Design, synthesis and biological evaluation of aryl pyrimidine derivatives as potential leishmanicidal agents

Article abstract of DOI:10.1016/j.bmcl.2013.06.060

A series of substituted aryl pyrimidine derivatives was synthesized and evaluated in vitro for their antileishmanial potential against intracellular amastigotes of Leishmania donovani using reporter gene luciferase assay. Among all, 8 compounds showed promising IC₅₀ values ranging from 0.5 to 12.9 μM. Selectivity indices (S.I.) of all these compounds are far better than reference drugs, sodium stibogluconate (SSG) and miltefosine. On the basis of good S.I., compounds were further screened for their in vivo antileishmanial activity against L. donovani/hamster model. Compounds 2d, 4a and 4b have shown significant inhibition of parasitic multiplication that is 88.4%, 78.1% and 78.2%, respectively at a daily dose of 50 mg/kg × 5 days, when administered intraperitoneally. Compound 2d is most promising one, which may provide a new lead that could be exploited as a new antileishmanial agent.

Full text of DOI:10.1016/j.bmcl.2013.06.060

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5235–5238
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of aryl pyrimidine
derivatives as potential leishmanicidal agents q
a
b
a
a
S. N. Suryawanshi a, , Santosh Kumar , Rahul Shivahare , Susmita Pandey , Avinash Tiwari ,
⇑
Suman Gupta b
a Division of Medicinal Chemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, M.G. Road, Lucknow 226001, India
b Division of Parasitology, CSIR-Central Drug Research Institute, Chattar Manzil Palace, M.G. Road, 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 series of substituted aryl pyrimidine derivatives was synthesized and evaluated in vitro for their antile-
Received 3 April 2013
Revised 7 June 2013
Accepted 21 June 2013
Available online 29 June 2013
ishmanial potential against intracellular amastigotes of Leishmania donovani using reporter gene lucifer-
ase assay. Among all, 8 compounds showed promising IC50 values ranging from 0.5 to 12.9 lM. Selectivity
indices (S.I.) of all these compounds are far better than reference drugs, sodium stibogluconate (SSG) and
miltefosine. On the basis of good S.I., compounds were further screened for their in vivo antileishmanial
activity against L. donovani/hamster model. Compounds 2d, 4a and 4b have shown significant inhibition
of parasitic multiplication that is 88.4%, 78.1% and 78.2%, respectively at a daily dose of 50 mg/
kg  5 days, when administered intraperitoneally. Compound 2d is most promising one, which may pro-
vide a new lead that could be exploited as a new antileishmanial agent.
Keywords:
Pyrimidines
Leishmania donovani
Hamster
Ó 2013 Elsevier Ltd. All rights reserved.
In vitro
In vivo
Luciferase assay
Leishmaniasis, a vector-borne parasitic disease, is a major cause
of concern in developing countries. The disease is caused by more
than 20 species of protozoan Leishmania and transmitted by the
bite of female phlebotomine sand flies. Leishmaniasis has tradi-
tionally been classified into three major clinical forms: visceral
leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutane-
ous leishmaniasis (MCL) which differs in immunopathologies and
degree of morbidity and mortality. VL caused by Leishmania dono-
vani is the most severe form of leishmaniasis and is usually fatal in
the absence of treatment.1 Most of the first line drugs available for
the treatment of leishmaniasis such as sodium stibogluconate,
meglumine antimoniate, pentamidine, etc. cause serious side ef-
fects and toxicity.2–4 Although drugs like amphotericin B and its li-
pid complex5 are quite effective but they are expensive and out of
reach of poor people.6 Newly introduced first orally active drug
miltefosine7 is quite effective but shows teratogenic effects there-
fore its use is strictly prohibited in pregnant women. Moreover,
resistance developing to first line agents is now increasing even
up to an extent of >60% of the patient in some endemic areas.1
Thus, new, safer, affordable drugs are urgently required for the
treatment of leishmaniasis. Various classes of natural products
such as licochalcone8 A, quinoline alkaloids9 have shown
promising antileishmanial activity. Some biochemical targets
trypanothione reductase,10 cysteine peptidases,11 sterol biosynthe-
sis,12 dihydrofolate reductase13 (DHFR) and ornithine decarboxyl-
ase14 are also under investigation to generate novel small
molecules as lead compounds.15 Pyrimidine16 derivatives are
known DHFR inhibitors13 and have been evaluated for the design
of novel antileishmanial agents. In continuation of our efforts to
generate novel synthetic molecules as antileishmanial agents cou-
pled with encouraging results of 1, 2 and 3 (Fig. 1), we have de-
signed and synthesized novel pyrimidine derivatives. The
synthesized compounds were evaluated for their in vitro/in vivo
antileishmanial activity against amastigote form of L. donovani
and the results are reported in this manuscript.
a
-Oxoketene dithioacetals of aromatic substrates are very useful
synthons in the synthesis of variety of heterocyclic and carbocyclic
compounds.17 Considering their importance in the synthesis of var-
ious heterocycles, a novel series of 4-S-substituted pyrimidine
derivatives (2a–i) and 4-N-substituted pyrimidine derivatives (4a,
b) has been synthesized to assess their antileishmanial potential.
The protocol for the synthesis of pyrimidine derivatives begins with
the synthesis of substituted aryl ketene dithioacetals as described in
our previous articles.18a,b The ethanolic solution of these aryl dithio-
acetals (1a–i) on reaction with guanidine hydrochloride at 130 °C
gave the desired compounds (2a–i) in quantitative yield (Scheme 1).
Synthesis of 4-N-substituted pyrimidine derivatives (4a, b) was per-
formed in two steps. In the first step, dithioacetals (1d, 1g) were
q
CDRI Communication No. 8489.
⇑
Corresponding author. Tel.: +91 522 221 2411 18; fax: +91 522 222 3405.
E-mail address: shivajins@yahoo.co.in (S.N. Suryawanshi).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.060

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S. N. Suryawanshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5235–5238
NH2
N
di- and tri-methoxy substituted aryl ring containing 4-S-substi-
tuted pyrimidine derivatives (2a–c), compound 2b, having
dimethoxy aryl substitution, have shown better antileishmanial
in vitro activity with IC50 2.0 lM. No regular order of decrease
Cl
NH2
N
Cl
H3CO
H3CO
NH2
N
N
N
NH2
H2N
N
H2N
CH3
OCH3
or increase in the activity among these three derivatives was
observed. Rest of the compounds (2e, g and i) have shown good
to moderate in vitro activity (IC50 value 12.9, 3.6, 6.3 lM,
3
2
1
NH2
NH2
respectively). Almost, all the 4-S-substituted pyrimidine deriva-
tives were found better than reference drugs against intracellu-
lar amastigotes in vitro. In order to assess the effect of –SMe
group on the activity of 4-S-substituted pyrimidine derivatives,
we undertook some modification on the pyrimidine ring. 4-O-
substituted terpenyl pyrimidines displayed moderate antileish-
manial activity profile.19 Therefore, we decided to introduce a
nitrogen group at the 4-position of pyrimidine ring which is re-
quired for the DHFR inhibition. The synthetic strategy adopted
to have these changes is depicted in Scheme 2. The synthesized
4-N-substituted pyrimidine derivatives (4a, b) were screened
for their antileishmanial in vitro activity. These 4-N-substituted
pyrimidine derivatives displayed far better in vitro activity as
compared to their 4-S-substituted analogs. All the synthesized
compounds were found non-toxic to mammalian cell line
OMe
N
N
N
N
R4
R3
R4
R3
N
H
SMe
R1
R1
R2
R2
4(a-b)
2(a-i)
Figure 1. Structures of pyrimidines: trimethoprim 1, pyrimethamine 2, cycloguanil
3 and synthesized compounds 2(a–i) & 4(a–b) as antileishmanial agents.
reacted with anisidine to yield intermediate compound (3a, b). In
second step, these intermediates were allowed to react with guani-
dine hydrochloride at 135 °C to give the target compounds (4a, b)
(Scheme 2).The structures of all the synthesized compounds were
determined on the basis of their spectroscopic data and microanal-
ysis. The IR spectra of the compounds, in general, exhibited the
absorption band at around 3451 cmÀ1 indicating the presence of
heterocyclic amino group, absorption band of methyl group ap-
peared at around 2918 cmÀ1. The 1H & 13C NMR spectra are consis-
tent with the proposed structure. The 1H NMR spectra of a prototype
molecule 2d displayed exchangeable N–H proton signal as a singlet
at d 4.90 ppm. The two proton of benzyloxy methylene showed a
singlet at 5.09 ppm. and one multiplet at 7.39–7.47 (5H, C6H5) the
aromatic protons showed two doublets at 6.98 and 7.49 ppm with
coupling constant 9.00 Hz. The two trans olefinic protons showed
doublet at 6.69 and 7.66 ppm with coupling constant 16.00 Hz.
13C NMR spectra, exhibited the three aromatic carbons appeared
as signals at 128 and 129 ppm for aromatic carbons and styryl car-
bons at 124 and 134 ppm. Almost similar patterns were observed in
1H NMR and 13C NMR spectra of the rest of the compounds of series.
All the synthesized compounds were screened for their
in vitro antileishmanial activity against intramacrophagic L.
donovani amastigotes20,21. Antileishmanial in vitro screening re-
sults of all the synthesized compounds (2a–i and 4a, b) have
been reported in Table 1. Among the 4-S-substituted pyrimidine
derivatives (2a–i), compound 2d, having benzyloxy aryl substi-
tution, was found the most promising one with IC50 value of
(CC50 ranging from 57.8 to 375.9 lM) at activity concentration.
All the synthesized 4-S- and 4-N-substituted pyrimidine deriv-
atives, screened for their in vitro potential were also tested
in vivo against L. donovani/hamster model at dose of 50 mg/
kg  5 days by intraperitoneal route. There were no toxic symp-
toms observed during treatment of leishmania infected animals.
Among all, compound 2d has shown very promising in vivo
efficacy22 with 88.4% inhibition of parasite multiplication which
is comparable to the reference drug sodium stibogluconate
(88.5% inhibition) and slightly less to the miltefosine (98.1%
inhibition). However, compounds 2b, 2c, 2e, 2g and 2i have
shown moderate in vivo inhibition (in the range of 33–55%)
of parasitic growth as compared to the untreated control.
Although majority of the synthesized compounds have shown
better in vitro activity profile as compared to reference drugs
but the same trend could not be resulted in in vivo efficacy
of these compounds. To understand this discrepancy in both
in vitro and in vivo results, further pharmacokinetic and phar-
macodynamic studies are in progress in our lab and shall be
communicated in our future communication.
In summary, synthesis and biological evaluation of these 4-S-
substituted and 4-N-substituted pyrimidine derivatives led us to
discover compound 2d which showed very promising in vitro
2.0 lM and selectivity index (S.I.) of 188. Among the mono-,
NH2
O
SMe
SMe
N
N
R4
R3
CHO
R1
R4
R3
R4
R3
O
SMe
SMe
SMe
(i)
R1
+
R1
R2
R2
R2
2(a-i)
1(a-i)
Scheme 1. Synthesis of pyrimidine derivatives (2a–i). Reagents and conditions: (i) guanidine hydrochloride in isopropanol, ethanol, heat in steel bomb, 130 °C for 24 h.
OMe
NH2
OMe
N
N
O
HN
R4
R3
R4
R3
(ii)
(iii)
N
H
1d & 1g
SMe
R1
R1
R2
R2
4(a-b)
3(a-b)
Scheme 2. Synthesis of pyrimidine derivatives (4a and b). Reagents and conditions: (i) prim. amines, Ethanol, reflux, 9h; (ii) guanidine hydrochloride, isopropanol, heat in
steel Bomb at 135 °C, for 18 h.

S. N. Suryawanshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5235–5238
5237
Table 1
In vitro and in vivo antileishmanial activity and cytotoxicity results of synthetic pyrimidine derivatives 2a–i and 4a, b
NH2
NH2
OMe
N
N
N
N
R4
R3
R4
R3
SMe
N
H
R1
R1
R2
R2
2(a-i)
4(a-b)
Compd. No.
R1
R2
R3
R4
In vitro assessment
Selectivity Index (S.I.)
CC50/IC50
In vivo activity (Dose- 50 mg/kg  5 days, ipb)
% Inhibition SD
IC50
>40
(
l
M)
CC50
ND⁄
(lM)
2a
2b
2c
2d
2e
2f
2g
2h
H
H
H
H
H
H
H
H
H
H
H
—
—
OCH3
OCH3
H
H
H
OCH3
H
H
H
H
H
—
—
OCH3
OCH3
OCH3
OBn
OTHP
OH
OTHP
OH
OSO3Na
OBn
OTHP
—
OCH3
H
H
H
H
H
H
H
H
H
H
—
—
0
NA⁄
62
27
188
19
NA
34
NA
48
116
128
>7
NI⁄
2.0 0.2
9.1 0.8
2.0 0.1
12.9 1.2
ND
124.8 14.8
249.7 7.0
375.9 5.1
248.2 7.9
ND
47.1 28.7
54.4 12.5
88.4 10.6
33.5 29.4
ND
3.6 0.7
ND
121.8 4.8
ND
54.4 13.2
ND
2i
4a
6.3 0.6
0.5 0.1
2.7 0.5
59.8 7.5
12.5 0.9
301.1 10.9
57.8 5.9
345.4 19.6
55.1 10.5
78.1 17.7
78.2 4.4
88.5 4.4
98.1 1.0
4b
SSGa
>400
0
Miltefosinec
—
54.7 6.9
4
IC50 and CC50 values are the mean SD of two independent experiments.
See References and notes section for experimental details.
The selectivity index (S.I.) is defined as the ratio of CC50 on vero cells to IC50 on L. donovani intramacrophagic amastigotes.
ND⁄ = not done, NA⁄ = not available, NI⁄ = no inhibition.
a
SSG = Sodium stibogluconate (40 mg/kg  5 d, ip).
b
ip = Intraperitonial.
c
Miltefosine (30 mg/kg  5 d, po) used as a reference drugs.
12. Roberts, C. W.; Leod, R. M.; Rice, D. W.; Ginger, M.; Chance, M. L.; Goad, L. J. Mol.
Biochem. Parasitol. 2003, 126, 129.
13. Gilbert, I. H. Biochem. Biophys. Acta 2002, 1587, 249.
14. Muller, S.; Coombs, G. H.; Walter, R. D. Trends Parasitol. 2001, 17, 242.
15. Croft, S. L.; Coombs, G. H. Trends Parasitol. 2003, 11, 502.
16. Chandra, N.; Ramesh; Ashutosh; Goyel; Suryawanshi, S. N.; Gupta, S. Eur. J.
Med. Chem. 2005, 40, 552.
17. Suresh, J. R.; Kumar, U. K. S.; Junjappa, H. I.; Junjappa, H. Tetrahedron 2001, 57,
781.
antileishmanial activity against intracellular amastigotes and have
potential in vivo efficacy in the golden hamster model of VL. More-
over, it displayed no toxicity for macrophages and vero cells, and its
high value of selectivity index was much better than other current
antileishmanials. These findings revealed that these 4-S-substituted
and 4-N-substituted pyrimidine derivatives can be very useful for
further optimization work in antileishmanial chemotherapy.
18. (a) Pandey, S.; Surawanshi, S. N.; Gupta, S.; Srivastava, V. M. L. Eur. J. Med. Chem.
2005, 40, 751; (b) Kumar, S.; Tiwari, A.; Suryawanshi, S. N.; Mittal, M.;
Vishwakarma, P.; Gupta, S. Bioorg. Med. Chem. Lett. 2012, 22, 6728.
19. Pandey, S.; Surawanshi, S. N.; Gupta, S.; Srivastava, V. M. L. Eur. J. Med. Chem.
2004, 39, 969.
Acknowledgments
We gratefully acknowledge to the Division of Sophisticated
Analytical Instrument Facility (SAIF), CSIR-CDRI for providing spec-
tral and elemental data. Financial support from CSIR, New Delhi is
also gratefully acknowledged.
20. In vitro antileishmanial assay: In order to assess the activity of aryl pyrimidines
against amastigote stage of the parasite, the murine macrophage cell line (J-
774A.1) infected with luciferase-transfected promastigotes was used. Briefly,
cells (4 Â 103/100
lL) were seeded in 96-well plates. After 24 h incubation in
CO2 incubator, cells were infected with promastigotes (4 Â 104/100
lL).
Supplementary data
Promastigotes invade the macrophage and are transformed into amastigotes.
The test compounds in appropriate concentration (0.62–40
and plates were incubated in CO2 incubator for 72 h. After incubation, the
compound-containing medium was aspirated and 50 L PBS was added in
lM) were added
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
06.060.
l
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 (Pandey et al.,
2007). 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. (Pandey, Susmita;
Suryawanshi, S.N.; Nishi; Goyal, Neena; Gupta, Suman. Eur. J. Med. Chem. 2007,
42, 669).
References and notes
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21. Cytotoxicity assay: The cytotoxicity of compounds was determined by following
the method of Mosmann (1983) with some modifications. The monkey kidney
fibroblast cells (Vero cell line) were incubated with test compounds for 72 h
and MTT was used as reagent for detection of viable cells. Fifty percent
cytotoxic concentration (CC50) values were estimated as described by Huber &
Koella (1993). (Mosmann, T. J. Immunol. Methods 1983, 65, 55) (Huber, W.;
Koella, J. C.; Acta Trop. 1993, 55, 257).
22. In vivo antileishmanial assay: The in vivo antileishmanial activity was carried
out in golden hamsters infected with L. donovani as described by Kumar et al.
(2012). Briefly, golden hamsters (inbred strain) of either sex weighing 40–45 g
were infected intracardiacally with 1 Â 107 amastigotes per animal. Pre-
treatment spleen biopsies were performed after 15–20 days to assess the
degree of infection. The animals with +1 infection (5–15 amastigotes/100 liver
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11. Sajid, M.; Mckerro, J. R. Mol. Biochem. Parasitol. 2002, 120, 1.

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S. N. Suryawanshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5235–5238
cell nuclei) were included in the in vivo trials. After establishment of infection,
Giemsa staining. Intensity of infection in both, treated and untreated animals,
and also the initial count in treated animals was compared and the efficacy was
expressed in terms of per cent inhibition (PI) (Kumar, S; Tiwari, A;
Suryawanshi, S.N.; Mittal, M; Vishwakarma, P. and Gupta, S. Bioorg. Med.
Chem. Lett. 2012, 22, 6728).
drug treatment by either intraperitoneal (ip) or per oral (p.o.) route was
initiated for 5 consecutive days. Sodium stibogluconate (SSG) and miltefosine
are used as reference drugs. Post-treatment biopsies were done on day 7 after
the last dose of drug administration and amastigote counts are assessed by