Synthesis of 9-anilinoacridine triazines as new class of hybrid antimalarial agents

Article abstract of DOI:10.1016/j.bmcl.2009.10.010

There is challenge and urgency to synthesize cost-effective chemotherapeutic agents for treatment of malaria after the widespread development of resistance to CQ. In the present study, we synthesized a new series of hybrid 9-anilinoacridine triazines usin

Full text of DOI:10.1016/j.bmcl.2009.10.010

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6996–6999
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 9-anilinoacridine triazines as new class of hybrid
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 226 001, India
b Division of Parasitology, Central Drug Research Institute, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
There is challenge and urgency to synthesize cost-effective chemotherapeutic agents for treatment of
malaria after the widespread development of resistance to CQ. In the present study, we synthesized a
new series of hybrid 9-anilinoacridine triazines using the cheap chemicals 6,9-dichloro-2-methoxy acri-
dine and cyanuric chloride. The series of new hybrid 9-anilinoacridine triazines were evaluated in vitro
for their antimalarial activity against CQ-sensitive 3D7 strain of Plasmodium falciparum and their cytotox-
icity were determined on VERO cell line. Of the evaluated compounds, two compounds 17
(IC50 = 4.21 nM) and 22 (IC50 = 4.27 nM) displayed two times higher potency than CQ (IC50 = 8.15 nM).
Most of the compounds showed fairly high selectivity index. The compounds 13 and 29 displayed
>96.59% and 98.73% suppression, respectively, orally against N-67 strain of Plasmodium yoelii in swiss
mice at dose 100 mg/kg for four days.
Received 20 August 2009
Revised 17 September 2009
Accepted 5 October 2009
Available online 29 October 2009
Keywords:
Antimalarial
9-Anilinoacridine triazine
Plasmodium falciparum
Ó 2009 Elsevier Ltd. All rights reserved.
The burden of malaria on the health and economics of the sub-
tropical countries is increasing due to the rapid spread of resis-
tance to the most of the classical drugs in use. It is estimated
that malaria affects the 500 million people across the world, causes
approximately 2.5 million deaths every year especially among the
children under the age of five years.1 Plasmodium falciparum is the
most virulent form of parasite among the four plasmodium species
infecting humans and is responsible for most of mortality.2 Cur-
rently an effective therapeutic option for the treatment of resistant
malaria is combination therapy based on the natural endoperoxide
artemisinin and its semisynthetic derivatives.3 The combination
therapy is a viable option as the combination of drugs delays the
selection of resistance to the parasite but the cost of treatment is
much higher than monotherapy.4 So there is an urgent need for
new safer, more efficacious and affordable antimalarial agents as
the disease is prevalent mainly in the poor subtropical countries.
The antimalarial drugs based on 4-aminoquinoline scaffold
have been the mainstay of the malaria chemotherapy (Fig. 1) but
the emergence of resistance to most commonly employed drugs
like chloroquine (CQ), quinine, and mefolquine has limited their
use in treatment.5 So it is imperative to investigate other biologi-
cally important nucleus for the development of new antimalarial
agents. In this context acridine nucleus offers an alternative to
quinoline moiety. Quinacrine was the first synthetic antimalarial
used before chloroquine. Pyronaridine is a new highly active blood
schizonticidal mannich base antimalarial developed in china, effec-
tive against chloroquine resistant strain of P. falciparum.6 Over the
last few years acridine derivative mainly based on 9-aminoacridine
and 9-anilinoacridine scaffold have been reported for antimalarial
activity.7–10 Moreover, acridine moiety possess a wide range of bio-
logical activities including antitumor,11 antiprion,12 anti-alzhei-
mer,13 antileishmanial, and antitrypanosomal14 activity. Various
CH3
N
N
HN
N
HN
N
OMe
Cl
Cl
1
2
R1
N
N
N
N
H
N
OH
R2
N
HN
HN
O
O
Cl
N
Cl
N
3
* 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).
11-32
Figure 1. Structure of CQ, quinacrine, pyronaridine and title compounds.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.010

A. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6996–6999
6997
modes of action of acridine derivatives for their antimalarial effect
have been proposed including DNA intercalation,15 binding to
heme,16 and inhibition of the enzyme topoisomerase II.17
4,6-dichloro-6-substituted-[1,3,5]triazine 5 and 6 were synthe-
sized by nucleophilic substitution of cyanuric chloride with
amines. Commercially available 6,9-dichloro-2-methoxyacridine
7 was condensed with p-phenylenediamine to give the N-(6-
Designing hybrid drugs with multiple effects is a common strat-
egy in today’s search for new treatment of malaria. In recent years,
various structurally diverse hybrid molecules were reported for the
antimalarial activity. It included the 4-aminoquinoline-based isat-
in derivatives,18 the 4-aminoquinoline-based b-carbolines,19 the
peroxide-based trioxaquine derivatives,20,21 ferrocene–chloro-
quine analogues,22 the 4-aminoquinolines based on inhibitors of
a neutral zinc aminopeptidase,23 the 4-aminoquinoline-based pyr-
azole analogues,24 the 4-aminoquinoline-based tetraoxanes,25 and
the clotrimazole-based 4-aminoquinolines.26 More recently, a new
dual function acridones as a new antimalarial chemotype having
the heme-targeting character of acridones along with a chemosen-
sitizing component able to reverse the CQ-resistance were
discovered.27
Triazine and dihydrotriazine derivatives have been reported to
possess antimalarial activity.28–31 Today, cycloguanil used for pro-
phylaxis and treatment of malaria lost its efficacy due to emer-
gence of resistance which warrants further investigation for
developing new antimalarials. As part of our research efforts de-
voted to synthesis of nitrogen heterocycles as antimalarial agents,
our group have identified trisubstituted triazines, pyrazole based
triazines and hybrid 4-aminoquinoline triazines as antimalarials.32
We envisaged that combining triazine with 9-anilinoacridine
would lead to develop more efficacious antimalarial molecules.
In this Letter, we report the synthesis and evaluation for in vitro
and in vivo antimalarial activity of a series of hybrid 9-anilinoacri-
dine triazines as well as determination of their cytotoxicity.
The synthesis of title compounds 11–32 were achieved by a
synthetic protocol as depicted in Scheme 1. To begin with
chloro-2-methoxy-acridinyl-9-yl)-benzene-1,4-diamine
8 using
the p-TSA as a catalyst.33 The compound 8 was refluxed with 4,6-
dichloro-6-substituted-[1,3,5]triazines in THF to yield the corre-
sponding 6-chloro-N-[4-(6-chloro-2-methoxy-9-acrdinyl-9-ylami-
no)-phenyl]-N0-substituted-[1,3,5]triazine-2,4-diamine 9 and 10.
The compounds 9 and 10 were subjected to nucleophilic substitu-
tion with various amine to give the title compounds 11–32 (Table
1). All the synthesized compounds were well characterized by IR,
mass, NMR, and elemental analysis.34
All the synthesized compounds were evaluated in vitro for their
antimalarial activity against CQ-sensitive strain 3D7 of P. falcipa-
rum using the described standard protocol.35 The activity results
are summarized in Table 1. Variations of different substituents
on the triazine nucleus around position-4 and position-6 have been
explored to ascertain the structure–activity relationship among the
synthesized compounds of the series. Among the 22 evaluated
compounds of the series, two compounds showed the two times
higher antimalarial potency than CQ, three compounds displayed
the antimalarial activity comparable to CQ, 13 compounds exhib-
ited the IC50 values ranging from 15.08 to 68.39 nM and the four
compounds showed the IC50 values ranging from 115.8 to
365.29 nM. While keeping the aniline or p-fluoroaniline as substi-
tuent at position-4, various amines were placed at position-6 to
study the effect of substituent on the antimalarial potency. Consid-
ering the importance of fluorine in medicinal chemistry, aniline
was replaced with p-fluoroaniline at position-4 around the triazine
nucleus. However, activity results clearly suggest that the com-
pound bearing aniline at position-4 around triazine nucleus were
Cl
N
Cl
Cl
N
R1
a
N
N
N
N
Cl
4
H
N
Cl
5; R1 =
6; R1 =
H
N
F
R1
N
N
N
H
NH2
OCH3
N
Cl
HN
N
Cl
OCH3
HN
N
OCH3
b
c
Cl
N
Cl
Cl
7
H
8
9; R1 =
N
H
10; R1 =
N
F
R1
N
N
H
N
N
R2
HN
N
OCH3
d
Cl
11-32
Scheme 1. Reagents and conditions: (a) aniline/p-fluoroaniline, THF, 0 °C temp, 2 h; (b) p-phenylenediamine, p-TSA, absolute ethanol, reflux, 3 h; (c) monosubstituted
triazines, dry THF, reflux, 10 h; (d) various amines, dry THF, 65 °C, 4 h.

6998
A. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6996–6999
Table 1
In vitro antimalarial activity of title compounds against CQ-sensitive strain 3D7 of P. falciparum and their cytotoxicity on VERO cell line
Compound
R1
R2
IC50 (nM)
SI
146.41
164.46
2896.02
61.38
159.75
136.85
295.02
138.19
5008.61
754.62
17.45
315.39
448.48
210.17
193.17
28.12
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
CQ
Aniline
Aniline
Aniline
Aniline
Aniline
Aniline
Aniline
Aniline
Aniline
Aniline
Aniline
Aniline
N-Methyl piperazine
N-Ethylpiperazine
22.10
19.17
6.97
15.08
7.95
21.78
4.21
289.17
17.51
9.46
4-(2-Aminoethyl)morpholine
4-(3-Aminopropyl)morpholine
N,N-Dimethylethylenediamine
N,N-Diethylethylenediamine
N,N-Dimethylpropylenediamine
n-Butylamine
Cyclopentylamine
2-Amino-1-ethanol
3-Amino-1-propanol
Hydrazine
Ammonia
365.29
4.27
Aniline
68.39
49.44
65.61
40.06
27.88
35.17
20.15
45.13
119.31
115.83
8.15
p-Fluoro-aniline
p-Fluoro-aniline
p-Fluoro-aniline
p-Fluoro-aniline
p-Fluoro-aniline
p-Fluoro-aniline
p-Fluoro-aniline
p-Fluoro-aniline
p-Fluoro-aniline
N-Methyl piperazine
N-Ethylpiperazine
N,N-Dimethylethylenediamine
N,N-Diethylethylenediamine
N,N-Dimethylpropylenediamine
4-(2-Aminoethyl)morpholine
4-(3-Aminopropyl)morpholine
Ammonia
155.29
99.82
3088.52
1558.49
37.13
Methylamine
335.72
8983
IC50: concentration corresponding to 50% growth inhibition of the parasite.
Selectivity index (SI) = IC50 values of toxic activity/IC50 values of antimalarial activity.
Table 2
more active than compounds with p-fluoroaniline. In case of com-
pounds with aniline as common substituent, the compounds 11
and 12 having N-methyl piperazine and N-ethylpiperazine at posi-
tion-6 displayed the much higher antimalarial potency than the
compounds 25 and 26 bearing the p-fluoroaniline. Compound 13
bearing the aniline and 4-(2-aminoethyl) morpholine showed the
three times higher potency than compound 29 having the p-fluoro-
aniline. Replacing the 4-(2-aminoethyl) morpholine with 4-(3-
aminopropyl) morpholine, antimalarial activity was reduced two
times (13, IC50 = 6.97 nM; 14, IC50 = 15.08 nM). This activity
pattern was also consistent in compounds 29 and 30 with p-fluoro-
aniline (29, IC50 = 20.15 nM; 30, IC50 = 45.13 nM). Compound 15
bearing aniline and N,N-dimethylethylenediamine had an
IC50 value of 7.45 nM while compound 16 with aniline and N,N-
diethylethylenediamine displayed an IC50 value of 21.78 nM. On
substituting the N,N-dimethylethylenediamine with N,N-dimethyl-
propylenediamine 17 (IC50 = 4.21 nM), the antimalarial activity
was significantly increased. Compound 15 was five times more po-
tent than compound 26 while the compound 17 was eight times
more active than compound 28. Compound bearing aniline and
cyclopentylamine 19 had an IC50 value of 17.51 nM while substi-
tuting the cyclopentylamine with n-butylamine 18 led the dra-
matic decrease in antimalarial potency. Compound 22 having
aniline and hydrazine as substituents displayed an IC50 value of
4.27 nM whereas compound 23 having aniline and ammonia as
substituents showed an IC50 value of 68.39 nM. This observation
indicates that hydrazine being the more basic favors the antimalar-
ial activity. Compound 31 bearing the p-fluoroaniline and ammo-
nia as substituents had an IC50 value of 119.31 nM while the
compound 32 with p-fluoroaniline and methylamine exhibited an
IC50 value of 115.83 nM. Structure–activity relationship studies
based on the activity results revealed that aniline at position-4
and substituents N-ethylpiperazine, N,N-dimethylethylenedi-
amine, N,N-dimethylpropylenediamine, 4-(2-aminoethyl)morpho-
line, 4-(3-aminopropyl)morpholine, 2-amino-1-ethanol, hydra-
zine and cyclopentylamine at position-6 were well tolerated for
antimalarial activity.
In vivo antimalarial activity against chloroquine resistant strain N-67 of P. Yoelii in
swiss mice at dose 100 mg/kg for 4 days by oral route
Compound
% Suppression on day 4
13
15
17
20
27
29
CQ
96.59
49.00
95.70
58.52
95.42
98.73
99.9
assay.36 Tested compounds were endowed with selectivity index
ranging from 17.45 to 5008.61. The most of the compounds having
good in vitro activity have shown fairly high selectivity index.
Compound 19 having an IC50 value of 17.51 nM have shown the
highest selectivity index among the evaluated compounds.
Amongst the most potent compounds of the series, compounds
13, 17, and 22 with the IC50 values of 6.97, 4.21, and 4.27 nM dis-
played the selectivity index 2896.02, 295.02, and 31.39, respec-
tively, thus demonstrating good activity profile.
The compounds with good in vitro activity were further sub-
jected to in vivo study against CQ-resistant N-67 strain of Plasmo-
dium yoelii orally in swiss mice at dose 100 mg/kg for four days
(Table 2).37 The compounds 13, 17, 27, and 29 exhibited >95% sup-
pression but could not provide significant protection to the treated
mice in 28 days survival assay. Further structural optimization of
9-anilinoacridine triazine analogues may lead to the development
of the more potent molecules.
In conclusion, there is challenge and urgency to synthesize cost-
effective chemotherapeutic agents for treatment of malaria after
the widespread development of resistance to CQ. As part of our re-
search devoted to develop heterocycles as antimalarial, a series of
22 hybrid 9-anilinoacridine triazine molecules were synthesized.
Compounds 13, 15, 17, 20, and 22 exhibited the good in vitro anti-
malarial activity with high selectivity index. Moreover, the com-
pounds 13 and 29 displayed 96.59% and 98.73% suppression,
respectively, against CQ-resistant N-67 strain of P. yoelii by oral
The cytotoxicity of the all the synthesized hybrid 9-anilinoacri-
dine triazine were determined against VERO cells using MTT

A. Kumar et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6996–6999
6999
Bioorg. Med. Chem. Lett. 2005, 15, 4957; (c) Kumar, A.; Srivastava, K.; Raja
Kumar, S.; Puri, S. K.; Chauhan, P. M. S. Bioorg. Med. Chem. Lett. 2008, 18, 6530.
33. Hoglund, I. P. J.; Silver, S.; Engstrom, M. J.; Salo, H.; Tauber, A.; Kyyronem, H. K.;
Saarenketo, P.; Hoffren, A. M.; Kokko, K.; Pohjanoksa, K.; Sallinen, J.; Savola, J.
M.; Wurster, S.; Kallatsa, O. A. J. Med. Chem. 2006, 49, 6351.
administration. Activity data suggests that fine-tuning of substitu-
ent at position-4 and position-6 around the triazine nucleus might
yield the more potent antimalarial molecules.
34. General procedure for the synthesis of title compounds (11–32): The solution of
compound (11–32, 1.0 equiv) and different amines (2.0 equiv) in dry THF
(25 ml) in the presence of K2CO3 was refluxed for 4 h. The solvent was removed
under reduced pressure and the resultant residue was dissolved in chloroform
(50 ml). The organic phase was washed with water (3 ꢀ 20 ml), dried over
anhydrous Na2SO4. The solution was concentrated and purified with column
chromatography using chloroform/methanol (100:2) to afford the compound
(11–32). Spectroscopic data for 13: MS: 648 (M+1); 1H NMR (CDCl3, 300 MHz):
d (ppm) 8.21–8.06 (m, 2H, Ar-H), 7.98 (d, 1H, J = 9.24 Hz, Ar-H), 7.56 (d, 2H,
J = 7.72 Hz, Ar-H), 7.46–7.43 (m, 3H, Ar-H), 7.33–7.26 (m, 4H, Ar-H), 7.14 (br s,
1H, NH), 7.04 (t, 1H, J = 7.32 Hz, Ar-H), 6.84 (br s, 2H, NH), 6.81 (d, 2H,
J = 8.76 Hz, Ar-H), 5.54 (br s, 1H, NH), 3.77 (s, 3H, OCH3), 3.72 (t, 4H, J = 4.72 Hz,
O–CH2), 3.43 (t, 2H, J = 5.82 Hz, NH–CH2), 2.57 (t, 2H, J = 5.82 Hz, N–CH2), 2.50
(t, 4H, J = 4.72 Hz, N–CH2); 13C NMR (CDCl3, 50 MHz): d 164.38, 164.80, 156.93,
148.11, 139.38, 135.45, 133.52, 129.13, 126.01, 125.71, 125.41, 123.38, 122.42,
121.49, 120.79, 119.27, 118.48, 100.52, 57.67, 56.48, 55.80, 37.60, 30.09. Anal.
Calcd for C35H34ClN9O2: C, 64.86; H, 5.29; N, 19.45. Found: C, 64.73; H, 5.17; N,
19.63; 17: MS: 620 (M+1); 1H NMR (CDCl3 + CD3OD, 300 MHz): d (ppm) 8.02–
7.92 (m, 3H, Ar-H), 7.51–7.44 (m, 4H, Ar-H), 7.37 (dd, 1H, J = 2.58 Hz,
J = 9.39 Hz, Ar-H), 7.25–7.18 (m, 4H, Ar-H), 6.99 (t, 1H, J = 6.96 Hz, Ar-H),
6.87 (d, 2H, J = 8.43 Hz, Ar-H), 3.77 (s, 3H, OCH3), 3.43 (t, 2H, J = 6.3 Hz, NH–
CH2), 2.62 (t, 2H, J = 6.72 Hz, CH2), 2.42 (s, 6H, CH3), 1.85 (quint., 2H, J = 6.72 Hz,
CH2); 13C NMR (CDCl3 + CD3OD, 50 MHz): d 166.24, 164.52, 156.45, 146.90,
140.71, 139.20, 136.15, 129.00, 126.04, 125.34, 123.43, 122.49, 121.08, 120.26,
119.59, 117.93, 101.29, 56.88, 55.72, 44.67, 29.99, 26.78. Anal. Calcd for
C34H34ClN9O: C, 65.85; H, 5.53; N, 20.33. Found: C, 65.98; H, 5.48; N, 20.46; 26:
MS: 624 (M+1); 1H NMR (CDCl3, 300 MHz): d (ppm) 8.16–8.05 (m, 2H, Ar-H),
7.98 (d, 1H, J = 9.00 Hz, Ar-H), 7.46–7.43 (m, 5H, Ar-H), 7.33–7.30 (m, 2H, Ar-
H), 7.14 (br s, 1H, NH), 6.98 (t, 2H, J = 8.61 Hz, Ar-H), 6.86 (br s, 2H, NH), 6.83 (d,
2H, J = 8.76 Hz, Ar-H), 5.59 (br s, 1H, NH), 3.77 (s, 3H, OCH3), 3.49 (t, 2H,
J = 5.82 Hz, NH–CH2), 2.50 (t, 2H, J = 5.82 Hz, N–CH2), 2.26 (s, 6H, CH3); 13C
NMR (CDCl3 + DMSO-d6, 50 MHz): d 171.27, 169.55, 160.46, 150.41, 144.32,
139.93, 136.24, 134.73, 130.46, 126.98, 126.83, 124.06, 122.92, 121.75, 120.26,
119.82, 101.37, 63.72, 60.67, 50.66, 43.62. Anal. Calcd for C33H31ClFN9O: C,
63.51; H, 5.01; N, 20.20. Found: C, 63.42; H, 5.13; N, 20.03.
Acknowledgements
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. C.D.R.I. communication No. 7850.
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dilutions of all compounds in screening medium were prepared and 50
highest starting concentration (500 ng/ml) was dispensed in duplicate wells in
row of 96-well tissue culture plate. The highest concentration for
chloroquine was 25 ng/ml. Subsequently twofold serial dilutions were
prepared up to row (seven concentrations). Finally 50 of 2.5%
ll of
B
H
ll
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 one hour 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.
36. 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
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)
dose–response curves and represented the concentration of compound
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37. 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
inoculated with 1 ꢀ 106 parasitized RBC on day
0
and treatment was
administered to
a group of five mice from day 0 to 3, once daily. The
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80. The efficacy of test compounds was evaluated at 100 mg/kg/day and
required daily dose was administered in 0.1 ml volume via oral route.
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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.
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