Synthesis of 2-[3,5-substituted pyrazol-1-yl]-4,6-trisubstituted triazine derivatives as antimalarial agents

Article abstract of DOI:10.1016/j.bmcl.2005.08.023

A series of 22 compounds were synthesized and screened against Plasmodium falciparum NF-54 strain. Of the screened compounds, 6 compounds showed MIC in the range between 1 and 2 μg/mL. These compounds are 32 times more potent than the cycloguanil which wa

Full text of DOI:10.1016/j.bmcl.2005.08.023

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4957–4960
Synthesis of 2-[3,5-substituted pyrazol-1-yl]-4,6-trisubstituted
triazine derivatives as antimalarial agents
Sanjay Babu Katiyar,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 17 June 2005; revised 3 August 2005; accepted 4 August 2005
Available online 15 September 2005
Abstract—A series of 22 compounds were synthesized and screened against Plasmodium falciparum NF-54 strain. Of the screened
compounds, 6 compounds showed MIC in the range between 1 and 2 lg/mL. These compounds are 32 times more potent than the
cycloguanil which was used as the standard drug.
Ó 2005 Elsevier Ltd. All rights reserved.
About one-third of the worldꢀs population are currently
living with a serious risk of contracting malaria.1 It is
estimated that there are approximately 2–3 million
deaths every year from malaria,2 and in sub-Saharan
part of Africa alone about 1–2 million children below
the age of 5 years die of malaria every year.3 Despite
continuous research efforts of more than two decades,
no vaccine is yet discovered for effective control of
malaria. Treatment of malaria is becoming more diffi-
cult due to the spreading resistance of the parasite to
standard antimalarial drugs, in particular to chloro-
quine (CQ), which had been the affordable and effective
antimalarial mainstay for more than 50 years,4,5 to the
resistance of mosquitoes to insecticides, and due to cli-
matic changes that have enlarged areas of disease trans-
mission. It is commonly known that infection caused by
Plasmodium falciparum is frequently more fatal in chil-
dren than in adults, producing respiratory distress, neu-
rological problems, and severe anemia, and leading to
death in 5–35% of severe infections. 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 per-
manent resistance against the different subclasses, there
is a great urge to develop new and effective drugs.6
Cycloguanil and pyrimethamine are specific inhibitors
of the plasmodial DHFR, which is one of the important
targets for drugs against malaria.
The role of DHFR is to catalyze the NADPH-depen-
dent reduction of dihydrofolate to give tetrahydrofolate,
a central component in the single carbon metabolic
pathway. The tetrahydrofolate is methylated to methy-
lene tetrahydrofolate, which is directly involved in thy-
midine synthesis (assisting in the methylation of
deoxyuridine monophosphate to give thymidine mono-
phosphate) and implicated in the metabolism of amino
acids and purine nucleotide. Inhibition of DHFR thus
prevents biosynthesis of DNA, leading to cell death.
The design of novel chemical entities specially affecting
these targets could lead to better drugs for the treatment
of malaria.7,8
Previously, we had reported the antimalarial activity in
the substituted triazines, pyrimidines, indoles, and quin-
olines.9 Here, we report the antimalarial activity of new
substituted pyrazolyl triazine derivatives.
N
N
Cl
H2N
N
NH2
Cycloguanil
The synthesis of targeted compounds 4–25 is shown
in Scheme 1. The compound 3,3-bis-methylsulfanyl-1-
(substituted-phenyl)-propenone (1) was synthesized by
the reaction of substituted acetophenone with CS2 in
the presence of NaH followed by methylation with
Keywords: Dihydrofolate reductase; Antimalarial; Pyrazolyl triazine.
*
Corresponding author: Tel.: +91 522 2262411x4332; fax: +91 522
2623405; e-mail: Prem_chauhan_2000@yahoo.mail.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.08.023

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S. B. Katiyar et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4957–4960
H3CO
H3CO
H3CO
H3CO
SCH3
SCH3
H3CO
H3CO
CH3
O
I
II
O
N
N
H
SCH3
R
R
R
1
Substituted acetophenone
2
III
H3CO
H3CO
IV
H3CO
H3CO
N
N
N
N
N
SCH3
N
SCH3
Cl
R
R
N
N
N
R1
R1
Cl
N
3
4-25
Scheme 1. Reagents and conditions: (I) CS2, NaH, MeI, THF, 0 °C-rt (II) NH2NH2ÆH2O, MeOH, reflux. (III) Cyanuric chloride, K2CO3, THF,
reflux. (IV) Various amines, K2CO3, THF, reflux.
methyl iodide.10,11 The compound 1 was reacted with
hydrazine hydrate12,13 in methanol to obtain 5-meth-
ylsulfanyl-3-(substituted-phenyl)-1H-pyrazole (2). The
compound 2 was reacted with cyanuric chloride (2,4,
6-trichloro-1,3,5-triazine) in the presence of K2CO3 to
obtain 2,4-dichloro-6-[5-methylsulfanyl-3-(substituted-
phenyl)-pyrazol-1-yl]-[1,3,5]-triazine (3), which was sub-
jected to nucleophilic substitution with different amines
to afford the final targeted compounds 4–25 (Table 1).
All the synthesized compounds were well characterized
by spectroscopic methods such as IR, mass, NMR,
and elemental analysis.17
All the synthesized compounds 4–25 were evaluated for
their antimalarial activity against P. falciparum strain
NF-54 and the activity results are described in Table
1. Out of the 22 screened compounds, 6 compounds
(4, 7, 12, 13, 16, and 24) have shown MIC in the range
between 1 and 2 lg/mL concentrations and 6 com-
pounds (14, 15, 17, 19, 20, and 25) have shown MIC
of 10 lg/mL. The compound 4 has 3,4,5-trimethoxy
phenyl group at position 3 of the pyrazole ring and O-
tolyl amino group at positions 4 and 6 of the triazine
ring, while the compound 15 having the same substitu-
ents at positions 4 and 6 of the triazine ring and 3,4-di-
methoxy phenyl group at position 3 of the pyrazole ring
reduced the activity from 1 to 10 lg/mL. The in vitro
antimalarial result suggested that the O-tolyl amino
group at positions 4 and 6 of the triazine ring and
3,4,5-trimethoxy phenyl substituent at position 3 of the
pyrazole ring is very important in exhibiting antimalar-
ial activity. The compound 5 showed MIC of 50 lg/mL
having the 3,4,5-trimethoxy phenyl substituent at posi-
tion 3 of the pyrazole ring and N-benzyl piperazino
group at positions 4 and 6 of the triazine ring, while
the compound 16 has shown MIC of 2 lg/mL having
the same groups at positions 4 and 6 of the triazine
nucleus and 3,4-dimethoxy phenyl group at position 3
of the pyrazole ring. The compound 7, having 3,4,5-tri-
methoxy phenyl substituent at position 3 of the pyrazole
ring and benzyl amino group at positions 4 and 6 of the
triazine ring, has shown MIC of 2 lg/ mL, while the
compound 18 showed MIC of >50 lg/mL having benzyl
amino group at positions 4 and 6 of the triazine ring and
3,4-dimethoxy phenyl group at position 3 of the pyra-
zole ring; this indicates that the presence of 3,4-dime-
thoxy group at position 3 of the pyrazole ring, activity
was reduced. The compound 12 showed MIC of 2 lg/
mL, while the compound 23 showed MIC of 50 lg/
mL, both having the cyclohexyl amino group at
positions 4 and 6 of the triazine ring and different sub-
stituents at position 3 of the pyrazole ring. Compound
12 has 3,4,5-trimethoxy phenyl and compound 23 has
3,4-dimethoxy phenyl group at position 3 of the pyra-
zole ring. The compounds 13 and 14 have shown MIC
The in vitro antimalarial assay was carried out in 96-
well microtiter plates according to the micro assay of
Rieckmann.14 The culture of P. falciparum NF-54
strain is routinely maintained in RPMI-1640 medium
supplemented with 25 mM Hepes, 1% D-glucose,
0.23% sodium bicarbonate, and 10% heat-inactivated
human serum.15 The asynchronous parasite of P. fal-
ciparum was synchronized after 5% D-sorbitol treat-
ment to obtain parasitized cells harboring only the
ring stage.16 For carrying out the assay, an initial ring
stage parasitemia of ꢀ1% at 3% hematocrit in a total
volume of 200 lL of RPMI-1640 medium was uni-
formly maintained. The test compound in 20 lL vol-
ume at the required concentration (ranging between
0.25 lg and 50 lg/mL) in duplicate wells was incubat-
ed with parasitized 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 re-
cord maturation of ring stage parasites into trophozo-
ites and schizonts in the presence of different
concentrations of compounds. The tested concentra-
tion that inhibits the complete maturation into schizo-
nts was recorded as the minimum inhibitory
concentration (MIC). Cycloguanil was used as the
standard reference drug.
Activities of all the tested compounds are shown in
Table 1.

S. B. Katiyar et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4957–4960
Table 1. Antimalarial in vitro activity against P. falciparum
4959
of 1 and 10 lg/mL, respectively. Both have 3,4,5-tri-
methoxy phenyl substituent at position 3 of the pyrazole
ring and ethyl amino morpholino and propyl amino
morpholino groups, respectively, at positions 4 and 6
of the triazine ring. Compounds 24 and 25 having 3,4-di-
methoxy phenyl group at position 3 of the pyrazole ring
and ethyl amino morpholino and propyl amino mor-
pholino groups, respectively, at positions 4 and 6 of
the triazine ring have shown MIC of 2 and 10 lg/mL,
respectively. The antimalarial activity results have sug-
gested that increase in the chain length of the appropri-
ate amines decreases the activity of compounds.
Compound
R
R1
MIC (lg/mL)
H3C
4
OCH3
1
-HN
5
OCH3
50
N
N
CH2-Ph
6
7
OCH3
OCH3
N
>50
2
-HNH2C
8
9
OCH3
OCH3
OCH3
50
>50
50
N
N
N Ph
SAR results indicate that the 3,4,5-trimethoxy phenyl
and 3,4-dimethoxy phenyl groups at position 3 of the
pyrazole ring and O-tolyl amino, benzyl amino, cyclo-
hexyl amino N-ethyl amino morpholine, and N-benzyl
piperazino groups at positions 4 and 6 of the triazine
ring are crucial for antimalarial activity.
O
H3CO
10
N
N
N
C
H2
A series of 22 compounds were synthesized and screened
against P. falciparum NF-54 strain. Of the screened
compounds, 6 compounds have shown MIC in the range
between 1 and 2 lg/mL. These compounds are 32 times
more potent than the cycloguanil which was used as
standard drug. These molecules can be useful for further
lead optimization work in malaria chemotherapy.
11
12
13
14
15
OCH3
OCH3
OCH3
OCH3
H
>50
2
HN
1
HN(H2C)2
N
N
O
O
10
10
HN(H2C)3
H3C
Acknowledgments
S.B.K. 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., CDRI,
Lucknow, for providing spectroscopic data. This is
CDRI communication No. 6800.
-HN
16
H
2
N
N CH2-Ph
17
18
H
H
10
N
References and notes
>50
-HNH2C
N
1. Rogers, D. J.; Randolph, S. E. Science 2000, 289, 1763.
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chonwongpaisan, S.; Sirawarapornc, W.; Yuthavong, Y.
Bioorg. Med. Chem. 2003, 11, 217.
19
20
21
22
H
H
H
H
10
10
50
50
N
Ph
N
O
N
8. Zolli-Juran, M.; Cechetto, J. D.; Hartlen, R.; Daigle, D.
M.; Brown, E. D. Bioorg. Med. Chem. Lett. 2003, 13,
2493.
23
24
25
H
H
H
>50
2
HN
9. (a) Agarwal, A.; Srivastava, K.; Puri, S. K.; Chauhan, P.
M. S. Bioorg. Med. Chem. Lett. 2005, 15, 531; (b) Agarwal,
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HN(H2C)2
HN(H2C)3
N
N
O
O
10
MIC, minimum inhibiting concentration for the development of ring
stage parasite into the schizont stage during 40 h incubation.
MIC of cycloguanil = 64 lg/mL.

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129.88, 127.98, 120.69, 117.55, 104.32, 103.56, 61.23,
57.37, 56.42, 49.86, 44.06, 18.45. Anal. Calcd for
C38H45N9O3S: C, 64.47; H, 6.41; N, 17.81. Found: C,
64.73; H, 6.55; N, 17.67%; Spectroscopic data for
compound 15. Yield: 65%; mp 190–192 °C; FAB-MS:
540 (M+1); IR (KBr): 3311, 3035, 2961, 2833, 1588,
10. Thuillier, A.; Vialle, J. Bull. Chem. Soc. Fr. 1959, 13, 98.
11. Shahak, I.; Sasson, Y. Tet. Lett. 1973, 1, 4207.
12. Gompper, R.; Topfl, W. Chem. Ber. 1962, 95, 2881.
13. Chauhan, S. M. S.; Junjappa, H. Synthesis 1975, 798.
14. Rieckmann, K. H.; Sax, L. J.; Campbell, G. H.; Mrema,
J. E. Lancet 1978, 1, 22.
1505, 1434, 1404, 1375 cmÀ1
;
1H NMR (200 MHz,
CDCl3): d (ppm) 7.80 (d, 2H, J = 6.80 Hz, Ar-H), 7.58
(d, 1H, J = 1.68 Hz, Ar-H), 7.38 (dd, 1H, J = 1.66,
8.28 Hz, Ar-H), 7.25–7.10 (m, 6H, Ar-H), 6.90 (d, 1H,
J = 8.30 Hz, Ar-H), 6.37 (s, 1H, pyrazole-H), 3.99 (s,
3H, OCH3), 3.92 (s, 3H, OCH3), 2.43 (s, 3H, SCH3),
2.33 (s, 6H, Ar-CH3); 13C NMR (50 MHz, CDCl3): d
(ppm) 165.66, 162.95, 153.82, 154.75, 150.26, 149.39,
144.72, 146.42, 136.24, 139.41, 130.90, 126.78, 125.36,
119.78, 111.23, 109.76, 103.72, 56.48, 56.30, 18.59, 18.12.
Anal. Calcd for C29H29N7O2S: C, 64.54; H, 5.42; N,
18.17. Found: C, 64.45; H, 5.65; N, 18.24%; Spectro-
scopic data for compound 16. Yield: 62%; mp 195–
198 °C; FAB-MS: 678 (M+1); IR (KBr): 3015, 2936,
15. Trager, W.; Jensen, J. B. Science 1979, 193, 673.
16. Lambros, C.; Vanderberg, J. P. J. Parasitol. 1979, 65, 418.
17. Spectroscopic data for compound 4 Yield: 65%; mp
196–198 °C; FAB-MS: 570 (M+1); IR (KBr): 3365,
3065, 2935, 2828, 1578, 1509, 1428, 1382 cmÀ1
;
1H
NMR (300 MHz, CDCl3):
d (ppm) 7.81 (d, 2H,
J = 6.80 Hz, Ar-H), 7.40–7.10 (m, 8H, Ar-H), 6.37 (s,
1H, pyrazole-H), 3.99 (s, 6H, OCH3), 3.92 (s, 3H,
OCH3), 2.52 (s, 3H, SCH3), 2.32 (s, 6H, Ar-CH3); 13C
NMR (50 MHz, CDCl3):
d (ppm) 165.66, 162.95,
154.75, 153.19, 151.66, 149.49, 146.31, 139.44, 136.24,
130.90, 126.77, 125.36, 111.23, 104.66, 103.72, 61.55,
56.48, 18.59, 18.12. Anal. Calcd for C30H31N7O3S: C,
63.25; H, 5.48; N, 17.21. Found: C, 63.15; H, 5.57; N,
17.45%; spectroscopic data for compound 5. Yield: 61%;
mp 190–192 °C; FAB-MS: 707 (M+1); IR (KBr): 3031,
1
2810, 1592, 1501, 1396, 1354 cmÀ1; H NMR (200 MHz,
CDCl3): d (ppm) 7.52 (d,1H, J = 1.80 Hz, Ar-H), 7.50–
7.28 (m, 11H, Ar-H), 6.88 (d, 1H, J = 8.30 Hz, Ar-H),
6.40 (s, 1H, pyrazole-H), 3.95 (s, 3H, OCH3), 3.90 (s,
3H, OCH3), 3.90–3.82 (m, 8H, NCH2), 3.55 (s, 4H,
NCH2), 2.50–2.42 (m, 11H, SCH3, NCH2); 13C NMR
2933, 2803, 1621, 1578, 1501, 1419, 1370 cmÀ1
;
1H
NMR (200 MHz, CDCl3): d (ppm) 7.35–7.26 (m, 10H,
Ar-H), 7.11 (s, 2H, Ar-H), 6.37 (s, 1H, pyrazole-H),
4.01–3.85 (m, 8H, NCH2), 3.92 (s, 6H, OCH3), 3.87 (s,
3H, OCH3), 3.55 (s, 4H, NCH2), 2.50–2.39 (m, 11H,
NCH2, SCH3); 13C NMR (50 MHz, CDCl3): d (ppm)
165.44, 162.92, 154.30, 153.88, 151.68, 145.42, 139.33,
(50 MHz, CDCl3): d (ppm) 166.28, 162.15, 154.12,
150.09, 149.59, 145.58, 139.69, 128.85, 128.26, 127.52,
125.71, 119.49, 111.75, 109.85, 103.66, 56.23, 56.68,
44.28, 46.97, 45.85, 18.32. Anal. Calcd for
C37H43N9O2S: C, 65.56; H, 6.39; N, 18.60. Found: C,
65.45; H, 6.56; N, 18.72%.