Antimalarial activity of 2,4,6-trisubstituted pyrimidines

Article abstract of DOI:10.1016/j.bmcl.2005.02.015

A series of 2,4,6-trisubstituted pyrimidines (3a-o) was synthesized and evaluated for their in vitro antimalarial activity against P. falciparum. Out of the 15 compounds synthesized 11 compounds showed MIC in the range of 0.5-2 μg/mL. These compounds are

Full text of DOI:10.1016/j.bmcl.2005.02.015

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1881–1883
Antimalarial activity of 2,4,6-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 22 December 2004; revised 1 February 2005; accepted 4 February 2005
Abstract—A series of 2,4,6-trisubstituted pyrimidines (3a–o) was synthesized and evaluated for their in vitro antimalarial activity
against P. falciparum. Out of the 15 compounds synthesized 11 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
The design of novel chemical entities specially affecting
these targets could lead to better drugs for the treatment
of malaria.5,6
Malaria is one of the most widespread diseases in the
world. Each year, more than 100 million people are in-
fected and close to two million die, because they do
not receive adequate treatment.1–3 Malaria is caused
by different species of Plasmodium, of which P. falcipa-
rum is the most vicious one. It causes Malaria tropica,
which without treatment, is often lethal for the infected
patient. There are a number of effective drugs available
that interact in different ways with the biochemical life
cycle of the parasite (quinine, chloroquine, primaquine,
cycloguanil, pyrimethamine and proguanil), but as the
parasites rapidly develop permanent resistance against
the different subclasses, there is a great urge to develop
new and effective drugs.4 Pyrimethamine is a specific
inhibitor of the plasmodial DHFR, which is one of the
important target for drugs against malaria. The role of
DHFR is to catalyze the NADPH dependent reduction
of dihydrofolate to give tetrahydrofolate, a central com-
ponent in the single carbon metabolic pathway. The
tetrahydrofolate is methylated to methylene tetra-
hydrofolate, which is directly involved in thymidine
synthesis (assisting the methylation of deoxyuridine
monophosphate to give thymidine monophosphate)
and implicated in the metabolism of amino acids and
purine nucleotide. Inhibition of DHFR thus prevents
biosynthesis of DNA, leading to cell death.
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 and quinolines.8 In this study we report a
new prototype, having pyridine moiety along with
pyrimidine moiety as a new class for antimalarial
activity.
2. Chemistry
To synthesize the 2,4,6-trisubstituted pyrimidine com-
pounds (3), 4-acetylpyridine was reacted with different
aldehydes (a–o) in 10% aq NaOH and methanol to yield
the corresponding chalcones 2(a–o).9 Morpholine-4-carb-
oxamidine hydrochloride was synthesized by refluxing
morpholine with S-methylisothiourea sulfate in water
according to a reported procedure.10a The chalcones
2a–o were further cyclized with morpholine-4-carbox-
amidine hydrochloride in the presence of sodium iso-
propoxide (synthesized in situ by adding sodium metal
in isopropanol) to afford the 2,4,6-trisubstituted pyrimi-
dines 3(a–o) as shown in Scheme 1. In the cyclization
C2H5
N
Keywords: Dihydrofolate reductase; Antimalarial; Pyrimidine.
*
Cl
NH2
N
Corresponding author. Tel.: +91 522 2612411x4332; fax: +91 522
2623405; e-mail addresses: prem_chauhan_2000@yahoo.com;
premsc58@hotmail.com
H2N
1
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.015

1882
A. Agarwal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1881–1883
O
O
NH
a
.HCl
O
N
+
R
CHO
(a-o)
+
N
N
R
CH3
NH2
2(a-o)
NH
c
MeS
.H2SO4
2
b
R
N
N
O
NH2
R
N
N
H
N
N
N
H
N
N
O
A
3(a-o)
O
Scheme 1. Reagents and conditions: (a) different aldehydes, 10% aq NaOH, methanol, 0 °C to rt, 30 min; (b) (i) morpholine, S-methylisothiourea
sulfate, water, reflux, 15 min; (ii) barium chloride, reflux, 15 min; (c) morpholine-4-carboxamidine. HCl, sodium isopropoxide, isopropanol, reflux,
8 h.
Table 1. Antimalarial in vitro activity against P.falciparum
initially dihydropyrimidine (A) is formed, which is oxi-
dized in the presence of air to form the pyrimidine.10b
All the synthesized compounds were well characterized
by spectroscopic methods as IR, mass, NMR and ele-
mental analysis.14
Compd no.
R
ClogPa
MIC
3a
3b
3c
3d
3e
3f
C6H5
3.07
3.11
3.11
2.56
2.61
3.17
2.31
2.52
3.57
3.67
4.02
4.28
3.78
2.82
2.75
2
4-OMe-C6H4
3-OMe-C6H4
2,3-DiOMe-C6H3
2,5-DiOMe-C6H3
3,5-DiOMe-C6H3
2,4,5-TriOMe-C6H2
3,4,5-TriOMe-C6H2
4-Me-C6H4
2
2
1
1
2
3. Biological activity
3g
3h
3i
0.5
10
10
10
1
The in vitro antimalarial assay was carried out in 96 well
microtiter plates according to the micro assay of Rieck-
mann.11 The culture of P. falciparum NF-54strain is
routinely being maintained in medium RPMI-1640 sup-
plemented with 25 mM HEPES, 1% D-glucose, 0.23%
sodium bicarbonate and 10% heat inactivated human
serum.12 The asynchronous parasite of P. falciparum
was synchronized after 5% D-Sorbitol treatment to ob-
tain parasitized cells harbouring only the ring stage.13
For carrying out the assay, an initial ring stage parasita-
emia of ꢀ1% at 3% hematocrit in total volume of
200 lL of medium RPMI-1640 was uniformly main-
tained. The test compound in 20 lL volume at the re-
quired concentration (ranging between 0.25 lg and
50 lg/mL) in duplicate wells, were incubated with para-
sitized cell preparation at 37 °C in candle jar. After 36–
40 h incubation, the blood smears from each well were
prepared and stained with giemsa stain. The slides were
microscopically observed to record maturation of ring
stage parasites into trophozoites and schizonts in pres-
ence of different concentrations of compounds. The
tested concentration, which inhibits the complete matu-
ration 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.
3j
4-SMe-C6H4
3k
3l
3,4-DiMe-C6H3
4OMe-naphthyl
4Cl-C6H4
1
3m
3n
3o
1
10
1
3NO2-C6H4
2-Thiophene
1
10
MIC = Minimum inhibiting concentration for the development of ring
stage parasite into the schizont stage during 40 h incubation.
1: standard drug, pyrimethamine.
a ClogP value were calculated using the software chemdraw.
Phenyl substituted compound (3a) having a ClogP value
of 3.07, showed a MIC of 2 lg/mL. Substituting the phe-
nyl ring with methoxy group at the 4position ( 3b) and 3
position (3c) showed a ClogP value of 3.11 and a MIC
of 2 lg/mL. In dimethoxy substituted compounds, 3,5-
dimethoxy substituted compound (3f) has a ClogP value
of 3.17 and a MIC of 2 lg/mL. 2,3-Dimethoxy substi-
tuted (3d) and 2,5-dimethoxy substituted (3e) com-
pounds have a ClogP value of 2.56 and 2.61,
respectively, and a MIC of 1 lg/mL. The 2,4,5-trimeth-
oxy substituted compound (3g) have ClogP of 2.31 and
a MIC of 0.5 lg/mL. All these results emphasize that
with decrease in ClogP value activity increases. An ex-
ception to this, the 3,4,5-trimethoxy substituted compound
(3h), showed a ClogP value of 2.52 and a MIC of 10 lg/
mL. On substituting the phenyl ring with methyl (3i) and
S-methyl group (3j) the ClogP value increased to 3.57
and 3.67, respectively, thus showing a MIC value of 10
lg/mL. Exceptions to this were compounds having R
as 3,4-dimethylbenzene (3k) and 4-methoxynaphthalene
(3l), having a ClogP value of 4.02 and 4.28, respectively,
showing a MIC of 1 lg/mL. Substitution of chloro group
at 4position ( 3m) increased the ClogP value to 3.78
and thus the compound showed a MIC of 10 lg/mL.
4. Results and discussion
Among all the 15 compounds tested, 1 compound
showed MIC of 0.5 lg/mL whereas 6 compounds
showed MIC of 1 lg/mL and 4compounds have shown
MIC, 2 lg/mL. The compounds showed a good struc-
ture–activity relationship. Most of the compounds
showed a good correlation with ClogP value. In general
compounds showed increase in activity with the decrease
in ClogP value (lipophilicity).

A. Agarwal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1881–1883
1883
Agarwal, S. K.; Bhaduri, A. P.; Singh, S. N.; Fatima, N.;
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2000, 10, 1409.
Substitution of nitro group at 3 position (3n) reduced
the ClogP value to 2.82, thus showing a MIC of 1 lg/
mL. Substituting the benzene ring with thiophene ring
(3o) also reduced the ClogP value to 2.75 having a
MIC of 1 lg/mL. In general the compounds having
ClogP value in the range of 2.56–2.82 showed a MIC
of 1 lg/mL, whereas compounds having ClogP in the
range 3.07–3.17 showed a MIC of 2 lg/mL. Compounds
having ClogP value in the range of 3.57–3.78 showed a
MIC value of 10 lg/mL. The interaction of the drug
with the target has a complex nature so besides lipophili-
city various other factors also contribute to the activity
of the compound.
8. (a) Agarwal, A.; Srivastava, K.; Puri, S. K.; Chauhan, P.
M. S. Bioorg. Med. Chem. Lett. 2005, 15, 531; (b)
Shrivastava, S.; Tiwari, S.; Chauhan, P. M. S.; Puri, S.
K.; Bhaduri, A. P.; Pandey, V. C. Bioorg. Med. Chem.
Lett. 1999, 9, 653; (c) Shrivastava, S.; Tiwari, S.; Shriv-
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E. Lancet 1978, 1, 22.
5. Conclusion
The fifteen 2,4,6-trisubstituted-pyrimidines (3a–o) were
synthesized as pyrimethamine analogs. Out of the synthe-
sized compounds one compound have shown MIC of
0.5 lg/mL. Six compounds showed MIC of 1 lg/mL,
whereas four compounds showed MIC of 2 lg/mL.
These compounds are 5–20 times more potent than pyri-
methamine. The present study suggested that the newly
synthesized 2,4,6-trisubstitued pyrimidines are new leads
in antimalarial chemotherapy. These molecules can be
very useful for further optimization work in malarial
chemotherapy.
12. Trager, W.; Jensen, J. B. Science 1979, 193, 673.
13. Lambros, C.; Vanderberg, J. P. J. Parasitol. 1979, 65, 418.
14. Spectroscopic data for 3e MS: 379 (M+1), mp 142–144 °C;
IR (KBr) 2932, 1645, 1574, 1484, 1319, 1280 cmÀ1 1H
;
NMR (CDCl3, 200 MHz)
d (ppm) 8.77 (d, 2H,
J = 5.9 Hz), 7.95 (d, 2H, J = 5.9 Hz), 7.38 (s, 1H), 7.32
(s, 1H), 7.22 (d, 1H, J = 6.8 Hz), 6.71 (d, 1H, J = 6.8 Hz),
4.01 (t, 4H, J = 4.6 Hz), 3.87 (t, 4H, J = 4.6 Hz), 3.84 (s,
3H, OMe), 3.82 (s, 3H, OMe). 13C (CDCl3, 50 MHz):
164.9, 162.6, 162.2, 154.3, 152.9, 150.8, 146.1, 128.3, 121.6,
116.9, 116.6, 113.7, 107.9, 67.3, 56.9, 56.3, 44.8. Anal.
Calcd for C21H22N4O3: C, 66.65 H, 5.86; N, 14.81. Found:
C, 66.78; H, 5.98; N, 14.72. Spectroscopic data for 3g MS:
409 (M+1), mp 153–155 °C; IR 2926, 1638, 1580, 1480,
Acknowledgements
A.A. thanks the Council of Scientific and Industrial Re-
search (India) for the award of Senior Research Fellow-
ship. We are also thankful to S.A.I.F. Division, CDRI,
Lucknow for providing spectroscopic data. C.D.R.I.
Communication No. 6702.
1325, 1272 (KBr) cmÀ1 1H NMR (CDCl3, 200 MHz) d
;
(ppm) 8.75 (d, 2H, J = 6.1 Hz), 7.95 (d, 2H, J = 6.1 Hz),
7.80 (s, 1H), 7.72 (s, 1H), 6.62 (s, 1H), 3.97 (s, 3H, OMe),
4.02 (t, 4H, J = 4.6 Hz), 3.93 (s, 6H, 2OMe), 3.81 (t, 4H,
J = 4.6 Hz). 13C (CDCl3, 50 MHz): 164.5, 162.6, 162.1,
154.1, 152.3, 150.8, 146.3, 143.9, 121.6, 118.9, 114.2, 107.6,
98.3, 67.3, 57.2, 57.0, 56.6, 44.8. Anal. Calcd for
C22H24N4O4: C, 64.69; H, 5.92; N, 13.72. Found: C,
64.74; H, 5.86; N, 13.62. Spectroscopic data for 3k MS:
347 (M+1), mp 194–196 °C; IR 2948, 1636, 1584, 1486,
References and notes
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7. (a) Srivastava, S. K.; Chauhan, P. M. S.; Agarwal, S. K.;
Bhaduri, A. P.; Singh, S. N.; Fatima, N.; Chatterjee, R.
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Srivastava, S. K.; Agarwal, A.; Chauhan, P. M. S.;
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Srivastava, S. K.; Agarwal, A.; Chauhan, P. M. S.;
1325, 1265 (KBr) cmÀ1 1H NMR (CDCl3, 200 MHz) d
;
(ppm) 8.77 (d, 2H, J = 6.1 Hz), 7.97 (d, 2H, J = 6.1 Hz),
7.87 (s, 1H), 7.82 (d, 1H, J = 6.9 Hz), 7.42 (s, 1H), 7.23 (d,
1H, J = 6.9 Hz), 4.04 (t, 4H, J = 4.7 Hz), 3.84 (t, 4H,
J = 4.7 Hz), 2.37 (s, 3H), 2.34 (s, 3H). 13C (CDCl3,
50 MHz): 166.6, 162.9, 162.7, 150.8, 145.9, 140.2, 137.4,
135.6, 130.5, 128.6, 125.0, 121.5, 102.6, 67.4, 44.8, 20.3,
20.1. Anal. Calcd for C21H22N4O: C, 72.81; H, 6.40; N,
16.17. Found: C, 72.68; H, 6.22; N, 16.44. Spectroscopic
data for 3l MS: 399 (M+1), mp 160–162 °C; IR (KBr)
2936, 1642, 1578, 1488, 1324, 1284 cmÀ1 1H NMR
;
(CDCl3, 200 MHz) d (ppm) 8.76 (d, 2H, J = 6.1 Hz),
8.37–8.32 (m, 2H), 7.96 (d, 2H, J = 6.1 Hz), 7.70 (d, 1H,
J = 8.0 Hz), 7.55–7.52 (m, 2H), 7.30 (s, 1H), 6.92 (d, 1H,
J = 8.0 Hz), 4.07 (s, 3H, OMe), 4.02 (t, 4H, J = 4.6 Hz),
3.82 (t, 4H, J = 4.6 Hz). 13C (CDCl3, 50 MHz): 165.3,
162.5, 162.2, 161.9, 150.9, 145.5, 133.2, 129.9, 128.7, 127.6,
127.1, 125.9, 125.5, 122.9, 121.5, 107.6, 103.7, 67.4, 56.1,
44.9. Anal. Calcd for C24H22N4O2: C, 72.34; H, 5.57; N,
14.06. Found: C, 72.45; H, 5.65; N, 14.25.