition CT DNA. The calcu-
lated values of Kb for the complexes at 20 lM were
found to be 3.4 ꢀ 104 (1), 8.5 ꢀ 103 (2), 4.25 ꢀ 103 (3),
1.2 ꢀ 103 (4), 4.01 ꢀ 104 (6), 1.7 ꢀ 104 (7), 1.1 ꢀ 104 (8).
The Kb values obtained for complex 1 and 6 are high-
est and close to the previously reported Ru(II) com-
plexes [25c]. The trend in the binding constant value
ꢀ
interaction (distance 3.49 A) results in a single helical
motif (Figs. 3(b) I and II).
3.2. Thermal denaturation
In general unwinding of DNA is accompanied by
lowering in melting temperature ðTmÞ, the temperature
at which a DNA double strand unwinds to two single
strands. Metal ions which interact through the phos-
phate groups of the DNA lead to an increase in the
melting temperature ðTmÞ, while those interacting
through both the phosphate group and nitrogen bases
lead to a decrease in the value of melting temperature
ðTmÞ [24]. In the present study, we found that there is
insignificant change of 4 °C (ꢆ2.0) in the Tm value,
which is of least information but supports for interca-
lative mode of interaction.
3.3. DNA binding
It is well established that, intercalation is not re-
stricted to only completely flat or square planar com-
plexes, but partial intercalation is possible with the
ligands coordinated to octahedral metal centers [25a].
Such types of complexes bind to DNA by two non-co-
valent modes. One is ascribed as an intercalative inter-
action in the major groove, and the other to groove
bound interaction in the minor groove of the helix
[25b,25c]. It is essential to mention here that molecular
geometry of the complex 1 (obtained from the single
crystal data) is exhibiting a plane made up of uncoor-
dinated pyridyl ring and azine nitrogen. The same type
of structure is obtained in the complex 6 also where
uncoordinated tptz pyridyl and triazine rings are planar.
The possibility of partial intercalation of these com-
plexes with the nucleotides can not be ruled out. It has
been shown that the Ru(II) poly-pyridyl complexes
having octahedral geometry interact with the DNA non-
covalently through any of these modes: (i) electrostati-
cally, (ii) hydrophobic interactions of the ligands with
the minor groove of the DNA and (iii) by partial
intercalation of the uncoordinated planar part of the

2264
M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
Fig. 5. The cleavage reaction was conducted by incubating
pBR322DNA with topo II. Lane 1 pBR322 DNA alone, lane 2 en-
zyme + DNA, lane 3 complex 1, lane 4 complex 2, lane 5 complex 3,
lane 6 complex 6 and lane 7 complex 7.
Smearing of DNA by complexes 1–3, 6, and 7 (Fig. 5)
supports the cleavage of DNA in presence of complexes.
The formation of enzyme–drug–DNA complex can be
seen from the appearance of linear DNA which results
from DNA strand breaks caused by dissociation of the
topo II homo-dimer by SDS. Thus the poly-pyridyl
Ru(II) hydrido carbonyl complexes under study could
be taken as both topo II inhibitor as well as cleaving
agent.
Further interaction behaviour is well supported by
the inhibitory effect of metal complexes on DNA topo II
activity of filarial parasite S. cervi (Figs. 6(a) and (b)).
The enzyme was incubated with metal complexes for 10
min at 37 °C and reaction was started by the addition of
pBR322 DNA. Enzyme inhibitory activity of complex 1
and 6 were found to be 90% and 95% while, the com-
plexes 2–4, 7 and 8 showed 75%, 80%, 75%, 65% and
55% inhibitory activity at 10 lg per reaction mixture
concentration (Table 5). It is clear from the available
data that mononuclear complexes are exhibiting higher
activity in comparison to binuclear systems. This may
result from structural changes discussed above. The li-
gands being used in the present study contain both the
Fig. 4. Absorption spectra of the complexes 3 (a) and 6 (b) in buffer A
in the presence of increasing amount of CT (complex concentration
10ꢁ5 M and CT DNA in buffer, 0–280 lM for complex 3 and 0–320
lM for complex 6).
corroborated well with conclusions drawn from inhibi-
tion data of topo II activity.
An experiment was carried out to support DNA
cleavage by the synthesized complexes. It has been
shown that topo II is an essential enzyme, which dis-
plays an important role in DNA replication, repair and
transcription. Complexes that block the overall catalytic
activity of topo II can be divided into two classes. First
category includes topo II poisons that trap the topo II–
DNA cleavage complex, generating high level of DNA
breaks. The second category includes topo II inhibitors,
which inhibit the overall catalytic activities without in-
ducing DNA breaks. The cleavage experiments were
carried out following with a little modification to the
method reported by Mark and co-workers [7c]. The
ability of these complexes to cause drug induced sta-
bilization of DNA–topo II complex was studied.
Fig. 6. (a) DNA topisomerase II activity of S. cervi of a filarial parasite
and effect of metal complexes on the enzyme activity. Lane 1,
pBR322DNA alone; lane 2, S. cervi enzyme; lane 3, complex 1; lane 4,
complex 3. (b) Lane 1, S. cervi enzyme; lane 2, complex 4; lane 3,
complex 2; lane 4, complex 7; lane 5, complex 8; lane 6, complex 6.

M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
2265
Table 5
Effect of Ru(II) hydrido carbonyl complexes on DNA topo II activity
of S. cervi
bition towards filarial parasite enzyme is dependent upon:
(i) number and type (pyridyl or azine) of coordinated and
uncoordinated nitrogen atoms and (ii) mononuclear and
binuclear nature of complexes. Finally results obtained
from absorption titration and gel electrophoresis
experiment suggest these complexes bind to DNA with
moderate strength most probably by an intercalative
mode.
S. No.
1.
Complex No.
Concentration of
complex (lM)
% inhibition
Nil
DNA topo II
enzyme
–
2.
3.
4.
5.
6.
7.
8.
1
2
3
4
6
7
8
0.105
0.099
0.092
0.056
0.094
0.102
0.096
90
75
80
75
95
65
55
4. Supporting information available
Setaria cervi with enzyme was incubated with the compounds 10
min. at 37 °C. The reaction was started by addition of pBR322 DNA.
Crystallographic data for the structural analysis in
CIF format have been deposited to CCDC and the de-
position code is 229574.
pyridyl and the azine type of nitrogen donor atoms. It is
observed that the inhibitory activity is dependent upon
number of coordinated and uncoordinated donor ni-
trogen atoms. In the complex 6, there are six-pyridyl
nitrogen atoms out of which, only two are involved in
coordination with the metal center and other four ni-
trogen atoms are uncoordinated. It exhibited maximum
inhibition activity (95%). Similarly, in complexes 7 and 8
there are four pyridyl nitrogen atoms but only two of the
pyridyl nitrogen atoms are uncoordinated showing 65%
and 55% inhibition. It therefore appears that the num-
ber of uncoordinated N-atom decides the inhibition
activity, the higher the number, higher the activity.
Complexes 1–4 have both pyridyl and azine nitrogen
atoms in it and each of them have only four nitrogen
atoms. In the mononuclear complexes 1–3, out of four
nitrogen atoms one of the pyridyl and an azine nitrogen
atom are involved in coordination with the metal center
and other two are uncoordinated. They exhibited in-
hibitory activity in the range 75–90%, where as, the bi-
nuclear complex 4 in which all the four nitrogen atoms
are coordinated, showed very poor activity. The inhib-
itory activity of binuclear complex 5 is the least and it is
not discussed here.
In the present work, we have prepared and character-
ized some mono and binuclear Ru(II) pyridyl-azine
complexes. Molecular structure of the representative
mononuclear complex [RuH(CO)(PPh3)2(paa)]BF4 have
been determined by single crystal X-ray studies. Anti-
filarial activity of the complexes has been studied. The
possible efficacy of some complexes [RuH(CO)(PPh3)2-
(paa)]BF4. [RuH(CO)(PPh3)2(paa)]PF6[RuH(CO)-
(PPh3)2(pbp)]PF6, [RuH(CO)(PPh3)2(-l-pbp)RuH(CO)-
(PPh3)2](BF4)2, [RuH(CO)(PPh3)2(-l-bbp)RuH(CO)–
(PPh3)2](BF4)2, [RuH(CO)(PPh3)2(g2-tptz)]BF4, [RuH-
(CO)(PPh3)2(g2-bppz)]BF4 and [RuH(CO)(PPh3)2(g2-
bppz)]PF6 as anti-filarial drugs has been examined. These
complexes are inhibitors of topo II as well as cleaving
agent. These studies suggest that the activity or the inhi-
Acknowledgements
Thanks are due to University Grants Commission,
New Delhi for providing financial assistance (F-12-21/
97). M.C. and A.N.S acknowledge CSIR, New Delhi for
awarding SRF and SRA, respectively. Thanks are also
due, to RSIC, Central Drug Research Institute, Luc-
know, for providing analytical and spectral facilities,
National Diffraction Facility, X-ray Division, IIT
Mumbai for single crystal data collection and the Head,
Department of Chemistry, Awadhesh Pratap Singh
University, Rewa for extending laboratory facilities.
Special thanks are due to Prof. U.C. Agarwala, De-
partment of Chemistry, Indian Institute of Technology,
Kanpur and Prof. P. Mathur, Department of Chemistry,
Indian Institute of Technology, Mumbai for their kind
help and encouragement.
References
[1] (a) K. Kalyanasundaram, M. Gratzel, Photosensitization and
Photocatalysis using Inorganic and Organometallic Compounds,
Kluwer Academic, Dordrecht, 1993;
(b) A. Juris, V. Balzani, F. Barigelletti, S. Campagna, P. Belser,
A. Von Zelewsky, Coord. Chem. Rev. 84 (1988) 85;
(c) T.J. Meyer, Pure Appl. Chem. 58 (1986) 1193;
(d) N.H. Dam Rauer, G. Cerullo, A. Yeh, T.R. Boussie, C.V.
Shank, J.K. McCusker, Science 275 (1997) 54.
[2] (a) N. Gupta, N. Grover, G.A. Neyhart, P. Singh, H.H. Thorp,
Inorg. Chem. 32 (1993) 310;
(b) E. Brauns, W. Sumner, J.A. Clark, S.M. Molnar, Y.
Kawanishi, K.J. Brewer, Inorg. Chem. 36 (1997) 2861;
(c) J.-D. Lee, L.M. Vrana, E.R. Bullock, K.J. Brewer, Inorg.
Chem. 37 (1998) 3575;
(d) C. Ceroni, F. Paolucci, S. Roffia, S. Serroni, S. Campagna,
A.J. Bard, Inorg. Chem. 37 (1998) 2829;
(e) A. Klein, V. Kasack, R. Reinhardt, S. Torsten, T. Scheiring, S.
Zalis, J. Fiedler, W. Kaim, J. Chem. Soc., Dalton Trans. (1999)
575;

2266
M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
(f) P. Paul, B. Tyagi, A.K. Bilakhia, D. Parthsarthi, E. Suresh,
Inorg. Chem. 39 (2000) 14;
[10] (a) S. Mackay, W. Dong, C. Edwards, A. Henderson, C. Gilmox,
N. Stewart, K. Shankland, A. Donald, University of Glasgow,
Scotland, 1999;
(g) G. Albano, P. Belser, C. Dual, Inorg. Chem. 40 (2001) 1408.
[3] (a) M. Ghedini, M. Longeri, F. Neve, J. Chem. Soc., Dalton
Trans. (1986) 2669;
(b) G.M. Sheldrick, SHELXS-97: Program for Structure Solution
and Refinement of Crystal Structure, Gottingen, Germany,
1997;
(b) M. Ghedini, M. Longeri, F. Neve, A.M.M. Lanfredi, A.
Tiripicchio, J. Chem. Soc., Dalton Trans. (1989) 1217;
(c) D.S. Pandey, R.L. Mishra, U.C. Agrawala, Ind. J. Chem. A 30
(1991) 41;
(c) G.M. Sheldrick, Shelxl-97: program for structure refinement,
Gottingen, Germany, 1997;
(d) C.K. Jhonson, ORTEPIII; Report ORNL-5138, Oak Ridge
National Laboratory, Oak Ridge, TN, 1976;
(e) PLATON A.L. Spek, Acta Crystallogr., Sect. A 46 (1990)
C31.
(d) J. Saroja, V. Manivannan, P. Chakraborty, S. Pal, Inorg.
Chem. 34 (1995) 3099;
(e) J. Granifo, M.E. Vargas, E.S. Dodsworth, D.H. Farrar, S.S.
Fielder, A.B.P. Lever, J. Chem. Soc., Dalton Trans. (1996)
4369;
[11] (a) M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am.
Chem. Soc. 76 (1954) 3074;
(b) J. Marmur, J. Mol. Biol. 3 (1961) 208.
(f) Z. Xu, S. White, L.K. Thompson, D.O. Miller, M. Ohba, H.
Okawa, C. Wilson, J.A.K. Howard, J. Chem. Soc., Dalton Trans.
(2000) 1751;
[12] (a) M. Ghedini, M. Longeri, F. Nerve, J. Chem. Soc., Dalton
Trans. (1986) 2669;
(g) S. Pal, S. Pal, Inorg. Chem. 40 (2001) 4807.
[4] (a) K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev. 99 (1999)
2777;
(b) M. Ghedini, A.M.M. Lanfredi, F. Nerve, A. Tiripicchio, J.
Chem. Soc., Chem. Commun. (1987) 847.
[13] (a) D.K. Gupta, O.S. Sisodia, A.N. Sahay, D.S. Pandey, Synth.
React. Inorg. Metal Org. Chem. 28 (1998) 355;
(b) M. Chandra, A.N. Sahay, D.S. Pandey, M.C. Puerta, P.
Valerga, J. Organometal. Chem. 648 (2002) 39–48.
[14] P. Didier, I. Ortmans, A. Kirsch- De Mesmaeker, R. Watts, J.
Inorg. Chem. 34 (1993) 5239.
(b) C.S. Allardyce, P.J. Dyson, Platinum Metals Rev. 45 (2)
(2001) 62;
(c)
S.R. Korupoju, M. Nagarathinam, P.S. Zacharias, J.
Mizuthani, H. Nishihara, Inorg. Chem. 41 (2002) 4099;
(d) H. Chen, J.A. Parkinson, S. Parsons, R.A. Coxall, R.O.
Gould, P.J. Sadler, J. Am. Chem. Soc. 124 (2002) 3064;
(e) C.S. Allardyce, P.J. Dyson, D.J. Ellis, S.L. Heath, Chem.
Commun. (2002) 1396.
[15] R.M. Berger, D.D. Elis II, Inorg. Chim. Acta 241 (1996) 1.
[16] B.P. Sullivan, D.J. Salmon, T.J. Meyer, Inorg. Chem. 17 (1978)
3334.
[5] (a) A.M. Pyle, J.K. Barton, in: S.J. Lippard (ed.), Progress in
Inorganic Chemistry: Bioinorganic Chemistry, vol. 38, 1990, p.
413;
[17] R. Rillema, K.B. Mack, Inorg. Chem. 21 (1982) 3849.
[18] (a) G. Giuffrida, S. Campagna, Coord. Chem. Rev. 135–136
(1994) 517;
(b) R.E. Holmlin, P.J. Dandliker, J.K. Barton, Angew. Chem.,
Int. Ed. Engl. 36 (1997) 2714;
(b) P. Ceroni, F. Paolucci, C. Paradisi, A. Juris, S. Roffia, S.
Serroni, S. Campagna, A.J. Bard, J. Am. Chem. Soc. 120 (1998)
5480.
(c) T.W. Hambley, A.R. Jones, Coord. Chem. Rev. 212 (2001) 35;
(d) J.-G. Liu, Q.-L. Zhang, X.-F. Shi, L.-N. Ji, Inorg. Chem. 40
(2001) 5045;
[19] P. Ghosh, A. Chakravorty, Inorg. Chem. 36 (1997) 64, and
references therein.
(e) D. Ossipov, P.I. Pradeepkumar, M. Holmer, J. Chat-
topadhyaya, J. Am. Chem. Soc. 123 (2001) 3551;
(f) Y. Jenkins, A.E. Friedman, N.J. Turro, J.K. Barton, Bio-
chemistry 31 (1992) 10809;
[20] (a) M.F. McGuiggan, L.H. Pignolet, Inorg. Chem. 21 (1982)
2523;
(b) A. Sahajpal, S.D. Robinson, M.A. Mazid, M. Motevalli, M.B.
Hursthouse, J. Chem. Soc., Dalton Trans. (1990) 2119.
[21] (a) K. Osakada, T. Yamamoto, A. Yamamoto, A. Takenaka, Y.
Sasada, Inorg. Chim. Acta 9 (1979) 105;
(g) E.M. Proudfoot, J.P. Mackay, P. Karuso, Biochemistry 40
(2001) 4867;
(h) H.D. Stoeffer, N.B. Thornton, S.L. Temkin, K.S. Sxchanze, J.
Am. Chem. Soc. 117 (1995) 7119
(b) A.C. Skapski, P.G.H. Throughton, J. Chem. Soc., Chem.
Commun. (1968) 1230;
z(i) A. Wolfe, G.H. Shimer, T. Meehan, Biochemistry 26 (1987)
6392;
(c) M.R. Churchill, K.M. Keil, F.V. Bright, S. Pandey, G.A.
Baker, J.B. Keister, Inorg. Chem. 39 (2000) 5807.
[22] (a) W.S. Sheldrick, H.S. Hagen-Eckhard, S. Heeb, Inorg. Chim.
Acta 206 (1993) 15;
(j) M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111
(1989) 8901;
(k) B.T. Farrer, H.H. Thorp, Inorg. Chem. 39 (2000) 44;
(l) P.J. Carter, C.-C. Cheng, H.H. Thorp, J. Am. Chem. Soc. 120
(1998) 632.
(b) H. Brunner, R. Oeschey, B. Nuber, J. Chem. Soc., Dalton
Trans. (1996) 1499;
(c) M.E. Gress, C. Creutz, C.O. Quicksall, Inorg. Chem. 20 (1981)
1522;
[6] E. Michael, D.A.P. Bundy, Parasitol. Today 13 (1997) 472.
[7] (a) U. Pandya, J.K. Saxena, S.M. Kaul, P.K. Murthy, R.K.
Chatterjee, R.P. Tripathi, A.P. Bhaduri, O.P. Shukla,, Med. Sci.
Res. 27 (1999) 103;
(d) W. Luginbuhl, P. Zbinden, P.A. Pittet, T. Armbruster,
H.-B. Burgi, A.E. Merbach, A. Ludi, Inorg. Chem. 30 (1991)
2350;
(b) R.P. Tripathi, S.K. Rastogi, B. Kundu, J.K. Saxena, V.J.M.
Reddy, S. Shrivastav, S. Chandra, A.P. Bhaduri, Comb. Chem.
Throughput Screen. 4 (2001) 237;
(e) A.J. Davenport, D.L. Davies, J. Fawcett, S.A. Garratt, D.R.
Russell, J. Chem. Soc., Dalton Trans. (2000) 4432;
(f) D.H. Gibson, J.G. Andino, S. Bhamidi, B.A. Sleadd, M.S.
Mashuta, Organometallics 20 (2001) 4956.
(c) G. Marx, H. Zhou, D.E. Granes, N. Oshroff, Biochemistry 63
(1997) 15884.
[23] (a) W. Saenger, Principles of Nucleic Acid Structures, Springer-
Verlag, New York, 1984, p. 132;
[8] D.D. Perrin, W.L.F. Armango, D.R. Perrin, Purification of
Laboratory Chemicals, Pergamon, Oxford, V.K., 1986.
[9] (a) W.J. Stratton, D.H. Busch, J. Am. Chem. Soc. 80 (1958)
1286;
(b) L.P.G Wakelin, Med. Res. Rev. 6 (1986) 275;
(c) D. Ranganathan, V. Haridas, R. Gilardi, I.L. Karle, J. Am.
Chem. Soc. 120 (1998) 10793;
(b) M.-A. Haga, K. Koizumi, Inorg. Chim. Acta 104 (1985) 47;
(c) N. Ahmad, J.J. Levison, S.D. Robinson, M.F. Uttley, Inorg.
Synth. 15 (1974) 45.
(d) G.R. Desiraju, T. Steiner, The Weak Hydrogen Bond in
Structural Chemistry and Biology, Oxford University Press,
Oxford, 1999.

M. Chandra et al. / Journal of Organometallic Chemistry 689 (2004) 2256–2267
[24] (a) A. Wolfe, G.H. Shimer Jr., T. Meehan, Biochemistry. 26 [25] (a) J.K. Barton, Science 233 (1986) 727;
2267
(1987) 6392;
(b) J.K. Barton, J.J. Dannesberg, A.J. Raphel, J. Am. Chem. Soc.
104 (1982) 4967;
(b) A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar,
N.J. Turro, J.K. Barton, J. Am. Chem. Soc. 111 (1989)
3051;
(c) J.K. Barton, A.T. Danishefrky, J.M. Goldberg, J. Am. Chem.
Soc. 106 (1984) 2172;
(c) A. Ambriose, B.G. Maiya, Inorg. Chem. 39 (2000) 4264,
and references therein.
(d) A.H.J. Wang, J. Nathans, G.V.D. Marel, J.H.V. Boom, A.
Rich, Nature 276 (1978) 471.