Structure-activity relationships of antileishmanial and antimalarial chalcones

Article abstract of DOI:10.1016/S0968-0896(03)00233-5

A series of oxygenated chalcones which have been evaluated earlier for antimalarial activity (Plasmodium falciparum K1) were tested for antileishmanial activity against Leishmania donovani amastigotes. A comparison of structure-activity relationships reve

Full text of DOI:10.1016/S0968-0896(03)00233-5

Bioorganic & Medicinal Chemistry 11 (2003) 2729–2738
Structure–Activity Relationships of Antileishmanial and
Antimalarial Chalcones
Mei Liu,a Prapon Wilairat,b Simon L. Croft,c
Agnes Lay-Choo Tand and Mei-Lin Goa,*
aDepartment of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore
bDepartment of Biochemistry, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
cDepartment of Infectious andTropical Diseases, London School of Hygiene andTropical Medicine,
London WC1E 7HT, UK
dInstitute of Cell andMolecular Biology, National University of Singapore, 30 Medical Drive,
Singapore 117609, Singapore
Received 20 January 2003; accepted 3 April 2003
Abstract—A series of oxygenated chalcones which have been evaluated earlier for antimalarial activity (Plasmodium falciparum K1)
were tested for antileishmanial activity against Leishmania donovani amastigotes. A comparison of structure–activity relationships
reveal that different physicochemical and structural requirements exist for these two activities. Antileishmanial activity is associated
with less lipophilic chalcones, in particular those with 40-hydroxyl-substituted B rings and hetero/polyaromatic A rings. In contrast,
chalcones with good antimalarial activity have alkoxylated B rings and electron-deficient A rings. Visualization of the steric and
electrostatic fields generated from comparative molecular field analysis (CoMFA) indicate that the ring A of chalcones make a more
significant contribution to antileishmanial activity while both rings A and B are important for antimalarial activity. Despite differ-
ent requirements, two alkoxylated chalcones (8, 19) were identified which combined good antimalarial and antileishmanial activ-
ities.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
The antileishmanial activity of several chalcones have
been reported in the literature.1,8À10 The most promising
member to date is licochalcone A (Fig. 1), an oxyge-
nated chalcone isolated from the roots of Chinese
liquorice and presently thought to exert its action by
inhibiting fumarate reductase, a selective target present
in the parasite mitochondria.10 The activity of licochal-
cone A has stimulated interest in the antileishmanial
potential of other chalcones from natural and synthetic
sources, and several members have been identified for
lead development.1,8,9,11 Antimalarial activity has also
been found in licochalcone A12 and other oxygenated
chalcones.2,3 However, reports on chalcones that com-
bine activities against both protozoal infections are
lacking, though the example of licochalcone A suggests
that this is not an unreasonable expectation.
For a structurally simple group of compounds, chalcones
have displayed an impressive array of pharmacological
activities, among which anti-protozoal,1À3 anti-inflam-
matory,4 immunomodulatory,5 nitric oxide inhibition,6
anticancer7 activities have been cited in the literature.
Chalcones that combine one or more types of activities
may be advantageous or otherwise. A combination of
activities can enhance the likelihood of undesirable side
effects during therapy. On the other hand, it may trans-
late into a successful dual-acting drug, especially if clo-
sely related activities (for example, antiprotozoal) are
involved. Whether the objective of drug design is exclu-
sion or inclusion of more than one kind of activity, it
would have to start with defining the structure–activity
relationships for each pharmacophore.
In this study, we have taken a group of oxygenated
chalcones that have previously been evaluated for anti-
malarial activity and tested them for antileishmanial
properties. Activities were analyzed by conventional
*Corresponding author. Tel.: +65-68742654; fax: +65-67791554;
e-mail: phagoml@nus.edu.sg
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00233-5

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M. Liu et al. / Bioorg. Med. Chem. 11 (2003) 2729–2738
Regression analyses show that antimalarial and antil-
eishmanial activities are not correlated in any way
(r2=0.05 for linear regression). It is evident that chalcones
have different structural requirements for antimalarial
and antileishmanial activities. Based on the compounds
with very good (A) antimalarial and antileishmanial
activities, dual-acting chalcones are more likely to be
found among methoxylated than hydroxylated chalcones.
Indeed, the only two chalcones with combined category A
activities are alkoxylated chalcones (20, 40-dimethoxy-4-
ethylchalcone 8, 40-methoxy-4-hydroxychalcone 19).
Previous structure–activity studies on the antileishmanial
activity of chalcones have emphasized the importance of
ring B for activity. In particular, para-substitution of ring
B with oxygenated and non-bulky substituents have been
reported to be desirable.1,8 The good activity seen here
with the 40-hydroxychalcones is in keeping with these
findings.
Figure 1. Structure of licochalcone A.
structure–activity relationship (SAR) approaches and
by comparative molecular field analysis (CoMFA)
with the aim of establishing the relevant SARs and to
see if these are mutually exclusive or share some ele-
ments of similarity. Such information would be useful
in either fine-tuning one activity to the exclusion of
the other, or in developing dual-acting drugs against
both infections.
Among the different structural classes, the 40-hydroxy-
chalcones have yielded the largest number of category
A antileishmanial compounds, with nearly half of the
available members possessing ED50 values of less than
5 mM. Unfortunately, many of these compounds are
also toxic against macrophages (Table 1). The toxicity
of oxygenated chalcones against murine bone marrow-
derived macrophages have been reported.8 Further
structural modifications to reduce toxicity while main-
taining antileishmanial activity would be necessary
before 40-hydroxychalcones can be useful agents.
Results and Discussion
Table 1 summarizes the antileishmanial and anti-
malarial activities of the chalcones expressed in terms of
ED50 and IC50 values, respectively. The antimalarial
activities have been reported previously (with the
exception of the 20-hydroxychalcones) but the antil-
eishmanial activities are reported for the first time. It is
noted that the ED50 values for antileishmanial activity
span a narrow 30-fold range, in contrast to a wider
300-fold spread for antimalarial activity.
In chalcones, ring A and its substitution pattern are
generally considered less important for antileishmanial
activity compared to ring B.1,8 However, this was not
observed in our study where notwithstanding the nature
of ring B, very good antileishmanial activity was
observed when ring A is 1-naphthalenyl (110, 205, 212,
242), 2-pyridinyl (207, 214, 241) and 4-quinolinyl (28,
30, 34, 215). The position of attachment of ring A to the
ab unsaturated carbonyl linkage is also critical, as seen
from the poor activities of the 3-quinolinyl (213, 29)
versus 4-quinolinyl (215, 30), and 2-naphthalenyl (216,
243) versus 1-naphthalenyl (212, 242) derivatives. This is
unlike antimalarial activity where better activities are
associated with 3-quinolinyl (27, 29, 31, 33) than 4-qui-
nolinyl (28, 30, 32, 34 ) derivatives.3 This departure
from earlier reports1,8 may be due to the fact that Ring
A in the present series of chalcones has not been
restricted to substituted benzene rings (as in earlier
studies) but includes heteroaromatic and polycyclic ring
systems which were not previously investigated.
To facilitate discussion, the chalcones are classified into
seven groups according to the substitution on the B
ring, namely 20,30,40-trimethoxy (n=18), 20,40-dimethoxy
(n=16), 40-methoxy (n=14), 40-ethoxy (n=13), 20,40-
dihydroxy (n=12), 40-hydroxy (n=16) and 20-hydroxy
(n=13). In addition, activities are arbitrarily classified
into 5 categories (A–E) for comparison. For antil-
eishmanial activity, compounds with ED50 values
<5 mM are classified as A (very good); 5–10 mM=B
(good); 10–20 mM=C (fair); 20–30 mM=D (poor) and
>30 mM=E (very poor). IC50 values for antimalarial
activities are similarly classified: A<10 mM, B:
10–20 mM, C: 20–50 mM, D: 50–100 mM and
E>100 mM.
The distribution of the chalcones according to this clas-
sification is shown in Figure 2. It can be seen that
methoxylated chalcones (ring B substituted with tri-
methoxy, dimethoxy or methoxy substituents) are
represented in every category (A–E) of antimalarial and
antileishmanial activities. The ethoxychalcones are an
exception, being notable in having poor antimalarial
activity. Hydroxylated chalcones are better antil-
eishmanial agents: they are not represented in category
A antimalarial activity but many members are found to
have category A antileishmanial activity.
Correlation analysis show that the activities of chal-
cones with very good (A) antileishmanial properties are
inversely correlated to lipophilicity (ClogP) (Pearson
correlation À0.443, p=0.034, n=23). This differs from
the general opinion that lipophilicity is an important
determinant of antileishmanial activity.8 However, a
statistically significant correlation with ClogP could not
be obtained by linear regression (r2=0.196, n=23),
possibly because of the narrow range of antileishmanial
activity (30-fold) associated with our compounds.

M. Liu et al. / Bioorg. Med. Chem. 11 (2003) 2729–2738
2731
Table 1. Antileishmanial, antimalarial activities and toxicity against mice macrophages of chalcones
Compd
Ring B
20,30,40-
Ring A
ED50 IC50
Toxicity to Compd Ring B
Ring A
ED50 IC50 (mM)b Toxicity to
macro-phages
(mM)a (mM)b macro-phages
(mM)c
(mM)a
(mM)c
3
4
6
11
12
2,4-Dichloro
13.24
7.81 18.0
6.82 3.0
5.4
30
10
10
30
30
10
10
10
30
10
30
10
—
30
10
30
10
10
—
—
30
—
10
30
3
39
121
122
123
124
125
126
127
136
41f
40-Ethoxy
4-Fluoro
2,4-Dichloro
4-Trifluoromethyl >30
2,4-Dimethoxy 24.17
4-Methyl
4-Nitro
4-Dimethylamino 29.93
4-Cyano
H
2,4-Dimethoxy
2,4-Dichloro
3-Quinolinyl
2,4-Difluoro
2,4-Dimethoxy 12.12
1-Naphthalenyl 4.03
4-Trifluoromethyl 8.07
2-Pyridinyl
2-Naphthalenyl >30
>30
>30
24.10
96.00
24.00
30.00
38.00
39.00
30.00
540.00
43.00
108.00
68.5
16.1
16.0
56.4
24.8
26.5
19.7
—
—
30
—
—
—
—
30
30
30
30
—
10
30
10
10
3
-Trimethoxy 4-Dimethylamino
4-Trifluoromethyl
2,4-Dimethoxy
4-Methyl
13.15 16.5
14.15 25.6
6.98 16.5
7.95 2.0
4.38 60.0
>30
25.23
13
4-Ethyl
27d
28d
35
3-Quinolinyl
4-Quinolinyl
4-Methoxy
4-Fluoro
4-Phenyl
2,4-Difluoro
4-Nitro
3,4-Dichloro
4-Chloro
2-Chloro
3-Chloro
H
2,4-Dichloro
13.92
11.23
>30
23.66
>30
>30
13.57 25.0
8.13
36
40
9.5
21.52 26.2
4.38 18.5
40-Butoxy
20,40-
201
128
129
130
131
132
133
134
1
2
5
7
8
29d
30d
101
102
104
105
106
107
108
109
110d
19
22
23
31d
32d
38
111
112
113
114
115
116
117
135
25
202d -Dihydroxy
203
>30
22.5
12.33 14.5
4.89 14.5
9.09 41.5
4.39 24.4
6.82 15.8
204
205d
206
207d
208d
209d
210d
211
2.23
20.0
121.6
92.8
12.30
ND
39.9
—
30
ND
10
10
3
20,40-
>30
-Dimethoxy 4-Trifluoromethyl >30
18.75
5.85
6.23
2.10
2.42
2.16
27.00
4-Pyridinyl
4-Quinolinyl
4-Chloro
10.99
ND
4.36
8.02
2,4-Difluoro
2,4-Dimethoxy >30
4-Ethyl
3-Quinolinyl
4-Quinolinyl
4-Methyl
4-Methoxy
4-Fluoro
4-Chloro
13.16
245
4-Methyl
4.38
12.46
1.5
212d 40-Hydroxy 1-Naphthalenyl 1.38
213d
214d
215d
216d
217
220
221
222
224
225
226
227
228
229
230
231
232
233d
234
235
236
237
238
239
241d
242d
243d
244d
3-Quinolinyl
2-Pyridinyl
4-Quinolinyl
>30
1.48
3.83
41.0
16.3
51.0
27.5
38.0
32.2
25.4
25.8
30.4
20.4
21.7
18.4
17.70
24.50
29.6
35.45
188.00
28.00
12.85
62.50
61.50
25.50
35.50
47.00
31.00
32.50
29.50
ND
—
3
6.82 93.75
12.23 128.50
10
30
—
—
30
30
30
—
10
10
30
30
10
10
ND
30
—
30
ND
—
—
10
30
—
—
30
10e
10
—
10
10
10
10
10
—
3
—
10
10
10
30
—
30
10
30
30
30
—
30
3
2-Naphthalenyl >30
>30
>30
322.00
342.00
4-Chloro
4-Methoxy
4-Methyl
3-Methyl
6.98
5.77
4.4
4-Bromo
23.38 542.50
11.89 600.00
11.27 297.50
2-Chloro-4-fluoro
3,4-Dichloro
4-Nitro
1-Naphthalenyl
4-Hydroxy
2,4-Difluoro
4-Methoxy
3-Quinolinyl
4-Quinolinyl
4-Fluoro
2,4-Dichloro
4-Trifluoromethyl >30
2,4-Dimethoxy
4-Methyl
4-Nitro
4-Dimethylamino >30
4-Cyano
H
2,4-Difluoro
4-Methoxy
3-Quinolinyl
4-Quinolinyl
4.19
4-Trifluoromethyl 4.18
>30
415.00
4.37 320.00
4-Nitro
4-Fluoro
3,4-Dichloro
>30
2.6
>30
40-
-Methoxy
4.18
7.00
12.51 26.75
13.13 21.70
6.82 4.83
8.13 43.00
ND 14.40
12.38 16.00
4-Dimethylamino 8.13
2,4-Dichloro
H
2,4-Dichloro
4.39
4.41
11.74
20-Hydroxy
4-Dimethylamino >30
3-Quinolinyl
4-Chloro
4-Methyl
4-Methoxy
2,4-Dimethoxy 10.85
4-Trifluoromethyl 29.31
4-Fluoro
2-Pyridinyl
1-Naphthalenyl 4.07
2-Naphthalenyl 11.73
13.16
6.82
12.05
13.16
19.00
6.40
23.38
ND
>30
70.00
100.00
70.00
6.91 94.50
55.50
28.10
29.31
1.57
12.2
>30
40-Ethoxy
10
30
30
26
29.37 33.00
9.99 24.85
4.39 100.00
33d
34d
4-Quinolinyl
12.63
aIn vitro sensitivity against L. donovani (L82) amastigotes determined in a mouse peritoneal macrophage model.20 Compounds with ED50 values
>30 mM are considered inactive. ED50 for pentosam1 (sodium stilbogluconate) is 0.6 mM. Compounds were tested in triplicates.
bInhibition of [3H] hypoxanthine uptake into P. falciparum K1.3 IC50 for chloroquine=0.27 mM.
cConcentration at which toxicity to mouse macrophages was observed. (—) no toxicity observed at 30 mM. ND, not done.
dRing A=heterocyclic or polyaromatic.
eCompound 34 is toxic at 10 mM, but parasites are present at this concentration.
fCompound 41 (20,40-dimethoxy-40-butoxychalcone) has been reported to have antimalarial and antileishmanial activities.9,12 Low levels of activities
observed here may be due to different strains of parasites being used.

Figure 2. Distribution of antimalarial and antileishmanial activities of chalcones classified according to nature of Ring B. Antileishmanial activities & ED50 values: <5 mM (A), 5–10 mM (B), 10–20 mM
(C), 20–30 mM (D), >30 mM (E). Antimalarial activities & IC50 values: <10 mM (A), 10–20 mM (B), 20–50 mM (C), 50–100 mM (D), >100 mM (E).

M. Liu et al. / Bioorg. Med. Chem. 11 (2003) 2729–2738
2733
The SAR for antimalarial activity has been reported
earlier.3 Using multivariate analysis, it was shown that a
sterically larger ring B and a polar ring A had a positive
influence on activity.
accessible surface area of the molecule. In view of this
relationship, the CoMFA was re-done with the inclu-
sion of log A. A statistically better model (r 2 cv=0.655;
SEPcv=0.227, three components) was obtained, with
contributions by the steric, electrostatic and log A
parameters at 33.6, 40.1 and 26.3%, respectively. In
view of this improvement, the new model incorporating
log A is adopted for subsequent discussion.
In the next stage of analysis, the three-dimensional struc-
ture–activity relationship study for antimalarial and
antileishmanial activities were carried out using compara-
tive molecular field analysis (CoMFA). Various CoMFA
models were generated using all compounds, compounds
classified according to B ring substitution pattern or
according to activity levels (A–E). The best model
(r2cv=0.547, standard error of prediction, cross validated
(SEPcv)=0.218, three components) for antimalarial activ-
ity was obtained using ‘active’ chalcones, namely alkoxy-
lated chalcones with IC50 <10mM, and hydroxylated
chalcones with IC50 <20mM. Coincidentally, these were
the same compounds that had been successfully analyzed
using multivariate analysis in an earlier study, with activ-
ity represented by the following equation:3
The best CoMFA model for antileishmanial activity was
found for chalcones with hydroxylated B rings (n=40)
covering the entire range of antileishmanial activity
(A–E). However, this model had low predictability
(r2cv=0.358, SEP CV=0.383, four components), with
balanced steric (48.8%) and electrostatic (51.2%) con-
tributions. In an attempt to improve the CoMFA model,
ClogP was included as an additional parameter, since
earlier results showed good antileishmanial activity to be
inversely correlated to hydrophobicity. However, no
significant improvement to the model was evident upon
inclusion of the hydrophobic parameter (r2 cv=0.352,
SEPCV=0.373). Thus, ClogP was not included in the
CoMFA model for antileishmanial activity.
ꢀ log IC50 ¼ 6:4 log A ꢀ 10:6
n ¼ 19; r2 ¼ 0:690; r2cv ¼ 0:616
Visualization of the steric and electrostatic contours of
the two CoMFA models was carried out using
The equation shows a good correlation to Connolly
surface area (log A) which is a measure of the solvent
1-(40-hydroxy)-3-(2-pyridinyl)-2-propen-1-one (214),
a
Figure 3. Steric and electrostatic contour maps of CoMFA models for antimalarial and antileishmanial activities, showing the orientation of 1-(40-
hydroxy)-3-(2-pyridinyl)-2-propen-1-one (214) in the antimalarial model (a,b) and antileishmanial model (c,d). In (a), the steric contours show that a
large sized B ring (green) and a smaller sized A ring (yellow) is preferable. In (b), the blue contours indicate that an electron-deficient A ring is
preferred. The steric (c) and electrostatic (d) contours in the antileishmanial model are concentrated around ring A, indicating the relative impor-
tance of this ring compared to ring B. Steric contours are colour-coded yellow (where bulk is undesirable) and green (where bulk is acceptable). The
electrostatic contours are colour-coded blue (where positive charge is desirable) and red (where negative charge is preferred).

2734
M. Liu et al. / Bioorg. Med. Chem. 11 (2003) 2729–2738
representative compound with an ED50 of 1.48mM and
IC50 of 16.25 mM for antileishmanial and antimalarial
activities, respectively. Steric contours in the CoMFA
model are colour-coded yellow (where bulk is undesirable)
and green (where bulk is acceptable). The electrostatic
contours are colour-coded blue (where positive charge is
desirable) and red (where negative charge is preferred).
The orientation of 214 in the steric (Fig. 3a) contours of
the CoMFA model for antimalarial activity shows a
concentration of green zones around ring B, indicat-
ing that more bulk is tolerated or even preferred in
this region. Unfortunately, the solitary 4-hydroxyl
substituent on ring B of 214 did not project into the
green zones. An alkoxylated B ring would be more
advantageously placed in the green zones and this
was indeed observed when the steric contours
around the 20,40 dihydroxy chalcone (202) and its
20,40-dimethoxy analogue (29) were compared (Fig.
4). The distribution of steric contours in this model
would explain why alkoxylated chalcones are gen-
erally better antimalarials than their hydroxylated
counterparts.
Unlike the preference for more bulk around ring B, the
large yellow zone in the vicinity of ring A would suggest
some degree of steric restriction in this region (Fig. 3a).
The small-sized pyridine ring in 214 is still well-accom-
modated in this region, but so were the bigger sized
quinoline and naphthalene rings.
The strong blue contours (preference for positive
charged entities) around ring A suggests that the elec-
tronic characteristics of ring A would be important for
activity. (Fig. 3b). Thus, the p deficient carbon atoms of
the pyridine ring of 214 would be suitably placed in this
region, as would the carbon atoms of the p deficient
quinoline ring.
In summary, the CoMFA model for antimalarial activity
indicates a preference for a sterically large (alkoxylated)
ring B and an electron deficient Ring A. These conclu-
sions compare favourably with those derived from mul-
tivariate analysis (large size ring B and polar ring A).3
Examination of the steric and electronic contours
around 214 in the CoMFA model for antileishmanial
activity shows that both contours are concentrated
around ring A (Fig. 3c and d). Ring A may thus have a
greater influence than ring B in the activity of
hydroxylated chalcones. The interspersing of red and
blue zones around ring A suggest that the ring can be
electron-deficient or electron-rich.
Smaller electronic contours are found around Ring B
and a careful examination shows that the good activity
of 40-hydroxylated chalcones may be traced to the
favourable orientation of the 40-hydroxyl group away
from a blue zone (preference for positive charge) and
towards a negative charge-seeking red zone, as seen in
Figure 3d for 214. When the B ring carries 20,40-dihy-
droxyl substituents, the 20-hydroxyl group impinges into
the blue zone (undesirable) although some members
(205, 207) have their B rings twisted so as to avoid this
interaction. Similarly, some 20-hydroxychalcones (241,
242) orient their hydroxyl groups away from this blue
zone. These members manifest better antileishmanial
activity than those that do not adopt a twisted B ring
conformation (235, 238, 239). This is shown in Figure 5,
using 207, 241 and 238 as examples. The reason as to
why some hydroxylated chalcones adopt a twisted
Figure 4. Orientation of 1-(20,40-dihydroxy)-3-(3-quinolinyl)-2-propen-
1-one (202) and its dimethoxy analogue (29) in the steric contours of
the CoMFA model for antimalarial acitivty. The methoxy groups on
ring B of 29 are directed towards the green zone (where bulk is desir-
able), unlike the hydroxyl groups of 202. This may account for the
generally better antimalarial activities of alkoxylated chalcones.

M. Liu et al. / Bioorg. Med. Chem. 11 (2003) 2729–2738
2735
Figure 5. Orientation of 1-(20,40-dihydroxy)-3-(2-pyridinyl)-2-propen-1-one (207), 1-(20-hydroxy)-3-(2-pyridinyl)-2-propen-1-one (241) and 20hydroxy-
4-trifluoromethylchalcone (238) in the electrostatic contours of the CoMFA model for antileishmanial activity. The B rings of 207 and 241 are ‘twisted’,
and their 20-hydroxyl groups (electron rich oxygen) do not point towards the blue zone. In 238, the B ring is not twisted and the 20-hydroxyl group points
towards the blue zone which is undesirable. ED50 of 238 is 29.3 mM compared to 2.23 and 1.57 mM for 207 and 241, respectively.
versus non-twisted B ring conformation is not known,
but these differences do exist.
CoMFA models for antimalarial and antileishmanial
activities. For antileishmanial activity, the steric and
electronic characteristics of ring A appear to be more
important than that of ring B. Ring A could be
electron-rich or electron poor. The good antileishma-
nial activities of the naphthalenyl (110, 205, 212, 242)
and pyridinyl (207, 214, 241) derivatives suggest that
considerable tolerance for the size of ring A exists.
For antimalarial activity, the size characteristics of
ring B (large, alkoxylated) and the electronic char-
acteristics of ring A (electron deficient) are important.
However, one shortcoming of these CoMFA models
is that they have been derived from different subsets
of compounds. The antimalarial model was obtained
using alkoxylated and hydroxylated chalcones with
category A activities, while the antileishmanial model
was based only on hydroxylated chalcones exhibiting
the full range (A–E) of activity. This may create
Conclusion
The present study shows that different physicochem-
ical and structural requirements exist for the anti-
malarial and antileishmanial activities of chalcones.
Conventional structure activity relationships show
that antileishmanial activity is favoured by chalcones
with more hydrophilic character, with the most active
members found among 40-hydroxychalcones. In con-
trast, good antimalarial activity is found among
alkoxylated chalcones with polar A rings, in parti-
cular those substituted with electron withdrawing
groups or replaced by quinoline rings. The relative
contributions of rings A and B were assessed by

2736
M. Liu et al. / Bioorg. Med. Chem. 11 (2003) 2729–2738
some biase in the interpretation of the results.
Despite this limitation, CoMFA confirms the differing
requirements for antimalarial and antileishmanial
activities. Notwithstanding these differences, two chal-
cones (8, 19) have been identified which have good in
vitro antimalarial and antileishmania activities
(<10 mM) and moderate toxicities (observed at 10 mM)
against mice macrophages. 8 is of particular interest as
it has been shown to increase the survivability of P.
berghei ANKA infected mice at a dose of 100 mg/kg.3
trol. Damage to macrophages was concurrently assessed
in order to determine toxicity. Where antileishmanial
activity was detected at 30 mM, the compound was
retested at an appropriate range of concentrations to
obtain its ED50, which was calculated by linear regres-
sion with P95 confidence limits.
The in vitro antimalarial activities of the chalcones were
evaluated by determining the concentration required to
inhibit the uptake of [3H] hypoxanthine into a chlor-
oquine-resistant strain of P. faciparum (K1) as reported
earlier.3
Experimental
Synthesis
Correlation and regression analyses
The chalcones were synthesized by a base-catalyzed
Claisen–Schmidt condensation of an aromatic aldehyde
with the appropriate ketone. The syntheses of the chal-
cones, except the 2-hydroxychalcones, have been repor-
ted earlier.3 The 20-hydroxyl chalcones (231–239,
241–244) were synthesized without protection of the
2-hydroxy group in 20-hydroxyacetophenone, following
the method of Nielsen et al.1 Briefly, the method
involved adding 20-hydroxyacetophenone (10 mmol,
10 mL methanol) dropwise to a stirred mixture of the
aldehyde (10 mmol) and NaOH (3% w/v) in methanol
(20 mL) and stirring at room temperature (28 ꢀC) for
4–18 h. Stirring in an ice bath was necessary if heat was
generated during addition. The solid formed was
removed by filtration, washed with cold methanol and
recrystallized. When no precipitate was obtained, the
solution was diluted with water, neutralized with HCl,
and extracted with ethyl acetate. The organic layer was
dried with anhydrous Na2SO4 and removed by eva-
poration under reduced pressure to give either a solid
or liquid residue. The solid residue was treated as
above. The liquid residue was passed through a column
of silica gel (230–400 mesh ASTM) and eluted with
chloroform or chloroform–hexane. Recrystallization
was done twice and purity checked by TLC before
characterization by 1H NMR, IR, accurate mass
determination and elemental analyses (Table 2). The
20-hydroxyl chalcones (231–239, 241–244) have been
reported in literature13À19 and a comparison of the
physical and spectroscopic data of the synthesized
compounds with reported values (where available)
showed good agreement.
The following parameters were determined from the
force-field minimized geometries of the chalcones using
the SYBYL 6.6 force field MMFF94 (Tripos Associates,
St Louis, MO, USA), with calculations continued until
the root mean square (rms) gradient was less than
0.001 kcal molÀ1 A: ClogP (Version 3), molecular
refractivity, total dipole moment, Connolly surfaces
(volume and surface area, calculated from MOLCAD in
SYBYL), negative charge on carbonyl oxygen calcu-
lated using the Gasteiger–Huckel method, HOMO and
LUMO (MOPAC, QCPE program 455, version 6.0,
interfaced with SYBYL). Multiple regression and Pear-
son correlation analyses of these properties were carried
out using SPSS 10 (SPSS Inc., Chicago, IL, USA). The
regression equations were assessed by the following sta-
tistical parameters: r2 (explained variance), standard
error (SE), cross validated r2 and cross validated SE
(determined using the QSAR module of SYBYL).
Comparative molecular field analysis (COMFA)
To obtain the CoMFA for antimalarial chalcones, the
energy-minimized conformations of the chalcones were
aligned using the geometry of the ab unsaturated car-
bonyl linkage of the most potent member for antimalarial
activity (27). In the case of antileishmanial activity, the
process was repeated using the most active antileishma-
nial chalcone (212). Both compounds have a trans orien-
tation of rings A and B, and comparable torsion angles
(9–11ꢀ) for the carbon–carbon single bond in the –C(O)–
CH¼CH– linkage.
CoMFA analysis was carried out using the QSAR mod-
ule of SYBYL 6.6 with the following settings: 2 A grid
spacing, 4 A extension of the region beyond the van der
Waals volume of the molecules, sp3 carbon probe atom
Antimalarial and antileishmanial activity
The in vitro sensitivity of Leishmania donovani (L82)
amastigotes to the test compounds were determined in a
mouse peritoneal model.20 Briefly, peritoneal macro-
phages were isolated from CD1 mice and maintained in
culture for 24 h before being infected with L. donovani
amastigotes isolated from the spleen of an infected
hamster. The test compounds were then added to the
infected macrophages 24 h later, with each compound
tested in quadruplicate at 30 mM. After 5 days of incu-
bation, parasite growth is assessed microscopically after
staining the cells with 10% Giemsa solution. The per-
centage of infected cells was determined and expressed
as% reduction in parasite burden compared to the con-
À
Á
with +1 charge and a distance dependent 1=r2 dielec-
tric constant. Computation of atomic charge was carried
out using the Gasteiger–Huckel method. The steric and
electrostatic CoMFA fields were scaled using default set-
tings which gives identical weight to CoMFA fields and
additional variables. Column filtering was set at 2.0 kcal
and steric/electrostatic cutoff values at 30 kcal/mol. The
CoMFA QSAR equations were derived using Partial
Least Squares (PLS) ‘leave-one-out’ cross validation
procedure and the models were evaluated on the basis of
cross validated r2, optimum number of components and
cross validated standard error of prediction (SEPcv).

Table 2. Physical and analytical data of 20-hydroxychalcones
Compd
Melting point (ꢀC)a
Yield (%)
Elemental analysis
C, H,
Accurate mass (M+1)
1H NMR (d ppm)
292.0067
(C15H10O2Cl2=292.0058)
In DMSO, 12.65 (s, OH) 8.26 (s, J=15.44 Hz, bH) 7.62 (s, J=15.44 Hz, aH)
7.88–6.95 (m, 20H, 30H, 50H, 60H, 3H, 5H, 6H)
231
232
176–178
69–71
10.0
25.0
Cl: calcd 23.95, found 24.33
C, H,
267.1251
(C17H17O2N=267.1259)
In DMSO, 12.72 (s, OH) 7.95 (s, J=15.07 Hz, bH) 7.59 (s, J=15.07 Hz, aH)
7.95–6.68 (m, 20H, 30H, 50H, 60H, 2H, 3H, 5H, 6H) 3.03 (s, –N–(CH3)2)
N: calcd 5.24, found 5.39
C, H,
233
196–198
32.6
275.0938
(C18H13O2N=275.0946)
In DMSO, 12.42 (s, OH), 9.20 (d, 2H) 8.26 (s, J=15.83 Hz, bH), 7.53
(s, J=15.82 Hz, aH), 8.30–6.82 (m, 4H, 5H, 6H, 7H, 8H, 20H, 30H, 50H, 60H)
N: calcd 5.09, found 5.18
C: calcd 69.76, found 70.04
258.0443
(C15H11O2Cl=258.0447)
In DMSO, 12.77 (s, OH), 7.84 (m, J=15.83 Hz, bH), 7.61 (m, J=15.45 Hz,
aH), 7.89–6.94 (m, 2H, 3H, 5H, 6H, 20H, 30H, 50H, 60H)
234
235
149–150
48.1
44.6
H: calcd 4.30, found 4.75
Cl: calcd 13.55, found 13.28
C, H
114–115
120–121b
238.0986
(C16H14O2=238.0994)
In DMSO, 12.88 (s, OH), 7.91 (s, J=15.45 Hz, bH), 7.62 (d, J=15.45 Hz,
aH), 7.92–6.94 (m, 2H, 3H, 5H, 6H, 20H, 30H, 50H, 60H), 2.36 (s, CH3)
236
89–91
91–92b
35.4
C, H
C,
254.0946
(C16H14O3=254.0943)
In DMSO, 12.95 (s, OH), 7.90 (s, J=15.45 Hz, bH), 7.54 (s, J=15.45 Hz,
aH), 7.92–6.95 (m, 2H, 3H, 5H, 6H, 20H, 30H, 50H, 60H), 3.82 (s, OCH3)
284.1049
(C17H16O4=284.1049)
In DMSO, 12.90 (s, OH), 7.96 (d, J=15.8 Hz, bH), 7.65 (d, J=15 Hz, aH),
7.98–6.57 (m, 2H, 3H, 5H, 6H, 20H, 30H, 50H, 60H), 3.90 (s, OCH3), 3.82
(s, OCH3)
237
238
111–113
25.0
46.4
H: calcd 5.68, found 5.47
C: calcd 65.74, found 64.95
292.0704
(C16H11O2F3=292.0711)
In DMSO, 12.69 (s, OH), 8.03 (m, J=15.82 Hz, bH), 7.70 (m, J=15.45 Hz,
aH), 8.11–6.90 (m, 2H, 3H, 5H, 6H, 20H, 30H, 50H, 60H)
<50
H: calcd 3.80, found 3.78
F: calcd 19.51, found 19.71
C,
242.0732
(C15H11O2F=242.0743)
In DMSO, 12.37 (s, OH), 7.88 (m, J=15.45 Hz, bH), 7.64 (d, J=15.45 Hz,
aH), 8.08–6.88 (m, 2H, 3H, 5H, 6H, 20H, 30H, 50H, 60H)
239
<50
43.5
H: calcd 4.58, found 4.50
F: calcd 7.85, found 7.62
C,
225.0785
(C14H11O2N=225.0790)
In DMSO, 12.73 (s, OH), 8.43–8.38 (d, J=15.45 Hz, bH), 7.77–7.72
(d, J=15.45 Hz, aH), 8.82–6.93 (m, 3H, 4H, 5H, 6H, 20H, 30H, 50H, 60H)
241
242
89–91
25.3
41.1
H: calcd 4.93, found 5.03
N: calcd 6.22, found 6.38
C, H
106–108
274.0993
In DMSO, 12.42 (s, OH), 8.69 (d, J=15.45 Hz, bH), 7.73 (d, J=15.44 Hz,
aH) 8.50–6.70 (m, 2H, 3H, 4H, 5H, 6H, 7H, 8H, 20H, 30H, 50H, 60H)
In DMSO, 12.16 (s, OH), 8.07–8.02 (d, J=15.44 Hz, bH), 7.76–7.71 (d, J=
15.45 Hz, aH) 8.40–6.83 (m, 1H, 3H, 4H, 5H, 6H, 7H, 8H, 20H, 30H, 50H,
60H)
(C19H14O2=274.0994)
274.1003
(C19H14O2=274.0994)
C,
243
244
146–148
151–153
39.6
11.2
H: calcd 5.15, found 5.10
C,
275.0960
(C18H13O2N=275.0946)
In DMSO, 12.39 (s, OH), 8.48–8.43(d, J=15.45 Hz, bH), 8.37–8.31 (d, J=
15.45 Hz, aH), 9.27–6.93 (m, 2H, 3H, 5H, 6H, 7H, 8H, 20H, 30H, 50H, 60H)
H: calcd 4.76, found 4.89
N: calcd 5.09, found 5.21
aRecrystallized from methanol.
bCited in product catalog (2000/2001) of Indofine Chemical Company, Inc. (Somerville, NJ, USA).

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M. Liu et al. / Bioorg. Med. Chem. 11 (2003) 2729–2738
7. Ducki, S.; Forrest, R.; Hadfield, J. A.; Kendall, A.; Lawr-
Acknowledgements
ence, N. J.; McGown, A. T.; Rennison, D. Bioorg. Med.
Chem. Lett. 1998, 8, 1051.
Mei Liu gratefully acknowledges the National Uni-
versity of Singapore (NUS) for granting her a research
scholarship. This work has been supported by RP-140-
000-016-112 (NUS) and Thailand Research Fund. The
authors gratefully acknowledge Miss Kate De Luca-
Fradley of the London School of Hygiene and Tropical
Medicine for carrying out the antileishmanial assays.
SLC received financial support from the UNDP/World
Bank/WHO Special Program for Research and Training
in Tropical Diseases.
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