Synthesis and evaluation of 2,4,6-trisubstituted pyrimidine derivatives as novel antileishmanial agents

Article abstract of DOI:10.1007/s00044-012-0167-y

A series of new 2,4,6-trisubstituted pyrimidine derivatives 8(a-j) were synthesized by reacting substituted chalcones containing imidazole 6(a-d) and benzimidazole 7(a-f) with guanidine hydrochloride in the presence of strong base. Substituted chalcones were synthesized by reacting 4-(1H-imidazol-1-yl) benzaldehyde or 4-(1H-benzo[d]imidazol-1-yl)benzaldehyde with different substituted acetophenones in the presence of 40 % NaOH in methanol. The synthesized compounds were confirmed by IR, ¹HNMR, and mass spectral data and screened for antileishmanial activity. Antileishmanial activity was performed against Leishmania donovani parasite, and percentage lysis inhibition were calculated by meglumine antimoliate taking a positive control and chloroform (0.1 % CHCl₃) treatment served as control. Among all the compounds, 8h and 8j exhibited 50-57 % inhibition against promastigotes, thus providing new structural lead for antileishmanials.

Full text of DOI:10.1007/s00044-012-0167-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:1756–1761
DOI 10.1007/s00044-012-0167-y
ORIGINAL RESEARCH
Synthesis and evaluation of 2,4,6-trisubstituted pyrimidine
derivatives as novel antileishmanial agents
•
•
•
S. K. Patle N. Kawathekar M. Zaveri
P. Kamaria
Received: 9 September 2011 / Accepted: 28 June 2012 / Published online: 21 July 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A series of new 2,4,6-trisubstituted pyrimidine
derivatives 8(a–j) were synthesized by reacting substituted
chalcones containing imidazole 6(a–d) and benzimidazole
7(a–f) with guanidine hydrochloride in the presence of
strong base. Substituted chalcones were synthesized by
reacting 4-(1H-imidazol-1-yl)benzaldehyde or 4-(1H-benzo
[d]imidazol-1-yl)benzaldehyde with different substituted
acetophenones in the presence of 40 % NaOH in methanol.
The synthesized compounds were confirmed by IR,
1HNMR, and mass spectral data and screened for antile-
ishmanial activity. Antileishmanial activity was performed
against Leishmania donovani parasite, and percentage lysis
inhibition were calculated by meglumine antimoliate tak-
ing a positive control and chloroform (0.1 % CHCl3)
treatment served as control. Among all the compounds, 8h
and 8j exhibited 50–57 % inhibition against promastigotes,
thus providing new structural lead for antileishmanials.
leishmaniasis are present in the world (Bouhlel et al.,
2010). This disease is mostly endemic in 88 countries in
the world, and visceral form of leishmaniasis is the most
severe type among all types of leishmaniasis, known as
kala-azar, which is caused by Leishmania donovani and is
nearly always fatal, if untreated (Sunduru et al., 2009).
More than 12 million people were infected by this disease
around the world (Bhandari et al., 2010). Five lakh new
cases of visceral leishmaniasis or kala-azar were found
every year (Perez-Victoria et al., 2006). Currently available
drugs are ineffective due to emergence of resistance among
parasites; also, chemotherapy of leishmaniasis is quite
difficult due to significant toxicity, variable efficacy, lack
of oral bioavailability, and high cost of the therapeutic
agents. So novel antileishmanial agents are urgently needed
(Srinivas et al., 2009).
Mostly the nitrogen heterocycles such as quinolines,
acridines, phenothiazines, pyrimidines, purines, bis-benz-
amidines, pyrazolo[3,4b]pyridine, benzothiazoles, and im-
idazolidine were reported as antileishmanial agents
(Agarwal et al., 2009).
Keywords Synthesis ꢀ Chalcones ꢀ Pyrimidines ꢀ
Antileishmanial activity
Most of the clinically used DHFR inhibitor shows less
selectivity for leishmanial enzymes because the gene for
pteridine reductase (PTR1) is amplified in some leish-
manial mutants. Antifolate drugs simultaneously target
both DHFR and PTR1 to be successful antileishmanials
because PTR1 can reduce pterins and folates, and there-
fore, act as a bypass for DHFR inhibition. A number of
compounds having pyrimidine moiety is reported to be
potent inhibitor of PTR1 in Leishmania (Sunduru et al.,
2006).
Introduction
Leishmaniasis is a parasitic disease caused by 17 species of
the protozoan parasite Leishmania (Chandra et al., 2005)
and transmitted by the bite of a sand-fly genus Phleboto-
mus. Three different forms of the diseases: visceral leish-
maniasis, cutaneous leishmaniasis, and mucocutaneous
S. K. Patle ꢀ N. Kawathekar (&) ꢀ M. Zaveri ꢀ P. Kamaria
Department of Pharmacy, Shri G.S. Institute of Technology
& Science (An Autonomous Institution), 23 Park Road,
Indore 452003, MP, India
Based on the above observations, we have synthesized
hybrid derivatives of pyrimidine containing imidazole or
benzimidazole. The synthesized compounds act as an
e-mail: neha_kawathekar@yahoo.co.in
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Med Chem Res (2013) 22:1756–1761
1757
F
Scheme 1 Synthesis of 2,4,6-
trisubstituted pyrimidine
derivatives. Reagent and
conditions: (I) K2CO3/
DMF
N
N
DMF
N
CHO
N
or
HN
+
HN
N
(Ib)
(Ia)
N
5
4
C16H33(CH3)3N?Br-, 100 °C,
stirrer, 28 h; (II) MeOH/NaOH/
25 °C, stirrer, 50–60 min; (IIIa
& IIIb) EtOH/NaOH/90 °C,
reflux, 16 h
2
3
CHO
R-COCH3
(IIb)
OHC
1
R-COCH3
(IIa)
N
N
O
N
N
O
7(a-f)
6(a-d)
R
(IIIb)
Guanidine.HCl
Guanidine.HCl
R
(IIIa)
R
NH2
N
N
N
N
N
8(e-j)
N
N
N
H2
N
R
8(a-d)
Table 1 General structures of synthesized compounds and anti-leishmanial activity of with respect to control
NH2
N
R1
N
R
Compound code
Molecular
formula
R1
R
H
Dose (lg/
ml)
Biological
activity*
8a
C19H15N5
10
32.81
46.14
24.32
18.14
41.86
N
N
N
N
N
N
N
N
8b
8c
8d
8e
C19H14ClN5
C19H14BrN5
C20H17N5O
C23H17N5
4-Cl
4-Br
4-
10
10
10
10
OCH3
H
N
N
123

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Med Chem Res (2013) 22:1756–1761
Table 1 continued
Compound code
Molecular
formula
R1
R
Dose (lg/
ml)
Biological
activity*
8f
8g
8h
8i
C
23H16FN5
4-F
10
10
10
10
10
29.64
32.67
56.42
32.49
51.18
N
N
N
N
N
N
N
N
N
N
C23H16ClN5
C23H15Cl2N5
C23H16BrN5
C24H19N5O
4-Cl
2,4-Cl
4-Br
4-
8j
OCH3
* Percentage lysis with respect to control
inhibitor of PTR1 as well as an inhibitor of DHFR and thus
act as potential antileishmanial agents.
Scheme 1I, III); (Trivedi et al., 2008; Rashinkar et al.,
2009).
Chemistry
Result and discussion
N-arylation of imidazole or benzimidazole was carried
out with para-fluorobenzaldehyde using hexadecyl-
trimethylammonium bromide as a catalyst to yield
compounds 4-(1H-imidazol-1-yl) benzaldehyde (4) or
4-(1H-benzo[d]imidazol-1-yl)benzaldehyde (5). Compounds
4 and 5 were reacted with substituted acetophenones in the
presence of base (40 % NaOH in methanol) to afford substi-
tuted chalcones by Claisen Schmidt condensation (6a–d,
Scheme 1I, IIa and 7a–f, 1I, IIb), (Hussain et al., 2009).
The formation of pyrimidine ring from chalcone pro-
ceeds via Michael reaction followed by cyclization (Micky
et al., 2006). Different substituted chalcones were cyclized
by guanidine hydrochloride in the presence of base using
ethanol as a solvent to yield pyrimidine derivative (8a–j,
Chemistry
Absorbtion band at 3,323–3,487 cm-1 which indicates
the presence of aromatic primary amino group (-NH2). IR
data confirms the presence of specific functional groups
present in all the synthesized compounds. Absorbtion at
813–740 cm-1; 542–594 cm-1; 1,053–1,230 cm-1, and
1,232–1,255 cm-1 indicates the presence of C–Cl, C–Br,
C–F, and C–OCH3 substitution groups in synthesized
compound, respectively.
The presence of 7.26–7.15 d ppm (1H, s, pyrimidine)
indicates the formation of pyrimidine ring at synthesized
compound. The presence of 5.89–6.45 d ppm (2H, s, -
NH2) indicates the amino group (-NH2) at synthesized
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Med Chem Res (2013) 22:1756–1761
1759
compound. 3.84 d ppm values indicate the presence of
methoxy group in synthesized compound.
crushed ice (200 ml). Pale yellow crystals obtained were filtered,
dried, and recrystallized from methanol (Hussain et al., 2009).
Biological activity
Synthesis of 4-(1H-benzo[d]imidazol-1-
yl)benzaldehyde (5; Scheme 1I, Ib)
Biological evaluation was performed against L. donovani
strain, and percentage lysis inhibition was calculated by
maglumine antimoniate as positive control (Table 1). 4-Cl
substitution on aromatic phenyl ring increases the biologi-
cal activity and 2,4 dichloro substitution on aromatic phenyl
ring further increases the biological activity. A 4-OCH3
substituted benzimidazole derivative was found to be more
potent than 4-OCH3 substituted imidazole derivatives. 8h
and 8j were the most potent compounds of the series and
shows more than 50 % inhibition against promastigotes.
A mixture of benzimidazole 3 (11.8 g, 100 mmol), anhy-
drous potassium carbonate (13.80 g, 100 mmol), 4-fluoro-
benzaldehyde 1 (12.40 g, 100 mmol), hexadecyltrimethyl
ammonium bromide (20 mg), and DMF (50 ml) was stirred
for a period of 28 h at 100 °C, and after cooling to room
temperature, was poured on to crushed ice (200 ml). Pale
yellow crystals obtained were filtered, dried, and recrystal-
lized from methanol.
Synthesis of 3-(4-(1H-imidazol-1-yl)phenyl)-1-
phenylprop-2-en-1-one (6a–d; Scheme 1I, IIa)
Conclusion
A methanolic sodium hydroxide solution (40 %, 10 mmol)
was added dropwise to a mixture of 4-(1H-imidazol-1-
yl)benzaldehyde (10 mmol,), acetophenone (10 mmol),
and methanol over a period of 50–60 min with continuous
stirring till completion of reaction (as indicated by TLC).
Precipitates obtained were filtered and washed with cold
methanol–water mixture (1:10). Finally, the product was
recrystallized from methanol (Hussain et al., 2009).
2,4,6-Trisubstituted pyrimidine derivatives were synthe-
1
sized and confirmed by IR, H NMR and Mass spectral
data and screened for antileishmanial activity. Compounds
8h and 8j exhibited 56.42 and 51.18 % inhibition against
promastigotes, respectively, thus providing lead structure
for antileishmanials.
Experimental
Synthesis of 3-(4-(1H-benzo[d]imidazol-1-yl)phenyl)-
1-phenylprop-2-en-1-one (7a–f; Scheme 1I, IIb)
Melting points of synthesized compounds were identified
by open capillary method and were uncorrected. Infrared
(IR) spectra were recorded using Shimadzu FTIR (SGSITS
Indore). Nuclear magnetic resonance (1H NMR) spectra
were recorded on Bruker Avance II, 400 MHz Spectro-
photometer (Panjab University) using DMSO as a solvent
and TMS as internal standard (Chemical shift in d ppm).
Spin multiplates are given as s (singlet), d (doublet),
t (triplet), and m (multiplet). Mass spectra were recorded on
GCMS solution\system\tune1\Auto Tuning-EI (IICT
Hyderabad). Biological activity of synthesized compounds
was performed from Pharmbio Research Center Pvt. Ltd.,
Indore.
A methanolic sodium hydroxide solution (40 %, 10 mmol)
was added dropwise to a mixture of 4-(1H-benzo[d]imi-
dazol-1-yl)benzaldehyde
(10 mmol),
acetophenone
(10 mmol), and methanol over a period of 50–60 min with
continuous stirring till completion of reaction (as indicated
by TLC). Precipitates obtained were filtered and washed
with cold methanol–water mixture (1:10). Finally, the
product was recrystallized from methanol (Hussain et al.,
2009).
Synthesis of 4-(4-(1H-imidazol-1-yl)phenyl)-
6-phenylpyridine-2-amine (8a–d; Scheme 1I, IIIa)
Synthesis of 4-(1H-imidazol-1-yl)benzaldehyde
(4; Scheme 1I, Ia)
Guanidine hydrochloride (3 mmol) was dissolved in 12 ml
5 % ethanolic solution of NaOH. Synthesized chalcones
(6a–d) was added to it and refluxed for 16 h. Solution was
cooled at room temperature and poured into the crushed
ice. Precipitate was filtered and washed with cold water.
The crude product was dried and recrystalised from ethanol
than methanol (Rashinkar et al., 2009).
A mixture of imidazole 2 (6.8 g, 100 mmol), anhydrous potas-
sium carbonate (13.80 g, 100 mmol), 4-fluorobenzaldehyde 1
(12.40 g, 100 mmol), hexadecyltrimethylammonium bromide
(20 mg), and DMF (50 ml) was stirred for a period of 28 h at
100 °C, and after cooling to room temperature, was poured on to
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Med Chem Res (2013) 22:1756–1761
4-(4-(1H-imidazol-1-yl)phenyl)-
6-(4 chlorophenyl)pyrimidin-2-amine (8a)
8.38(2H, d, Ar–H), 8.33(1H, s, imidazole), 8.28(2H, d,
Ar–H), 8.20–8.16(2H, m, Ar–H), 7.84(1H, d, imidazole),
7.75(3H, t, Ar–H), 7.38–7.35(2H, m, Ar–H), 7.26(1H, s,
pyrimidine), 6.04(2H, s, -NH2). Mass (m/z): 381.
% Yield: 42. MP (°C):234–236. IR (KBr, cm-1): 3109, 3454,
1520, 1051, 813. 1H NMR (DMSO, 400 MHz) d: 8.33(2H, d,
Ar–H), 8.20(3H, t, Ar–H), 8.05(1H, s, imidazole),
7.71–7.64(3H, m, Ar–H), 7.51(2H, d, imidazole), 7.15(1H, s,
pyrimidine), 6.45(2H, s, -NH2). Mass (m/z): 347.
4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-
6-(4-bromophenyl) pyrimidin-2-amine (8f)
% Yield: 45. MP (°C): 138–140. IR (KBr, cm-1): 3086,
1
4-(4-(1H-imidazol-1-yl)phenyl)-6-phenylpyrimidin-
2-amine (8b)
3410, 1521, 1234, 594. H NMR (DMSO, 400 MHz) d:
8.41(3H, t, Ar–H), 8.12(2H, d, imidazole), 7.81–7.73(3H,
m, Ar–H), 7.67–7.63(4H, m, Ar–H), 7.38–7.31(2H, m,
Ar–H), 6.38(2H, s, -NH2). Mass (m/z): 442.
% Yield: 40. MP (°C): 136–138. IR (KBr, cm-1): 3128,
3371, 1543, 1053. 1H NMR (DMSO, 400 MHz) d: 8.36(2H,
d, Ar–H), 8.28(3H, t, Ar–H), 8.23(1H, s, imidazole),
7.72(2H, d, imidazole), 7.66–7.61(4H, m, Ar–H), 7.26(1H,
s, pyrimidine), 5.89(2H, s, -NH2). Mass (m/z): 313.
4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-6-
phenylpyrimidin-2-amine (8g)
% Yield: 45. MP (°C): 185–187. IR (KBr, cm-1): 3086,
3323, 1544, 1224. 1H NMR (DMSO, 400 MHz) d: 8.30(2H,
d, Ar–H), 8.19(1H, s, imidazole), 8.10–8.07(2H, m, Ar–H),
7.92–7.8(1H, m, Ar–H), 7.67(2H,d, imidazole), 7.53–7.39
(4H, m, Ar–H), 7.39–7.35(2H, m, Ar–H), 7.26(1H, s,
pyrimidine), 5.28(2H, s, -NH2). Mass (m/z): 363.
4-(4-(1H-imidazol-1-yl)phenyl)-
6-(4-bromophenyl)pyrimidin-2-amine (8c)
% Yield: 46. MP (°C): 230–233. IR (KBr, cm-1): 3113,
1
3458, 1519, 1051, 586. H NMR (DMSO, 400 MHz) d:
8.31(2H, d, Ar–H), 8.10(3H, t, Ar–H), 7.94(1H, s, imid-
azole), 7.67–7.59(5H, m, Ar–H), 7.16(1H, s, pyrimidine),
6.31(2H, s, -NH2). Mass (m/z): 391.
4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-6-(4-
chlorophenyl) pyrimidin-2-amine (8h)
% Yield: 46. MP (oC): 161–164. IR (KBr, cm-1): 3101,
3433, 1514, 1010, 742. 1H NMR (DMSO, 400 MHz)
d:8.40(3H, t, Ar–H), 8.18(2H, d, Ar–H), 7.93(1H, s,
imidazole), 7.79(2H, d, imidazole), 7.65 (2H, t, Ar–H),
7.50(2H,d, Ar–H), 7.36–7.33(2H, m, Ar–H), 6.33(2H,s,
-NH2). Mass (m/z): 397.
4-(4-(1H-imidazol-1-yl)phenyl)-
6-(4-methoxyphenyl)pyrimidin-2-amine (8d)
% Yield: 40. MP (°C): 205–208. IR (KBr, cm-1): 3107,
1
3454, 1514, 1051, 1255. H NMR (DMSO, 400 MHz) d:
8.33(3H, t, Ar–H), 8.18(2H, d, imidazole), 7.76(3H, d, Ar–
H), 7.63(1H, s, imidazole), 7.14(1H, s, pyrimidine),
7.02(2H, d, Ar–H), 6.51(2H, s, -NH2), 3.85(3H, s, -OCH3).
Mass (m/z): 343.
4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-
6-(4-methoxyphenyl) pyrimidin-2-amine (8i)
% Yield: 48. MP (°C): 186–189. IR (KBr, cm-1):3070,
3477, 1512, 1031, 1232. H NMR (DMSO, 400 MHz) d:
Synthesis of 4-(4-(1H-benzo[d]imidazol-1-yl)phenyl)-
6-phenylpyridine-2 amine (8e–j; Scheme 1I, IIIb)
1
8.80(3H, t, Ar–H), 8.14(2H, d, Ar–H), 7.79(1H, d), 7.7(2H,
d, imidazole), 7.76(1H, d, Ar–H),7.58 (1H, s, pyrimidine),
7.38–7.30(2H, m, Ar–H),7.02(2H, d, Ar–H), 6.29(2H, s,
-NH2), 3.86(3H, s, -OCH3). Mass (m/z): 393.
Guanidine hydrochloride (3 mmol) was dissolved in 12 ml
5% ethanolic solution of NaOH. Synthesized chalcones
(7a–f) were added to it and refluxed for 16 h. Solution was
cooled at room temperature and poured into the crushed
ice. Precipitate was filtered and washed with cold water.
The crude product was dried and recrystallized from eth-
anol then methanol (Rashinkar et al., 2009).
4-(4-(1H-benzo[d]imidazol-1-yl) phenyl)-6-(2,4-
dichlorophenyl) pyrimidin-2-amine (8j)
% Yield: 50. MP (°C):128–130. IR (KBr, cm-1): 3070,
3485, 1521, 1105, 736. 1H NMR (DMSO, 400 MHz)
d:8.49(1H, s, imidazole), 8.23(2H, d, imidazole), 7.78(3H,
t, Ar–H), 7.76(2H, t, Ar–H), 7.59 (1H, s, pyrimidine),
7.47(1H, d, Ar–H), 7.37–7.29(3H, m, Ar–H), 6.73(2H,
s,-NH2). Mass (m/z): 431.
4-(4-(1H-benzo[d]imidazol-1-yl)phenyl)-
6-(4 fluorophenyl) pyrimidin-2-amine (8e)
% Yield: 42. MP (°C): 182–184. IR (KBr, cm-1):3086,
1
3487, 1510, 1014, 1230. H NMR (DMSO, 400 MHz) d:
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Biological activity
The splenic culture L. donovani was made in Medium-199
(L-glutamine with Hepes buffer without NaHCO3) supple-
mented with 10% fetal bovine serum (pH 7.2). The loga-
rithm phases of promastigotes (2 9 106 cells/ml) were
incubated with or without the isolates along with Medium-
199 at 22 °C. The samples were dissolved in 0.2% DMSO
and then added to the culture in doses of 10 lg/ml. After
2 h of treatment, all tubes were centrifuged at 8,0009g for
10 min, the supernatant was decanted and the pellets were
washed with 20 mM phosphate buffer saline (PBS). Each
pellet was dissolved in 100 ll (2 mg/ml) of MTT in
phosphate buffer saline.
All the tubes were incubated at 22 °C for 4 h and then
centrifuged at 8,0009g for 10 min. All the pellets were
dissolved in 500 ll DMSO and assessed by UV spectro-
photometry (UV 2060 plus—Analytical India) at 570 nm.
Percent of lysis of promastigotes by the compounds was
calculated using the standard formula of Tim Mosmann.
Chloroform (0.1% CHCl3) treatment served as control.
Srinivas N, Palne S, Nishi, Gupta S, Bhandari K (2009) Aryloxy
cyclohexyl imidazoles: a novel class of antileishmanial agents.
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Chauhana MS (2006) Synthesis of 2,4,6-trisubstituted pyrimi-
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Trivedi AR, Dodiya DK, Ravat NR, Shah VH (2008) Synthesis and
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Lysis ð%Þ ¼ 100 ꢁ ðT ꢁ PCÞ=ðC ꢁ PCÞ ꢂ 100
T is the test isolate, PC is the positive control—meglumine
antimoliate, C is the control.
ð1Þ
Acknowledgment The authors are grateful to Government of India
(Ministry of Human Resource Development) for financial support.
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