In vitro and in vivo anti-Leishmania activity of polysubstituted synthetic chalcones

Article abstract of DOI:10.1016/j.bmcl.2009.11.033

The in vitro screening of 43 polysubstituted chalcones against Leishmania amazonensis axenic amastigotes, led to the evaluation of 9 of them in a macrophage-infected model with the two other most infectious Leishmania species prevalent in Peru (L. braziliensis and L. peruviana). The five most active and selective chalcones were studied in vivo, resulting on the identification of two chalcones with high reduction parasite burden percentages.

Full text of DOI:10.1016/j.bmcl.2009.11.033

Bioorganic & Medicinal Chemistry Letters 20 (2010) 100–103
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro and in vivo anti-Leishmania activity of polysubstituted synthetic
chalcones
José C. Aponte a, Denis Castillo b, Yannick Estevez c,d, German Gonzalez b, Jorge Arevalo b,
Gerald B. Hammond a, Michel Sauvain c,d,
*
a Department of Chemistry, University of Louisville, KY 40292, USA
b Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru
c IRD, Université de Toulouse III, UMR-152, Mission IRD, Lima, Peru
d Université de Toulouse, UPS, UMR-152 (Laboratoire de Pharmacochimie des Substances Naturelles et Pharmacophores Redox), 118, rte de Narbonne, F-31062 Toulouse Cedex
9, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The in vitro screening of 43 polysubstituted chalcones against Leishmania amazonensis axenic amastig-
otes, led to the evaluation of 9 of them in a macrophage-infected model with the two other most infec-
tious Leishmania species prevalent in Peru (L. braziliensis and L. peruviana). The five most active and
selective chalcones were studied in vivo, resulting on the identification of two chalcones with high reduc-
tion parasite burden percentages.
Received 16 September 2009
Revised 9 November 2009
Accepted 9 November 2009
Available online 14 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Anti-Leishmanial drugs
Chalcone
Leishmania amazonensis
Peruviana
Braziliensis
Leishmaniasis is a vector-borne disease caused by protozoan
parasites of the genus Leishmania. It is transmitted through the
bite of female phlebotomine sandflies and can range from mild
self-healing cutaneous lesions to lethal visceral leishmaniasis. Dif-
ferent clinical manifestations of the various forms of leishmaniasis
are given by different types of infected tissues, host genetic factors
and immune responses. Leishmaniasis is prevalent in 82 countries
and has infected about 12 million people worldwide.1 Recently,
more than a thousand Leishmania strains have been isolated and
identified from patients co-infected with HIV, which can exhibit
wider drug-resistance, and, interestingly, the majority of these
cases where found on individuals in industrialized countries such
France, Spain and Italy.2 The United States of America and Canada,
countries where leishmaniasis is not endemic, face canine leish-
maniasis as a public health problem;3 furthermore, in the year
2004, 522 cases of cutaneous leishmaniasis where confirmed by
the US Department of Defense, from military personnel who served
in Southwest/Central Asia.4 With no available vaccine, the only
control intervention chemotherapy against leishmaniasis involves
the use of highly toxic pentavalent antimonials, developed decades
ago and for which resistance has been extensively reported.5
Amphotericin B is used alternatively as second-line drug; however
it is highly toxic and expensive for routine use in resource-poor
countries.1e Clearly, the development of new effective drugs with
high selectivity, minimum side effects and low manufacture costs
is urgently required. The economical, facile and rapid synthesis of
chalcones,6a make them attractive as potential drug candidates to
fight leishmaniasis and some other of the so-called neglected dis-
eases that affect the populations of many countries in the Third
World.
Chalcones exhibit a wide variety of pharmacological activities,
among them their anticancer, antibacterial and antiprotozoal are
remarkable.6 Recently, we have reported the synthesis, cytotoxic-
ity and in vitro anti-Trypanosoma cruzi activity of 28 new chalcones
featuring hydroxyl and allyoxy moieties on the ring A.7 On this
communication, we want to report the in vitro anti-Leishmania
activity of 43 chalcones and the in vivo study of five of the ones
that exhibited the highest parasite-development inhibition and
the lowest cytotoxicity, namely, 21, 38, 41, 42 and 43.
The general method for the synthesis of chalcones is summa-
rized in Scheme 1; the preparation of compounds 1–40, has been
described previously.7 Synthesis of chalcones 41–43 was accom-
plished using previously reported methodology.8 All synthetic
compounds are described on Table 1. To the best of our knowledge
this is the first report for preparation of 13 compounds (25, 27, 32–
40 and 43). In this present work we have evaluated the activity of
* Corresponding author. Tel.: +51 14811794; fax: +51 14413223.
E-mail addresses: michel.sauvain@ird.fr, michel.sauvain1@orange.fr (M. Sau-
vain).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.033

J. C. Aponte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 100–103
101
OH
O
O
O
O
O
O
O
O
a
b
c
HO
OH
O
OH
HO
O
OH
O
f
d
O
O
O
O
R3
O
O
O
O
O
O
O
R3
2',4'-AC
3-23
d
O
OH
O
O
O
g
e
O
2'-EC
43
R3
g
O
O
O
R3
O
O
HO
OH
2',4'-OC
41-42
2',4'-HC
24-40
Scheme 1. Reagents and conditions: (a) K2CO3, (CH3)2SO4, (CH3)2CO, 65 °C, 6 h; (b) AlCl3, benzene, reflux, 1 h; (c) K2CO3, allyl bromide, DMF, rt, overnight; (d) Claisen–
Schmidt aldol condensation of an acetophenone with an aromatic aldehyde, KOH, H2O, CH3OH, rt, 1–48 h; (e) K2CO3, catalytic Pd(PPh3)4, MeOH, 60 °C, 1 h; (f) K2CO3,
(CH3)2CO, CH3OCH2Cl, reflux, 3 h; (g) HCl 3 N, reflux, CH3OH, 30 min.
43 substituted chalcones against axenic amastigotes of Leishmania
amazonensis (Table 1), and the anti-L. braziliensis and anti-L. peru-
viana activity of nine chalcones, using macrophages infected with
the corresponding parasites (Table 2). As shown in Table 1, the
great majority (74%) of the synthesized compounds exhibited an
parasitic activity for this set of compounds is independent of the
substitution pattern on the ring A, since the anti-Leishmanial activ-
ity is conserved for each analog pair. This appreciation is also appli-
cable for inactive compounds such 19 and 39, in which it can also
be seen that the conjugation of the double bond on the
urated bridge results on the lost of the bioactivity.
a,b-unsat-
IC50 value below 20
lM, while 21 of them (49%) an IC50 value be-
low 10 M; more interestingly, nine of the most active compounds
l
Table 2 shows the bioactivities of the nine compounds which
showed the highest SI on the axenic amastigotes assay. From this
macrophage-infected model, it can be observed that, in general,
L. peruviana was the species of parasites which showed the stron-
gest resistance towards all chalcones, whereas L. braziliensis and L.
amazonensis seemed to respond differently depending on the type
of chalcone administered. Compound 41 showed an interesting
selectivity against L. braziliensis. Although it showed selectivity
(21, 26, 30, 31, 38, 40–43) showed a selectivity index (SI) greater
the 10. Among the two largest series of chalcones (20,40-diallyl-
oxy-60-methoxychalcones, 20,40-AC, 3–23 and 20,40-dihydroxy-60-
methoxychalcones, 20,40-HC, 24–40), it was the 20,40-HC chalcone
series, which showed the better selectivity against the axenic
amastigotes of L. amazonensis, despite not being the series with
the most active derivatives.
The anti-Leishmania activity of chalcones with different substi-
tution patterns have been previously reported.6 Herein, we present
for the first time, the anti-Leishmania activity of 20,40-AC; 20,40-HC;
20,40-OC and 20-EC. Among compounds 1, 5, 26 and 41, which differ
only on the substitution pattern on ring A, it can be observed that
having a methoxymethyl substituent on the 20,40-position not only
maintains the activity of the molecule, but also greatly enhances its
selectivity against the parasite, when compared to its 20,40-allyoxy
and 20,40-hydroxy analogs. This observation suggests that there ex-
ists considerable tolerance for the size and substitution pattern on
ring A. In the same way, compounds 4, 7, 8, 10, 12, 18 and 23, all of
them having 20,40-diallyloxy moieties on the ring A, resulted inac-
against the L. amazonensis species (29
resulted highly selective on the axenic amastigote assay—resulted
inactive against L. braziliensis and L. peruviana (237 and 290 M,
respectively). On the other hand, compound 21, containing a pyrid-
inyl moiety, seemed to exert the highest bioactivity against all Leis-
lM), compound 31—which
l
mania species with values ranging from 0.9 to 4.0 lM.
Considering these in vitro results, as well as their synthetic and
physicochemical characteristics; we started an in vivo study using
compounds 21, 38, 41, 42 and 43. During the preparation of the
20,40-HC series, we did not find the isomeric flavanone that could
arise as a condensation product for any of the compounds in this
series. For the in vivo assay, a larger amount of sample was needed,
and in this regard the synthesis of compounds 26, 30 and 31 was
not as readily simple as for compounds 21, 41–43. Therefore, be-
sides compounds 26 and 30 showed moderate activities on the
macrophage-infected model, they were not selected for the
in vivo screening. However, the chemical functionalities on ring B
for 26 and 30, are in close relation to those on compounds 41–
43, furthermore; the solubility of the later is several times greater
in DMSO. The in vivo results are summarized on Table 3. From
these results, we can conclude that none of the five tested com-
pounds was as effective as the positive control (Gluc) to reduce
tive (IC50 values >80
substituted counterparts, compounds 25, 28, 29, 31, 33, 38 and 40
showed IC50 values ranging from 15.7 to 1.1 M. These results are
l
M) while their corresponding 20,40-dihydroxy
l
in agreement with the results reported by Liu and co-workers,6b
which showed that the anti-Leishmania activity of chalcones is en-
hanced by the presence of polar 20 and 40-hydroxy groups. How-
ever, when comparing the rest of active 20,40-AC series (3, 5, 6, 9,
11, 13–16) against their corresponding 20,40-HC series (24, 26, 27,
30, 32, 34–37), all of them bearing electron donating or electron
withdrawing groups on the ring B, it seems clear that the anti-

102
J. C. Aponte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 100–103
Table 1
In vitro activity of compounds 1–43 against axenic amastigotes of L. amazonensis and macrophage
O
O
β
1'
2'
1
α
R3
4
Ring B
Ring A
R2O
Ring B
OR1
4'
a
Compds
Ring A
IC50 (lM)
L. amazonensis
Macrophage
SIb
1
2
3
4
5
6
7
!8
9
20-Hydroxy,40-methoxy
20,40-Diallyloxy
4-Methoxy
3,4-Methylenedioxy
Benzyl
4-Methyl
4-Methoxy
17.2 12.3
>300
ndc
—
14.8 2.5
—
18.7 0.7
13.1 1.1
—
—
nd
—
—
—
9.4 1.5
79.8 5.2
5.5 1.0
8.7 0.8
85.2 3.3
107.2 5.1
6.6 4.2
134.5 9.7
8.2 3.9
127.6 5.6
9.4 4.3
9.5 1.8
9.5 0.8
7.9 2.4
8.3 1.0
>180
1.6
—
3.4
1.5
—
—
—
—
1.5
—
1.4
1.5
1.4
0.7
1.6
—
—
—
11.4
—
—
1.7
2.2
16.6
1.5
1.4
—
38.3
>50
—
2.0
1.7
2.4
1.4
1.4
10.5
—
83.5
>200
12.2
21.4
76.0
3-Methoxy
4-Hydroxy
4-Hydroxy-3-Methoxy
3,4-Methylenedioxy
2,4-Dimethoxy
3,4,5-Trimethoxy
4-Trifluoromethyl
4-Chloro
4-Fluoro
2-Fluoro
2-Bromo
4-Nitro
4-Bromo-3,5-Diallyloxy
Cinnamyl
4-Pyridinyl
2-Pyridinyl
2-Pyrrolyl
2-Funanyl
Benzyl
4-Methyl
4-Methoxy
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
12.0 0.6
—
13.0 2.0
13.8 0.9
13.3 5.3
5.4 1.0
13.2 0.4
—
—
nd
12.5 0.8
—
—
27.0 2.0
16.5 4.8
166.2 4.7
17.6 2.9
21.7 0.7
nd
157.2 0.9
>300
nd
17.1 1.8
19.0 4.2
28.8 2.6
16.0 1.1
14.6 5.5
11.5 2.0
—
384.3 13.2
>250
142.9 11.0
132.5 31.3
5.3 0.2
75.2 10.3
0.6 0.1
1.1 0.3
94.3 11.4
>300
20,40-Dihydroxy
15.9 1.7
7.4 1.9
10.0 0.1
12.0 0.2
15.7 3.6
14.9 1.7
4.1 1.2
6.4 0.9
9.7 1.2
8.6 2.0
11.2 1.6
11.8 0.3
11.8 0.5
10.6 2.4
1.1 0.1
52.6 4.8
4.6 1.1
1.3 0.2
11.7 4.1
6.2 0.7
0.1 0.01
3-Methoxy
4-Hydroxy
4-Hydroxy-3-Methoxy
3,4-Methylenedioxy
2,4-Dimethoxy
3,4,5-Trimethoxy
4-Trifluoromethyl
4-Chloro
4-Fluoro
2-Fluoro
2-Bromo
4-Bromo-3,5-Diallyloxy
Cinnamyl
2-Funanyl
4-Methoxy
3,4-Methylenedioxy
3,4,5-Trimethoxy
20,40-Dimethoxy methyl
20-Hydroxy-40-methoxymethyl
Amphotericin B
a
b
c
IC50: concentration that produces 50% inhibitory effect.
SI: selectivity index = IC50, macrophage/IC50,
Not determined.
.
L. amazonensis
Table 2
Activity of chalcones against macrophages infected with three Leishmania species
(values in M)
the lesion diameter (mice’s footpad measured with a caliper).
However, since these results depend on a series of parameters such
as immune response, inflammation process and parasite viru-
lence—which are not proportional to the parasite load and the par-
asite burden—we completed the measure of mice’s footpad by
counting the L. amazonensis amastigotes in the foot tissue using a
fluorescent probe.
From this count, compounds 42 and 43 (administered to the in-
fected mice in a concentration almost seven times lower than the
positive control) showed high reduction of the parasite burden,
with P = 0.0004 and P = 0.0019, respectively, after the initial four
weeks of treatment, meaning a reduction of the parasite burden
of 92% and 74%, respectively. These results were further confirmed
by the experiment at the seventh week in which they showed
l
Compds L. amazonensis Lma
L. braziliensis
L. peruviana
CL1
PER006
LCA08
21
26
30
31
38
40
41
42
0.9
14.3
16.6
28.5
4.1
16.9
12.1
24.0
29.7
0.4
1.4
7.6
8.6
237.0
14.4
30.4
5.2
17.2
13.1
0.1
4.0
23.0
19.7
290.2
ndb
34.2
110.8
34.8
19.3
0.1
43
Amp. Ba
a
Amphotericin B.
Not determined.
b
a
35% and 41% reduction of parasite burden, respectively.

J. C. Aponte et al. / Bioorg. Med. Chem. Lett. 20 (2010) 100–103
103
Table 3
In vivo activity of chalcones on L. amazonensis-infected BALB/c mice (n = 10)a
Supplementary data
Compds
Lesion diameter (mm)b
after 4 weeks
Reduction of parasite burden (%)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.11.033.
After 4 weeks
After 7 weeks
Control
21
38
41
42
2.8 0.4
3.0 0.3
3.1 0.3
2.8 0.1
2.9 0.2
3.5 0.5
2.1 0.1
—
ꢀ4
9
11
92
74
100
—
25d
8d
References and notes
17
35
41
100
1. (a) Alexander, J.; Russell, D. G. Adv. Parasitol. 1992, 31, 175; (b) Herwaldt, B. L.
Lancet 1999, 354, 1191; (c) Wiese, M.; Ilg, T.; Lottspeichl, F.; Overath, P. EMBO J.
1995, 14, 1067; (d) Dujardin, J.-C. Trends Parasitol. 2006, 22, 4; (e) Nwaka, S.;
Hudson, A. Nat. Rev. Drug Disc. 2006, 5, 941.
43
Gluc.c
a
b
c
2. Alvar, J.; Aparicio, P.; Aseffa, A.; Den Boer, M.; Canavate, C.; Dedet, J.-P.;
Gradoni, L.; Ter Horst, R.; Lopez-Velez, R.; Moreno, J. Clin. Microbiol. Rev.
2008, 21, 334.
3. (a) Oliva, G.; Scalone, A.; Foglia Manzillo, V.; Gramiccia, M.; Pagano, A.; Di
Muccio, T.; Gradoni, L. J. Clin. Microbiol. 2006, 44, 1318; (b) Duprey, Z. H.; Steurer,
F. J.; Rooney, J. A.; Kirchhoff, L. V.; Jackson, J. E.; Rowton, E. D.; Schantz, P. M.
Emerg. Infect. Dis. 2006, 12, 440.
4. (a) Aronson, N.; Ananthakrishnan, M.; Bernstein, W.; Hochberg, L.; Marovich, M.;
Ockenhouse, C.; Yoon, I.; Weina, P.; Benson, P.; Fischer, J.; Hack, D.; Hawkes, C.;
Polhemus, M.; Wortmann, G.; McEvoy, P.; Neafie, R.; Defraites, R.; Herwaldt, B. L.
J. Am. Med. Assoc. 2004, 291, 2188; (b) Coleman, R. E.; Burkett, D. A.; Putnam, J. L.;
Sherwood, V.; Caci, J. B.; Jennings, B. T.; Hochberg, L. P.; Spradling, S. L.; Rowton,
E. D.; Blount, K.; Ploch, J.; Hopkins, G.; Raymond, J.-L. W.; O’Guinn, M. L.; Lee, J.
S.; Weina, P. J. J. Med. Entomol. 2006, 43, 647.
5. (a) Brochu, C.; Wang, J.; Roy, G.; Messier, N.; Wang, X.-Y.; Saravia, N. G.;
Ouellette, M. Antimicrob. Agents Chemother. 2003, 47, 3073; (b) Mittal, M. K.; Rai,
S.; Ravinder, A.; Gupta, S.; Sundar, S.; Goyal, N. Am. J. Trop. Med. Hyg. 2007, 76,
681; (c) Croft, S. L.; Sundar, S.; Fairlamb, A. H. Clin. Microbiol. Rev. 2006, 19, 111.
6. (a) Yunes, R. A.; Chiaradia, L. D.; Leal, P. C.; Cechinel Filho, V.; Torres-Santos, E.
C.; Falcao, C. A. B.; Rossi-Bergmann, B. Curr. Trends Med. Chem. 2006, 4, 47; (b)
Liu, M.; Wilairat, P.; Croft, S. L.; Tan, A. L.-C.; Go, M.-L. Bioorg. Med. Chem. 2003,
11, 2729.
Effect of treatments after eight intralesional inoculations.
Compounds administrated at 5 mg/kg/day.
N-methylglucamine antimoniate, administrated at 33 mg/kg/day.
Compounds 21 and 38 were analyzed after 6 weeks treatment.
d
Compounds 42 and 43 did not exhibit any cutaneous toxicity at the
tested doses and they may not exhibit a reduction of the lesion
diameter, probably due to their lack of anti-inflammatory activity.
In conclusion we have screened 43 polysubstituted chalcones
against L. amazonensis amastigotes and further tested the most
selective against a macrophage-infected model using the three
most infectious parasites prevalent in Peru. After this large
in vitro screening, five of the best compounds were selected for
in vivo analysis, resulting on the identification of two chalcones
with high anti-parasitic potential.
Acknowledgment
7. Aponte, J. C.; Verástegui, M.; Málaga, E.; Zimic, M.; Quiliano, M.; Vaisberg, A. J.;
The authors are grateful to the FINCYT-BID (Banco Interameri-
cano para el Desarrollo) Program in Peru (Grant #PIBAB03-084 to
M.S.).
Gilman, R. H.; Hammond, G. B. J. Med. Chem. 2008, 51, 6230.
8. Du, Z.; She, X.; Ma, J.; Wang, Z.; Wu, H.; Li, Y.; Pan, X. J. Chem. Res.
2004, 1, 45.