Design, synthesis and evaluation of 5-substituted amino-2,4-diamino-8- chloropyrimido-[4,5-b]quinolines as novel antimalarials

Article abstract of DOI:10.1016/j.bmcl.2004.10.037

The design, synthesis and evaluation of 5-substituted amino-2,4-diamino-8- chloropyrimido-[4,5-b]quinolines as potent antimalarials is reported. Novel 5-substituted amino-2,4-diamino-8-chloropyrimido-[4,5-b]quinolines were designed based on a pharmacophore developed for potent antimalarial activity using Chem-X and MOE softwares. The designed molecules were synthesized by following a novel route and were evaluated by Rane's test for blood schizonticidal activity in mice infected by Plasmodium berghei. Based on the Mean Survival Time (MST) data, of the nine compounds evaluated, three had curative potential when compared with chloroquine.

Full text of DOI:10.1016/j.bmcl.2004.10.037

Bioorganic & Medicinal Chemistry Letters 15 (2005) 73–76
Design, synthesis and evaluation of 5-substituted
amino-2,4-diamino-8-chloropyrimido-[4,5-b]quinolines
as novel antimalarials
Advait A. Joshi, Sachin S. Narkhede and C. L. Viswanathan*
Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, Santacruz (E), Mumbai 400 098, India
Received 7 August 2004; accepted 12 October 2004
Available online 2 November 2004
Abstract—Novel 5-substituted amino-2,4-diamino-8-chloropyrimido-[4,5-b]quinolines were designed based on a pharmacophore
developed for potent antimalarial activity using Chem-X and MOE softwares. The designed molecules were synthesized by follow-
ing a novel route and were evaluated by RaneÕs test for blood schizonticidal activity in mice infected by Plasmodium berghei. Based
on the Mean Survival Time (MST) data, of the nine compounds evaluated, three had curative potential when compared with
chloroquine.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Malaria is a serious health problem and as per the recent
report of WHO, there are 300 million new cases of ma-
laria every year and about a million children die each
year of the disease.1 Moreover, drug resistance is a seri-
ous problem in malaria and it can be attributed to the
use of single drug (monotherapy) for treatment and to
the adaptation of the malarial parasite by developing
alternate pathways for survival. Hence, the present
strategy for new drug development is directed towards
identifying the essential enzyme systems in the parasite
and developing molecules to inhibit them. With this in
view, the inactivation of potent drugs like chloroquine
by malarial parasites has been studied in depth.2
schizonticide), quinacrine (gametocidal activity against
P. vivax and P. malariae), sulfadoxine (long duration
of action against blood schizonts) and pyronaridine
(inhibits P. falciparum topoisomerase II).3 The study
was carried out using Chem-X4 and Molecular Operat-
ing Environment (MOE)5 softwares on Pentium IV
1.6GHz computer. The conformers of each molecule
were generated by using dynamic simulations at the tem-
perature of 310ꢁC and the sampling time was taken as
5 · 10À4 s. The lowest energy conformations were identi-
fied for each molecule and were then used for arriving at
a three-point pharmacophore. The basic nucleus, 2,4,5-
triamino-8-chloropyrimido-[4,5-b]quinoline, was built
using the features of the pharmacophore.
The present work is aimed towards developing novel
molecules with improved potential for treating malaria
and with decreased probability for developing drug
resistance. It is proposed to achieve this by generating
a common pharmacophore from the structures of potent
antimalarials belonging to different classes, by designing
novel molecules, synthesizing and evaluating them.
Synthesis of the basic nucleus is outlined in Scheme 1.
Reaction of guanidine nitrate 1 with malononitrile 2 in
the presence of sodium alkoxide in dry ethanol or meth-
anol yielded 2,4,6-triaminopyrimidine 3 (mp 249ꢁC).
This was condensed with 2,4-dichlorobenzoic acid 4, in
the presence of activated copper bronze powder at
180–190ꢁC to yield N-(2,4-diamino-6-pyrimidino)-4-
chloroanthranilic acid 5 (mp 181ꢁC) which on cycliza-
tion using concentrated sulfuric acid yielded 5-oxo-
(10H)-2,4-diamino-8-chloropyrimido-[4,5-b]quinoline 6
(mp 160ꢁC). This was reacted with phosphorous oxy-
chloride to get 2,4-diamino-5,8-dichloropyrimido-[4,5-
b]quinoline 7 (mp 151ꢁC) which on stirring with liquor
ammonia at room temperature gave 2,4,5-triamino-8-
chloropyrimido-[4,5-b]quinoline 8 (mp 200ꢁC, dec.).
A three-point pharmacophore was developed by using
structures of potent antimalarials like chloroquine
(blood schizonticidal activity), pyrimethamine (tissue
Keywords: Malaria; Pyrimido-[4,5-b]quinolines; Antimalarials.
*
Corresponding author. Tel.: +91 22 26670871; fax: +91 22
26670816; e-mail addresses: chelakara_viswanathan@yahoo.com;
joshiadvait78@yahoo.com
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.037

74
A. A. Joshi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 73–76
NH2
NH
NH2
.HNO3
+
sodium alkoxide
CO2H
Cl ,
N
H2N
NC
CN
reflux, 6 h
H2N
N
NH2
2
1
Cl
3 (85%)
4
180-1900C,
12 h
activated copper
bronze powder
NH2
O
NH2
N
CO2H
conc. H2SO4
1000C, 8 h
N
Cl
N
H
N
NH2
Cl
N
H
N
NH2
135-1400C,
6 h
5 (64%)
6 (74%)
redistilled
POCl3
Cl
NH2
N
NH2 NH2
liq. NH3
R. T., 1 h
N
Cl
N
N
NH2
Cl
N
N
NH2
7 (65%)
8 (72%)
Scheme 1. Synthesis of 2,4,5-triamino-8-chloropyrimido-[4,5-b]quinoline 8.
Compound 8 was found to be active against P. berghei
infected mice in vivo at 160mg/kg.
pound 5 using phosphorous oxychloride in a single step
led to a drastic reduction in yield.
In order to achieve enhanced potency, compound 7 was
condensed with substituted aminobenzenesulfonamides
in the presence of dry pyridine to get compounds 9–11
(Scheme 2). Similarly, on condensing compound 7 with
3-substituted and 3,5-disubstituted 4-hydroxyanilines
gave compounds 12 and 14, and, 13 and 15, respectively
(Scheme 3). Finally reaction of compound 7 with
methyl- and ethyl-amines and then with formaldehyde
and t-butyl hydroperoxide gave compounds 16 and 17,
respectively (Scheme 4). All the compounds were puri-
fied by silica gel column chromatography. The physical
constants and spectral characteristics of the final com-
pounds are mentioned in Table 1.
The final compounds 8–17 were evaluated for antimalar-
ial activity in vivo against virulent strains of Plasmodium
berghei in mice using RaneÕs blood schizonticidal
method described by Osdene et al.6 Four-week old mice
weighing 18–20g each received intra-peritoneal inocu-
lum of 1 · 106 parasitized red blood cells. A group of
five infected mice were kept as the control group. The
test solutions of the synthesized compounds and chloro-
quine were prepared in water with the addition of one
drop of 1% Tween 80 and were injected to mice subcu-
taneously 72h post-infection. The dose range selected
was 20, 40, 80 and 160mg/kg for the test compounds
and 20mg/kg for chloroquine as the standard group
and five mice per dose were used. Mean Survival Time
(MST) was calculated for control, the standard and test
groups and based on this, compounds were classified as
active, curative and inactive (Table 2).
During the synthesis of compound 5, activated copper
bronze gave improved yields and reduced reaction time
when compared to the use of copper powder. Cycliz-
ation of compound 5 with conc. H2SO4 gave good qual-
ity product compared to reaction using polyphosphoric
acid. Attempts at cyclization and halogenation of com-
From the results it may be observed that among the
aminobenzenesulfonamides, the compound with bulky
R
NH
O2S
R
NH NH2
Cl
NH2
N
H2N
SO2 NH
,
N
anhydrous pyridine
6-9 h, reflux
Cl
N
N
NH2
Cl
N
N
NH2
7
9, R = H
10, R =
N
N
N
11, R =
H3CO
OCH3
Scheme 2. Synthesis of compounds 9–11.

A. A. Joshi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 73–76
75
R
R
HO
R1
H2N
OH
NH NH2
N
Cl
NH2
N
R1
Cl
N
N
NH2
Cl
N
N
NH2
7
12, R =
R1 = H
N
13, R and R1
=
=
N
14, R =
R1 = H
N
15, R and R1
N
Scheme 3. Synthesis of compounds 12–15.
O O
N
i) R-NH2
ii) t-butyl
hydroperoxide,
R
Cl
NH2
N
NH2
N
formaldehyde
Cl
N
N
NH2
Cl
N
N
NH2
16, R = -CH3
17, R = -C2H5
7
Scheme 4. Synthesis of compounds 16 and 17.
Table 1. Physical constants and spectral data of the final compounds
R
R1
N
N
NH2
N
Cl
N
NH2
Compd
R
R1
H
Melting point (ꢁC)
Yield (%)
60
1H NMR (d in ppm)
SO2NH2
9
159
d 2.5 (s, 1H, –NH), d 7.5–7.9 (m, 7H, Ar-H)
N
10
H
166
48
d 2.5 (s, 1H, –NH), 3.1 (s, 4H, –NH2), 7.5–7.9
(m, 11H)
SO2 NH
SO2 NH
N
N
11
12
H
H
178
165
72
65
d 2.5 (s, 1H, –NH), 3.6 (s, 3H, –OCH3), 3.9 (s, 3H,
–OCH3), 6.5 (s, 2H, Ar-H), 6.6 (s, 2H, Ar-H),
7.5–8.4 (m, 4H, Ar-H)
MeO
MeO
N
d 1.00 (t, 6H, –CH2–CH3), 2.3 (q, 4H, CH2–CH3),
2.5 (s, 1H, –NH), 3.6 (s, 4H, –CH2–), 4.9 (br s, 1H,
O–H), 7.5–7.9 (m, 6H, Ar-H)
OH
N
13
H
191
70
d 0.9 (t, 12H, –CH2–CH3), 2.2 (q, 8H, CH2–CH3),
2.5 (s, 1H, –NH), 3.4 (s, 4H, –CH2–), 5.0 (br s, 1H,
O–H), 7.5–7.9 (m, 5H, Ar-H)
OH
N
(continued on next page)

76
A. A. Joshi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 73–76
Table 1 (continued)
Compd
R
R1
H
Melting point (ꢁC)
Yield (%)
57
1H NMR (d in ppm)
N
14a
235
d 1.5 (s, 4H, –CH2– of pyrrolidine), 2.2 (s, 4H,
–CH2–N), 2.4 (s, 1H, –NH), 3.6 (s, 2H, –CH2–),
5.2 (br s, 1H, O–H), 7.4–7.9 (m, 6H, Ar-H)
OH
N
15
H
182
66
d 1.52 (s, 8H, –CH2– of pyrrolidine), 2.0 (s, 8H,
–CH2–N), 2.4 (s, 1H, –NH), 3.4 (s, 4H, –CH2–),
5.0 (br s, 1H, O–H), 7.4–7.9 (m, 5H, Ar-H)
OH
N
16
17
–CH3
210
232
72
77
d 1.2 (s, 9H, C–(CH3)3), 2.2 (s, 3H, N–CH3), 5.3
(s, 2H, N–CH2–O), 7.4, 7.6, 8.0 (s, 3H, Ar-H)
O
O
O
–C2H5
d 1.0 (q, 3H, N–CH2–CH3), 1.4 (s, 9H, C–(CH3)3),
3.2 (t, 2H, N–CH2–CH3), 5.3 (s, 2H, N–CH2–O),
7.4, 7.6, 8.0 (s, 3H, Ar-H)
O
a Isolated as hydrochloride salt.
Table 2. In vivo antimalarial activity of compounds against P. berghei in mice
Compd
Untreated control (days)
Mean survival time (days) at doses (mg/kg)
Remarks
20
15
40
16
80
18
160
8
10
11
10
10
11
13
12
11
14
10
10
20
14
Active (160mg/kg)
Inactive
Inactive
9
10
12
11
13
14
15
15
13
11
12
>60
12
>60
13
>60
15
>60
14
Curative (all doses)
Inactive
13
14
>60
15
>60
20
>60
25
>60
26
Curative (all doses)
Active (80, 160mg/kg)
Curative (all doses)
Active (20, 40mg/kg)
Inactive
15
16
>60
28
>60
30
>60
20
>60
14
17
Chloroquine
13
15a
12
14
15
—
—
—
Curative (20mg/kg)
a Only three mice died from this group.
substitution (compound 11) was curative while among
the substituted aminophenols the disubstituted com-
pounds (13 and 15) were curative. Hence it may be
concluded that the nature and size of the substituents
at the 5-amino function in the pyrimidoquinoline class
of compounds significantly influences the antimalarial
activity.
References and notes
1. WHO Report; World Health Organization: Geneva, Swit-
zerland, 1999.
2. Ridley, R. G.; Hofheinz, W.; Matile, H.; Jaquet, C.; Dorn,
A.; Masciadri, R.; Jolidon, S.; Richter, W. F.; Guenzi, A.;
Girometta, M.; Urwyler, H.; Huber, W.; Thaithong, S.;
Peters, W. Antimicrob. Agents Chemother. 1996, 40(8),
1846.
3. Murray, M. C.; Perkins, M. E. Annu. Rep. Med. Chem.
1996, 31, 141.
Acknowledgements
4. Chem-X; Chemical Design Ltd: Oxon, England, Jan. 1997.
5. Molecular Operating Environment (MOE) 2003.02; Chemi-
cal Computing Group: Quebec, Canada, 2003.
6. Osdene, T. S.; Russel, P. B.; Rane, L. J. Med. Chem. 1967,
10, 431.
The authors thank University Grants Commission
(UGC), New Delhi for the financial support and Dr.
(Mrs.) V. R. Kamath of Haffkine Institute, Parel, Mum-
bai 400012 for antimalarial evaluation.