Synthesis and biological evaluation of a novel class as antileishmanial agent

Article abstract of DOI:10.1007/s00044-010-9532-x

Leishmaniases are a group of diseases caused by the protozoan parasites of the genus Leishmania. Despite the tremendous progress made in the understanding of the biochemistry and molecular biology of the parasite, the first choice treatment for leishmaniases still relies on pentavalent antimonial developed more than 50 years ago. These drugs are potentially toxic and often ineffective. The spread of drug resistance, combined with other shortcoming of the available antileishmanial drugs, emphasizes the importance of developing new effective, and safe drugs against leishmaniasis. The study reported here was undertaken to examine the antileishmanial activity of novel substituted 1,2,3,4- tetrahydropyrimidine-5-carboxamide (I) or cabohydrazide (II) analogs both in vivo and in vitro against Leishmania major. All tested compounds showed in vitro antileishmanial activity, but only four compounds showed in vivo activity in Swiss strain albino mice model as revealed by clinical cure of the cutaneous lesion, parasitologically by marked reduction in parasitic load and by histopathological changes. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9532-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:257–267
DOI 10.1007/s00044-010-9532-x
ORIGINAL RESEARCH
Synthesis and biological evaluation of a novel class
as antileishmanial agent
•
M. N. S. Saudi M. M. A. El-Semary
•
•
R. Y. Elbayaa M. I. Jaeda M. M. Eissa
•
•
•
E. I. Amer N. M. Baddour
Received: 25 May 2009 / Accepted: 2 December 2010 / Published online: 25 December 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Leishmaniases are a group of diseases caused
by the protozoan parasites of the genus Leishmania.
Despite the tremendous progress made in the understanding
of the biochemistry and molecular biology of the parasite,
the first choice treatment for leishmaniases still relies on
pentavalent antimonial developed more than 50 years ago.
These drugs are potentially toxic and often ineffective. The
spread of drug resistance, combined with other shortcom-
ing of the available antileishmanial drugs, emphasizes the
importance of developing new effective, and safe drugs
against leishmaniasis. The study reported here was under-
taken to examine the antileishmanial activity of novel
parasitologically by marked reduction in parasitic load and
by histopathological changes.
R2
O
R1
O
R1
R2
HN
N
R3
NH
NH
NH
I
II
N
H
X
N
H
X
Keywords 1,2,3,4-tetrahydropyrimidine-5-carboxamide
or cabohydrazide ꢀ Antileishmanial agents ꢀ
substituted
1,2,3,4-tetrahydropyrimidine-5-carboxamide
Leishmania major ꢀ Histopathological study
(I) or cabohydrazide (II) analogs both in vivo and in vitro
against Leishmania major. All tested compounds showed in
vitro antileishmanial activity, but only four compounds
showed in vivo activity in Swiss strain albino mice model
as revealed by clinical cure of the cutaneous lesion,
Introduction
Leishmaniasis is group of disease caused by various spe-
cies and strains of Leishmania. It is a growing health
problem in many parts of the world mainly due to an
increasing frequency of drug-resistance and an enhanced
risk of co-infection with HIV (Alvard et al., 1997). A wide
spectrum of disease in humans, with many different clinical
presentations, appearing in three main forms: visceral,
cutaneous, and mucocutaneous (Handman, 1999). The
severity of the disease is largely dictated by the immuno-
logical status of the infected individual and by the species
of the Leishmania involved (Barret et al., 1999). Approx-
imately, 350 million people in 80 countries are estimated to
be threatened by the disease (Desjeux, 2001). Furthermore,
as some all types of leishmanial diseases (Barrel et al.,
1991). Despite the tremendous progress made in the
understanding of the biochemistry and molecular biology
of the parasite, the first choice treatment for the several
M. N. S. Saudi (&) ꢀ M. M. A. El-Semary ꢀ R. Y. Elbayaa
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Alexandria University, Alexandria, Egypt
e-mail: manalnsaudi@yahoo.com
M. M. Eissa ꢀ E. I. Amer
Department of Parasitology, Faculty of Medicine,
Alexandria University, Alexandria, Egypt
N. M. Baddour
Department of Pathology, Faculty of Medicine,
Alexandria University, Alexandria, Egypt
M. I. Jaeda
Department of Medicinal and Pharmaceutical Chemistry,
Faculty of Pharmacy, Al-Fateh University of Medical Sciences,
Tripoli, Libya
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Med Chem Res (2012) 21:257–267
forms of leishmanisis still relies on pentavalent antimoni-
als, which are the clinical drugs most frequently employed,
have been in use since the 1920s. However, antimoniates
have a narrow therapeutic window due to their toxicity, and
there are additional conditions, which permit the persis-
tence of Leishmania in the vertebrate host. When these
drugs are ineffective, or cannot be prescribed, treatment
with amphotericin B, pentamidine, or paramomycin is
indicated (Croft and Coombs, 2003). Miltefosine, an
inhibitor of sterol biosynthesis, has been recently approved
as the first oral drug for visceral leishmaniosis in India. It
yields cure rates of about 98%, has negligible side effects
and is used successfully to treat cases resistant to antimo-
nalis (Jha et al., 1999). Recently, miltefosine produced a
dramatic clinical and parasitological response in patients
with diffuse cutaneous leishmaniasis and improvement
continued during drug administration, but relapse occurred
after stoppage of the treatment (Zerpa et al., 2007). How-
ever, teratogenicity, gastrointestinal upset, potential of
resistance development, and a low therapeutic window
pose limitations on its use (Croft et al., 2006). A series of
1-[4-(40-sulfanilyl)phenyl]urea and its derivatives were
proved to be efficient in treatment of leishmaniasis (David,
1997). These findings encourage us to synthesize our target
compounds as they possess urea or thiourea moieties.
Therefore this study was under taken to examine the antile-
ishmanial activity of novel substituted 1,2,3,4-tetra-
hydropyrimidine-5-carboxamide (I) or 5-carbohydrazide
(II) analogs both in vitro and in in vivo against leishmania
major disease (Ponte-Sucre, 2003).
without separating out their intermediate 2d and 2c,
respectively. Chlorination of compound 3h using thionyl
chloride yielded the dichlorinated product 3i.
Experimental
Melting points were determined in open glass capillaries
and are uncorrected. I.R spectra were recorded for KBr disc
using Perkin-Elmer 421 spectrophotometer. 1HNMR
spectra were recorded on a Varian EM-390 (400 MHz)
spectrophotometer, using TMS as internal standard and
DMSO-d6 as solvent. The MS spectra were determined
with GC-MS 1000 Fx Shimadzu mass spectrometer.
Microanalyses were performed in the microanalytical unit,
University of Assute.
General procedure for synthesis of N-(4-
acetamidophenyl)-3-oxobutanamide (2a) and
N2-acetoacetyl-2 hydroxybenzohydrazide (2b)
A mixture of 4-aminoacetanilide (1a) or salicylic acid
hydrazide (1b) (10 mmol) and ethylacetoacetate (7.8 g,
60 mmol) was heated at 150°C for 1 h. After cooling, the
reaction mixture was triurated with ethanol (5 ml) and the
precipitated material was collected by filtration. The yields
and melting points of the synthesized compounds were
reported (Gionanni, 1941).
General procedure for synthesis of 4-aryl-6-
N-substituted-2-oxo or 2-thioxo-1,2,3,
4-tetrahydropyrimidine-5-carboxamides (3a–f)
Chemistry
To a stirred solution of urea or thiourea (10 mmol),
substituted benzaldehyde (10 mmol) and few drops of
conc. HCl in ethanol (15 ml), the appropriate ethylace-
toacetate derivatives (2a or 2b) (10 mmol) were added.
The reaction mixture was heated to reflux for 3–5 h and
was then allowed to stand overnight at room temperature.
The precipitated products were filtered, washed with eth-
anol, dried and crystallized from n-propanol.
The designed target compounds are depicted in Scheme 1.
The intermediates 2a and 2b were obtained by heating a
mixture of ethylacetoacetate and p-aminoacetanilide 1a or
salicylic acid hydrazide 1b in 6:1 ratio, respectively. On the
other hand, compound 2c was prepared also following the
previous procedure, for the synthesis of 2a and 2b, starting
with diethanolamine 1c and the further reaction was done
on the residue without separation. Whereas, the interme-
diate 2d was prepared by two reactions. The first one is
hydrazinolysis of crotonylester with excess hydrazine
hydrate for 1 h, then evaporation to dryness under vacuum.
The residue 1d was exposed to the second reaction directly
using the same procedure described for the synthesis of 2a
and 2b. The target compounds 3a–f have been synthesized
following the Biginelli reaction (Biginelli, 1893) by heat-
ing our intermediate 2a or 2b with the appropriate aldehyde
with urea or thiouea in ethanol in the presence of hydro-
chloric acid at reflux temperature. However, compounds 3g
and 3h have been also prepared by Biginelli reaction but
Synthesis of N-(but-2-enoyl)-1,2,3,4-tetrahydro-
6-methyl-4-(3-nitrophenyl)-2-thiopyrimidine-
5-carbohydrazide (3g)
Ethylcrotonate (1.1 g,10 mmol) and hydrazine hydrate
(2 ml) was refluxed for 1 h.After cooling,the reaction
mixture was evaporated to dryness under vaccum.The
residue (crotonyl hydrazide) was exposed to the next
reaction without separation by heating it with ethylace-
toacetate (7.8 g,10 mmol) at 150°C for 1 h.After cooling,
the semisolid residue (2d) was evaporated to dryness and
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Med Chem Res (2012) 21:257–267
259
Scheme 1
then involved in the second reaction by adding ethanol
(15 ml), thiourea (0.76 g,10 mmol),3-nitrobenzaldehyde
and few drops of conc. HCl. The reaction mixture was then
heated to reflux for 4 h.After cooling, the prepared com-
pound (3g) was separated out,filtered and crystallized from
n-propanol.
directly to the next reaction by adding ethanol (15 ml),
thiourea (0.76 g, 10 mmol), 3-nitrobenzaldehyde and few
drops of conc. HCl. The reaction mixture was then heated to
reflux for 5 h. After cooling, the prepared compound (3h)
was separated out, filtered, and crystallized from ethanol.
Synthesis of N,N-bis(2 chloroethyl)-1,2,3,4-tetrahydro-
6-methyl-4-(3-nitrophenyl)-2-thioxopyrimidine-
5-carboxamide (3i)
Synthesis of N,N-bis(2-hydroxyethyl)-
1,2,3,4tetrahydro-6-methyl-4-(3-nitrophenyl)-
2 thioxopyrimidine-5 carboxamide (3h)
A solution of SOCl2 (11.8,100 mmol) in dry benzene
(20 ml) was added dropwise, while stirring to a solution of
3h (3.8 g, 10 mmol) in dry benzene (10 ml).Stirring was
continued for 24 h at room temperature. The obtained
crystals were filtered, washed with dry benzene, dried and
A mixture of diethanolamine (1c) (1.1 g, 10 mmol) and
ethylacetoacetate (7.9 g, 60 mmol) washeated at 150°C for
1 h. After cooling, the reaction mixture was evaporated to
dryness under vacuum to give 2c which was exposed
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Med Chem Res (2012) 21:257–267
required for animal inoculation for performance of in
vivo study.
crystallized from benzene/ethanol. The yields, melting
points, and microanalytical data of the prepared com-
pounds were listed in Table 1. IR and 1HNMR spectral data
are recorded in Table 2.
Tested compounds
All the newly synthesized compounds (3a–i) were screened
for their antileishmanial activity both in vitro and in vivo.
Materials and methods
Leishmania strain and its maintenance
Experimental animals
The leishmania strain used in this study was L. major
(MHOM/IL/81/FEBNI). It was kindly provided by Prof.
Dr. Tamas Lasaky (Professor of immunology, Research
Unit, Institute for Medical Microbiology and Hygiene.
University of Lu¨beck, Germany) and further maintained in
the laboratory of parasitology Department, Faculty of
Medicine, Alexandria University, both in vitro on Tanabe’s
liquid medium and in vivo by serial passage in Swiss strain
Albino mice.
Laboratory bred Swiss albino mice 4- to 8-weeks old were
used. For animal inoculation, promastigotes of L. major
were harvested from N.N.N. medium. The parasites were
adjusted to the required concentration of 20 9 106, and
inoculated into the left hind footpad.
In vitro antileishmanial assay
The in vitro sensitivity of Leishmania promastigote to the
tested compounds was performed. The compounds were
added at a concentration of 10 lmol to equal volumes of
Leishmania promastigotes (106/ml). 0.5% DMSO and
medium alone were used as controls. Parasites were
examined microscopically immediately after the addition
of the compounds and every 10 min for 1 h, then they were
incubated at 23°C in a cooling incubator, parasites were
checked for viability and counted daily for 5 days in a
Culture media
(a) Tanabe’s medium (Tanabe, 1923) was used for in
vitro maintenance of the L. major strain.
(b) N.N.N. medium (McCartry-Burke et al., 1991) was
used for the in vitro antileishmanial assay of the tested
compounds and for mass cultivation of leishmania
Table 1 The physicochemical
properties and elemental
analyses of the new compounds
Comp. no.
Yield (%)
MP (°C)
Mol. formula
(mol. weight)
Analysis
Found/Cald (%)
C
H
N
3a
3b
3c
3d
3e
3f
53
58
62
68
56
70
65
75
60
[300
219
186
250
259
131
199
165
123
C22H25N5O3 (407.47)
64.85
64.61
61.60
61.31
59.28
58.95
55.47
55.17
53.39
53.62
55.92
55.63
51.19
51.32
50.52
50.41
46.05
46.35
6.18
6.42
5.66
5.43
5.45
5.13
4.17
4.42
4.01
4.32
5.12
5.45
4.56
5.82
5.30
5.23
4.35
4.61
17.19
17.32
17.10
17.06
16.40
16.32
17.02
16.99
16.38
16.52
11.86
12.01
18.66
18.91
14.73
14.52
13.43
13.73
C
C
C
C
C
C
C
C
21H23N5O4 (409.44)
21H23N5O3S (425.50)
19H17N5O6 (411.37)
19H17N5O5S (427.43)
22H24N4O6S (472.51)
16H17N5O4S (375.40)
16H20N4O5S (380.42)
16H18Cl2N4O3S (417.31)
3g
3h
3i
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261
1
Table 2 IR, HNMR and MS spectral data of the newly synthesized compounds
Comp. IR (cm)-1
no.
1H-NMR (d-ppm) (DMSO-d6)
MS (% relative abundance)
3a
3b
3420 (NH), 1710 (C=O), 2.11 (s, 3H, CH3–CO–NH–), 2.56 (s, 3H, CH3,
1680 (C=O)
pyrimidine-C6), 3.11 (s, 6H, (CH3)2N), 5.14 (d, 1H,
pyrimidine-H4) 6.92–8.39 (m, 9H, 8Ar–H and
pyrimidine N3–H) 9.10(s, 1H, pyrimidine N1–H,
exchangeable with D2O), 9.94 (s, 1H, –NH–CO–C5-
pyrimidine, exchangeable with D2O), 10.11 (s, 1H,
CH3–CO–NH-phenyl, exchangeable with D2O)
3350–3300(NH, OH), 2.36 (s, 3H, CH3, pyrimidine-C6), 3.23 (s, 6H,
1715 (C=O), 1580, 1235 (CH3)2N), 5.21 (d, 1H, pyrimidine-H4), 7.12–7.98
(C=S)
(m, 9H, 8Ar–H and pyrimidine N3–H), 8.1 (d, 1H,
j = 6.9, –NH–NH–CO-pyrimidine, exchangeable
with D2O), 9.68 (s, 1H, pyrimidine N1–H,
exchangeable with D2O), 10.75 (d, 1H, j = 6.9,
–NH–NH–CO-pyrimidine, exchangeable with D2O),
11.91 (s, 1H, –OH, exchangeable with D2O)
3d
3e
3f
3404–3355 (NH, OH),
1720 (C=O), 1685
(C=O)
2.31 (s, 3H, CH3, pyrimidine-C6), 5.25 (d, 1H,
pyrimidine-H4), 6.94–8.13 (m, 9H, 8Ar–H and
pyrimidine N3–H), 8.32 (d, 1H, j = 7.3, –NH–NH–
CO-pyrimidine, exchangeable with D2O), 9.71 (s, 1H,
pyrimidine N1–H, exchangeable with D2O), 10.81 (d,
1H, j = 7.3, –NH–NH–CO-pyrimidine, exchangeable
with D2O), 12.01 (s, 1H, –OH, exchangeable with
D2O)
3410–3355 (NH, OH),
1720 (C=O), 1689
(C=O) 1585,1235 (C=S) pyrimidine N3–H), 8.45 (d, 1H, j = 7.4, –NH–NH–
CO-pyrimidine, exchangeable with D2O), 9.71 (s, 1H,
pyrimidine N1–H, exchangeable with D2O), 10.92
(d, 1H, j = 7.4, –NH–NH–CO-pyrimidine,
2.29 (s, 3H, CH3, pyrimidine-C6), 5.31 (d, 1H,
pyrimidine-H4), 7.12–8.19 (m, 9H, 8Ar–H and
M?, 427 (3.02), 366 (4.23), 302 (574), 267 (5.74),
203 (3.32), 163 (6.65), 121 (81.2), 65 (100)
exchangeable with D2O), 12.14 (s, 1H, –OH,
exchangeable with D2O)
2.27 (s, 3H, CH3, pyrimidine-C6), 3.83 (s, 9H, 3OCH3), M?-84, 388 (67.63), 345 (23.60), 299 (5.83), 267
5.32 (d, 1H, pyrimidine-H4) 7.41–8.21 (m, 7H, 6Ar–H (1.84), 255 (2.90), 193 (33.80), 137 (26.86) 121
and pyrimidine N3–H), 8.51 (d, 1H, j = 6.9, –NH–
NH–CO-pyrimidine, exchangeable with D2O), 9.68
(s, 1H, pyrimidine N1–H, exchangeable with D2O),
10.89 (d, 1H, j = 6.9, –NH–NH–CO-pyrimidine,
exchangeable with D2O), 11.99 (s, 1H, –OH,
exchangeable with D2O)
3390–3365 (NH, OH,
1725 (C=O), 1685
(C=O), 1590, 1230
(C=S)
(100), 84 (2.08), 66 (58.97)
3g
3350 (NH), 1715 (C=O), 2.12 (s, 1H, CH3–CH=CH–), 2.39 (s, 3H, CH3,
1685 (C=O)
pyrimidine-C6), 5.62 (d, 1H, pyrimidine-H4), 6.11
(d, 1H, j = 7.4, –CO–CH=CH– CH3), 6.31 (d, 1H,
j = 7.4, –CO–CH=CH– CH3) 7.56–8.12 (m, 5H,
4Ar–H and pyrimidine N3–H), 8.91 (d, 1H, j = 7.3,
–NH–NH–CO-pyrimidine, exchangeable with D2O),
9.68(s, 1H, pyrimidine N1–H, exchangeable with
D2O), 10.94 (d, 1H, j = 7.3, –NH–NH–CO-
pyrimidine, exchangeable with D2O)
3h
3i
34104-0 (NH, OH), 1700 2.31 (s, 3H, CH3, pyrimidine-C6), 3.15 (t, 4H, N–(CH2– M?-1, 379 (12.30), 231 (9.09), 194 (11.23), 149
(C=O), 1590, 1235
(C=S)
CH2–OH)2, 3.61 (t, 4H, N–(CH2–CH2–OH)2, 4.78
(s, 2H, 2OH, exchangeable with D2O). 5.62
(d, 1H, pyrimidine-H4), 7.05–7.89 (m, 5H, 4Ar–H and
pyrimidine N3–H), 9.32 (s, 1H, pyrimidine N1–H,
exchangeable with D2O)
(54.01), 104 (62.03), 91 (19.79), 55(100)
3380- (NH), 1720 (C=O), 2.34 (s, 3H, CH3, pyrimidine-C6), 3.46 (t, 4H, N–(CH2–
1585, 1228 (C=S)
CH2–Cl)2, 3.72 (t, 4H, N–(CH2–CH2–Cl)2, 5.67
(d, 1H, pyrimidine-H4), 7.17–8.10 (m, 5H, 4Ar–H
and pyrimidine N3–H), 9.32 (s,1H, pyrimidine N1–H,
exchangeable with D2O)
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haemocytometer with a light microscope (LM) and
the inhibition was calculated: inhibition (in percent) =
100 - [(number of parasite remaining after treatment)/
number of parasites in control] 9 100 (Chan and Fong,
1990). Each assay was performed in duplicate and repeated
in separate experiments.
?2: About 50 amastigotes in most filed at
least 25 fields were scanned.
?1: One to 20 amastigotes in some but not all
fields. At least 50 fields were scanned.
: Very few amastigotes, but at least two
were seen. Preparation was scanned for
5–10 min.
–: No amastigotes seen after 10 min search.
In vivo antileishmanial assay
(c) Histopathological study: Portions of the cutaneous
lesions from all groups were fixed in 10% formalin,
embedded in paraffin and cut into sections of 5 lm
each. They were stained with haematoxylin and
eosin stain.
To examine the therapeutic efficacy of the tested com-
pounds, # Swiss albino mice infected with 20 9 106
promastigotes in the left hind footpad were used. They
were allocated into two main groups.
Group I: control group
It was further subdivided into:
Results
–
–
Group Ia: Infected untreated control.
In vitro antileishmanial assay: DSMO at a conc. of 0.5%
didn’t have any antileshmanial activity and therefore was
used as a control for the in vitro study. All tested com-
pounds that were dissolved in 0.5% DMSO showed antil-
eshmanial effect at a conc. of 10 mmol .Immediate
cessation of movement without change in morphology was
seen immediately after the addition of the tested com-
pounds, promastigotes exposed to all tested compounds
were examined frequently every 10 min under LM for an
hour, but no motility was seen and complete death occur.
Group Ib: DSMO control group (infected, received
local intralesional injection of 0.5% DSMO)
Group II: experimental group
It was further subdivided equally into nine subgroups each
group received one of the nine tested compounds. Treat-
ment was started 21 days post infection. Tested compounds
were infiltrated into the lesion at a dose of 20 lmol. Five
weeks after local treatment, animals were killed and the
effect of the tested compounds on the cutaneous leish-
manial lesion was assessed by the following parameters in
comparison to infected non-treated control (group I).
In vivo antileshmanial assay
Monitoring of the cutaneous lesion of infected control
groups (group Ia and Ib) showed redness and swelling of
the inoculated footpad. The swelling was visible from the
7th day post infection and progressed gradually with time
(Fig. 1) by the 5th week mice developed crust that
increased progressively in size by time (Fig. 2a, b), later on
gangrene started to develop in the crust and extended to the
digits and foot to end by autoamputation of the inoculated
footpad by the 8th week post infection (Fig. 3a–c). As
regards animals treated with the nine tested compounds by
local infiltration into the lesion, only those treated by
compounds 3d, 3e, 3g, and 3i showed cessation of the
clinical signs. The swelling and redness started to decrease
by time without a need for another injection as clinically
the footpad started to return back to the nearly normal
appearance by the end of the experiment (Fig. 4).
(a) Clinical evaluation: Animals of all groups were
subjected to examination of the inoculated footpad
for the progression of lesion.
(b) Parasitological study: The study includes
(i) Culture was performed to detect the presence of
viable parasites in the lesion. Parts of the cutaneous
leishmanial lesions from all groups were cultured
aseptically on Tanabe’s liquid medium and kept at
23–25°C with weekly examination. Culture was
recorded as negative if no parasite was observed
within 3 weeks.
(ii) Tissue impression smear: Impression smears were
made from the cutaneous lesions of all groups, fixed
in methanol, and stained with Giemsa stain. The
parasite density in the stained smear was examined
under oil immersion, and was recorded on empirical
scale as follows (Schnur et al., 1973):
On the other hand those treated with the rest of the
compounds namely 3a, 3b, 3c, 3f, and 3h showed pro-
gression of the cutaneous lesion but in slower rate than that
of the control. At the end of the experiment, while infected
?3: More than 150 amastigotes in virtually
every field. At least 25 fields were scanned.
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263
Fig. 4 Inoculated footpad of an infected mouse treated with
compound 3g at the end of the experiment showing almost normal
appearance
Fig. 1 Inoculated footpad of an infected mouse at the 3rd week post
infection showing redness and swelling
Fig. 2 a, b Inoculated footpad of infected mouse showing crust
formation
Fig. 5 Inoculated footpad of an infected mouse treated with
compound 3a showing black coloration of the inoculate footpad
Fig. 6 Inoculated footpad of an infected mouse treated with
compound 3f at the end of the experiment showing autoamputation
of the fingers
Fig. 3 a, b, c Inoculated footpad of an infected mouse at the 8th
week post infection showing autoamputation of the inoculated
footpad
untreated mice showed autoamputation of the inoculated
footpad, those treated with compound 3c showed only
swelling and redness. Those treated with compound 3a, the
footpad turned black in color (Fig. 5). Mice treated with
compound 3h showed crust formation whereas compound
3b and 3f failed to cessate the lesion development and
gangrene and autoamputation of the fingers of the inocu-
lated footpad was observed (Figs. 6, 7). This is evident that
these compounds were partially effective as they slower the
process of leishmanial lesion development when compared
to the infected control.
Fig. 7 Inoculated footpad of an infected mouse treated with
compound 3f at the end of the experiment showing autoamputation
of the fingers
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Parasitological study revealed living parasites in the
cutaneous lesion of mice of both infected and infected
treated group even those who received compounds 3d, 3e,
3g, and 3i and showed clinical cure as revealed by positive
culture of parts of the footpad on Tanabe’s medium and by
impression smear. The grading of parasite density in the
cutaneous lesion of the infected control group (group Ia
and Ib) was ?3 (Fig. 8). Compounds 3e, 3g, and 3i suc-
ceeded to drop the grade to
whereas compound 3d
reduces it to grade ?1 (Fig. 9). As regards other tested
compounds that failed to induce clinical cure the grade was
?2 in groups treated with compound 3a and 3c grade ?3 in
groups treated with compound 3b, 3f, and 3h.
Fig. 10 Section in the skin lesion of infected mouse showing chronic
inflammatory cellular infiltrate (H&E, 9100)
Histopathological study of the cutaneous lesion of
infected non-treated mice showed focal ulcer on the epi-
dermis, which overlies connective tissue heavily infiltrated
by acute and chronic non-specific inflammatory cells,
predominately composed of macrophages, lymphocytes,
and polymorphs with areas of necrosis (Figs. 10, 11) in
the adjacent intact epidermis showed hyperkeratosis,
Fig. 11 Higher magnification of Fig. 10 (H&E, 9200)
acanthosis, and parakeratosis (Fig. 12). Parasites were
found in large numbers in dermal macrophages, extracel-
lularly and in epidermal cell (langerhans dendritic cells)
(Figs. 13, 14). Gangrenous changes were seen. The
inflammatory reaction involved the dermis and hypoder-
mis. In animals that received compounds 3d, 3e, 3g, and 3i,
the intensity of inflammatory reaction was remarkably
diminished and leishmania parasites could hardly be
detected. A striking feature in cutaneous lesions treated
with compound 3d and 3e is the appearance of nest of
epithelial invading the dermis (Figs. 15, 16), indicating
sever irritation of the epidermis induced by these com-
pounds. This finding were more apparent in mice treated
with compound 3d than 3e and this finding raises the
possibility of the carcinogenicity of these two compounds.
In compound 3g and 3i which induced clinical cure
together with marked reduction of intensity of the inflam-
matory change, the epithelial changes were unremarkable
thus these two compounds seems to be promising antile-
ishmanial agents. As regards histopathological finding in
groups received compounds 3a, 3b, 3c, 3f, and 3h showed
the same histopathological findings as the infected control
group. Moreover, compound 3a appeared to be toxic and
Fig. 8 Impression smear from cutaneous lesion of an infected mouse
showing parasite density grade ?3 (Giemsa 91000)
Fig. 9 Impression smear from cutaneous lesion of an infected mouse
treated with compound 3d showing parasite density grade ?1
(Giemsa 91000)
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265
Fig. 12 Section in the skin lesion of infected mouse showing dys-
plastic changes in basal cell layer (H&E, 9400)
Fig. 15 Section in the skin lesion of infected mouse treated with
compound 3d showing downward growth of squamous epidermis into
dermis (H&E, 9200)
Fig. 13 Section in the skin lesion of infected mouse showing
numerous Leishmania amastigotes throughout the lesion (H&E,
91000)
Fig. 16 Section in the skin lesion of infected mouse treated with
compound 3d showing downward extension of epidermal nests into
the dermis with an intact basement membrane (H&E, 9400)
Discussion
To date, there are no vaccines against leishmaniasis, and
chemotherapy remains the mainstay for its control. The
drugs of choice used were significantly toxic, expensive,
and with a growing frequency of refractory infections.
Dramatic increase in the rates of infections, drug resis-
tance, and the non-availability of safe vaccines has
highlighted the need for identification for novel and inex-
pensive antileishmanial agents (Dutta et al., 2007).
Efforts to find new chemotherapeutics for leishmaniasis
have been ongoing for decades, and numerous potentially
new drug candidates and/or putative drug targets have
indeed been proposed (Croft and Yardley, 2002).
Fig. 14 Section in the skin lesion of infected mouse showing
numerous Leishmania amastigotes throughout the lesion (H&E,
91000)
In this study, the antileishmanial activity of the novel
compounds (3a–i) was evaluated both in vitro and in vivo
against L. major. In both the assays, DMSO was used a
solvent in a concentration of 0.5%. This concentration did
not show any antileishmanial activity, similarly, to what
was previously reported by Ma et al., (2004) and Tiuman
et al., (2005).
potentially carcinogenic as there was nest of epidermal
cells infiltrating the dermis, necrotic areas, subepidermal
and intradermal vesicles were seen. In addition, there was
vascular thrombosis with collection of haemosidrin in
macrophages.
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Med Chem Res (2012) 21:257–267
In vitro results revealed that all tested compounds
carcinogenicity of these compounds which need further
investigations. On the other hand, compounds 3a, 3b, 3c, 3f,
and 3h showed the same histopathological findings of con-
trol group. Furthermore, compound 3a appeared to be toxic
as vascular thrombosis with collection of haemosidrin in
macrophages was seen beside epidermal changes as what
observed with compound 3d and 3e which raise its possi-
bility of being also carcinogenic.
showed a dramatic and amazing antileishmanial activity
with immediate cessation of parasite movement with no
apparent morphological changes by LM. Elucidations of
mechanism(s) by which these compounds act on L. major,
and ultrastructural changes which could occur are points
which require further investigations.
Numerous studies reported potent in vitro antileishma-
nial activity of several agents such as a hypocrellin A, two
synthesized sterol families, crude ethanolic extract, lignoid
fraction, and the purified compound yangambin (Ma et al.,
2004; Bazin et al., 2006; Monte et al., 2007). Such an
activity justifies further in vivo antileishmanial evaluation.
This is because not all compounds that show in vitro effect
should show the same in vivo. Therefore, in this study, all
the tested compounds that showed potent antileishmanial
activity in vitro were subjected to in vivo evaluation in
experimental animals and evaluated clinically, parasito-
logical, and pathologically.
Thus it can be concluded that, in spite that all these
compounds showed in vitro antileishmanial activity, in
vivo activity was apparent only in compounds 3d, 3e, 3g,
and 3i. Compounds 3d and 3e appeared to be potentially
carcinogenic; however, compounds 3g and 3i appeared to
be safe. Thus, further studies and restricted toxicological
analysis should be performed on the promising compounds
3g and 3i for the possibility of their development as new
potent antileshimanial agents.
Clinical examination of infected untreated animals
showed that in the inoculated footpad, leishmanial lesion
developed and ends by autoamputation of the footpad. In
contrast, animals that received compounds 3d, 3e, 3g, and 3i
could achieve clinical cure. On the other hand, compounds
3a, 3b, 3c, 3f, and 3h were partially effective, they showed
different degrees of clinical presentation starting from
swelling and redness in animals treated with compound 3c to
autoamputation in animals received compound 3b.
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