Synthesis and bioevaluation of hybrid 4-aminoquinoline triazines as a new class of antimalarial agents

Article abstract of DOI:10.1016/j.bmcl.2008.10.049

The emergence and rapid spread of chloroquine resistant strains of Plasmodium falciparum has dramatically reduced the chemotherapeutic options. Towards this goal, a series of new class of hybrid 4-aminoquinoline triazines were synthesized and screened aga

Full text of DOI:10.1016/j.bmcl.2008.10.049

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6530–6533
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and bioevaluation of hybrid 4-aminoquinoline triazines as a new
class of antimalarial agents
Ashok Kumar a, Kumkum Srivastava b, S. Raja Kumar b, S. K. Puri b, Prem M. S. Chauhan a,
*
a Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow 226001, India
b Division of Parasitology, Central Drug Research Institute, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The emergence and rapid spread of chloroquine resistant strains of Plasmodium falciparum has dramati-
cally reduced the chemotherapeutic options. Towards this goal, a series of new class of hybrid 4-amino-
quinoline triazines were synthesized and screened against CQ sensitive strain 3D7 of P. falciparum in an
in vitro model. Compounds 65 and 69 exhibited more than 99% suppression on day 4 and on day 6 post
treatment, compound 69 showed impressive 99.11% suppression against CQ resistant strain N-67 of P.
yoelii in an in vivo assay.
Received 1 August 2008
Revised 11 September 2008
Accepted 10 October 2008
Available online 14 October 2008
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Antimalarial
4-Aminoquinoline
Triazine
P. falciparum
Malaria is a major tropical parasitic disease, currently killing
more people than any other communicable disease aside from
tuberculosis. Today, malaria is transmitted in more than 100 coun-
tries. Some 500 million people in Africa, India, Southeast Asia and
South America are infected with malaria, with an estimated 2.5
million deaths annually, and about a million of these being chil-
dren under the age of five.1 Of the four species, Plasmodium falcipa-
rum is responsible for the most severe form of malaria, cerebral
malaria, and cause the majority of morbidity case.2 Chloroquine
(CQ) has been the drug of choice for antimalarial chemotherapy
since its discovery as it was safe, affordable, and effective but the
emergence and rapid spread of resistance of P. falciparum to CQ
and other related antimalarials has dramatically reduced the ther-
apeutic options3 (Fig. 1). The combination therapy was identified
as a strategic and viable option in improving the efficacy, and
delaying development and selection of resistant parasite.4 In this
context, development of hybrid antimalarial agents, combining 4-
aminoquinoline with other chemical entity having the antimalarial
potency, might prove to be an effective approach as 4-aminoquin-
oline based hybrid molecules can overcome the problem of drug
resistance, and have the low potential to induce the resistance to
parasite.5
red blood cells. This binding inhibits the formation of hemozoin,
resulting the accumulation of heme to toxic level that leads to
the death of parasite.6 CQ resistance in Plasmodium falciparum is
associated with mutations in the digestive vacuole (DV) trans
membrane protein, P. falciparum resistance transporter (pfCRT).7
Excessive export of CQ from its site of action in the DV is thought
to result from these mutations. The mechanism of Chloroquine
resistance (CQR) in P. falciparum probably involves specific struc-
tural interactions between Chloroquine and amino acid substitu-
tions in pfCRT that were slow to evolve because of their
complexity. Therefore the heme is still an attractive drug target
for the development of 4-aminoquinoline based antimalarials
which are not easily recognized by CQR mechanism.8
The comprehensive structure activity relationship studies on
CQ–Hematin binding have been explored to identify the optimum
structural requirements for designing the new antimalarial agents.
It was established that 7-chloro-4-aminoquinoline is critical for
the antimalarial activity.9–11 To counter the problem of drug resis-
N
HN
CQ and other structurally related antimalarials exert their effect
by binding to heme molecules released from the hemoglobin that
is digested by malaria parasites as they grow within their host
N
N
Cl
N
H2N
N
NH2
1. Chloroquine
* Corresponding author. Tel.: +91 522 221 2411x4332; fax: +91 522 262 3405.
E-mail addresses: prem_chauhan_2000@yahoo.com, premsc58@hotmail.com
(P.M.S. Chauhan).
2. Cycloguanil
Figure 1. Structure of antimalarial chloroquine and cycloguanil.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.049

A. Kumar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6530–6533
6531
tance, a diverse functionalization of the lateral side chain of chlo-
roquine with other antimalarial moiety led to the identification
of new 4-aminoquinoline based antimalarials effective against
both Chloroquine sensitive and chloroquine resistant strains of P.
falciparum. It included the 4-aminoquinoline-based isatin deriva-
tives,12 the 4-aminoquinoline-based b-carbolines,13 the peroxide-
based trioxaquine derivatives,14,15 ferrocene–chloroquine ana-
logs,16 or the 4-aminoquinolines based on inhibitors of a neutral
zinc aminopeptidase.17 Additionally, a new hybrid chloroquine
reversal agents have also been developed through coupling of
imipramine (reversal agent) with 4-aminoquinoline.18
Plasmodium falciparum dihydrofolate reductase (pfDHFR) is an
essential enzyme in the folate pathway and exists as a bifunctional
enzyme linked to thymidylate synthase (TS), and has been an
important target for malarial therapy.19 Pyrimethamine and cyclo-
guanil, the active metabolite of proguanil, are potent DHFR inhibi-
tors and are clinically used for P. falciparum malaria treatment.
Cyclogunil represents the triazine chemical class (Fig. 1). Our group
has previously identified substituted triazines as potential antima-
larial agents.20
We envisioned that incorporation of triazine moiety in the side
chain attached to 4-aminoquinoline pharmacophore would lead to
develop the new antimalarial agents active against chloroquine
resistance strains of P. falciparum. It was well established that basic
nature of side chain of the 4-aminoquinoline was crucial for accu-
mulation of the drug within acidic food vacuole of the parasite
along the pH gradient.10 Considering the importance of basicity
for antimalarial activity, triazine functionalized with piperazine
was linked to 4-aminoquinoline component. Herein, we report
the synthesis and evaluation of the antimalarial activity of new hy-
brid 4-aminoquinoline triazines against both chloroquine sensitive
3D7 and chloroquine resistant N-67 strains of P. falciparum.
The target compounds (52–70) were synthesized by a synthetic
protocol as outlines in Scheme 1. The synthesis of 2,4,6-trisubsti-
tuted-[1,3,5]triazines were achieved by consecutive nucleophilic
substitution of cyanuric chloride. The sequence of addition of
amines to cyanuric chloride depends upon the strength and struc-
ture of nucleophile. First chlorine of cyanuric chloride (3) was dis-
placed with the moderately nucleophilic aromatic amines at 0 °C
for 1 h to give the monosubstituted triazines (4–10) followed by
the treatment with more nucleophilic amine to provide the disub-
stituted triazines (11–29). Final chlorine of disubstituted triazines
was replaced with excess of piperazine (3 times) at 0 °C to afford
the corresponding 2,4,6-trisubstituted triazines (30–48). Coupling
of 4,7-dichloroquinoline (49) with excess of 2-amino ethanol gave
the 2-(7-chloro-quinolin-4-ylamino)-ethanol (50) in
a good
yield.21 Chemoselective o-mesylation was achieved in pyridine at
0 °C for 5 h to yield the methanesulfonic acid 2-(7-chloro-quino-
lin-4-ylamino)-ethyl ester (51).22 Chemoselectivity in the o-mes-
yaltion may be attributed to the presence of poor nucleophilic 4-
amino (NH) group owing to the conjugation of the lone pair of elec-
trons into the quinoline ring. Compound (51) was subjected to
nucleophilic substitution with trisubstituted triazines to yield the
corresponding targeted compounds (52–70) under microwave
condition. All the synthesized compounds were well characterized
by IR, mass, NMR, and elemental analysis.26
The antimalarial activity of all the synthesized compounds was
evaluated in an in vitro malaria assay against a chloroquine sensi-
tive strain of P. falciparum 3D7, according to a described protocol23
(Table 1). Variation of different substituents on the triazine nucleus
around four and six position has been explored to identify the bet-
ter possible combination of substituents for the improvement of
antimalarial potency. Among the 19 evaluated compounds of the
series, three compounds showed the antimalarial potency compa-
rable to CQ, six compounds displayed IC50 values ranging from
10.49 to 21.27 ng/mL and the rest of the compounds showed IC50
values in the range from 37.98 to 255.66 ng/mL. To study the effect
of substituents on the antimalarial activity, variation of the R2 sub-
stituents at position-6 have been done while keeping the R1 substi-
tuent at position-4 fixed. When aniline was placed at position-4,
compounds having piperidine (52) and cyclohexylamine (53) at
position-6 displayed almost equal antimalarial potency. Com-
pound with morpholine (54) at position-4 showed an IC50
21.27 ng/mL, while placement of N-ethyl piperazine (56) signifi-
cantly decreased the inhibitory activity as compared to the 52
and 53. Putting the aniline at both position (55) had the adverse ef-
NH
R1
N
Cl
R1
N
Cl
R1
N
N
Cl
N
Cl
a
b
c
N
N
N
N
N
N
N
N
R2
Cl
R2
Cl
4-10
3
11-29
30-48
O
O S
O
OH
HN
Cl
N
HN
e
d
Cl
N
Cl
Cl
N
50
51
49
R1
N
N
N
N
HN
N
N
R2
f
Cl
52-70
Scheme 1. Reagents and conditions: (a) aromatic amines/secondary amines, 0 °C temp, 1 h, THF; (b) secondary amines/primary amines, room temp, THF, 2 h; (c) piperazine
(3 times), 0 oC, 2 h, THF; (d) 1-amino ethanol, n-butanol, 90 °C, 8 h; (e) methanesulfonyl chloride, pyridine, 0 °C, 3 h; (f) trisubstituted triazines (1.5 times), MW, 30 s, NMP.

6532
A. Kumar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6530–6533
Table 1
compounds. Compound with substituents morpholine and N-form-
ylpiperazine (70) exhibited an IC50 10.49 ng/mL. Structure–activity
relationship studies based on the activity results obtained revealed
that combination of piperidine, cyclohexylamine, p-fluoroaniline,
aniline and morpholine as substituents on triazine nucleus were
well tolerated for the antimalarial activity.
All the compounds were also tested for their cytotoxicity on
VERO cells using MTT assay.24 Tested compounds were endowed
with selectivity index ranging from 27.76 to 4105.82. Most of the
compounds possessing good in vitro inhibitory activity have
shown decent selectivity index. Compound (70) having an IC50
10.49 ng/mL showed the highest selectivity index amongst the
tested compounds and compound (69), the most potent com-
pounds of the series displayed the selectivity index 481.48, thus
demonstrating good activity profile.
The most active 4-aminoquinoline triazine hybrids based on
their in vitro activity were screened in an in vivo model against
chloroquine resistant N-67 strain of P. yoelii in swiss mice at
50 mg/kg/day for 4 days by intraperitoneal route (ip)25 (Table 2).
The evaluated compounds have shown significant suppression, in
particular compounds 65 and 69 exhibited more than 99% suppres-
sion on day 4. On day 6 post treatment, compound 69 showed
impressive 99.11% suppression but could not provide significant
protection to the treated mice in 28 days survival assay. Further
structural optimization of 4-aminoquinoline triazine analogs may
lead to the development of the more potent molecules.
In vitro antimalarial activity against chloroquine sensitive strain 3D7 of P. falciparum
and in vitro cytotoxicity of compounds on VERO cell line
Compound
R1 (4-position)
R2 (6-position)
IC50 (ng/mL)
SI
116.72
154.18
469.57
27.76
41.81
377.35
75.32
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
CQ
Aniline
Aniline
Aniline
Aniline
Piperidine
Cyclohexylamine
Morpholine
Aniline
N-Ethylpiperazine
Morholine
17.82
16.23
21.27
75.64
37.98
19.16
185.88
194.42
64.57
255.56
43.48
19.01
38.26
7.15
Aniline
o-Toluidine
o-Toluidine
o-Toluidine
p-Toluidine
p-Toluidine
p-Toluidine
p-Toluidine
p-Toluidine
p-Fluoroaniline
p-Fluoroaniline
o-Fluoroaniline
Piperidine
Piperidine
Morpholine
o-Toluidine
Cyclohexylamine
N-Phenylpiperazine
Cyclohexylamine
Morpholine
N-Ethylpiperazine
p-Toluidine
Piperidine
Cyclohexylamine
Piperidine
28.97
52.19
114.96
512.09
41.03
155.25
328.61
261.81
14.95
7.83
159.16
44.38
4.43
10.49
2.6
Piperidine
Cyclohexylamine
N-Formylpiperazine
53.17
481.48
4105.82
8983
IC50: concentration corresponding to 50% growth inhibition of the parasite.
IC50 values of toxic activity
IC50 values of antimalarial activity
SI ¼
.
Table 2
In vivo antimalarial activity against chloroquine resistant strain N-67 of P. yoelii in
In conclusion, a series of new class of hybrid 4-aminoquinolin-
etriazine derivatives have been identified as potential antimalarial
agents. Many of the evaluated compounds have shown significant
antimalarial potency against CQ sensitive 3D7 strain of P. falcipa-
rum in an in vitro model. Some of the selected compounds, in par-
ticular compound 65 and 69 exhibited a promising antimalarial
activity in vivo against P. yoelii by ip route at a dose of 50 mg/kg/
day. The synthesis of easily accessible and affordable hybrid mole-
cules can be utilized in the development of antimalarial agents.
swiss mice at dose 50 mg/kg/day by intraperitoneal route
Compound
% Suppression on day 4
% suppression on day 6
52
53
65
66
69
CQ
66.67
86.43
99.9
74.81
99.9
55.36
78.73
96.51
71.75
99.11
94.26
99.9
fect on the antimalarial potency. In case of compounds having o-
toluidine as R1 substituent, interesting activity pattern has been
observed, compound (57) having morpholine at postion-6 showed
an IC50 19.16 ng/mL, whereas the compound (58) having the o-
toluidine at both position exhibited remarkably decreased inhibi-
tory activity with IC50 185.88 ng/mL, compound bearing the cyclo-
hexylamine at position-6 (59) exhibited low antimalarial potency
as compared to 53. Compound (60) bearing the p-toluidine and
N-phenylpiperazine had an IC50 64.57 ng/mL while compound
(63) with the p-toluidine and N-ethylpiperazine showed an IC50
19.01 ng/mL. It seems that bulky phenyl group exerted negative ef-
fect on the antimalarial potency. Combination of p-toluidine and
cyclohexylamine as substituents (61) showed an IC50 255.56 ng/
mL, while the replacement of cyclohexylamine with morpholine
(62) led to the increased inhibitory activity having an IC50
43.48 ng/mL. Compound 64 having the p-toluidine at both posi-
tions exhibited an IC50 38.16 ng/mL. Compound 64 was more ac-
tive than compound 55 and 58. This observation indicated that
substituting methyl in the aryl ring at para-position was favorable
for the antimalarial activity while ortho-substitution in the aryl
ring had the detrimental effect on the antimalarial potency. Intro-
duction of the fluoro group at para-position in the phenyl ring (65
and 66) increased the activity while fluoro group at ortho-position
(67) in the phenyl ring reduced the activity dramatically. Com-
pound (66) was more active than compound (61). So it was evident
that the nature and position of the substituent in the phenyl ring
influence the inhibitory activity. Compound (68) bearing piperi-
dine at both positions 4 and 6 had an IC50 44.38 ng/mL while com-
pound (69) having piperidine at position-4 and cyclohexylamine at
position-6 exhibited lowest IC50 4.43 ng/mL amongst the evaluated
Acknowledgments
A. Kumar thanks the Council of Scientific and Industrial Re-
search, 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.: 7594.
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concentration (IC50
)
was determined using non-linear regression analysis
dose–response curves and represented the concentration of compound
required to kill 50% of the fibroblast cells. Mosmann T. J. Immunol. Methods.
1983, 65, 55.
18. Burgess, S. J.; Selzer, A.; Kelly, J. X.; Smilkstein, M. J.; Riscoe, M. K.; Peyton, D. H.
J. Med. Chem. 2006, 49, 5623.
25. The in vivo drug response was evaluated in Swiss mice infected with P. yoelii
(N-67 strain) which is innately resistant to CQ. The mice (22 2 g) were
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22. Lundt, L.; Madsen, R. Synthesis 1992, 1129.
23. The compounds were evaluated for antimalarial activity against CQ sensitive
3D7 strain of P. falciparum BY SYBER Green I-based fluorescence (MSF) assay1.
The compounds were dissolved in DMSO at 5 mg/mL. For the assays, fresh
inoculated with 1 ꢀ 1 parasitized RBC on day
0
and treatment was
administered to group of five mice from day
a
0 to 3, once daily. The
aqueous suspensions of compounds were prepared with a few drops of Tween
80. The efficacy of test compounds was evaluated at 50 mg/kg/day and
required daily dose was administered in 0.2 mL volume via intraperitoneal
route. Parasitaemia levels were recorded from thin blood smears between days
4 and 6. The mean value determined for a group of five mice was used to
calculate the percent suppression of parasitaemia with respect to the
untreated control group. Mice treated with CQ served as reference controls.
Puri, S. K.; Singh, N. Exp. Parasitol. 2000, 94, 8.
dilutions of all compounds in screening medium were prepared and 50
l
l of
26. Spectroscopic data for 63: MS: 587 (M+1); IR (KBr): 3408, 2937, 1582, 1369,
highest starting concentration (500 ng/mL except for compound nos. 65, 66, 69
the highest starting concentration was 100 ng/mL) was dispensed in duplicate
wells in row B of 96 well tissue culture plate. The highest concentration for
chloroquine was 25 ng/ml. Subsequently two fold serial dilutions were
1003 cmꢁ1
;
1H NMR (CDCl3, 300 MHz): d (ppm) 8.54 (d, 1H, J = 5.22 Hz, Ar–
H), 7.98 (d, 1H, J = 1.92 Hz, Ar–H), 7.69 (d, 1H, J = 8.94 Hz, Ar–H), 7.41–7.38
(m, 3H, Ar–H), 7.01 (d, 2H, J = 8.31 Hz, Ar–H), 6.58 (br s, 1H, NH), 6.39 (d, 1H,
J = 5.22 Hz, Ar–H), 5.94 (br s, 1H, NH), 3.86–3.81 (m, 8H, NCH2), 3.39 (t, 2H,
J = 5.59 Hz, NH–CH2), 2.85 (t, 2H, J = 5.52 Hz, NCH2), 2.58 (t, 4H, J = 4.2 Hz,
NCH2), 2.48–2.42 (m, 6H, NCH2), 2.32 (s, 3H, CH3), 1.14 (t, 3H, J = 7.11 Hz,
CH2–CH3); 13C (CDCl3, 50 MHz): 165.62, 165.46, 164.83, 152.48, 150.14,
149.47, 137.32, 135.30, 132.17, 129.61, 129.14, 125.83, 121.49, 120.22,
117.73, 99.71, 56.03, 53.12, 52.98, 52.86, 43.67, 43.51, 39.32, 21.16, 12.35;
Anal. Calcd for C31H39ClN10: Calcd C 63.41, H 6.69, N 23.85. Found: C 63.58, H
6.72. N 23.98. Compound 69: MS: 550 (M+1); IR (KBr): 3415, 1591, 1450,
prepared up to row
H (seven concentrations). Finally 50 ll of 2.5%
parasitized cell suspension containing 0.5% parasitaemia was added to each
well except four wells in row A which received non-infected cell suspension.
These wells containing non-infected erythrocytes in the absence of drugs
served as negative controls, while parasitized erythrocytes in the presence of
CQ served as positive control. After 72 h of incubation, 100 ll of lysis buffer
[20 mM tris (pH 7.5), 5 mM EDTA, 0.008% (wt/vol) saponin, and 0.08% (vol/vol)
Triton X-100] containing 1ꢀ concentration of SYBER Green I (Invitrogen) was
added to each cell. The plates were re-incubated for 1 h at room temperature
and examined for the relative fluorescence units (RFUs) per well using the
FLUOstar, BMG lab technologies. The 50% inhibitory concentration (IC50) was
determined using non-linear regression analysis dose–response curves.
Smilkstein, M.; Sriwilaijaroen, N.; Kelly, J. X.; Wilairat, P.; Riscoe, M.
Antimicrob. agents Chemother. 2004, 48, 1803–1806.
1015, 853 cmꢁ1 1H NMR (CDCl3, 300 MHz): d (ppm) 8.55 (d, 1H, J = 5.37 Hz,
;
Ar–H), 8.01 (d, 1H, J = 2.01 Hz, Ar–H), 7.41 (dd, 1H, J = 2.01, 8.91 Hz, Ar–H),
7.73 (d, 1H, J = 8.91 Hz, Ar–H), 6.42 (d, 1H, J = 5.37 Hz, Ar–H), 6.12 (br s, 1H,
NH), 4.82 (br s, 1H, NH), 3.83 (t, 4H, J = 4.85 Hz, NCH2), 3.71 (t, 4H, J = 5.53 Hz,
NCH2), 3.38 (t, 2H, J = 5.45 Hz, NH–CH2), 2.83 (t, 2H, J = 6.09 Hz, NCH2), 2.57
(t, 4H, J = 4.65 Hz, NCH2), 2.02–1.98 (m, 2H, CH2), 1.76–1.72 (m, 2H, CH2),
1.64–1.56 (m, 6H, CH2), 1.39–1.31 (m, 2H, CH2), 1.27–1.18 (m, 4H, CH2); 13C
(CDCl3, 75 MHz): 165.11, 151.21, 150.23, 148.17, 135.28, 127.94, 125.58,
121.38, 117.17, 99.15, 55.62, 52.65, 49.17, 44.14, 43.19, 39.05, 33.21, 25.80,
24.94; Anal. Calcd for C29H40ClN9: Calcd C 63.31, H 7.33, N 22.91. Found: C
63.42, H 7.21, N 22.79.
24. Cytotoxicity of the compounds was determined against VERO cell lines (C-
1008; Monkey kidney fibroblast cells) using MTT assay. A total of 1 ꢀ 104 cells/
well were incubated with varying concentrations of compound for 72 h. The
highest concentration of compound was 100 lg/mL. The 50% inhibitory