Syntheses of 2,4,6-trisubstituted triazines as antimalarial agents

Article abstract of DOI:10.1016/j.bmcl.2004.11.052

A series of 2,4,6-trisubstituted-1,3,5-triazines (2a-s) were synthesized and evaluated for their in vitro antimalarial activity against P. falciparum. Out of the 19 compounds synthesized eight compounds showed MIC in the range of 1-2 μg/mL. These compounds are in vitro several times more active than cycloguanil.

Full text of DOI:10.1016/j.bmcl.2004.11.052

Bioorganic & Medicinal Chemistry Letters 15 (2005) 531–533
Syntheses of 2,4,6-trisubstituted triazines as antimalarial agents
a,
*
a
Anu Agarwal, Kumkum Srivastava, S. K. Puri and Prem M. S. Chauhan
b
b
a
Division of Medicinal & Process Chemistry, Central Drug Research Institute, Lucknow 226001, India
b
Division of Parasitology, Central Drug Research Institute, Lucknow 226001, India
Received 11 August 2004; revised 18 November 2004; accepted 19 November 2004
Available online 13 December 2004
Abstract—A series of 2,4,6-trisubstituted-1,3,5-triazines (2a–s) were synthesized and evaluated for their in vitro antimalarial activity
against P. falciparum. Out of the 19 compounds synthesized eight compounds showed MIC in the range of 1–2 lg/mL. These com-
pounds are in vitro several times more active than cycloguanil.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Despite years of continual effort, malaria is still one of
the most deadly diseases affecting third world countries
1
–3
claiming more than 2 million lives annually. Plasmo-
dium falciparum, the causative agent of the most malig-
nant forms of malaria is a particularly resistant parasite,
which is known to have high adaptability by mutation.
This mutability makes quite likely the development of
resistance to chemotherapies currently being intro-
Figure 1.
2. Chemistry
4
duced. A major thrust in this initiative is the identifica-
The 2,4,6-trisubstituted-1,3,5-triazine compounds (2,
Fig. 1) are synthesized from 2,4,6-trichloro-1,3,5-tri-
azine (cyanuric chloride) by reacting them with different
nucleophiles. The mono substituted triazine was synthe-
sized by refluxing cyanuric chloride with p-nitro aniline
in the presence of potassium carbonate in tetrahydrofu-
ran (THF). Normally the first substitution in cyanuric
chloride takes place at 0 °C but the amino group was
highly deactivated due to the presence of nitro group
at para position. The trisubstituted triazines (2) were
synthesized by refluxing the monosubstituted triazine
with different nucleophiles (R, Fig. 2) in the presence
of potassium carbonate in THF. All the synthesized
compounds were well characterized by spectroscopic
tion of new targets that are critical to the disease process
or essential for the survival of the parasite. The dihydro-
folate reductase (DHFR) domain of P. falciparum is one
of the few well defined targets in malarial chemotherapy.
The enzyme catalyzes the nicotinamide adenine dinucle-
otide phosphate (NADPH) dependent reduction of
dihydrofolate to tetrahydrofolate. DHFR has received
considerable attention as it is the target of cycloguanil
(
1) and other antifolates used for prophylaxis and treat-
5
ment of P. falciparum infection. The rapid emergence of
antifolate resistance in P. falciparum has unfortunately
compromised the clinical use of the currently used drugs
and thus highlights the urgent need for new effective
6
,7
antifolate antimalarials. The design of novel chemical
entities specially affecting selective parasitic folate
metabolism could lead to better drugs for the treatment
of malaria.
11
data as IR, mass, NMR and elemental analysis.
3. Biological activity
The in vitro antimalarial assay was carried out in 96-
well microtitre plates according to the microassay of
Rieckmann et al. The culture of P. falciparum NF-54
strain was routinely maintained in medium RPMI-
1640 supplemented with 25 mM HEPES, 1% D-glucose,
Keywords: DHFR; Triazine; Antimalarial.
*
8
Corresponding author. Tel.: +91 522 221 2411x4332; fax: +91
22 262 3405; e-mail addresses: prem_chauhan_2000@yahoo.com;
premsc58@hotmail.com
5
0
960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.052

532
A. Agarwal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 531–533
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-
tration, which inhibits the complete maturation into
schizonts, was recorded as the minimum inhibitory con-
centration (MIC). Chloroquine and cycloguanil were
used as the standard reference drug. Activity of all the
tested compounds is shown in Table 1.
4. Results and discussion
Among all the compounds tested, eight compounds
showed MIC in the range of 1–2 lg/mL whereas seven
compounds showed a MIC of 10 lg/mL. The results
showed a good structure–activity relationship. Results
also showed good correlation with logP and pK values,
a
which emphasize the importance of lipophilicity and
basicity in the antimalarial activity of the synthesized
compounds (2a–s). 1,3,5-Triazine having R as N-methyl
piperazine 2a showed a MIC of 1 lg/mL whereas when
the methyl group was replaced with benzyl 2b and phen-
yl group 2c it showed a MIC of 2 lg/mL and 10 lg/mL,
respectively. Compound 2a has a logP value of 0.228
whereas compound 2b and 2c have shown logP values
0.92 and 1.64, respectively, which shows that activity de-
creases with increase in lipophilicity. Compound 2a, 2b
Figure 2.
0.23% sodium bicarbonate and 10% heat inactivated
9
human serum. The asynchronous parasite P. falciparum
was synchronized after 5% D-Sorbitol treatment to ob-
0
1
tain parasitized cells harbouring only the ring stage.
For carrying out the assay, an initial ring stage parasita-
and 2c showed pK values of 1.88, 2.12 and 2.86, respec-
a
emia of ꢀ1% at 3% haematocrit in total volume of
tively, which shows that activity decreases with increas-
ing pKa or decreasing basicity of the compounds.
Morpholine substituted compound 2p showed a MIC
of 10 lg/mL whereas when morpholine was replaced
with 4-(3-aminopropyl) morpholine 2e and 4-(2-amino-
ethyl) morpholine 2f MIC reduced to 2 lg/mL. When
the morpholine group in 2e was replaced with imidazole
2
00 lL of medium RPMI-1640 was uniformly main-
tained. The test compound in 20 lL volume at required
Table 1. Antimalarial in vitro activity against P. falciparum
Compd no.
MIC (lg/mL)
logP
0.228
pK
a
2
d the activity increased showing a MIC of 1 lg/mL.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
a
b
c
d
e
f
1
2
1.88
2.12
2.86
1.96
2.26
2.28
2.58
2.64
2.32
2.91
3.05
3.68
2.88
3.13
2.94
2.98
3.95
3.62
2.86
3.82
These results emphasize the role of short aliphatic chains
and better efficacy of imidazole moiety over morpholine
moiety in the antimalarial activity. In the compounds
0.92
1.64
10
1
0.345
À0.14
À0.11
0.356
0.372
0.396
1.96
2
p, 2e and 2f the same relation between lipophilicity
2
and basicity with activity is observed. These compounds
showed a negative logP value on introduction of oxygen
atom in the molecule, its lipophilicity decreased to a
greater amount but its basicity was not affected. These
results emphasize the importance of both lipophilicity
and basicity in the activity. With R as cyclohexylamine
2
g
h
i
1
2
2
j
10
10
50
10
10
10
10
50
50
50
64
k
l
2.06
2.84
2
g MIC was 1 lg/ml whereas slightly increasing the ring
m
n
o
p
q
r
1.68
1.94
size to cycloheptylamine 2h activity reduced slightly hav-
ing a MIC of 2 lg/mL. In 2g and 2h, on increasing the
ring size the lipophilicity and pKa value increased
slightly so the activity decreased. When the R group
was n-butylamine 2i it showed a MIC of 2 lg/mL
whereas when chain length was increased to n-octyl-
amine 2j activity decreased having a MIC of 10 lg/
mL. Branching in the alkyl chain also does not favour
the activity. When the n-butylamine was replaced with
diethylamine 2k and t-butylamine 2l it showed a
decrease in activity having a MIC of 10 and 50 lg/mL.
1.64
À0.06
2.54
2.46
s
À0.56
2.94
MIC = minimum inhibiting concentration for the development of ring
stage parasite into the schizont stage during 40 h incubation.
logP and pK
a
values of all the compounds were calculated using
ChemSilico software.

A. Agarwal et al. / Bioorg. Med. Chem. Lett. 15 (2005) 531–533
533
Compounds 2i, 2j, 2k and 2l also followed the same rela-
tion of logP and pK values with activity. When the
References and notes
a
chain length was reduced to ethanolamine 2s activity
decreased further having a MIC of 50 lg/mL. In 2s the
logP value decreased extensively to À0.56 because of
the introduction of polar hydroxyl group in the mole-
1. Wahlgren, M.; Bejarano, M. T. Nature 1999, 400, 506–
07.
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2
. (a) Kumar, A.; Katiyar, S. B.; Agarwal, A.; Chauhan, P.
M. S. Curr. Med. Chem. 2003, 10, 1137–1150; (b)
Shrivastava, S. K.; Chauhan, P. M. S. Curr. Med. Chem.
cule whereas it showed pK value of 2.86. This molecule
a
2
001, 8, 1535–1542.
showed low activity in spite of the low logP value or low
lypophilicity. This shows that some other factors such as
basicity, etc., also contribute to the activity of the mol-
ecule. With R being cyclic secondary amines as piperi-
dine 2q and pyrrolidine 2r it showed a MIC of 50 lg/mL.
These results suggest that an unbranched aliphatic chain
of medium size favours activity. When the chain length
is increased or decreased above a particular size activity
decreases.
3
4
. Kumar, A.; Katiyar, S. B.; Agarwal, A.; Chauhan, P. M.
S. Drugs Fut. 2003, 28(3), 243–255.
. Enserink, M. Science 2000, 287, 1956.
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Pteridines; Blakley, R. L., Benkovic, S. J., Eds.; Wiley:
New York, 1984; Vol. 1, pp 191–253.
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Thebtaranonth, Y.; Yuthavong, Y. Bioorg. Med. Chem.
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Lancet 1978, 1, 22.
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7
8
9
5. Conclusion
The nineteen 2,4,6-trisubstituted-1,3,5-triazines (2a–s)
were synthesized as cycloguanil analogues. Out of the
synthesized compounds eight analogues have shown
MIC in the range of 1–2 lg/mL, 32–64 times more
potent than cycloguanil. These identified triazines can
be new leads in antimalarial chemotherapy. These mol-
ecules are very useful for further optimization work in
malarial chemotherapy.
1
0. Lambros, C.; Vanderberg, J. P., Jr. Parasitol. 1979, 65,
18.
11. Spectroscopic data for 3a: MS: 414 (M ), IR (KBr)
4
+
1
À1
1
3420, 2932, 1571, 1484, 1319, 1280 cm
(CDCl , 200 MHz): d (ppm) 8.18 (d, 2H, J = 8.6 Hz),
7.84 (d, 2H, J = 8.6 Hz), 3.85 (t, 2H, J = 4.9 Hz), 2.59 (t,
;
H NMR
3
1
3
2
H, J = 4.9 Hz), 2.34 (s, 3H); C (CDCl
70.1, 169.4, 152.3, 146.1, 129.9, 123.6, 59.9, 51.3, 48.2.
3
, 50 MHz):
1
Anal. Calcd for C H N O : Calculated C: 55.19, H:
1
9
27
9
2
6
3
1
.58, N: 30.49, O: 7.74. Found: C: 55.31, H: 6.82, N:
0.38, O: 7.49; 3d: MS 464 (M ), IR (KBr) 3409, 2943,
+
1
À1
1
582, 1494, 1326 cm ; H NMR (CDCl , 200 MHz): d
3
Acknowledgements
(
7
ppm) 8.15 (d, 2H, J = 8.7 Hz), 7.91 (d, 2H, J = 8.7 Hz),
.58 (s, 2H), 7.47 (d, 2H, J = 4.9 Hz), 7.04 (d, 2H,
J = 4.9 Hz), 4.09 (t, 4H, J = 5.11 Hz), 3.37 (m, 4H), 2.17
A.A. thanks the Council of Scientific and Industrial Re-
search (India) for the award of Senior Research Fellow-
ship. Thanks are also due to Farhana Samrin for her
help in synthesizing the compounds. We are also thank-
ful to S.A.I.F. Division, CDRI, Lucknow for providing
spectroscopic data. CDRI communication No 6646.
13
(
t, 4H, J = 5.0 Hz); C (CDCl , 50 MHz): 171.3, 169.4,
3
1
3
52.9, 145.6, 142.7, 134.0, 129.9, 124.6, 123.7, 49.5, 42.8,
6.3. Anal. Calcd for C21 : Calculated C: 54.42,
25 11 2
H N O
H: 5.44, N: 33.24, O: 6.90. Found: C: 54.36, H: 5.52, N:
33.28, O: 6.84.