Benzoheterocyclic amodiaquine analogues with potent antiplasmodial activity: Synthesis and pharmacological evaluation

Article abstract of DOI:10.1016/j.bmcl.2012.06.010

The synthesis and evaluation of antiplasmodial activity of benzothiazole, benzimidazole, benzoxazole and pyridine analogues of amodiaquine is hereby reported. Benzothiazole and benzoxazole analogues with a protonatable tertiary nitrogen atom possessed excellent activity against the W2 and K1 chloroquine resistant strains of Plasmodium falciparum, with IC₅₀s ranging from 7 to 22 nM.

Full text of DOI:10.1016/j.bmcl.2012.06.010

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5046–5050
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Bioorganic & Medicinal Chemistry Letters
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Benzoheterocyclic amodiaquine analogues with potent antiplasmodial activity:
Synthesis and pharmacological evaluation
Dennis S. B. Ongarora a, Jiri Gut b, Philip J. Rosenthal b, Collen M. Masimirembwa c, Kelly Chibale a,d,
⇑
a Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
b Department of Medicine, University of California, San Francisco, CA 94143, USA
c African Institute of Biomedical Science and Technology, P.O. Box 2294, Harare, Zimbabwe
d Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch 7701, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and evaluation of antiplasmodial activity of benzothiazole, benzimidazole, benzoxazole
and pyridine analogues of amodiaquine is hereby reported. Benzothiazole and benzoxazole analogues
with a protonatable tertiary nitrogen atom possessed excellent activity against the W2 and K1 chloro-
quine resistant strains of Plasmodium falciparum, with IC50s ranging from 7 to 22 nM.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 10 May 2012
Revised 1 June 2012
Accepted 4 June 2012
Available online 9 June 2012
Keywords:
Benzothiazole
Benzoxazole
Amodiaquine
Benzoheterocyclic
Antiplasmodial
Reactive metabolite
Quinone imine
About half the world’s population is at risk of malaria. In 2010
alone, malaria afflicted an estimated 216 million people world-
wide, resulting in 655,000 deaths. Most fatalities occur in sub-Sah-
aran Africa, where a child dies from the disease every minute.1 The
rapid development of resistance against first line antimalarials
underscores the imperative for the discovery of new antimalarial
drugs.2 The use of existing antimalarials as starting leads remains
a useful approach in drug discovery.3 In this regard, the 4-amino-
quinoline antimalarial amodiaquine (1) has many favorable as-
pects including efficacy against most Plasmodium falciparum
strains that are resistant to chloroquine (2), a reasonable costing,
relatively easy synthesis, and ease of administration, with daily
dosing over three days.4 These factors make amodiaquine a good
lead compound for the synthesis of newer antimalarials.
Amodiaquine appears to act by the same mechanism as chloro-
quine, exerting its intra-erythrocytic antimalarial action by prevent-
ing heme detoxification to the pigment hemozoin.5 Amodiaquine is
rapidly absorbed and extensively metabolized after oral administra-
tion in humans, which leads to a low plasma exposure of the parent
drug. Desethylamodiaquine is the main metabolite of amodiaquine.
Other reported metabolites are bisdesethylamodiaquine and 2-
hydroxydesethylamodiaquine.6 The elimination of desethylamo-
diaquine is very slow, unlike that of amodiaquine, giving this metab-
olite a long terminal half-life. Both amodiaquine and desethyl-
amodiaquine have antimalarial activity.7 The main enzyme respon-
sible for amodiaquine deethylation is CYP2C8.8
It was observed through pharmacovigilance programs that
weekly prophylactic use of amodiaquine rarely caused severe side
effects including hepatitis, myelotoxicity and agranulocytosis.9
Consequently, its use is restricted to the treatment of acute malar-
ia.10 Currently, amodiaquine monotherapy is discouraged, and the
drug is principally used in combination with artesunate in one of
the artemisinin-based combination therapies currently recom-
mended by the World Health Organization for the treatment of fal-
ciparum malaria.11
The 4-aminophenol moiety of amodiaquine has been identified
as a ‘structural alert’ for toxicity. Bioactivation of amodiaquine to
reactive quinone imine (3) and aldehyde (4) metabolites has been
identified as the likely cause of amodiaquine induced hepatotoxic-
ity and agranulocytosis, respectively.12,13
Several analogues such as 5–8 (Fig. 1) have been designed that
block the formation of the quinone imine but not the aldehyde
metabolite. The strategy adopted involves designing out the 4-ami-
nophenol moiety or introducing bulky groups ortho to the phenolic
hydroxyl group.12,14–16
In an effort to discover new approaches to block the formation
of both the quinone imine and aldehyde metabolites, we hypothe-
sized that analogues of amodiaquine in which the hydroxyl group
⇑
Corresponding author.
E-mail address: Kelly.Chibale@uct.ac.za (K. Chibale).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.010

D. S. B. Ongarora et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5046–5050
5047
Figure 1. Chemical structures of amodiaquine (1), chloroquine (2), reactive amodiaquine metabolites (3–4) and analogues designed to prevent quinone imine formation (5–
8).
and the Mannich side chain are cyclized into a benzothiazole (9–
13), benzoxazole (14, 15) or benzimidazole (16–18) ring system
will not form reactive metabolites. In addition, analogues (19 and
20) in which the aromatic ring is converted to a pyridyl ring bear-
ing an aminoalkyl side chain but without a hydroxyl group were
envisaged as lacking an essential functional group for bioactiva-
tion. Accordingly, these analogues were designed, synthesized
and evaluated for antiplasmodial activity and cytotoxicty.
In the synthesis of the benzothiazole series, commercially avail-
able 21 was converted to the bromo intermediate, 22, by the Sand-
meyer reaction. This was coupled with the appropriate primary
amines by nucleophilic substitution to give 9a–13a. The nitro
group was reduced via Palladium catalyzed hydrogenation to ani-
line derivatives 9b–13b which were coupled with 4,7-dichloro-
quinoline to give target compounds 9–13. Similarly, the synthesis
of the benzimidazole and pyridine series proceeded via the bromo
intermediates (27 and 29), amination under microwave conditions
(16a–20a), reduction to aniline derivatives (16b–20b) and cou-
pling with 4,7-dichloroquinoline to obtain target molecules 16–
20. The benzoxazole series was realized via the cyclization of 2-
amino-4-nitrophenol 23 with potassium ethyl xanthate to give
benzoxazole thione, 24. This was converted to the methylthio ether
25 prior to amination under microwave conditions (14a–15a).
Reduction of the aromatic nitro group to 14b and 15b followed
by coupling with 4,7-dichloroquinoline afforded target compounds
14 and 15. All the target compounds 9–20 gave 1H-, 13C NMR and
Figure 2. Chemical structures of target benzoheterocyclic (9–18) and pyridyl (19–20) analogues.

5048
D. S. B. Ongarora et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5046–5050
Scheme 1. Reagents and conditions: (i) HBr, NaNO2, CuBr, H2O, 4 h, rt; (ii) Amine, THF, 40 °C, 15 h; (iii) H2, 10% Pd/C, rt, 8 h; (iv) 4,7-dichloroquinoline, acetonitrile, HCl,
reflux, 24 h; (v) Br2, 48% HBr, AcOH, 5 °C-rt, 4.5 h, rt À0 °C, H2O, NaOH, pH 4; (vi) Amine, EtOH, MW 100 °C, 60 min; (vii) Potassium ethyl xanthate, EtOH, reflux, 4.5 h, AcOH,
pH 5; (viii) MeI, DMF, 25 °C, 4 h.
Table 1
In vitro antiplasmodial activity against chloroquine-resistant P. falciparum K1 and W2 strains and cytotoxicity in rat L6 myoblasts
R
IC50
K1
(
lM)
Selectivity index
(L6/W2)
W2
L6
Amodiaquine
Chloroquine
0.005
0.049
0.008
0.344
9
1.158
0.012
0.917
0.014
4.126
3.563
10
5.118
3.539
426.500
11
12
0.013
0.040
0.007
0.025
272.231
116.750
4.67
13
14
0.011
0.491
nta
nta
—
0.921
16.352
33.303

D. S. B. Ongarora et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5046–5050
5049
Table 1 (continued)
R
IC50
K1
(lM)
Selectivity index
W2
L6
(L6/W2)
15
16
0.008
0.022
0.249
8.703
1087.875
0.501
4.580
5.713
5.25
11.403
1.146
17
18
0.090
0.827
0.200
15.284
76.420
19
20
0.021
0.041
0.092
0.073
18.593
47.412
885.381
1156.390
a
Not tested.
HRMS data consistent with the proposed structures (see Fig. 2 and
Scheme 1).
In conclusion, we have demonstrated a new class of amodia-
quine analogs that low levels potentially block the formation of
the toxic quinone imine and aldehyde metabolites using a benzo-
heterocyclic ring system attached to the 4-amino-7-chloroquino-
line ring, without compromising antiplasmodial activity. The
benzothiazole and benzoxazole analogues possessed excellent
activity against chloroquine resistant P. falciparum strains. Further
studies will focus on determining whether these analogues gener-
ate other reactive metabolites.
Antiplasmodial activity was determined against two chloro-
quine resistant Plasmodium falciparum strains, K1 and W2. Cyto-
toxicity against L6 (rat myoblast) cells was also determined. For
studies with the W2 and K1 strains as well as cytotoxicity, assays
were conducted as previously reported.17,18 Table 1 shows anti-
plasmodial activity, cytotoxicity and selectivity indices for com-
pounds 9–20. The results suggest that the presence of
a
protonatable amine is essential for high potency, for example as
illustrated by the low potency of compound 14 (W2 IC50
491 nM) compared to 15 (W2 IC50 8 nM). Additionally, the benz-
imidazole series represented by compounds 16–18 (W2 IC50
200–4580 nM) was less potent compared to the other series.
The pyridine analogues 19–20 exhibited moderate potency
against the W2 strain (IC50 21–41 nM) but lower activity against
the K1 strain (IC50 73–92 nM). Benzothiazole analogues 10, 11
and 13 as well as the benzoxazole analogue 15 had excellent
activity against both strains, with IC50 values ranging from 7 to
22 nM. Among the most potent compounds, the benzoxazole ana-
logue, 15, had the highest selectivity index (1088). The Resistance
Index (IC50 of the resistant strain/IC50 of the sensitive strain) is an
indicator of the likelihood of cross resistance. Although the two
strains tested were both chloroquine resistant, the W2 strain dis-
played greater sensitivity than K1. The resistance index for chloro-
quine and the pyrollidine analogues of the benzothiazole (10),
benzoxazole (15) and pyridine (19) series were 7.0, 1.2, 2.8 and
4.4, respectively. These results suggest that all the analogues have
low levels of cross resistance with the benzothiazole analogue
(10) exhibiting the lowest risk.
Acknowledgments
We thank the South African Medical Research Council/National
Research Foundation (MRC/NRF) and the Carnegie Corporation for
funding (DSBO). The University of Cape Town, South African MRC
and South African Research Chairs initiative of the Department of
Science and Technology administered through the South African
NRF is gratefully acknowledged for support (KC).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.010.
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