Leishmanicidal activity of some aliphatic diamines and amino-alcohols.

Article abstract of DOI:10.1016/S0960-894X(01)00837-X

A number of aliphatic diamines and amino-alcohols and several of their alkyl, acyl and carbamoyl derivatives, have been synthesised and evaluated in vitro on cultures of Leishmania spp. In general, diamine derivatives resulted to be more potent than their amino-alcohol or amino-ether analogues. Two diamine derivatives (8b and 9d) and one amino-alcohol (6a) showed a fair inhibition of parasite growth, at concentrations below 10 microg/mL, with potencies close to that of the reference drug, amphotericin B. Some SAR considerations have been deduced.

Full text of DOI:10.1016/S0960-894X(01)00837-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 659–662
Leishmanicidal Activity of Some Aliphatic Diamines and
Amino-Alcohols
a,
a
a
b
b
Esther del Olmo, * Mario Alves, Jose L. Lopez, Alba Inchaustti, Gloria Yaluff,
´ ´
b,
a
Antonieta Rojas de Arias * and Arturo San Feliciano
aDepartamento de Quı´mica Farmace´utica, Facultad de Farmacia, Univ. de Salamanca, 37007 Salamanca, Spain
bInstituto de Investigaciones en Ciencias de la Salud (IICS), Univ. Nacional de Asuncio´n, Paraguay
Received 3 September 2001; revised 12 November 2001; accepted 5 December 2001
Abstract—A number of aliphatic diamines and amino-alcohols and several of their alkyl, acyl and carbamoyl derivatives, have been
synthesised and evaluated in vitro on cultures of Leishmania spp. In general, diamine derivatives resulted to be more potent than
their amino-alcohol or amino-ether analogues. Two diamine derivatives (8b and 9d) and one amino-alcohol (6a) showed a fair
inhibition of parasite growth, at concentrations below 10 mg/mL, with potencies close to that of the reference drug, amphotericin B.
Some SAR considerations have been deduced. # 2002 Elsevier Science Ltd. All rights reserved.
Leishmaniasis, is a parasitic disease endemic to the
1
noids,12 quinones,13 certain amino acids, amides and
14
15
American, African and Asian tropical countries. It
affects some 12 million people around the world and
esters, in addition to alkyl-lysophospholipids and Pt
1
6
(II) and (IV) complexes, have also been reported in the
literature. In spite of the great number of compounds
synthesised, no better substances than those already
known have been introduced into the clinic. In this
paper, we describe the synthesis of some lipidic amino-
alcohols and diamines and their in vitro evaluation on
cutaneous, mucocutaneous and visceral strains of
Leishmania. These type of compounds, bearing a cova-
lently bonded, non-hydrolysable, fatty chain attached to
the ethanolamine or ethylenediamine fragments, were
considered able to interact with membrane lipids, also
to be transported into the cytoplasm and possibly, to
interfere with the lipid or polyamine transport or meta-
3
50 million are at risk of becoming infected, of whom
2
some 1.7 million will be infected each year. The most
common varieties of leishmaniasis are the cutaneous
and mucocutaneous ones, they are not directly lethal,
but provoke multiple granulomatose or diffuse, auto-
inoculable and even metastatic skin ulcers, resembling
3
leprosy. Drugs currently in use as the Antimony deri-
vative glucantime, the bis-amidines, pentamidine and
stilbamidine, and the glycomacrolide amphotericin B,
display high liver and heart toxicities, develop clinical
4
resistance after a few weeks of treatment and it has
been estimated that they contribute to increase co-
5
17,18
infections leishmaniasis—AIDS. For these reasons, it is
bolism of the parasite.
necessary to discover new agents, either natural or syn-
thetic, more potent and selective, for treating this
increasing parasitosis.
Chemistry
A great number of natural and synthetic compounds
have been tested in the past few years in anti-leishma-
nial assays. Their structures are very diverse. They often
The synthesis of the compounds to be tested is sum-
marised in Scheme 1. Details of the reactions, experi-
mental conditions and characterisation of the products,
will be reported in a complete publication. The Boc-
aminoacid 1, prepared from diethyl 2-acetamido-
6
7
contain nitrogen heterocycles as: quinolines, acridines,
10
8
9
phenothiazines, pyrimidines and purines, but other
1
1
19
types of compounds such as aniline derivatives, flavo-
malonate as reported by Gibbons et al., was trans-
formed into the Boc-aminoalcohol 2a, by treatment
with ethyl chloroformate followed by sodium borohy-
dride reduction of the intermediate mixed anhydride.
Compound 2a was benzylated to give the Boc-aminoether
*
2
Corresponding authors. Tel.: +34-923-29-4500x1825; fax: +34-923-
9-4515; e-mail: olmo@usal.es (E. del Olmo); sarias@conexion.com.py
(A. R. de Arias)
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00837-X

660
E. del Olmo et al. / Bioorg. Med. Chem. Lett. 12 (2002)659–662
3
, which was Boc-deprotected to give the aminoether 4.
these compounds with TMSCl/PhOH in HCCl3,21 fol-
lowed by chromatographic purification on silica gel,
yielded the intramolecularly transamidated compounds
11a and 11b, which were further treated with the appro-
priated acyl chloride to obtain the amidoesters 12a–c.
The benzylic aminoether 4, treated with 1.2 or 2.2 equiv
of ethyl bromide gave the mono alkylated or dialkylated
derivatives 5a and 5b, respectively, while treatment with
ethyl bromoacetate led to the glycine ester 5c. The
acylation of 4 with glutaric anhydride provided the
amidoacid 5d. The hydrogenolytic debenzylation of
compounds 5a–d gave the corresponding free alcohol
derivatives 6a–d, and the hydrolysis of 5c with 10%
Biological Assays
0
KOH/MeOH, yielded the glycine derivative 6c .
The isolation, cultivation and maintenance of Leishma-
nia promastigotes and the technique used have been
2
2
Compound 2a, through mesylation, followed by sub-
stitution with sodium azide and reduction with NaBH /
MeOH, in the presence of Pd–C, was converted into
the Boc-diamine 8, which on alkylation/acylation with
the above mentioned reagents, followed (or not) by Boc-
deprotection, yielded the diamines and diamine deriva-
previously described. Briefly, promastigote inhibition
studies were performed on L. amazonensis (IFLA/BR/
67/PH8), L. brasiliensis (MHOM/BR/75/M2903) and L.
4
2
0
ꢀ
donovani (MHOM/BR/74/PP75) grown at 25 C in
Schneider’s drosophila medium containing 20% foetal
bovine serum. Compounds were dissolved in 40 mL of
dimethyl sulphoxide (DMSO, final concentration of this
solvent 0.4%), then diluted in the medium and placed in
microtitre plates in triplicate, with a final compound
concentration of 100 mg/mL. The activity of compounds
was evaluated after 48 h by optical observation of a
drop of each culture using a microscope and compar-
ison with control cell cultures and with those treated
with the reference drug amphotericin B. Other con-
0
tives 8a–d and 9a–f, respectively. Compounds 9c and
0
9
d were obtained from 9c and 9d respectively, after
treatment with 10% KOH/MeOH. Reaction of 9b with
glutaric anhydride gave 9e.
The Boc-aminoalcohol 2a was treated with heptanoyl
chloride or palmitoyl chloride to give the esters 10a and
1
0b, respectively. The attempted Boc-deprotection of
Scheme 1. The synthesis of lipidic diamines, amino-alcohols and their derivatives. R=(CH
b) NaBH /MeOH; (ii) BnCl/NaH/DMF; (iii) HCl/THF/argon; (iv) EtBr (1.1 or 2.2 equiv) or EtOOCCH
anhydride/EtOAc; (vi) H /Pd–C/AcOH; (vii) 10% KOH/MeOH; (viii) (a) MsCl/Et N/CH Cl ; (b) NaN /DMF; (c) Pd-C/CHCl
ix) EtBr (1.1 or 2.2 equiv) or EtOOCCH Br (1.1 or 2.2 equiv)/Et N/DMF; (x) n-C 13COCl or n-C15 31COCl/Et O.
2
)
13CH
3
; (i) (a) EtOOCCl/N-methylmorpholine/THF;
Br (1.1 equiv)/Et N/DMF; (v) glutaric
, NaBH /MeOH;
(
4
2
3
2
3
2
2
3
3
4
(
2
3
6
H
H
2

E. del Olmo et al. / Bioorg. Med. Chem. Lett. 12 (2002)659–662
661
Table 1. In vitro activity of lipidic aminoalcohol derivatives on the growth of Leishmania promastigotes
Compd
L. amazonensis (PH8) (mg/mL)
L. brasiliensis (2903) (mg/mL)
L. donovani (PP75) (mg/mL)
1
00
50
25
10
+
100
50
25
10
+
100
50
25
10
1
++
++
++
+
++
++
++
+
++
++
++
+
2
2
3
4
5
5
5
5
6
6
6
6
6
1
1
1
1
1
a
b
+
+
+
a
b
c
+++
++
++
++
+++
+++
+++
+++
+++
++
+
+++
++
+++
++
++
++
+++
+++
+++
+++
+++
+
+++
++
+++
++
++
++
+++
+++
+++
+++
+++
++
+
+++
++
+
d
a
b
c
+++
+++
++
+
++
+++
+
+
+
+
++
+
+
+++
+++
++
+
++
+++
++
+
+
+
++
++
+
+++
+++
++
+
++
+++
+
+
+
+
++
+
+
0
c
d
+
+
+
1a
1b
2a
2b
2c
+
++
+
+
++
+
+
++
+
+
Amphotericin B
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++: Total lysis of parasites; ++: 80–90% lysis; +: <70%; blank: no lysis.
centrations (50, 25 and 10 mg/mL) were tested after the
total lysis of promastigotes was confirmed at 100 mg/
mL. At least two independent experiments performed in
triplicate with different stock solutions of samples were
carried out.
sites, at concentrations under 25 mg/mL and more than
the 80% of lysis at 10 mg/mL for all of them, except for
9b with value under 70%, after a relatively short period
(48 h) of incubation. Compounds of group 6 were more
potent than their corresponding benzylated analogues
(
5), revealing the convenience of the presence of a free
hydroxyl group for the activity. Small size alkyl sub-
stitution seems better than medium size (6a/6c) or
longer substitutions (11a/11b, 12a/12b and 12c). Finally,
a free carboxylic acid at the end of the chain was less
Results and Discussion
The results of the evaluation of amino-alcohols and
their derivatives are presented in Table 1 and those
corresponding to the diamines and their derivatives are
included in Table 2.
0
active than an ester function (6c /6c).
With respect to the diamines, most of them produced
total lysis of parasites at 50 mg/mL. In general Boc pro-
tected derivatives were more potent than those unpro-
tected (8a–8c/9a–9c), except for the diester derivative
9d. Certain selectivity of compound 8a respecting L.
As can be seen, the amino-alcohol 6a and the diamino
derivatives 8b, 9b and 9d provoked the total lysis of
promastigotes of the three mentioned strains of para-
Table 2. In vitro activity of lipidic diamines and derivatives on Leishmania promastigotes
Compd L. amazonensis (PH8) (mg/mL) L. brasiliensis (2903) (mg/mL)
L. donovani (PP75) (mg/mL)
100
50
25
10
100
50
25
10
100
50
25
10
7
7
8
8
8
8
9
9
9
9
9
9
9
9
a
b
a
b
c
++
++
+++
+++
+++
++
+++
+++
+++
++
+++
+++
+++
+++
++
++
+++
+++
+++
++
+++
+++
+++
++
+++
+++
+++
+++
++
++
+++
+++
+++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+
+++
+
+
++
+
+++
+++
++
+++
+++
+
++
++
+
+++
+++
++
+
+++
+
+
++
+
d
a
b
c
++
+++
+
+
+++
++
+
+++
+
+
+
+
++
+++
+
+
+++
++
+
+++
+
+
+
+
++
+++
+
+
+++
++
+
+++
+
+
+
+
0
c
d
d
e
f
+++
+
++
++
++
+++
+
++
++
++
+++
+
++
++
++
0
+++
+++
+
+
+++
+++
+
+
+++
+++
+
+
Amphotericin B
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++: Total lysis of parasites; ++: 80–90% lysis; +: <70%; blank: no lysis.

662
E. del Olmo et al. / Bioorg. Med. Chem. Lett. 12 (2002)659–662
brasiliensis (2903) strain could be noticed: >80% lysis
versus <70% lysis for the other two strains, at 10 mg/
mL. As it was appreciated for amino-alcohols and di-
esters, large groups at both amine ends are not
recommended for the activity (9b/9e) and similarly the
free acids are less potent than their corresponding esters
5. World Health Organization (OMS). Rapport sur la Sante´
dans le Monde; WHO: Geneve, 1998.
. Chakrabarti, G.; Basu, A.; Manna, P. P.; Mahato, S. B.;
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`
6
1
999, 43, 359.
. Gamage, S. A.; Figgit, D. P.; Wojcik, S. J.; Ralph, R. K.;
7
Ransijn, A.; Mauel, J.; Yardley, V.; Snowdon, D.; Croft, S. L.;
0 0
9c /9c and 9d /9d). However the most potent mono-
(
Denny, W. A. J. Med. Chem. 1997, 40, 2634.
8
substituted compound 9f is nearly as potent as the dis-
ubstituted 9d, perhaps due to the presence of the amide
group or to the longer separation between both fun-
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than those monosubstituted (8b/8a, 9b/9a, 9d/9c),
excepting the Boc diester 8d.
. Khan, M. O.; Austin, S. E.; Chan, C.; Yin, H.; Marks, D.;
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1
1
1
1
119.
We have found, therefore, a new leishmanicidal mol-
ecular prototype, less toxic (data not shown) than the
reference drug Amphotericin B and displaying as
the main structural features a fatty 1,2-diamine with the
0. Aronov, A. M.; Gelb, M. H. Bioorg. Med. Chem. Lett.
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1
-NH group disubstituted by small alkyl or alkoxy-
2
carbonylmethyl groups and with the NH2 group at
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1
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`
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´
pez-Perez, J. L.;
´ ´
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4