Antimalarial activity and synthesis of new trisubstituted pyrimidines

Article abstract of DOI:10.1016/j.bmcl.2005.04.014

A series of 2,4,6-trisubstituted-pyrimidines was synthesized and evaluated for their in vitro antimalarial activity against Plasmodium falciparum. Out of the 30 compounds synthesized 21 compounds showed MIC in the range of 0.5-2 μg/mL. These compounds are

Full text of DOI:10.1016/j.bmcl.2005.04.014

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3130–3132
Antimalarial activity and synthesis of new trisubstituted pyrimidines
Anu Agarwal,a Kumkum Srivastava,b S. K. Purib and Prem M. S. Chauhana,*
aDivision of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226001, India
bDivision of Parasitology, Central Drug Research Institute, Lucknow 226001, India
Received 24 February 2005; accepted 8 April 2005
Available online 4 May 2005
Abstract—A series of 2,4,6-trisubstituted-pyrimidines was synthesized and evaluated for their in vitro antimalarial activity against
Plasmodium falciparum. Out of the 30 compounds synthesized 21 compounds showed MIC in the range of 0.5–2 lg/mL. These com-
pounds are in vitro several folds more active than pyrimethamine.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
essential for the DNA synthesis. The design of novel
chemical entities specially affecting these targets could
lead to better drugs for the treatment of malaria.4–6
Malaria is one of the major problems of many tropical
and subtropical countries in the world. It is estimated
that with 40% of the worldÕs population exposed to the
threat of malaria, there are an estimated 500 million clin-
ical cases per year and 2 million deaths. Malaria is caused
by protozoan parasites, namely Plasmodium falciparum,
Plasmodium vivax, Plasmodium ovale and Plasmodium
malariae, and is transmitted to humans by female mos-
quitoes belonging to the genus Anopheles. Endemic maps
indicates that P. falciparum and P. vivax account for 95%
of malaria infections.1,2 There are a number of effective
drugs available that interact in different ways with the
biochemical life cycle of the parasite (quinine, chloro-
quine, primaquine, cycloguanil, pyrimethamine, and
proguanil), but as the parasites rapidly develop perma-
nent resistance against the different subclasses, there is
a great urge to develop new and effective drugs.3 One
of the targets for drugs against malaria is the enzyme
dihydrofolate reductase (DHFR). Pyrimethamine (I) is
a specific inhibitor of the plasmodial DHFR, which is
As part of our ongoing program devoted to the synthesis
of diverse heterocycles as anti-infective agents,7 we had
previously reported antimalarial activity in substituted
triazines, pyrimidines, and quinolines.8
2. Chemistry
To synthesize the 2,4,6-trisubstituted-pyrimidine com-
pounds (3), 4-acetylpyridine (1) was reacted with differ-
ent aldehydes (a–j) in 10% aq-NaOH and methanol to
yield the corresponding chalcones 2(a–j).9 Methyl, ben-
zyl, and phenyl substituted piperazine-1-carboxamidine
hydrochloride (7x) were synthesized by refluxing either
methyl, benzyl, and phenyl piperazine (6x) with S-
methyl-isothiourea sulfate in water according to a re-
ported procedure.10 The chalcones (2a–j) were further
cyclized with different imidine hydrochlorides (7x) in
the presence of sodium isopropoxide (synthesized in situ
by adding sodium metal in isopropanol) to afford the
2,4,6-trisubstituted pyrimidines 3(a–j), 4(a–j), and 5(a–
j) as shown in Scheme 1. All the synthesized compounds
were well characterized by spectroscopic data as IR,
mass, NMR, and elemental analysis.14
3. Biological activity
Keywords: Dihydrofolate reductase; Antimalarial; Pyrimidine.
*
Corresponding author. Tel.: +91 522 221 2411x4332; fax: +91 522
262 3405; e-mail addresses: prem_chauhan_2000@yahoo.com;
premsc58@hotmail.com
The in vitro antimalarial assay was carried out in 96
well micro-titre plates according to the micro-assay of
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.014

A. Agarwal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3130–3132
3131
Scheme 1. Reagents and conditions: (a) Different aldehydes, 10% aq-NaOH, methanol, 0 °C–rt, 30 min. (b) (i) Different substituted piperazines,
S-methyl-isothiourea sulfate, water, reflux, 15 min; (ii) Barium chloride, reflux, 15 min. (c) Substituted piperazine carboxamidine, HCl,
sodiumisopropoxide, isopropanol, reflux, 8 h.
Rieckmann.11 The culture of P. falciparum NF-54 strain
is routinely being maintained in medium RPMI-1640
supplemented with 25 mM HEPES, 1% D-glucose,
0.23% sodium bicarbonate, and 10% heat inactivated
human serum.12 The asynchronous parasite of P. falci-
parum was synchronized after 5% D-Sorbitol treatment
to obtain parasitized cells harboring only the ring
stage.13
tration, which inhibits the complete maturation into
schizonts, was recorded as the minimum inhibitory
concentration (MIC). Pyrimethamine was used as the
standard reference drug. Activity of all the tested
compounds is shown in Table 1.
4. Results and discussion
For carrying out the assay, an initial ring stage parasita-
emia of ꢀ1% at 3% haematocrit in total volume of
200 lL of medium RPMI-1640 was uniformly main-
tained. The test compound in 20 lL volume at required
concentration (ranging between 0.25 lg and 50 lg/mL)
in duplicate wells, were incubated with parasitized cell
preparation at 37 °C in candle jar. After 36–40 h incuba-
tion, the blood smears from each well were prepared and
stained with giemsa stain. The slides were microscopi-
cally observed to record maturation of ring stage para-
sites into trophozoites and schizonts in presence of
different concentrations of compounds. The test concen-
Among all the 30 compounds tested, 2 compounds
showed MIC of 0.5 lg/mL whereas 9 compounds
showed MIC of 1 lg/mL and 10 compounds have shown
MIC, 2 lg/mL. The compounds showed a good struc-
ture activity relationship. When R was phenyl group
then compounds 3a, 4a, and 5a showed inhibition at a
concentration of 1 lg/mL, 2 lg/mL, and 10 lg/mL,
respectively. The activity was highest when the X group
was methyl. On substituting the methyl group (3a) with
benzyl group (4a), the MIC reduced slightly to 2 lg/mL
where as on substitution with phenyl group (5a) there
was a sharp decrease in the activity and the MIC
dropped to 10 lg/mL. Substitution on the phenyl ring
with methyl group (3b), thiomethyl group (3c), and
methoxy group (3e) at 4-position the activity was re-
tained at 1 g/mL. When the phenyl ring was di substi-
tuted with methyl group at 3 and 4 position (3d) or at
2 and 5 positions with methoxy group (3f) the activity
increased having a MIC of 0.5 lg/mL. This shows that
disubstitution on the phenyl ring favors the activity.
Where as when the phenyl ring was triply substituted
with methoxy group at 2,4,5 positions (3g) or 3,4,5 posi-
tions (3h) the activity reduced to 2 lg/mL and 10 lg/mL,
respectively. This emphasizes the fact that trisubstitu-
tion do not favor the activity. Substituting the phenyl
ring with nitro group at 3 position (3i) and chloro group
at 4 position (3j) showed a MIC of 1 lg/mL and 2 lg/
mL, respectively. In most of the cases (few exceptions
as 5b) compounds having X as methyl, benzyl, and
phenyl group the activity reduced when the methyl
group was substituted with benzyl group and further de-
creased when it was replaced with phenyl group.
Table 1. Antimalarial in vitro activity against P. falciparum
S. no.
R
MIC (lg/mL)
4(a–j)
X = CH3 X = CH2Ph X = C6H5
3(a–j)
5(a–j)
a
b
c
d
e
f
C6H5
4-Me-C6H4
1
2
2
10
1
1
4-SMe-C6H4
3,4-DiMe-C6H3
4OMe-C6H4
2,5-diOMe-C6H3
2,4,5-triOMe-C6H2
1
2
10
2
0.5
1
1
1
2
0.5
2
1
2
g
h
i
2
10
50
10
10
3,4,5-triOMe-C6H2 10
10
2
3NO2-C6H4
4Cl-C6H4
1
2
j
10
I
Pyrimethamine
10
MIC = Minimum inhibiting concentration for the development of ring
stage parasite into the schizont stage during 40 h incubation.
I: standard drug, pyrimethamine.

3132
A. Agarwal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3130–3132
2275–2279; (e) Tiwari, S.; Chauhan, P. M. S.; Bhaduri, A.
5. Conclusion
P.; Fatima, N.; Chatterjee, R. K. Bioorg. Med. Chem. Lett.
2000, 10, 1409–1412.
The thirty 2,4,6-trisubstituted-pyrimidines 3(a–j), 4(a–j),
and 5(a–j) were synthesized as pyrimethamine ana-
logues. Out of the synthesized compounds, two com-
pounds have shown MIC of 0.5 lg/mL. Nine
compounds showed MIC of 1 lg/mL, whereas 10 com-
pounds showed MIC of 2 lg/mL. These compounds
are 5–20 times more potent than pyrimethamine. These
identified pyrimidines are new lead in antimalarial che-
motherapy. These molecules can be very useful for fur-
ther optimization work in malarial chemotherapy.
8. (a) Agarwal, A.; Srivastava, K.; Puri, S. K.; Chauhan, P.
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Bhaduri, A. P.; Pandey, V. C. Bioorg. Med. Chem. Lett.
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Acknowledgements
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A.A. thanks the Council of Scientific and Industrial
Research (India) for the award of Senior Research
Fellowship. We are also thankful to S.A.I.F. Division,
CDRI, Lucknow for providing spectroscopic data.
CDRI Communication No. 6712.
14. Spectroscopic data for 3e: MS: 362(M+1); M.P. 152–
154 °C; IR (KBr) 2932, 1645, 1574, 1484, 1319, 1280 cmÀ1
;
1H NMR (CDCl3, 200 MHz) d (ppm) 8.75 (d, 2H, J =
5.9 Hz), 8.09 (d, 2H, J = 8.4 Hz), 7.95 (d, 2H, J = 5.9 Hz),
7.35 (s, 1H), 7.01 (d, 2H, J = 8.4 Hz), 4.05 (t, 4H,
J = 4.8 Hz), 3.88 (s, 3H, OMe), 2.54 (t, 4H, J = 4.8 Hz),
2.37 (s, 3H, NMe). 13C (CDCl3, 50 MHz): 165.8, 162.8,
162.6, 162.2, 150.8, 146.1, 130.6, 129.0, 121.5, 114.5, 101.6,
55.8, 55.5, 46.7, 44.2. Anal. Calc for C21H23N5O: Calcu-
lated C: 69.78, H: 6.41, N: 19.38. Found: C: 69.92, H: 6.54,
N: 19.51. Spectroscopic data for 4e: MS: 438(M+1); M.P.
148–150 °C; IR (KBr) 2926, 1638, 1580, 1480, 1325,
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1
1272 cmÀ1; H NMR (CDCl3, 200 MHz) d (ppm) 8.74 (d,
2H, J = 6.0 Hz), 8.09 (d, 2H, J = 8.8 Hz), 7.94 (d, 2H,
J = 6.0 Hz), 7.36 (s, 1H), 7.33–7.25 (m, 5H), 6.99 (d, 2H,
J = 8.8 Hz), 4.03 (t, 4H, J = 4.8 Hz), 3.87 (s, 3H, OMe),
3.58 (s, 2H), 2.57 (t, 4H, J = 4.8 Hz). 13C (CDCl3,
50 MHz): 165.8, 162.8, 162.6, 162.2, 150.8, 146.2, 138.4,
130.6, 129.6, 129.0, 128.7, 127.6, 121.5, 114.5, 101.5, 63.6,
55.8, 53.5, 44.3. Anal. Calc for C27H27N5O: Calculated C:
74.12, H: 6.22, N: 16.01. Found: C: 74.25, H: 6.34, N:
16.15. Spectroscopic data for 5e MS: 424(M+1); M.P.
158–160 °C; IR (KBr) 2948, 1636, 1584, 1486, 1325,
6. Rastelli, G.; Sirawaraporm, W.; Sompornpisut, P., et al.
Bioorg. Med. Chem. 2000, 8, 1117–1128.
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Agarwal, S. K.; Bhaduri, A. P.; Singh, S. N.; Fatima, N.;
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Srivastava, S. K.; Agarwal, A.; Chauhan, P. M. S.;
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1
1265 cmÀ1; H NMR (CDCl3, 200 MHz) d (ppm) 8.76 (d,
2H, J = 6.0 Hz), 8.11 (d, 2H, J = 8.7 Hz), 7.95 (d, 2H,
J = 6.0 Hz), 7.37 (s, 1H), 7.26 (d, 2H, J = 8.4 Hz), 7.01 (d,
2H, J = 8.7 Hz), 6.93–6.86 (m, 3H), 4.18 (t, 4H,
J = 4.7 Hz), 3.88 (s, 3H, OMe), 3.31 (t, 4H, J = 4.7 Hz).
13C (CDCl3, 50 MHz): 165.9, 162.9, 162.6, 162.3, 151.9,
150.8, 146.1, 130.5, 129.6, 129.1, 121.5, 120.6, 116.9, 114.5,
101.9, 55.8, 49.9, 44.3. Anal. Calc for C26H25N5O:
Calculated C: 73.74, H: 5.95, N: 16.54. Found: C: 73.88,
H: 5.87, N: 16.71.