Leishmanicidal activity of some stilbenoids and related heterocyclic compounds

Article abstract of DOI:10.1016/S0960-894X(01)00387-0

We have evaluated the leishmanicidal activity of some natural and semisynthetic dihydrostilbenoids and several compounds of other series of dihydrostilbamides, isoindoles, phthalazinones, imidazoisoindoles and pyrimidoisoindoles. The evaluation was performed in vitro, on cultures of cutaneous, mucocutaneous and visceral strains of Leishmania spp. The most potent and selective compounds of these series were the dihydrostilbene piperidides.

Full text of DOI:10.1016/S0960-894X(01)00387-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2123–2126
Leishmanicidal Activity of Some Stilbenoids and Related
Heterocyclic Compounds
Esther del Olmo,a,* Marlon Garcıa Armas,b Jose Luis Lopez-Perez,a Victoria Munoz,c
Eric Deharoc and Arturo San Felicianoa
a
´
´
Departamento de Quımica Farmaceutica, Facultad de Farmacia, 37007 Salamanca, Spain
bUniversidad P. Antenor Orrego, Trujillo, Peru
´
Instituto Boliviano de Biologıa de la Altura, La Paz, Bolivia
c
Received 13 February 2001; revised 7 May 2001; accepted 21 May 2001
Dedicated to the memory of Professor Joaquın De Pascual Teresa
Abstract—We have evaluated the leishmanicidal activity of some natural and semisynthetic dihydrostilbenoids and several com-
pounds of other series of dihydrostilbamides, isoindoles, phthalazinones, imidazoisoindoles and pyrimidoisoindoles. The evaluation
was performed in vitro, on cultures of cutaneous, mucocutaneous and visceral strains of Leishmania spp. The most potent and
selective compounds of these series were the dihydrostilbene piperidides. # 2001 Elsevier Science Ltd. All rights reserved.
Natural stilbenoids and their synthetic analogues, in
addition to their formerly reported therapeutic uses,
mainly based on their hormonal/antihormonal activ-
ities, are presently well known by other kinds of bioac-
tivity, such as antitumoral and antileukemic, platelet
antiaggregation,1 anti-inflammatory and antihistaminic2
activities. A number of recently described representative
reports can be found in the literature, for example,
piceatannol,3 lavendustin A,4 resveratrol,5 etc. Never-
theless, a computerized literature search looking for
descriptions of antiparasitic activities of stilbenoids,
revealed the inexistence of reports on this subject, with
the only exception of one paper published by one of us,
in this journal, dealing with the in vitro leishmanicidal
activity of combretastatins and some heteroanalogues.6
L. amazonensis and L. aethiopica, are not directly lethal,
but provoke multiple granulomatose or diffuse, auto-
inoculable and even metastatic skin ulcers, resembling
leprosy lesions. On the other hand, visceral leishma-
niasis, produced by L. donovani, localized in blood,
liver, spleen and other vital organs and tissues, is fatal in
more than 90% of untreated cases.9 Drugs currently in
use as the antimony derivative glucantine, the bis-ami-
dines, pentamidine and stilbamidine or the glycomacro-
lide amphotericin B, display high liver and heart
toxicities, develop clinical resistance after a few weeks of
treatment10 and currently contribute to increase co-
infections leishmaniasis-AIDS in some countries.11 For
these reasons it becomes necessary to discover new,
natural or synthetic, more potent and selective agents
for treating this increasing parasitosis. This paper
describes the in vitro anti-leishmanial evaluation of
some natural stilbenoids and some derivatives as well as
of some related condensed heterocyclic derivatives, on
cutaneous, mucocutaneous and visceral strains of
Leishmania.
Leishmaniasis is a parasitic disease endemic to the
American, African and Asian tropical countries.7 It
affects some 12 million people around the world and
350 million live on risk of becoming infected, of whom
some 2 million will actually be infected each year.8 The
most common varieties of leishmaniasis, the cutaneous
and mucocutaneous ones, produced by Leishmania tro-
pica, L. major, L. mexicana, L. braziliensis, L. peruviana,
Chemistry
The structural similarity of isonotholaenic acid (1a)
with combretastatins prompted us to submit the com-
pound to a number of tests of bioactivity and, among
*Corresponding author. Tel.: +34-823-29-4500x1825; fax: +34-823-
2945-15; e-mail: olmo@gugu.usal.es
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00387-0

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E. del Olmo et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2123–2126
them, those on Leishmania, Trypanosoma and Plasmo-
dium growth. Compound 1a displayed some activity on
Leishmania spp and Plasmodium falciparum and much
less activity on Trypanosoma cruzi. This finding promp-
ted us to prepare and evaluate the new substances
depicted in Scheme 1, with the aim of obtaining
improved antiparasitic agents.
tural fragments, as the common natural trimethoxy-
phenyl fragment and a masked amidine moiety, whose
presence in antiprotozoal and other antimicrobial
agents has proven to be useful clinically. The detailed
description of synthetic procedures, physicochemical
and spectroscopic data and structure assignments for
these compounds will be reported in a complete paper.
Compounds 1 are natural products or were obtained
through simple chemical transformations from iso-
notholaenic acid (1a), the main component of dichloro-
methane extracts from the Andean fern, Notholaena
nivea var. nivea.12 Compounds 3–9 were prepared by
total synthesis, through condensation of phthalic anhy-
dride with several substituted phenylacetic acids, to give
the corresponding benzalphthalide intermediates 2,
which were subsequently treated with piperidine,
hydrazine derivatives or diamines, to obtain, respec-
tively, the piperidides (3 and 4), isoindolinones (5),
phthalazinones (6 and 7), imidazo- (8) and pyrimido-
isoindoles 9 shown above. Compounds 4 could prob-
ably be formed through the autoxidation of a piperidine
enamine intermediate.13 Indeed, the proportion 3/4 can
be modulated by controlling the reaction conditions. All
the compounds retained the main stilbenoid skeleton of
isonotholaenic acid (1a) and incorporated some struc-
Biological Assays
The isolation, culture and maintenance of promastigote-
stage parasites have been previously described.14 Pro-
mastigotes inhibition studies were performed on L.
amazonensis (IFLA/BR/67/pH8), L. braziliensis
(MHOM/BR/75/M2903) and L. donovani (MHOM/BR/
74/pp75) grown at 27 ꢀC in Schneider’s drosophila
medium containing 10% of fetal bovine serum. Com-
pounds were dissolved in dimethyl sulphoxide (DMSO)
(final concentration of DMSO less than 0.1%) and
liquid medium placed in microcells Titertek 96 (Flow
Laboratories) to obtain final concentration of 100, 50,
25 and 10 mg/mL. All assays were done in triplicate.
Promastigote cultures in the logarithmic phase were
transferred at the level of 105 parasites per cell. The
activity of the compounds was evaluated after
Scheme 1. Natural and synthetic derivatives tested as anti-leishmanial agents.

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2123–2126
2125
incubation during 72 h, by optical observation and cell
counting, on a drop of each culture, with an inverted
microscope and comparison with control cells without
product and other containing the reference drug,
pentamidine (10 mg/mL).
In the case of piperidides (3 and 4), some main con-
siderations can be made. The presence of two carbonyl
groups at the ethylenic bridge seems to induce some
enhancement of the activity, in relation with those less
oxidized compounds, having only one keto group (4a vs
3a, 4b/3b and 4f/3f), except for 3e, the most active
compound of all the series evaluated. In parallel with
phthalide 2e, the piperidide 4e displayed certain selec-
tivity against the PH8 strain. Compound 3e was simi-
larly selective against M2903 and PH8 strains, with
potency close to that of pentamidine. The chloro derivative
4f also was active against the three strains, with some
selectivity for L. amazonensis and L. donovani. It must be
noted that the presence of the piperidide (1e, 3e, 4e) or the
N,N-diethylamide moities (4b2) are very important for
the anti-leishmanial activity, and that the other type of
amide like pyrrolidide seems to be less significant.
Results and Discussion
The results of the anti-leishmanial evaluation are shown
in Table 1. Within the group of natural products and
derivatives, isonotholaenic acid (1a) provoked the total
lysis of the three strains of Leishmania parasites at the
concentration of 50 mg/mL (IC50=32 mg/mL), while the
masking of its phenolic group as acetate (1b) or the
carboxylic acid as its methyl ester (1c), fairly decreased
or fully eliminated the activity, respectively. Elimination
of the carboxylic function also led to an inactive pro-
duct (1d), while the transformation into an amide (1e)
improved the activity of 1a four times, attaining a
potency only one order lower than that of the reference
drug, pentamidine.
Only a few compounds with structures 5 to 9 (Table 1)
displayed some activity against Leishmania; namely, the
chlorobenzylphthalazinone derivative 7f and the unsub-
stituted benzyl derivatives of imidazo- (8a) and pyr-
imido-isoindole (9a). They seem do not merit further
consideration.
Benzalphthalide intermediates, 2, were in general less
active, with the exception of compounds 2a (unsub-
stituted phenyl ring), 2e (3,4,5-trimethoxyphenyl) and 2f
(p-chlorophenyl). It is to be noted that while 2a showed
similar potency against the three Leishmania strains,
compounds 2e and 2f displayed some selectivity for one
of the strains, mucocutaneous PH8 in the case of 2e and
visceral PP75 in that of 2f.
On the basis of the results obtained for the piperidide
series, a number of different electron donating or with-
drawing substituents are being incorporated on the
starting anhydride, and further chemical and evaluation
studies, focused to enhance the selectivity and potency of
these new types of anti-Leishmanial agents, will be done.
Table 1. In vitro activity of stilbenoid derivatives on Leishmania spp. promastigotes
Type
Compound
L. braziliensis
(M2903)
L. amazonensis
(PH8)
L. donovani
(PP75)
Dihydrostilbenoid
1a
1b
1c, 1d
1e
32
62
I
32
62
I
32
62
I
10
7
7
Benzalphthalide
Piperidide
2a
2b–2d
2e
45
I
I
45
I
52
I
45
I
I
52
2f
I
3a–3d
3e
3f
4a
I
7
I
75
80
I
75
I
I
10
I
75
I
I
75
I
I
20
I
75
80
I
75
I
52
28
4b
(Pyrrolidide)
(Diethylamide)
4b1
4b2
4c, 4d
4e
52
52
28
25
4f
Isoindolinone
5a, 5b
I
I
I
Phthalazinone
Methylphthalazinone
6a–6f
7a–7e
7f
I
I
I
I
I
I
I
I
64
Imidazo-isoindole
Pyrimido-isoindole
Reference
8a
8b–8f
9a
60
I
45
I
60
I
45
I
60
I
45
I
9b–9f
Pentamidine
1.3
1.3
1.3
IC50 values (mg/mL); Iꢁ100 mg/mL (inactive).

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E. del Olmo et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2123–2126
J. M. Drugs Exp. Clin. Res. Commun. 1999, 25, 65. (c) Lu, R.;
Serrero, G. J. Cell. Physiol. 1999, 179, 297.
Acknowledgements
6. Del Rey, B.; Ramos, A. C.; Caballero, E.; Inchaustti, A.;
Yaluff, G.; Medarde, M.; Rojas de Arias, A.; San Feliciano,
A. Bioorg. Med. Chem. Lett. 1999, 9, 2711.
7. World Health Organization. Tropical Disease Research.
Progress 1975–1994. Highlights 1993–1994; UNDP/World
Bank/WHO: Geneva, 1995; p 137.
Financial support came from Spanish DGICYT (SAF
98/0103). M.G.-A. thanks the ICI-AECI (Spain) for a
fellowship. Collaborative work was performed under
the auspices of the Iberoamerican Program of Science and
Technology for Development (CYTED), Sub-Program X.
´
8. World Health Organization (OMS). Rapport sur la Sante
dans le Monde, 1998; WHO: Geneve, 1998; p 48.
`
9. Berkow R. The Merck Manual, 16th ed.; Merck Res. Lab.,
Rahway, 1992; p 232.
References and Notes
10. (a) Croft, S. L. Trends Pharmacol. Sci. 1988, 9, 376. (b)
Berman, J. D. Rev. Infect. Dis. 1988, 10, 560.
´
11. World Health Organization (OMS). Rapport sur la Sante
1. Ko, S. K.; Lee, S. M.; Whang, W. K. Arch. Pharm. Res.
1999, 22, 401.
dans le Monde, 1998; WHO: Geneve, 1998; p 59.
`
2. Matsuda, H.; Hiroshi, S.; Masayuki, Y. Bioorg. Med.
Chem. 1999, 7, 1445.
12. Wollenweber, E.; Doerr, M.; Waton, H.; Bonvin, J. F.
Phytochemistry 1993, 33, 611.
3. (a) Geahlen, R. L.; McLaughlin, J. L. Biochem. Biophys.
Res. Commun. 1989, 165, 241. (b) Zheng, J.; Ramirez, V. D.
Biochem. Biophys. Res. Commun. 1999, 261, 499.
4. Onoda, T.; Inuma, H.; Sasaki, Y.; Hamada, M.; Isshiki, K.;
Naganawa, H.; Takeguchi, T. J. Nat. Prod. 1989, 52, 1252.
5. (a) Chun, Y. J.; Kim, M. Y.; Guenagerich, F. P. Biochem.
Biophys. Res. Commun. 1999, 262, 20. (b) Jang, M.; Pezzuto,
13. Rappoport, Z. The Chemistry of Enamines, Part 2; J.
Wiley & Sons: Chichester, UK, 1994; p 924.
14. (a) Mesa-Valle, C. M.; Rodrıguez-Cabezas, M. N.; Mor-
aleda-Lindez, V.; Craciunescu, D.; Sanchez-Moreno, M.;
Osuna, A. Pharmacology 1998, 57, 160. (b) Canto-Cavalheiro,
M. M.; Echevarrıa, A.; Araujo, C. A.; Bravo, M. F.; Santos,
L.; Jansen, A. M.; Leon, L. L. Microbios. 1997, 90, 51.