A natural product inspired hybrid approach towards the synthesis of novel pentamidine based scaffolds as potential anti-parasitic agents

Article abstract of DOI:10.1016/j.bmcl.2012.10.101

A natural product inspired molecular hybridization approach led us to a series of novel pentamidine based pyrimidine and chalcone scaffolds. All the hybrids were evaluated for their anti-leishmanial potential. Most of the screened compounds have showed significant in vitro anti-leishmanial activity with less cytotoxicity in comparison to the standard drugs (pentamidine, sodium stibogluconate, and miltefosine). Additionally, anti-malarial screening of these compounds was also done and four compounds have shown superior activity against chloroquine resistance strain (K1) of Plasmodium falciparum.

Full text of DOI:10.1016/j.bmcl.2012.10.101

Bioorganic & Medicinal Chemistry Letters 23 (2013) 291–296
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A natural product inspired hybrid approach towards the synthesis of novel
pentamidine based scaffolds as potential anti-parasitic agents
Vikas Tyagi a, Shahnawaz Khan a, Rahul Shivahare b, Khushboo Srivastava b, Suman Gupta b,
Saqib Kidwai b, Kumkum Srivastava b, S. K. Puri b, Prem M. S. Chauhan a,
⇑
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226001, India
b Parasitology Division, CSIR-Central Drug Research Institute, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A natural product inspired molecular hybridization approach led us to a series of novel pentamidine
based pyrimidine and chalcone scaffolds. All the hybrids were evaluated for their anti-leishmanial poten-
tial. Most of the screened compounds have showed significant in vitro anti-leishmanial activity with less
cytotoxicity in comparison to the standard drugs (pentamidine, sodium stibogluconate, and miltefosine).
Additionally, anti-malarial screening of these compounds was also done and four compounds have shown
superior activity against chloroquine resistance strain (K1) of Plasmodium falciparum.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 16 August 2012
Revised 26 September 2012
Accepted 23 October 2012
Available online 1 November 2012
Keywords:
Pyrimidine
Chalcone
Pentamidine
Anti-leishmanial activity
Anti-malarial activity
Nowadays, neglected tropical disease (NTDs) affect more than
one billion people worldwide, causes over 550,000 deaths annu-
ally.1 Research projects aiming to discover new drugs for NTDs
have discouraged drug companies from investing due to the low
returns of investment.2 Owing to this, drug discovery pipeline is
still almost dry for NTDs. Among NTDs, Chagas disease, sleeping
sickness, malaria, and leishmaniasis are the major NTDs with the
highest rates of death.3 In particular, leishmania is responsible for
cutaneous and visceral infections, endemic in 88 countries in the
Horn of Africa, South Asia, and Latin America.4 Leishmaniasis is a
vector born disease caused by different species belonging to the
genus Leishmania, a protozoa transmitted by the bite of a tiny long
(2–3 mm) insect vector, the phlebotomine sandfly.5 Leishmania is
manifested in four major clinical forms i.e. cutaneous leishmania-
sis, mucocutaneous leishmaniasis, visceral leishmaniasis, and post
kala-azar dermal leishmaniasis or PKDL. Among all the four forms,
visceral leishmaniasis (VL), caused by the parasite Leishmania
donovani, is nearly always fatal if not treated.6
and pancreatitis.7 Pentamidine, miltefosine, and amphotericin-B,
are the second line drugs for the treatment of VL, which also suffer
from moderate to severe side effects.8 Pentamidine, an aromatic
diamidine, is orally inactive and may demonstrate renal, hepatic
and pancreatic toxicity beside with hypotension and dysglycemia.9
Amphotericin-B and its lipid complex is a most useful alternative,
however, major draw backs associated with amphotericin-B, such
as its high cost leave out of the reach of poor people.10
Furthermore, recently introduced first orally active drug Mil-
tefosine, a phosphocholine analogue, has a long half-life (100–
200 h) in humans and a low therapeutic ratio, presenting severe
gastrointestinal problems and also shows teratogenic effects and
cannot be used in the pregnant women.11 Since the chemotherapy
against leishmaniasis is still inefficient, as a result the finding of
more effective and safer drug for treating leishmaniasis remains
desirable.
The natural product inspired molecular hybridization approach
has been emerged as a powerful tool for tackling the problems
associated with the standard drugs.12 Recently, some hybrid mole-
cules of pentamidine with other heterocycles have been synthe-
sized, which showed potent anti-leishmanial activity with low
cytotoxicity.13 Furthermore, following the same approach some
pyrimidines and chalcone based hybrids were synthesized and
evaluated for their anti-parasitic potential.14
The first line drugs for the treatment of leishmania are pentava-
lent antimonial compounds, which were discovered almost
70 years ago, and generally require high dose of parental adminis-
tration. Moreover, this class of drugs need long-term treatment
associated with severe side effects including cardiac arrhythmia
Annomontin, a pyrimidine-b-carboline alkaloid, has showed
antileishmanial activity 34.8 1.5 against the Leishmania brazilien-
sis. On the other hand, licochalcone A, is a well known anti-parasitic
⇑
Corresponding author. Tel.: +91 522 2262411x4470; fax: +91 522 2623405.
E-mail addresses: premsc58@hotmail.com, prem_chauhan_2000@yahoo.com
(P.M.S. Chauhan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.101

292
V. Tyagi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 291–296
natural phenol.15 In the continuation of our ongoing programme to
develop new hybrid molecules as potent anti-parasitic agents,16
and inspired by the antileishmanial and antimalarial activity of
pyrimidine and chalcone based natural product annamontine and
licochalcone A, we herein report our work on the design, synthesis
and anti-protozoal evaluation of novel pentamidine based pyrimi-
dine and chalcone hybrids (Fig. 1).
The detailed synthetic route to synthesize compounds (7a–i) is
outlined in scheme 1. The synthesis was achieved by the coupling
of substituted pyrimidine with the pentamidine fragment. In this
context, pyrimidine derivatives (2a–i) were obtained by the conden-
sation of 2,4-dichloro-6-methylpyrimidine with various primary or
secondary amines under basic conditions. The pentamidine
fragment has been synthesized via a condensation of 1,5-dibromo-
pentane with N-(4-hydroxyphenyl)acetamide to furnish the inter-
mediate 5 under basic condition, followed by the deprotection of
amine in presence of 20% aq NaOH furnished the pentamidine frag-
ment 6 in 74% yield. Finally, pentamidine fragment 6 was condensed
with pyrimidine derivatives (2a–i) to afford the targeted pentami-
dine-pyrimidine hybrids (7a–i) in good yields (scheme 1).
In the second phase of our endeavour, we synthesized
chalcones-pentamidine hybrid (10a–f, scheme 2). In this contin-
uation intermediate 9 has been synthesized by replacing both
bromo groups of 1,5-dibromopentane via 4-hydroxy benzalde-
hyde. Intermediate 9 was condensed with various acetophenon-
es under the reported protocols. The use of 1 equiv of
acetophenones leads to chalcone (10d–f), while the use of
2 equiv of acetophenones furnished the bis-chalcones (10a–c).
All compounds were characterized using 1H NMR, 13C NMR,
mass spectrometry and IR spectroscopy (see Supplementary
data).17 The purity of these compounds was ascertained by
TLC and spectral analysis.
All the synthesized compounds were evaluated in vitro against
transgenic Leishmania donovani amastigotes.18 All the pyrimidine–
pentamidine hybrids (7a–i) (Table 1) showed very good inhibitory
activity with the IC50 values in the range of 0.30–1.72 lM and SI
O
OMe
O
O
N
N
5
H
H2N
NH2
NH
HO
OH
CH3
N
NH
H2C
CH3
N
H2N
Pentamidine
Licochalcone A
Annomontin
O
O
O
5
O
N
N
N
N
5
HN
NH
H2N
NH2
O
O
Pyrimidine-pentamidine hybrid
Chalcone-pentamidine hybrid
Figure 1. A natural product inspired hybrid approach to the synthesis of anti-parasitic agent.
Cl
Cl
a
2a, R = benzylamine
2f, R = 1-methylpiperazine
2g, R = 1-ethylpiperazine
2h, R = 1-phenylpiperazine
2i, R = morpholine
N
N
N
N
2b, R = p-methoxybenzylamine
2c, R = p-fluorobenzylamine
2d, R = cyclopropylamine
2e, R = cyclopentylamine
R
Cl
2a-i
1a-i
O
OH
O
5
b
Br
Br
NH
O
HN
O
+
5
5
3
HN
O
c
4
O
O
O
5
5
O
d
HN
NH
NH2
H2N
N
N
N
N
6
R
R
7a-i
Yield= 57-89%
Scheme 1. Synthesis of pyrimidine–pentamidine hybrids (7a–i). Reagents and conditions: (a) amines, DIPEA, EtOH, rt, 2 h (b) K2CO3, acetone, reflux, 10 h (c) 20% aq NaOH,
H2O:EtOH (1:1), 90 °C, 5 h (d) 2a–i, DIPEA, DMF, MW, 200 °C, 30 min.

V. Tyagi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 291–296
293
O
O
a
5
Br
Br
CHO
+
HO
OHC
CHO
O
5
9
3
8
b
R2
R1
O
O
O
R1
O
R2
O
10a
O
O
5
10d
MeO
H
R1
R2
MeO
10b
O
O
O
10a-f
Yield= 69-93%
MeO
MeO
MeO
MeO
10e
10f
H
O
Br
Cl
O
O
10c
H
Cl
Cl
Scheme 2. Synthesis of chalcone-pentamidine hybrids (10a–f). Reagents and conditions: (a) K2CO3, acetone, reflux, 10 h (b) 10% aq KOH, MeOH, rt, 2 h
Table 1
In vitro anti-leishmanial activity against intracellular amastigotes of pyrimidine–pentamidine hybrids
Compd no.
R
Antiamastigote activity IC50
(
lM)
Cytotoxicity CC50
26.04
(
lM)
Selectivitya index (SI)
N
H
7a
1.67
15.60
3.70
N
H
7b
7c
0.57
1.41
2.11
8.68
MeO
F
N
H
6.16
7d
7e
0.42
1.53
2.68
1.69
6.38
1.11
NH
NH
N
7f
0.81
0.30
2.93
3.65
3.62
N
N
7g
12.17
N
N
N
7h
1.72
>400
>232
N
7i
1.18
5.51
4.67
O
Pentamidine
SSG
Miltefosine
20.43
71.90
12.5
52.70
397.60
3.2
2.58
5.53
0.25
SSG = sodium-stibo-gluconate (IC50 & CC50 in
l
g/mL)
a
Selectivity index (SI) defined by the ratio CC50 (in KB cell lines)/IC50 (Intracellular leishmania amastigotes).
values in the range of 1.11–>232, when compared to the standard
drugs like Pentamidine (IC50 = 20.43 M, SI = 2.58), SSG
(IC50 = 71.90 g/mL, SI = 5.53) and miltefosine (IC50 = 12.5 M,
SI = 0.25). Among these pyrimidine-pentamidine hybrids, com-
pound 7b having p-methoxy benzyl amine functionality showed
times more active and less toxic than the standard drugs. Encour-
aging by the in vitro activity of pentamidine–pyrimidine hybrids,
we have also screened chalcones-pentamidine hybrids against L.
donovani, but only some of these showed good inhibition Table 2.
It was observed that compound 10a is the most active with
l
l
l
more activity with IC50 = 0.57
lM than the compounds 7c having
IC50 = 1.65
lM, and SI = 242.42 than the compound 10d with
p-fluoro benzyl amine functionality (IC50 = 1.41
lM) whereas com-
IC50 = 28.4
lM. Furthermore, the most active bis-chalcone-pentamidine
pound 7c showed less toxicity (SI = 6.16) than the 7b (SI = 3.70).
Moreover, in case compounds 7d and 7e having cyclopropyl and
cyclopently amine functionalities showed IC50 = 0.42, 1.53 lM
and SI = 6.38, 1.11, respectively. Next, we introduced the aliphatic
cyclic heterocyclic amines (7f–7i), the most active compound
found to be 7g having ethyl piperazine substitution showed high
hybrid 10a has been utilised for the synthesis of pyrimidine–
pentamidine hybrid 11 by the reaction of guanidine hydrochloride
in the presence of NaH (scheme 3). To our delight, this
pyrimidine–pentamidine hybrid showed potential activity with
IC50 = 0.96 lM and SI = 242.42 against the L. donovani, which was
manifold times better active and less toxic than the used
inhibitory activity against L. donovani with IC50 = 0.30 lM and
standards.
selectivity index (SI) = 12.17, but compound 7h having phenyl
piperazine substitution proved to be the most promising com-
pound of the series IC50 = 1.72 lM and SI >232 which was manifold
Additionally, the anti-malarial activity of pentamidine was al-
ready reported over many years ago, but presence of other suitable
drugs stuck its anti-malarial development.13 Due to the, growing

294
V. Tyagi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 291–296
Table 2
Anti-leishmanial in vitro activity against intracellular amastigotes of chalcone-pentamidine hybrids
Compd no.
R1
R2
Antiamastigote activity IC50
(
lM)
Cytotoxicity CC50
>400
(
lM)
Selectivity index a(SI)
>242.42
O
O
10a
1.65
O
O
10b
10c
10d
NI
ND
ND
—
MeO
MeO
MeO
O
MeO
O
—
—
Cl
Cl
O
H
O
28.39
13.86
0.49
MeO
MeO
O
H
O
O
10e
10f
NI
—
ND
—
ND
—
Br
Cl
O
H
Pentamidine
SSG
Miltefosine
20.43
71.90
12.5
52.70
397.60
3.2
2.58
5.53
0.25
SSG = sodium stibo-gluconate (IC50 & CC50 in lg/mL).
a
Selectivity index (SI) defined by the ratio CC50 (in KB cell lines)/IC50 (Intracellular leishmania amastigotes). NI = no inhibition, ND = not determine, NA = not available.
N
O
N
O
NH2
O
O
O
5
NaH, DMF
reflux, 18 h
11, yield = 71%
NH2
NH2.HCl
IC50 = 0.96, cytotoxicity = >400
SI = >242.42
N
N
HN
NH2
O
Scheme 3. Synthesis of pyrimidine-pentamidine hybrid 11 using chalcone-pentamidine hybrid 10a as precursor.
drug resistance of malaria, there are urgent needs for novel classes
of compounds that are effective against sensitive as well as resis-
tant Plasmodium falciparum strains.
(IC50 = 6.25 ng/mL) and K1 (IC50 = 11.26 ng/mL) (Table 3). Com-
pounds 7b, 7g and 7h having p-methoxy benzyl amine, N-ethyl
piperazine, and N-phenyl piperazine units respectively showed
good activity against the K1 cell line (Table 3).
In previous studies, pyrimidine and chalcone have also been
showed a therapeutic effect on Plasmodium falciparum strains.14
Prompted by the anti-malarial activity of pyrimidines, chalcones,
and pentamidine, the synthesized hybrid compounds were also
screened against Plasmodium falciparum.18 Among the pyrimi-
dine–pentamidine hybrids, some of these showed very good
in vitro activity against the 3D7 (chloroquine-sensitive strain)
and K1 (chloroquine-resistant strain), while the chalcone based hy-
brids of pentamidine were found to be inactive against Plasmodium
falciparaum. Moreover, compounds 7d having cyclopropyl amine
functionalities showed very good activity against the 3D7
In conclusion, a series of highly potent and less toxic hybrids
of pentamidine as anti-parasitic agents were designed and syn-
thesized. The synthesized hybrid molecules exhibited promising
in vitro antileishmanial activity, whereas several compounds
showed remarkable antimalarial potency in vitro, especially
against CQ-R Pf (K1) strain. Due to the better in vitro anti-
leishmanial activity and less toxicity than the standard drug
(pentamidine, SSG, and miltefosine), these compounds can be
very valuable for further optimization work in anti-leishmanial
chemotherapy.

V. Tyagi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 291–296
295
Table 3
Anti-malarial in vitro activity of pyrimidine–pentamidine hybrids against the sensitive 3D7 and resistance K1 cell lines of Plasmodium falciparum
Compd
no.
R
Anti-malarial activity 3D7 IC50 b(ng/
mL)
Anti-malarial activity K1 IC50 (ng/
mL)
Cytotoxicity CC50 (ng/
mL)
Selectivity index
c(SI)
N
7a
7b
51.45
11.69
382.59
23.61
22080
3370
429.15
288.28
H
N
H
MeO
F
N
7c
H
39.41
358.43
41440
1051.51
7d
7e
6.25
11.26
NDd
560
89.6
NH
NH
313.29
18080
57.71
N
7f
245.0
45.15
ND
10800
7130
43.97
157.9
N
N
7g
22.13
N
N
N
7h
59.24
53.06
100000
1688
N
7i
263.69
ND
2060
7.82
O
aCQ
2.45 1.03
141.52 25.6
75000
30612
a
b
c
CQ = Chloroquine, 3D7 (CQ-sensitive strain) and K1 (CQ-resistant strain).
IC50 (ng/mL): concentration corresponding to 50% growth inhibition of the parasite.
Selectivity index(SI) defined by the ratio: CC50 (in Viro cell lines) /IC50 values of antimalarial activity against 3D7 cell line).
ND = not done.
d
Chem. Lett. 2012, 20, 2971; (b) Devakaram, R.; Black, D. S.; Choomuenwai, V.;
Davis, R. A.; Kumar, N. Bioorg. Med. Chem. Lett. 2012, 20, 1527; (c) Nazarian, Z.;
Emami, S.; Heydari, S.; Ardestani, S. K.; Nakhjiri, M.; Poorrajab, F.; Shafiee, A.;
Foroumadi, A. Eur. J. Med. Chem. 2010, 45, 1424; (d) Barbosa, T. P.; Sousa, S. C.
O.; Amorim, F. M.; Rodrigues, Y. K. S.; de Assis, P. A. C.; Caldas, J. P. A.; Oliveira,
M. R.; Vasconcellos, M. L. A. A. Bioorg. Med. Chem. Lett. 2011, 19, 4250; (e)
Boeck, P.; Falcão, C. A. B.; Leal, P. C.; Yunes, R. A.; Filho, V. C.; Torres-Santosc, E.
C.; Rossi-Bergmannc, B. Bioorg. Med. Chem. Lett. 2006, 14, 1538.
Acknowledgments
V.T. and S. K. are thankful to University Grant Commission, New
Delhi, for financial support in the form of SRF. The authors also
acknowledge SAIF-CDRI for providing spectral and analytical data.
The CDRI Communication Number is 8343.
13. (a) Bakunova, S. M.; Bakunov, S. A.; Patrick, D. A.; Kumar, E. V. K. S.; Ohemeng,
K. A.; Bridges, A. S.; Wenzler, T.; Barszcz, T.; Jones, S. K.; Werbovetz, K. A.; Brun,
R.; Tidwell, R. R. J. Med. Chem. 2009, 2016, 52; (b) Bakunov, S. A.; Bakunova, S.
M.; Bridges, A. S.; Wenzler, T.; Barszcz, T.; Werbovetz, K. A.; Brun, R.; Tidwell, R.
R. J. Med. Chem. 2009, 52, 5763.
14. (a) Gupta, L.; Sunduru, N.; Verma, A.; Srivastava, S.; Gupta, S.; Goyal, N.;
Chauhan, P. M. S. Eur. J. Med. Chem. 2010, 45, 2359; (b) Kumar, R.; Khan, S.;
Verma, A.; Srivastava, S.; Viswakarma, P.; Gupta, S.; Meena, S.; Singh, N.;
Sarkar, J.; Chauhan, P. M. S. Eur. J. Med. Chem. 2010, 45, 3274; (c) Sunduru, N.;
Chauhan, P. M. S. Bioorg. Med. Chem. Lett. 2006, 14, 7706; (d) Agarwal, A.;
Chauhan, P. M. S. Bioorg. Med. Chem. Lett. 2005, 13, 6678; Sunduru, N.; Nishi;
Palne, S.; Chauhan, P. M. S.; Gupta, S. Eur. J. Med. Chem. 2009, 44, 2473; (f)
Sunduru, N.; Agarwal, A.; Katiyar, S. B.; Nishi; Goyal, N.; Gupta, S.; Chauhan, P.
M. S. Bioorg. Med. Chem. Lett. 2006, 14, 7706.
15. (a) Costa, E. V.; Pinheiro, M. L. B.; Xavier, C. M.; Silva, J. R. A.; Amaral, A. C. F.;
Souza, A. D. L.; Barison, A.; Campos, F. R.; Ferreira, A. G.; Machado, G. M. C.;
Leon, L. P. L. J. Nat. Prod. 2006, 69, 292; (b) Sairafianpour, M.; Kayser, O.;
Christensen, J.; Asfa, M.; Witt, M.; Stærk, D.; Jaroszewski, J. W. J. Nat. Prod. 2002,
65, 1754.
16. Porwal, S.; Chauhan, S. S.; Chauhan, P. M. S.; Shakya, N.; Verma, A.; Gupta, S.
J. Med. Chem. 2009, 52, 5793; (b) Sharma, M.; Chauhan, K.; Chauhan, S.;
Kumar, A.; Singh, S. V.; Saxena, J. K.; Agarwal, P.; Srivastava, K.; Kumar, S. R.;
Puri, S. K.; Shah, P.; Siddiqi, M. I.; Chauhan, P. M. S. Med. Chem. Commun.
2012, 3, 71.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.
101.
References and notes
1. WHO report, 2009, Available at: <http://www.who.int/leishmaniasis/burden/
en.; (b) Hotez, P. J.; Molyneux, D. H.; Fenwick, A.; Ottesen, E.; Ehrlich, S. S.;
Sachs, J. D. PLoS Med. 2006, 3, 102.
2. Pink, R.; Hudson, A.; Mouries, M. A.; Bendig, M. Nat. Rev. Drug Disc. 2005, 4, 727.
3. Hotez, P. J.; Molyneux, D. H.; Fenwick, A.; Kumaresan, J.; Sachs, S. E.; Sachs, J. D.;
Savioli, L. N. Eng. J. Med. 2007, 1018, 357.
4. Bern, C.; Maguire, J. H.; Alvar, J. PLoS Neglected Trop. Dis. 2008, 2, 313.
5. (a) Akopyants, N. S.; Kimblin, N.; Secundino, N.; Patrick, R.; Peters, N.; Lawyer,
P.; Dobson, D. E.; Beverley, S. M.; Sacks, D. L. Science 2009, 324, 265; (b) Rogers,
M.; Kropf, P.; Choi, B. S.; Dillon, R.; Podinovskaia, M.; Bates, P.; Muller, I. PLoS
Pathog. 2009, 5, 1000555.
6. (a) http://www.who.int/tdr/diseases/leish/diseaseinfo.htm.; (b) Sharma, U.;
Singh, S. Indian J. Exp. Biol. 2009, 47, 412.
7. (a) Murray, H. W.; Berman, J. D.; Davies, C. R.; Saravia, N. G. Lancet 2005, 366,
1561; (b) Chappuis, F.; Sundar, S.; Hailu, A.; Ghalib, H.; Alvar, J.; Boelaer, M. Nat.
Rev. Microbiol. 2007, 5, 873.
8. (a) Poola, N. R.; Kalis, M.; Plakogiannis, F. M.; Taft, D. R. J. Antimicrob. Chemother.
2003, 52, 397; (b) Bhattacharya, S. K.; Sinha, P. K.; Sundar, S.; Thakur, C. P.; Jha,
T. K.; Ganguly, N. K. J. Infect. Dis. 2007, 196, 591.
17. (a) Spectroscopic data for 7h: yellow solid, yield = 79%, mp = 107–110 °C, FT-IR
(KBr)
m
(cmÀ1): 3420, 2918, 1591, 1507, 1443, 1229, 923, 826, 758; 1H NMR
(300 MHz, CDCl3):d 7.50 (d, J = 8.7 Hz, 4H), 7.34 (d, J = 8.1 Hz, 4H), 6.99–6.88
(m, 12H), 5.94 (s, 2H), 4.03 (t, J = 6.3 Hz, 4H), 3.79 (br s, 8H), 3.27 (br s, 8H), 2.30
(s, 6H), 1.91–1.86 (m, 4H), 1.70–1.68 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3): d
165.8, 163.4, 159.6, 153.5, 134.7, 120.6, 114.7, 93.3, 68.0, 66.3, 29.0, 24.1,
22.8 ppm, ESI-MS (m/z): 790.4 (M+H)+; Anal. Calcd for C47H54N10O2: C, 71.37;
H, 6.88, N, 17.71. Found: C, 71.29; H, 6.86, N, 17.68.
9. Mcgregor, A. Lancet 1998, 351, 575.
10. (a) Croft, S. L.; Yardley, V. Curr. Pharm. Des. 2002, 8, 319; (b) Sundar, S.; Rai, M.
Curr. Opin. Infect. Dis. 2002, 15, 593; (c) Pink, R.; Hudson, A.; Mouriès, M.-A.;
Bendig, M. Nat. Rev. Drug Disc. 2005, 4, 727.
11. Herwaldt, B. L. N. Eng. J. Med. 1840, 1999, 341.
12. (a) Sashidhara, K. V.; Kumar, M.; Modukuri, R. K.; Srivastava, R. K.; Soni, A.;
Srivastava, K.; Singh, S. V.; Saxena, J. K.; Gauniyal, H. M.; Puri, S. K. Bioorg. Med.
(b) Spectroscopic data for 10a: white solid, yield = 81%, mp = 130–134 °C, FT-IR
(KBr)
m
(cmÀ1): 3447, 1654, 1598, 1510, 1291, 1216, 1018, 764, 670, 571; 1H
NMR (300 MHz, CDCl3):d 8.04 (d, J = 7.2 Hz, 3H), 7.86–7.78 (m, 3H), 7.63–7.40
(m, 9H), 7.10 (s, 2H), 7.03–6.94 (m, 5H), 4.13–4.04 (m, 4H), 1.92 (br s, 4H),
1.73–1.66 (m, 2H) ppm, 13C NMR (75 MHz, CDCl3): d 191.2, 188.9, 163.5, 160.8,
144.0, 137.8, 132.9, 131.8, 130.8, 129.4, 128.6, 128.4, 127.1, 119.3, 114.8, 67.9,

296
V. Tyagi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 291–296
67.6, 28.2, 22.0 ppm, ESI-MS (m/z): 516.2 (M+H)+; Anal. Calcd for C35H32O4: C,
81.37; H, 6.24. Found: C, 81.32; H, 6.22.
antimalarial assay: The compounds were evaluated for antimalarial activity
against both 3D7 (CQ- sensitive) and K1 (CQ-resistant) strains of Plasmodium
18. Bioevaluation methods In vitro antileishmanial assay: The anti-leishmanial
evaluation of these hybrids was performed on luciferase-transfected L.
donovani promastigotes maintained in our laboratory (Sunduru et al, 2009).
In order to assess the activity of compounds against the amastigote stage of the
parasite, the mouse macrophage cell line (J-774A.1), infected with
promastigotes was used. Cells were seeded in a 96 well plate at a density of
falciparum using Malaria SYBR Green I nucleic acid staining dye based
fluorescence (MSF) assay as mentioned by Singh et al (2011)1. The stock
(5 mg/ml) solution was prepared in DMSO and test dilutions were prepared in
culture medium (RPMI-1640-FBS). Chloroquine was used as reference drug.
The compounds were tested in 96well plate (in duplicate wells). 1.0%
parasitized cell suspension containing 0.8% parasitaemia was used. The
plates were incubated at 37 °C in CO2 incubator in an atmosphere of 5% CO2
4 Â 103 cells/100
lL/well in RPMI-1640 containing 10% fetal calf serum and the
plates were incubated at 37 °C in a CO2 incubator. After 24 h, the medium was
and air mixture. After 72 hours 100 ll of lysis buffer containing 2Â
replaced with fresh medium containing stationary phase promastigotes
concentration of SYBR Green-I (Invitrogen) was added to each well and
incubated for another one hour at 37 °C. The plates were examined at
485 20 nm of excitation and 530 20 nm of emission for relative
fluorescence units (RFUs) per well using the fluorescence plate reader (FLUO
star, BMG lab technologies). The IC50 values were obtained by Logit regression
analysis using pre-programmed Excel spreadsheet (S. Singh, R. K. Srivastava, M.
Srivastava, S. K. Puri, K. Srivastava, Experimental Parasitology., 2011, 127, 318).1
Cytotoxicity of the compounds was carried out using KB cell line (oral
carcinoma cell line) and Vero cell line (C1008; Monkey kidney fibroblast)
following the method of Mosmann (1983) with certain modifications.2 The
cells were incubated with compound dilutions for 72 h and MTT was used as
(4 Â 104/100
lL/well). Promastigotes were phagocytised by the macrophage
and transformed into amastigotes. The test compounds were added in up to 7
concentrations in fresh complete medium starting from 40
and the plate were incubated at 37 °C in a CO2 incubator for 72 h. After
incubation, the drug containing medium was decanted and 50 PBS
lM concentration,
l
L
(Phosphate Buffer Saline) was added in each well and mixed with an equal
volume of Steady GloÒ reagent. After gentle shaking for 1–2 min, the reading
was taken in a luminometer (Porwal et al, 2009). The values are expressed as
relative luminescence units (RLU). Data were transformed into graphical
program (Excel) and the inhibition of parasitic growth is determined by
comparison of the luciferase activity of drug treated parasites with that of
untreated controls. The 50% inhibitory concentration (IC50) for anti-leishmanial
activity was calculated by logit regression analysis. (Sunduru, N.; Nishi; Palne,
S.; Chauhan, P. M. S.; Gupta, S. Eur. J. Med. Chem. 2009, 44, 2473). In vitro
reagent for the detection of cytotoxicity. 50% cytotoxic concentration (CC50
)
was determined using nonlinear regression analysis using pre-programmed
Excel spreadsheet. Selectivity Index was calculated as SI = CC50/IC50. (T.J.
2
Mosmann, Immunol. Methods., 1983, 65, 55).