Application of multi-component reactions to antimalarial drug discovery. Part 1: Parallel synthesis and antiplasmodial activity of new 4-aminoquinoline Ugi adducts

Article abstract of DOI:10.1016/j.bmcl.2004.05.063

The synthesis of a new class of Ugi adducts incorporating the 4-aminoquinoline moiety is described. The novel compounds are active against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum with the best compound showing

Full text of DOI:10.1016/j.bmcl.2004.05.063

Bioorganic & Medicinal Chemistry Letters 14(2004) 3901–3905
Application of multi-component reactions to antimalarial drug
discovery. Part 1: Parallel synthesis and antiplasmodial activity
of new 4-aminoquinoline Ugi adducts
Chitalu C. Musonda,a Dale Taylor,b Julie Lehman,c Jiri Gut,c Philip J. Rosenthalc
and Kelly Chibalea,*
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
bDivision of Pharmacology, Department of Medicine, University of Cape Town Medical School, Observatory, 7925 South Africa
cDepartment of Medicine, University of California, San Francisco, CA 94143-0811, USA
Received 4 May 2004; revised 24 May 2004; accepted 24 May 2004
Available online 17 June 2004
Abstract—The synthesis of a new class of Ugi adducts incorporating the 4-aminoquinoline moiety is described. The novel com-
pounds are active against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum with the best
compound showing an IC50 value of 73 nM against a resistant K1 strain.
Ó 2004Elsevier Ltd. All rights reserved.
At present malaria is considered to be the world’s most
important tropical parasitic disease, afflicting 300–500
million people and killing 1–2 million annually.1 It is
estimated that nearly 40% of the world’s population
lives in malaria endemic regions. Of the four species of
the disease-causing parasites, Plasmodium falciparum (P.
falciparum) is the most dangerous form, accounting for
up to 95% of malaria-related deaths. The emergence of
widespread resistance in the parasite to contemporary
antimalarial drugs such as chloroquine, CQ (1), has
given a new dimension to the malaria problem. This
resistance has prompted the quest for new active com-
pounds against the parasite.
sity in the final products in a few reaction steps. Com-
pared with conventional organic reactions, MCRs are
advantageous in being highly convergent and in
requiring minimum effort to achieve. Several MCRs are
known to date among which the Ugi 4component
condensation (U-4CC)7 of amine, oxo compound, car-
boxylic acid and isocyanide, and its variants remains, by
far, the most documented and most versatile. Coupled
with combinatorial chemistry, MCRs have been used to
generate chemical diversity in a few reaction steps and
usually in a one-pot operation.8 Our interest in applying
MCRs to antimalarial drug discovery stems from this
attractive feature and the realization that the rational
choice of inputs based on known antimalarial pharma-
cophores could lead to the generation of diverse bio-
logically rich libraries designed to explore rapid
structure–activity relationships (SARs). We report in
this communication the synthesis and biological evalu-
ation of a new class of Ugi adducts based on the ami-
noquinoline antimalarial pharmacophore. To the best of
our knowledge this is the first report in which the Ugi
isocyanide MCR (IMCR) chemistry has been used to
synthesize antimalarial aminoquinoline analogues.
The advent of combinatorial chemistry has in the recent
past emerged as a powerful tool for the rapid genera-
tion, identification and optimization of lead compounds
in the drug discovery process.2–4 Multi-component
reactions (MCRs),5;6 reactions in which more than two
starting materials all present together in one reaction
vessel combine to form a final product, have in this
regard been used to efficiently generate chemical diver-
In the context of our on-going research, we wished to
synthesize novel compounds for screening against
malaria parasites and to this end we initially targeted
a-acylamino amides accessible by way of the Ugi 4CC
Keywords: Ugi isocyanide multi-component reactions; Malaria;
4-Aminoquinolines.
* Corresponding author. Tel.: +27-21-650-2553; fax: +27-21-689-7499;
e-mail: chibale@science.uct.ac.za
0960-894X/$ - see front matter Ó 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.063

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C. C. Musonda et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3901–3905
HO
N
N
HO
N
H
HN
N
MeO
N
CF3
Cl
N
CF3
2
3
1
Figure 1. Chemical structures of chloroquine (1), mefloquine (2) and quinine (3).
reaction. We initially designed our exploratory library in
such a way as to incorporate the quinoline substructure
as part of our amine input in the U-4CC reactions. The
basis of the design was that the quinoline moiety is
present as the pharmacophore in a number of known
antimalarial drugs including chloroquine, CQ, (1),
mefloquine (2) and quinine (3) (Fig. 1), where it is be-
lieved to inhibit haemozoin formation in the malaria
parasite.9–11 The initial choice of hydroxyl-containing
carboxylic acids was made on the basis of the ability of
the hydroxyl functionality to chelate iron,12 while it was
reasoned that the presence of a nitrogen atom in the
carboxylic acid would present a second protonatable
site, which would aid in compound accumulation within
the acidic parasite food vacuole via pH trapping,13 as
well as to improve the aqueous solubility of the com-
pounds via salt formation. The initial use formaldehyde
as the aldehyde input was governed by our desire to
obtain low molecular weight compounds14 while the
choice of isocyanides was limited by their commercial
availability.
Synthesis of the target compounds was then achieved in
methanol at room temperature in parallel array format
(Scheme 2).
Condensation of stoichiometric amounts of amine,
aldehyde, carboxylic acid and isocyanide at room tem-
perature in anhydrous methanol afforded mixtures of
the Ugi adducts. All the reactions with tert-butyl iso-
cyanide went to completion within 36 h, while those with
cyclohexylisocyanide took longer reaction times (up to
48 h). Figure 2 provides a summary of the target com-
pounds synthesized.
The use of aldehydes besides formaldehyde in the
U-4CC reaction would give rise to either enantiomers or
diastereomers depending on the absence or presence of
chirality in the other inputs. The use of formaldehyde in
combination with achiral substrates thus eliminated the
possibility of obtaining diastereomeric mixtures so that
purification was not made a tedious effort. Purification
was thus achieved simply by means of preparative thin
layer chromatography following solvent removal under
reduced pressure to afford the products in modest to
good yields (Table 1).
The quinoline-containing amines that were required for
the U-4CC were synthesized by way of Scheme 1. Thus
commercially available 4,7-dichloroquinoline 4 was re-
acted with excess ethylene or propane diamine in the
neat at 80 °C for 1 h, then at 135–140 °C for 3 h to afford
5a and 5b in excellent yields.
Initial characterization of compounds 9–18 by 1H NMR
was complicated by duplication of each of the individual
peaks. Rotation about the tertiary amide bond in the
NH2
Cl
( )n
HN
N
NH2
( )n
H2N
Cl
N
i, ii
Cl
4
n = 1, Ethylene diamine
n = 2, Propane diamine
5a, n = 1, 91%
5b, n = 2, 86%
Scheme 1. Synthesis of amines. Reagents and conditions: (i) 80 °C, 1 h; (ii) 135–140 °C, 3 h.
R1
HN
O
O
5a, b
N
CH2O
i
( )n
HN
N
COOH
6
+
R
R1NC
8
R
Cl
9-18
n = 1, 2
7
Scheme 2. Synthesis of target compounds. Reagents and conditions: (i) MeOH, rt, 36–48 h.

C. C. Musonda et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3901–3905
3903
NH
O
NH
NH
O
NH
O
O
O
O
N
O
N
N
N
O
HN
N
HN
N
HN
N
N
HN
N
OH
N
Cl
Cl
N
Cl
Cl
N
OH
10
11
9
12
O
NH
O
N
NH
O
NH
HN
N
HN
N
HN
N
N
O
O
O
N
N
Cl
N
HO
OH
N
Cl
Cl
15
13
14
NH
O
N
NH
O
N
NH
O
O
HN
N
N
HN
N
HN
N
O
O
N
N
N
N
Cl
N
Cl
Cl
16
17
18
Figure 2. Structures of U-4CC reaction adducts synthesized.
Table 1. Isolated yields of compounds 9–18
according to the methods of Trager and Jensen15 in O+
human erythrocytes. Growth medium was supple-
mented with gentamicin (1 mg/mL) and 0.5% (w/v)
Albumax II. Parasites were synchronized at the tro-
phozoite stage using the protocol of Lambros and
Compound
Yield (%)
9
65
69
68
76
70
65
63
60
74
72
10
11
12
13
14
15
16
17
18
Vandenberg16 with 5% (w/v)
of chloroquine diphosphate were made in Milli-Q water.
Constituents for growth medium, CQ and -sorbitol,
D-sorbitol. Stock solutions
D
were obtained from Sigma. Albumax II was purchased
from GIBCO/Invitrogen. The parasite lactate dehydro-
genase (pLDH) assay was used to measure drug activity
in vitro.17 The CQ IC50 was determined via regression
analysis using Prism v4.0 (GraphPad Software, Inc.
5755 Oberlin Drive, #110 San Diego, CA 92121 USA).
W2-strain P. falciparum parasites were cultured in
RPMI-1640 medium with 10% human serum, following
standard methods, and parasites were synchronized with
Ugi adducts gave rise to two rotamers (major and
minor) whose chemical shift values differed markedly.
This phenomenon of conformational rotation was con-
firmed by the fact that duplicate peaks possessed the
same coupling constants and also by running variable
temperature (VT) NMR between 60 and 90 °C in deu-
terated DMSO, in which case coalescence of the dupli-
cating peaks was observed at the higher temperature.
5%
D
-sorbitol.18 Beginning at the ring stage, microwell
cultures were incubated with different concentrations of
compounds for 48 h. The compounds were added from
DMSO stocks; the maximum concentration of DMSO
used was 0.1%. Controls without inhibitors included
0.1% DMSO. After 48 h, when controls had progressed
to new rings, the culture medium was removed, and
cultures were incubated for 48 h with 1% formaldehyde
in PBS, pH 7.4, at room temperature. Fixed parasites
were then transferred into 0.1% Triton X-100 in PBS
Three culture-adapted isolates of P. falciparum, K1 (CQ
resistant; IC50 568 nM), W2 (CQ resistant; IC50 240 nM)
and D10 (CQ sensitive; IC50 37 nM) were used in this
study. For K1 and D10 cultures were maintained

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C. C. Musonda et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3901–3905
Table 3. Comparison of resistance indexes of CQ with compounds 9–
18
containing 1 nM YOYO-1 dye (Molecular Probes).
Parasitemia was determined from dot plots (forward
scatter vs fluorescence) acquired on a FACSort flow
cytometer using CellQuest software (Beckton Dickin-
son). IC50 values for growth inhibition were determined
from plots of percent control parasitemia over inhibitor
concentration using the Prism 3.0 program (GraphPad
Software), with data from duplicate experiments fitted
by nonlinear regression, as previously described.19
Compound
RIa
RIb
CQ
9
15.5
1.040.93
1.60
1.02
0.98
2.29
ndc
6.49
10
11
12
13
14
15
16
17
18
1.67
0.73
1.15
3.55
nd
1.66
0.31
2.15
nd
1.20
6.60
3.62
nd
In order to ascertain their intrinsic antiplasmodial
activity, compounds 9–18 were screened against three
strains of P. falciparum, viz CQ-sensitive D10, and CQ-
resistant K1 and W2. The antiplasmodial activities were
determined as inhibitory concentrations at 50% parasite
survival (IC50) in the three strains and are tabulated in
Table 2. For comparative purposes the antimalarial
activity of CQ (1) in all three strains is also reported.
a K1 resistance index ¼ K1 IC50/D10 IC50
.
b W2 resistance index ¼ W2 IC50/D10 IC50
.
c Not determined.
The compounds displayed moderate to significant anti-
malarial activity in all three strains of the malaria par-
asite with activity averaging in the single micromolar
range. Compound 17 was generally the most active
against all three strains. On the other hand 16 showed
very high activity in the D10 and K1 strains (0.237 and
0.073 lM, respectively) but was not as active in the W2
strain (IC50 ¼ 1.566 lM). Compound 12, the most active
in W2 (IC50 ¼ 0.619 lM) was nearly as active in the
resistant strains as in the CQ-sensitive strain. An in-
crease in the size of the chain length in moving from
compound 12 to 16 resulted in a marked increase in
activity, whereas the reverse was observed with a similar
change in moving from compounds 9 to 14 and 10 to 15.
A more general trend was however more observable
with the change in the carboxylic acid input used. In this
case the activity decreased for compounds based on
isonicotinic acid (9 and 16), 2-pyrazinoic acid (10 and
17) and 2,4-dihydroxy benzoic acid (11 and 13) in this
order for both sizes of methylene spacers in D10 and K1
but less so in W2. Although some general trends were
observable from the compound activities, it must be
emphasized that the effects of increasing the chain
lengths beyond those presented herein need to be ex-
plored further before definite conclusions could be
made. The varying results across different strains
underscores the importance of testing compounds
against multiple parasites with varying degrees of sen-
sitivity and resistance as indeed the phenomenon of
antimalarial drug resistance is often compound specific.
Despite their low antiplasmodial activities in compari-
son to CQ, compounds 9–18 possess favourable resis-
tance indexes (Table 3). Whereas CQ has resistance
indexes of 15.5 and 6.49 in K1 and W2, respectively,
compounds 9–18 generally have values far below that of
the former, implying reduced cross-resistance between
the CQ-sensitive and CQ-resistance strains. The only
exception to this observation is compound 16 in W2.
It can be seen from the Table 3 that compounds 9, 11
and 12 with RI values close to 1 were as active in the
CQ-sensitive strains as in the CQ-resistant strains.
In summary we have synthesized new aminoquinoline-
containing a-acylamino amides that are active against
CQ-sensitive and CQ-resistant strains of P. falciparum
Although the in vitro antiplasmodial activities of the
aminoquinoline Ugi adducts described in this commu-
nication are not in the desirable low IC50 nanomolar
range, we have demonstrated a simple new approach to
novel analogues of existing aminoquinoline antimalarial
drugs. We believe this approach will greatly assist in
rapid SAR studies in this and other classes of antima-
larial agents. Since the SAR libraries are already en-
riched in antimalarial pharmacophores, they need not be
large in this approach. Thus the need for large libraries
is precluded and the high cost often associated with such
libraries significantly reduced. Combining antimalarial
pharmacophores with other inputs, which may or may
not be antimalarial pharmacophores themselves, offers
many attractive features for accelerating antimalarial
drug discovery in a rational manner. For example,
MCR inputs can be designed in such a way that the final
product is aimed at multiple targets within the malaria
parasite. This approach could be useful in potentially
slowing down or even overcoming the emergence of
drug resistance and is analogous to conventional com-
bination therapy wherein two or more antimalarial
drugs, with different mechanisms of action, are co-
administered.
Table 2. Antimalarial activity of compounds 9–18
Compound
IC50 D10 lM
IC50 K1 lM
IC50 W2 lM
CQ
9
0.037
1.0641.105
0.712
5.655
0.540
1.672
>20
0.568
0.991
1.133
0.24
10
11
12
13
14
15
16
17
18
1.190
4.145
0.619
5.936
3.806
5.786
0.531
3.826
18.15
1.379
0.237
0.242
0.793
2.2941.652
0.073
0.521
1.566
0.877
>10
Although the U-4CC reaction leads to the highly flexible
aminoquinoline amides, which are not very attractive
chemotherapeutic targets owing to the low bioavail-
1.350

C. C. Musonda et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3901–3905
3905
ability associated with such compounds, the versatility
References and notes
of this reaction allows for the synthesis of more con-
strained, biologically relevant structures with improved
stability and bioavailability. Efforts to synthesize anti-
malarial heterocycles based on the U-4CC20 and other
MCRs, incorporation of other antimalarial pharmaco-
phores (and/or combinations thereof) as well as work to
rotate the functionalities of the inputs of the Ugi 4CC in
this context is currently underway in our laboratories.
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(NH), 1674(C @O), 1567 (C@C); dH (300 MHz, DMSO-
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Acknowledgements
This material is based upon work supported by the
National Research Foundation of South Africa under
grant number 2053362 (K.C.).
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