Oxygenated chalcones and bischalcones as a new class of inhibitors of DNA topoisomerase II of malarial parasites

Article abstract of DOI:10.1007/s00044-007-9057-0

The DNA topoisomerase (topo) II of chloroquine-sensitive and chloroquine-resistant strains of the rodent malaria parasite P. berghei were utilized as a target for testing of antimalarial compounds. Compounds belonging to the bischalcone and chalcone serie

Full text of DOI:10.1007/s00044-007-9057-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2008) 17:234–244
DOI 10.1007/s00044-007-9057-0
ORIGINAL RESEARCH
Oxygenated chalcones and bischalcones as a new class
of inhibitors of DNA topoisomerase II of malarial
parasites
Shalini Srivastava Æ Shweta Joshi Æ Alok R. Singh Æ
Sarika Yadav Æ A. S. Saxena Æ V. J. Ram Æ
Subhash Chandra Æ J. K. Saxena
Received: 6 November 2007 / Accepted: 12 November 2007 / Published online: 11 December 2007
¨
Ó Birkhauser Boston 2007
Abstract The DNA topoisomerase (topo) II of chloroquine-sensitive and chloro-
quine-resistant strains of the rodent malaria parasite P. berghei were utilized as a
target for testing of antimalarial compounds. Compounds belonging to the bischal-
cone and chalcone series significantly inhibited enzyme activity and percentage
parasitaemia of chloroquine-sensitive and chloroquine-resistant strains of P. berghei.
Compounds 1a, 1b, 2a, 2b, and 2c showed 100% inhibition while compounds 2h and
2i showed 60% and 63% inhibition of topoisomerase II activity of the chloroquine-
sensitive strain, respectively. Compounds 2a, 2b, and 2d significantly inhibited the
topo II activity of chloroquine-resistant strain. Compounds 2g and 2e specifically
inhibited the topo II activity of the chloroquine-resistant strain of P. berghei with no
effect on the chloroquine-sensitive strain. The in vitro topo II inhibition by chalcone
and bischalcone analogs can be correlated with their in vivo antimalarial activity, as
compounds 2c and 2h inhibited both in vitro activity of topo II and in vivo para-
sitaemia of the chloroquine-sensitive strain of P. berghei. In the chloroquine-resistant
strain, compounds 2c, 2e, 2g, and 2i inhibited activity against both in vitro topo II and
parasitaemia in vivo. The significant inhibition of topo II in the chloroquine-resistant
strain by some of the analogs suggests the utilization of these structures for the
synthesis of compounds active against chloroquine-resistant malarial parasites.
Keywords DNA Á Topoisomerases Á Chalcones Á Bischalcones Á
Plasmodium berghei Á Topoisomerase inhibitors Á Chloroquine-resistant strain
S. Srivastava Á S. Chandra
Divisions of Parasitology, Central Drug Research Institute, Lucknow 226001, India
A. S. Saxena Á V. J. Ram
Medicinal Chemistry, Central Drug Research Institute, Lucknow 226001, India
S. Joshi Á A. R. Singh Á S. Yadav Á J. K. Saxena (&)
Biochemistry, Central Drug Research Institute, Lucknow 226001, India
e-mail: jkscdri@yahoo.com

Med Chem Res (2008) 17:234–244
235
Introduction
Topoisomerases are ubiquitous enzymes involved in solving topological problems
arising during the processes of DNA metabolism including transcription, recom-
bination, replication, and chromosome partitioning during cell divisions (Wang
1985;Austin and Fisher, 1990; Osheroff et al.,1991; Gasser et al., 1992). As a result
of performing this role, topoisomerases are essential for the viability of all
organisms from unicellular bacteria to humans. According to their catalytic
mechanism two categories of enzymes have been found in a variety of organisms.
Type I enzymes transiently cut a single DNA strand, pass an intact single strand of
DNA through the broken strand and then reseal the break. Type II enzymes make
transient double-strand breaks in DNA and pass an intact duplex through the broken
DNA before resealing the break. It has been found that the level of topoisomerase II
activity is increased during rat liver regeneration after partial hepatectomy (Duguet
et al., 1983) and in rapidly proliferating cells such as tumor cells (Riou et al., 1985).
In recent years, DNA topoisomerases from parasites have been the focus of
considerable study, not only because they are intrinsically interesting but also
because they may provide a target for much needed new antiparasitic chemotherapy
(Nenortas et al., 1998).The protozoan parasites of the genus Plasmodium, the
causative agent of malaria in human beings and other vertebrates, undergo a
complex evaluative cycle inside the red blood cells (RBC) of their host and
proliferate rapidly, with more than 70% of red blood cells infested within a few
days. The quinolones are the most widely used inhibitors of bacterial topoisome-
rases. It has also been reported that reactions catalyzed by topoisomerase extracted
from rat liver and trypanosomes are inhibited by various drugs at low concentrations
(Douc-Rasy et al., 1983; DoucRasy et al., 1984). Fluoroquinolone antibiotics are
cytotoxic to malaria parasites in vitro (Krishna et al., 1988; Divo et al., 1988) and
have efficacy in patients with malaria (Sarma, 1989). An assortment of novel DNA
intercalating agents has been synthesized and tested for activity against malarial
parasites. These include the 9-anilinoacridines, which stabilize cleavable complex
formation in vivo in L. chagazi and are more cytotoxic to Plasmodium falciparum
than to mammalian cells (Werbovetz et al., 1992; Werbovetz, 1993; Chavalitshe-
winkoon et al., 1993; Gamage et al., 1994).
Naturally occurring chalcones such as licochalcone A, isolated from Chinese
licorice root, are known to have antimalarial activity against chloroquine-sensitive
(3D7) and chloroquine-resistant (Dd2) isolates of Plasmodium falciparum (Khar-
azmi et al., 1997). Recently an analog of the licochalcones, 2,4-dimethoxy-4-
butylchalcone was found to exhibit potent activity against the human malaria
parasite Plasmodium falciparum in vitro and the rodent parasites P. berghei and P.
yoelii in vivo (Chen et al., 1997). Structurally, bischalcones have the same
pharmacophore as that of flavones and isoflavones, which are potent inhibitors of
DNA topoisomerase II and are therefore considered to be good antitumor and anti-
HIV agents (Wang et al.,1997). These findings prompted us to design and
synthesize a new series of oxygenated chalcones and bischalcones for the inhibition
of the DNA topoisomerase II of the chloroquine-sensitive and chloroquine-resistant
strains and also the malaria parasite in vivo.

236
Med Chem Res (2008) 17:234–244
Materials and methods
Chemistry
Bischalcones were synthesized by base-catalyzed condensation of bis(4-acetyl-
phenoxy) alkanes and the appropriate aldehyde (Fig. 1). To our surprise, a
condensation of (4-acetylphenoxy) alkanes with 3,4-methylenedioxy benzaldehyde
under similar conditions always yielded monochalcone structure 1 (Ram et al.,
2000). All the synthesized compounds were purified by column chromatography
and characterized by spectroscopic and elemental analysis.
O
OH
O
O
COMe
ii
O
COCH=CH
COMe
i
(CH2)nBr2
(CH2)n
(CH2)n
O
COMe
O
COMe
1
2
3
a) n=3
b) n=6
iii
Ar
N
O
CH3
O
N
O
COCH=CHAr
N
O
(CH2)n
iv
CH3
Ar
N
(CH2)n
O
O
O
COCH=CHAr
N
N
O
CH3
4
5
a) n =3
b) n =4
Ar=3,4 (CH3O)2C6H3
H3C
4
a
b
c
d
e
f
Ar
n
1
3,4-(CH3O)2C6H3
2,4-(CH3O)2C6H3
3,4-(CH3O)2C6H3
2,4-(CH3O)2C6H3
3-CH3O.C6H4
1
3
3
3
4
4
4
6
6
3,4-(CH3O)2C6H3
2,4-(CH3O)2C6H3
3-CH3O.C6H4
g
h
i
3,4-(CH3O)2C6H3
3--CH3O.C6H4
j
Fig. 1 Scheme for the synthesis of chalcones and bischalcones. Reagents and conditions were: (i)
K2CO3/acetone/reflux; (ii) piperonal/alc. KOH/RT; (iii) aryl aldehyde/alc. KOH/RT; (iv) NaOEt/6-
amino-1,3-dimethyluracil/reflux

Med Chem Res (2008) 17:234–244
237
Biology
DNA topoisomerase estimation
Supercoiled DNA pBR322 and all other chemicals used were obtained from Sigma
Chemical Co., St. Louis, USA.
P. berghei chloroquine-sensitive and chloroquine-resistant strains were separated
from the infected mice according to the method of Riou et al. (1986). DNA
topoisomerase II from malarial parasites was partially purified according to the
method of Pandya et al. (1999).
The enzyme activity was monitored by relaxation of supercoiled DNA pBR322
and separation of topological isomers of DNA by gel electrophoresis as reported
earlier (Pandya et al., 1999). Briefly, for the activity measurements pBR322 DNA
(0.25 lg) was incubated in a reaction mixture containing 50 mM Tris-HCl, pH 7.5,
50 mM KCl, 10 mM MgCl2, 1 mM Adenosine triphosphate (ATP), 0.1 mM
Ethylene diamine tetra acetic acid (EDTA), 0.5 mM Dithiothreitol (DTT), 0.44 lM
Bovine serum albumin (BSA), and enzyme protein. The reaction volume was 20 ll.
Adding pBR322 DNA and incubating at 37°C for 30 min started the reaction. The
reaction was stopped by adding 5 ll stop buffer. The samples were electrophoresed
on 1% agarose gel in Tris-acetate buffer for 18 h at 20 volts. Gels were stained with
ethidium bromide (0.5 lg/ml), visualized, and photographed on a UVP GDS 7500
ultraviolet (UV) transilluminator.
The effect of inhibitors on the enzyme activity was measured by incubating
enzyme with inhibitor for 10 min at 37°C and starting the reaction by the addition of
pBR322. The percentage relaxation was measured by microdensitometry of the gel
by using the Gel Base/Gel Blot Pro gel analysis software programme.
In vivo screening
Maintenance of Plasmodium berghei infection (chloroquine-sensitive strain)
Blood from orbital plexus of heavily infected animals was drawn under aseptic
conditions and mixed with 3.8 % sodium citrate to obtain a final dilution of
approximately 5 million parasitized erythrocytes per milliliter of diluate. The
counting of RBCs was done with the help of a hemocytometer. Each animal was
then injected intraperitoneally with 0.2 ml of this suspension containing approx-
imately 1 9 106 parasitized erythrocytes.
Development of the chloroquine-resistant strain of P. berghei
Chloroquine resistance was developed with a slight modification of the technique
described by Warhurst and Folwell (1968). 1 9 106 parasitized RBCs were
inoculated in a group of five animals and after 2 h these mice were given
chloroquine diphosphate (15 mg/kg) for four consecutive days, when these animals

238
Med Chem Res (2008) 17:234–244
developed about 2% parasitaemia; the infected blood from these animals was used
to infect healthy animals that were also given chloroquine. This process was
repeated several times with P. berghei kept under continuous drug pressure. The
line has approximately seven times higher chloroquine tolerance than the
chloroquine-sensitive strain. This resistant line was used for the screening and
biochemical studies.
Preparation of synthetic compounds
Synthetic compounds employed for screening were prepared by grinding accurately
weighed compound with distilled water when the compounds were soluble in water.
Poorly water-soluble compounds were ground up with dimethyl sulfoxide (DMSO).
Finally, distilled water was added to obtain the desired concentration of the
compound.
Method of screening
Chloroquine-sensitive strain
The suppressive method of screening of Peters (1965) was adopted. Male mice
(Park strain) weighing 20
2 g were infected intraperitoneally with standard
inoculums of 1 million parasitized RBCs (1 9 106 PRBC). The animals were
treated with the compounds 2-3 hrs post inoculation. For treatment the inoculated
animals were divided into two groups with five animals in each group. All five
animals in one group received the same dose of a compound intraperitoneally for
four consecutive days, while infected animals without treatment served as control.
Chloroquine-resistant strain
In the resistant group the animals were infected as described for the sensitive strain
with one group receiving chloroquine plus compound and the other group receiving
the compound only; chloroquine was injected in the third group, and the fourth
group of infected animals without any treatment served as control. The treatment
was given to group one to three for four consecutive days.
Results and discussion
DNA topoisomerases are enzymes that alter the topological state viz., underwind-
ing, knotting, and tangling of nucleic acids by generating transient breaks in DNA.
In order to maintain the integrity of the genetic material during this process,
topoisomerases form covalent bonds with the DNA termini created by their actions.
Topoisomerase proteins characterized from lower eukaryotes appear to share many

Med Chem Res (2008) 17:234–244
239
characteristics associated with their human homologs, but striking differences,
including different enzyme activity requirements, variable catalytic sites, and
different sensitivities to topoisomerase poisons, provide insight for the development
of topoisomerase-directed antiparasitic therapeutics. It has been established by
several studies that the inhibitors of topoisomerases convert these essential enzymes
into intracellular proliferating cell toxins and thereby provide a good tool for
preferentially killing the highly replicative parasite cells within the host. Attempts
have been made to evaluate topo II poisons as potential antimalarial agents viz., 9-
anilinoacridines, which inhibited parasite metabolism and topo II activity (Chav-
alitshewinkoon et al., 1993). A related antimalarial agent with similar properties,
pyronaridine (White and Kilbey, 1996), has been shown to be active against
chloroquine-resistant strains of the parasite (Fu and Xiao, 1991).
Three compounds of the chalcone group and nine compounds of the bischalcone
group were tested for their topoisomerase II inhibitory activity in chloroquine-
sensitive (CQS) and chloroquine-resistant (CQR) strains of P. berghei. Compounds
1a, 1b, 2a, 2b, and 2c showed 100% inhibition of topoisomerase II activity while
compounds 2h and 2i produced 60% and 63% inhibition, respectively, in the
chloroquine-sensitive strain of P. berghei (Table 1).
Compounds 2a and 2c when tested at 4 lg concentration did not inhibit
topoisomerase II activity in chloroquine-sensitive strain (Fig. 2). In the chloroquine-
resistant strain compounds 2a, 2b, and 2d inhibited topoisomerase II activity by
71%, 98%, and 74%, respectively. Compounds 2b and 2d at the lower concentration
of 4 lg (per 20 ll reaction volume) inhibited topo II activity by 70% and 50%,
Table 1 Effect of chalcones (compounds 1a–c) and bischalcones (compounds 2a–i) on DNA topoiso-
merase II of sensitive and resistant strains of P. berghei
No. Struct. no. Compd. no. Concentration (lg/reaction mixture) DNA topoisomerase II % inhibition
CQS strain
CQR strain
1
2
3
4
5
6
7
8
9
1a
1b
1c
2a
2b
2c
2d
2e
2f
98/260
98/261
98/262
98/371
98/372
98/257
98/373
98/263
98/258
98/264
98/259
98/265
40 lg
40 lg
40 lg
40 lg
40 lg
40 lg
40 lg
40 lg
40 lg
40 lg
40 lg
40 lg
100
100*
Nil*
100*
100
100*
42
41*
51*
17
71*
98
50*
74
Nil*
Nil
54*
7*
10 2g
11 2h
12 2i
Nil
66
60*
63
34*
50
The values are means of three experiments in duplicate. The enzymes from drug-sensitive and drug-
resistant strains were incubated at 37°C for 10 min. The OD of relaxed DNA without any compound was
184. * Compound showed complex formation with DNA

240
Med Chem Res (2008) 17:234–244
Fig. 2 Inhibition of DNA Topoisomerase II activity of CQS P. berghei by chalcones and bischalcones.
Lane 1: pBR322 DNA alone; lane 2: topo II activity of CQS P. berghei; lane 3: identical to lane 2 +
compound 98/257 (40 lg); lane 4: identical to lane 2 + compound 98/258 (40 lg); lane 5: identical to
lane 2 + compound 98/259 (40 lg); lane 6: identical to lane 2 + compound 98/260 (40 lg); lane 7:
identical to lane 2 + compound 98/261 (40 lg); lane 8: identical to lane 2 + compound 98/262 (40 lg);
lane 9: identical to lane 2 + compound 98/263 (40 lg); lane 10: identical to lane 2 + compound 98/264
(40 lg); lane 11: identical to lane 2 + compound 98/265 (40 lg); lane 12: identical to lane 2 + compound
98/257 (4 lg); lane 13: identical to lane 2 + compound 98/371 (4 lg); lane 14: identical to lane 2 +
compound 98/371 (40 lg); lane 15: identical to lane 2 + compound 98/372 (40 lg); lane 16: identical to
lane 2 + compound 98/373 (40 lg)
respectively (Fig. 3). Compounds 2g and 2e caused 66% and 54% inhibition of
topoisomerase II activity of the CQ-resistant strain of P. berghei, respectively.
However, the same compounds (2g and 2e) showed no inhibitory effects on the
chloroquine-sensitive strain of P. berghei. Compounds 1a, 1b, 1c, 2a, 2c, 2e, 2f and
2h also showed complex formation with DNA (Figs. 2 and 3).
As reported earlier (Ram et al. 2000) compounds 2c, 2e, and 2f showed 77%,
55%, and 75% inhibition of parasitaemia in the CQS strain of P. berghei up to day
11, respectively (Table 2). In a resistance reversal test chalcones and bischalcones
showed better inhibition of parasitaemia than was observed with chloroquine-
sensitive P. berghei. Compounds 2a, 2i, 2f, 1c, and 2e showed 90%, 74%, 66%,
60%, and 53% inhibition of parasitaemia, respectively, up to day 11, while
compounds 2g, 2c, and 1a also inhibited parasitaemia by 57%, 52%, and 50%,
respectively, until day 9 (Table 3).
Compounds 1a, 1b, 1c, 2a, 2c, 2e, 2f, and 2h also showed complex formation
with the supercoiled DNA as indicated by the low level of migration of DNA in the
treated lane as compared to the control. The stabilization of covalent complexes
between topoisomerase II and DNA by these compounds might be responsible for
the observed inhibitory effect.
It has been found that in CQS P. berghei the compounds 2c and 2h showed 100%
and 60% topo II inhibition, respectively. These compounds also showed 77% and

Med Chem Res (2008) 17:234–244
241
Fig. 3 Inhibition of DNA Topoisomerase II activity of CQR P. berghei by chalcones and bischalcones.
Lane 1: pBR322 DNA alone; lane 2: topo II activity of CQR P. berghei; lane 3: identical to lane 2 +
compound 98/257 (40 lg); lane 4: identical to lane 2 + compound 98/258 (40 lg); lane 5: Identical to
lane 2 + compound 98/259 (40 lg); lane 6: identical to lane 2 + compound 98/260 (40 lg); lane 7:
identical to lane 2 + compound 98/261 (40 lg); lane 8: identical to lane 2 + compound 98/262 (40 lg);
lane 9: identical to lane 2 + compound 98/263 (40 lg); lane 10: identical to lane 2 + compound 98/264
(40 lg); lane 11: identical to lane 2 + compound 98/265 (40 lg); lane 12: identical to lane 2 + compound
98/373 (40 lg); lane 13: identical to lane 2 + compound 98/372 (40 lg); lane 14: identical to lane 2 +
compound 98/371 (40 lg); lane 15: identical to lane 2 + compound 98/372 (4 lg); lane 16: identical to
lane 2 + compound 98/373 (4 lg)
47% inhibition of parasitaemia in vivo in the CQS strain. In the resistance reversal
test compounds 2e, 2i, and 2a showed 53%, 74%, and 91% inhibition of
parasitaemia until day 11, respectively. The same compounds showed 54%, 50%,
and 71% topo II inhibition, respectively.
Pyronaridine has been shown to be an effective gametocytocidal against drug-
resistant P. falciparum and exhibited both schizontocidal and gametocytocidal
activity. Norfloxacin, amsacrine, and etoposide, the DNA topo inhibitors were
effective against asexual but not sexual stages of the malaria parasite (Chavalit-
shewinkoon et al., 2000). Chloroquine, quinine, and related 4-aminoquinolines are
effective drugs against malarial infection, and the ability of chloroquine to form
complexes with DNA is well documented (Blodgett and Yielding, 1968; Yielding
et al., 1971). Chloroquine, mepacrine, and other quinoline derivatives are potent
inhibitors of DNA and RNA polymerase (O’Brien et al., 1966; Whichard et al.,
1972), as well as DNA and RNA synthesis in P. knowlesi (Gutteridge et al., 1972).
Chloroquine and quinine inhibited 50% of DNA topoisomerase II activity of drug-
sensitive and drug-resistant P. berghei at a 40 lg/reaction mixture concentration.
Flavones and isoflavones are known to display significant topoisomerase II
inhibitory activity due to oxygenated substituents and the presence of the CO–
CH = CH pharmacophore. Chalcones and bischalcones both meet these require-
ments and thus are expected to display this type of activity. In vitro, topoisomerase

242
Med Chem Res (2008) 17:234–244
Table 2 In vivo activity of chalcones (1a–c) and bischalcones (2a–i) (50 mg/kg/day) against the CQS
strain of P. berghei in mice
No. Struct. no.
Code no. Dose (mg/kg) Inhibition of Parasitaemia (%) on days
5
7
9
11
1. Control (Avg. % parasitaemia)
2.2
4.2
74
5.5
45
22
7
7.3
31
21
10
D
2. 1a
3. 1b
4. 1c
5. 2a
6. 2b
7. 2c
8. 2d
9. 2e
10. 2f
11. 2g
12. 2h
13. 2i
98/260
98/261
98/262
98/371
98/372
98/257
98/373
98/263
98/258
98/264
98/259
98/265
100
100
100
100
100
100
100
100
100
100
100
100
100
25
28
25
20
79
96
16
67
38
10
N.I.
D
28
93
81
D
77
33
100
100
7
60
77
9
55
75
8
100
28
100
25
88
58
3
47
N.I.
15
Route = ip (intraperitoneal) 9 4 days
D: death of animal, N.I.: no inhibition
Table 3 In vivo effect of chloroquine (CQ, 15 mg/kg/day), chalcones (1a–c), bischalcones (2a–i) (50
mg/kg/day), and their combination against the CQR strain of P. berghei in mice
No.
Struct. no.
Code no.
Dose (mg/kg)
Inhibition of parasitaemia (%) on days
5
7
9
11
1. Control (avg. % parasit)
2. CQ (avg. % parasit)
-
3.2
0.2
27
100
45
99
42
99
40
100
68
100
9
5.4
1.3
40
99
39
66
44
65
28
100
20
57
57
N.I.
57
75
17
90
8.1
3.3
45
50
43
42
43
62
16
96
50
52
23
22
38
53
37
55
14.6
5.7
N.I.
34
15
3.
4.
5.
6.
7.
8.
9.
10.
1a
98/260
50
1a + CQ
1b
98/260 + CQ
98/261
50 + 15
50
30.
33
1b + CQ
1c
98/261 + CQ
98/262
50 + 15
50
46.
60
1c + CQ
2a
98/262 + CQ
98/371
50 + 15
50
N.I..
90
2a + CQ
2c
98/371 + CQ
98/257
50 + 15
50
13
2c + CQ
2d
98/257 + CQ
98/373
50 + 15
50
N.I.
49
2d + CQ
2e
98/373 + CQ
98/263
50 + 15
50
16
42
97
50
91
23
43
2e + CQ
2f
98/263 + CQ
98/258
50 + 15
50
53
23
2f + CQ
98/258 + CQ
50 + 15
66

Med Chem Res (2008) 17:234–244
243
Table 3 continued
No.
Struct. no.
Code no.
Dose (mg/kg)
Inhibition of parasitaemia (%) on days
5
7
9
11
11.
12.
13.
2g
98/264
50
37
52
16
57
50
58
N.I.
98
30.
47
2g + CQ
2h
98/264 + CQ
98/259
50 + 15
50
73
27
77
50
35
2h + CQ
2
98/259 + CQ
98/265
50 + 15
50
100
28
98
50
N.I.
100
N.I.
74
2i + CQ
98/265 + CQ
50 + 15
100
Route = ip (intraperitoneal) 9 4 days, N.I.: no inhibition
II inhibitory activity of bischalcones can be correlated with in vivo antimalarial
activity. Methylene chain length in bischalcones seems to be crucial for exhibiting
topoisomerase inhibitory activity. As the chain length increases from CH2 to
(CH2)4, inhibitory activity decreases. Secondly compounds with methoxy substit-
uents at position 2 and 4 of the phenyl ring are more active than the compounds
having methoxy substitution at positions 3 and 4 of the phenyl ring.
The results of the present study show that DNA topoisomerase II of P. berghei
can be utilized as a target for the synthesis of new antimalarials based on the
structure of bischalcones. The specific inhibition of topo II by some compounds in
drug-resistant malarial parasites can also be utilized for the synthesis of compounds
specifically active against drug-resistant malaria parasites.
Acknowledgement The authors are grateful to the director of the CDRI for providing necessary
facilities for this work. The authors SS and ASS are indebted to the ICMR for providing research
fellowships. SJ, SY, and ARS are indebted to the CSIR for the award of JRFs. CDRI communication no.
6049.
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