Synthesis and in vitro anti-leishmanial activity of 1-[5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl]- and 1-[5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines

Article abstract of DOI:10.1016/j.bmc.2008.02.052

The synthesis and anti-leishmanial activity of nitroheteroaryl-1,3,4-thiadiazole-based compounds including 1-[5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines and 1-[5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines were described. Most of the synthesized compounds exhibited potent anti-leishmanial activity against both promastigote and amastigote forms of Leishmania major at non-cytotoxic concentrations. In general, 5-nitrofuran derivatives were more active than the corresponding 5-nitrothiophene analogues.

Full text of DOI:10.1016/j.bmc.2008.02.052

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4509–4515
Synthesis and in vitro anti-leishmanial activity of
1-[5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl]- and
1-[5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines
Mina Behrouzi-Fardmoghadam,a Fatemeh Poorrajab,b Sussan Kaboudanian Ardestani,b
Saeed Emami,c Abbas Shafieea and Alireza Foroumadia,*
aDepartment of Medicinal Chemistry, Faculty of Pharmacy & Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences, Tehran 14174, Iran
bInstitute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran, PO Box 13145-1384, Tehran, Iran
cDepartment of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
Received 10 January 2008; revised 14 February 2008; accepted 15 February 2008
Available online 20 February 2008
Abstract—The synthesis and anti-leishmanial activity of nitroheteroaryl-1,3,4-thiadiazole-based compounds including 1-[5-(5-nitro-
furan-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines and 1-[5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines were
described. Most of the synthesized compounds exhibited potent anti-leishmanial activity against both promastigote and amastigote
forms of Leishmania major at non-cytotoxic concentrations. In general, 5-nitrofuran derivatives were more active than the corre-
sponding 5-nitrothiophene analogues.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
A great number of natural and synthetic compounds
have been tested in the recent years in anti-leishmanial
assays.6–8 The use of nitroheterocycles such as 5-nitrofu-
rans and 5-nitrothiophenes as antibacterial and antipro-
tozoal is well established.9,10 On the other hand, the
antiparasitc property of 1,3,4-thiadiazoles is well docu-
mented and their attachment with other heterocycles
often ameliorates or diminishes the bioresponses,
depending upon the type of substituent and position of
attachment.11,12 Various 2,5-disubstituted-1,3,4-thiadia-
zole analogues have been previously synthesized and
some of them showed excellent leishmanicidal activ-
ity.5,13,14 All of these compounds had a nitrogen hetero-
cycle linked to the C-5 position of 1,3,4-thiadiazole ring
through the heterocyclic nitrogen. Previous results
demonstrated that C-5 substituent is the most adaptable
site for chemical change and is an area where it deter-
mines the potency and physicochemical properties of
2-(nitroaryl)-1,3,4-thiadiazoles. Accordingly, in the pre-
ceding papers we described a number of 2-(nitroaryl)-
1,3,4-thiadiazoles bearing a piperazin-1-yl substituent
at the C-5 position of 1,3,4-thiadiazole nucleus. The
biological activity of 1-[5-(nitroaryl)-1,3,4-thiadiazol-2-
yl]piperazines indicated that the piperazine moiety of
these compounds possesses enough structural flexibility
Parasitic diseases such as leishmaniasis have significant
impacts on the world; especially in developing countries
with infections spread over several hundred millions of
people and are one of the significant causes of morbidity
and mortality.1
The treatment options for leishmaniasis are limited and
involve the administration of pentavalent antimonial
agents as first line and amphotericin B and pentamidine
as second line drugs.2,3 These drugs are expensive and
potentially toxic and require long-term treatment. In
addition the development of drug resistance by the
pathogens especially in HIV leishmania co-infected pa-
tients has aggravated public health risk.4 The develop-
ment of new effective, cheap and safe drugs for the
treatment of leishmaniasis is therefore an urgent task.5
Keywords: 1,3,4-Thiadiazole; Nitrothiophene; Nitrofuran; Leishmania
major; Anti-leishmanial activity; Promastigote; Amastigote.
*
Corresponding author. Tel.: +98 21 66954708; fax: +98 21
66461178; e-mail: aforoumadi@yahoo.com
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.052

4510
M. Behrouzi-Fardmoghadam et al. / Bioorg. Med. Chem. 16 (2008) 4509–4515
to allow product optimization.5,14 These facts motivate
our concern to N-4 substituent of 1-[5-(nitroaryl)-
1,3,4-thiadiazol-2-yl]piperazines. Thus, we report here
the synthesis and in vitro anti-leishmanial activity of
The nitrofuran and nitrothiophene analogues 6–12a,b
were tested for their in vitro activity against the promas-
tigote form of the Leishmania major strain (MRHO/IR/
75/EꢁR) along with meglumine antimonate (Glucan-
time ), using MTT assay.17 The IC50 values (in lM)
against promastigotes, in comparison with Glucantime,
are listed in Table 3. The most potent compounds
against the promastigote form of L. major were found
to be nitrofuran analogues 6a, 7a, 8a, 11a and 12a with
IC50 values between 10.73 0.69 and 13.19 0.96 lM.
The activity profile of these compounds against prom-
astigotes demonstrated that there is a small difference
in their IC50 values. In particular, compound 7a de-
signed as a nitrofuran derivative containing 2-chloro-
benzoyl on the piperazine ring, with the
IC50 = 10.73 lM, was found to be the most active com-
pound. Although halogen substitution (Cl or Br) on
thiophen-2-carbonyl moiety seems to increase the activ-
ity in both nitrofuran and nitrothiophene analogues
(compounds 10a and 10b when compared with 11–12a
and 11–12b, respectively) but chloro-substitution on
benzoyl containing compounds (6a and 6b) cannot im-
prove anti-leishmania activity. The effect of positional
substitution was investigated by preparing all three pos-
sible chloro-substitutions on benzoylpiperazine moiety.
The better results were achieved with 2-chloro- in nitro-
furan series, and 4-chloro- in nitrothiophene derivatives.
some
1-[5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl]-4-
aroylpiperazines 6–12a and 1-[5-(5-nitrothiophen-2-yl)-
1,3,4-thiadiazol-2-yl]-4-aroylpiperazines 6–12b.
2. Chemistry
The synthesis of 1-[5-(5-nitrofuran-2-yl)-1,3,4-thia-
diazol-2-yl]-4-aroylpiperazines 6–12a and 1-[5-(5-nitro-
thiophen-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines
6–12b was achieved with an efficient synthetic strategy
outlined in Figure 1. Intermediates 2-amino-1,3,4-thi-
adiazoles 3a,b15,16 were obtained from 5-nitro-2-arylid-
ene diacetates 1. Thus, treatment of compound 1 with
thiosemicarbazide in the presence of HCl in ethanol
afforded thiosemicarbazone which cyclized using
NH4Fe(SO4)2 to give 2-amino-1,3,4-thiadiazoles 3a,b.
Diazotization of amine 3 in hydrochloric acid in the
presence of copper powder gave the 2-chloro-1,3,4-thia-
diazole 4. The reaction of compounds 4a,b with pipera-
zine in refluxing EtOH gave the corresponding
N-piperazinyl compounds 5a,b. N-aroylation of the
piperazine analogues 5a,b with appropriate thiophen-2-
carbonyl chlorides or benzoyl chlorides afforded target
compounds 6–12a,b. The synthesis of compounds 6a
and 6b has been reported previously.14 Physicochemical
and spectral data of compounds 7–12a,b are shown in
Tables 1 and 2, respectively.
Comparison between IC50 values of nitrofurans 6–12a
and nitrothiophene analogues 6–12b against promastig-
otes revealed that nitrofurans possessed better activity
with respect to corresponding nitrothiophenes, with
the exception of 4-chlorobenzoyl derivatives (9a and
9b), which nitrothiophene analogue 9b showed slightly
better activity than nitrofuran counterpart 9a.
3. Results and discussion
The leishmania life cycle consists of two developmental
forms: promastigotes, flagellated extracellular parasites
of the digestive tract of sand flies, and amastigotes,
non-flagellated, non-motile stages that is more sensitive
and live inside parasitophorous vacuoles in macro-
phages of mammalian hosts.2 In the present study we
describe the evaluation of anti-leishmania activity of tar-
get compounds on both Leishmania stages.
Compounds 6a, 7a, 8a, 11a and 12a that exhibited po-
tent activity (IC50 6 13.19 lM) against promastigotes
were also assessed for their activity against the amasti-
gote form of L. major in marine peritoneal macrophages
(Fig. 2).18 All selected compounds significantly reduced
intracellular amastigotes. Among them, compounds 6a
and 11a were found to be more efficient in reducing
intracellular amastigotes (Fig. 2A). Moreover, all se-
N N
iii
i
ii
NH2
CH(OAc)2
O2N
CH=NNHCSNH2
2a, b
O2N
S
X
X
X
O2N
1a, b
3a, b
a: X = O
b: X = S
N N
N N
N N
iv
v
N
N
R
Cl
S
S
S
NH
N
X
X
X
O2N
O2N
O2N
O
4a, b
5a, b
6a-12a
6b-12b
Figure 1. Synthesis of compounds 6–12a,b. Reagents and conditions: (i) thiosemicarbazide, EtOH, HCl, reflux; (ii) ammonium ferric sulfate, H2O,
reflux; (iii) NaNO2, HCl, Cu; (iv) piperazine, EtOH, reflux; (v) appropriate thiophen-2-carbonyl chlorides or benzoyl chlorides, benzene, pyridine, rt.

M. Behrouzi-Fardmoghadam et al. / Bioorg. Med. Chem. 16 (2008) 4509–4515
Table 1. Structures and physicochemical data of compounds 7–12a,b
4511
N N
S
N
R
N
X
O2N
O
Compound
X
O
R
Mp (ꢂC)
Yielda (%)
MW
Formula
Cl
7a
225–227
78
419.84
C17H14ClN5O4S
C17H14ClN5O4S
C17H14ClN5O4S
C15H13N5O4S2
Cl
8a
O
O
O
O
O
S
217–219
244–246
239–241
223–225
241–243
243–245
250–252
260–262
247–249
250–252
261–263
80
83
81
73
69
73
78
81
82
73
70
419.84
419.84
391.42
425.87
470.32
435.91
435.91
435.91
407.49
441.94
486.39
9a
Cl
Cl
10a
11a
12a
7b
S
C15H12ClN5O4S2
C15H12BrN5O4S2
C17H14ClN5O3S2
C17H14ClN5O3S2
C17H14ClN5O3S2
C15H13N5O3S3
S
Br
S
Cl
Cl
8b
S
9b
S
Cl
Cl
10b
11b
S
S
S
C15H12ClN5O3S3
C15H12BrN5O3S3
S
12b
S
Br
S
a Yields of the final step.
lected compounds significantly decreased the percentage
of macrophage infectivity (Fig. 2B), and infectivity in-
dex (Fig. 2C).
It is believed that the lipophilic character of the molecule
plays an essential role in producing the biological effect.
The permeability of the cell to the test compounds is
one of the factors determining their activity. Drugs cross
biological barriers most frequently through passive trans-
port, which strongly depends on their lipophilicity. There-
fore, hydrophobicity is one of the most important
physical properties of biologically active compounds.
Thisthermodynamic parameter describes the partitioning
of a compound between an aqueous and an organic phase,
and is characterized by the octanol/water partition coeffi-
cient.19 In this context the balance of hydrophobicity
would be important for such activity. LogP, that is, the
logarithm of the partition coefficient for n-octanol/water,
was calculated using the program HyperChem ver. 7.0.
The cytotoxicity of target compounds was also assessed
using MTT colorimetric assay on macrophage cells.
Macrophage cells were treated with synthesized com-
pounds in the IC50 for 24 h, and side by side these were
treated with the standard drug Glucantime. The results
(Table 3) indicated that these compounds in the IC50
concentrations (against promastigote form of the L. ma-
jor) are not remarkably toxic towards marine peritoneal
macrophage cells (cell death <10%). Whereas, the refer-
ence drug Glucantime showed about 40% toxicity in
host cells.

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M. Behrouzi-Fardmoghadam et al. / Bioorg. Med. Chem. 16 (2008) 4509–4515
Table 2. Spectral data of compounds 7–12a,b
Compound
IR (KBr); mmax
1H NMR (80 MHz, DMSO-d6) d (ppm)
MS (m/e, %)
7a
1629 (C@O), 1542 and 1342 cmÀ1 (NO2)
7.85 (d, 1H, H4-furan, J = 4.0 Hz), 7.35–
7.55(m, 5H, H3-furan and phenyl), 3.60–
3.80 (m, 8H, piperazine).
421 ([M++2], 7), 419 (M+, 18),
308 (10), 263 (28), 166 (24), 139
(100), 110 (50), 68 (40), 55 (60).
421 ([M++2], 4), 419 (M+, 10),
401 (43), 376 (25), 308 (100), 196
(43), 138 (20), 90 (50), 56 (57).
8a
1634 (C@O), 1511 and 1342 cmÀ1(NO2)
7.85 (d, 1H, H4-furan, J = 4.0 Hz), 7.43–
7.58 (m, 4H, phenyl), 7.4 (d, 1H, H3-
furan, J = 4.0 Hz), 3.65–3.75 (m, 8H,
piperazine).
9a
1634 (C@O), 1512, 1352 cmÀ1 (NO2)
7.85 (d, 1H, H4-furan, J = 4.0 Hz), 7.54
(s, 4H, phenyl), 7.42 (d, 1H, H3-furan,
J = 4.0 Hz), 3.69 (br s, 8H, piperazine).
7.86 (d, 1H, H4-furan, J = 4.0 Hz), 7.6–
7.8 (m, 1H, H5-thiophene), 7.41 (d, 1H,
H3- furan, J = 4.0 Hz), 7.45–7.55 (m, 1H,
H3-thiophene), 7.10–7.25 (m, 1H, H4-
thiophene), 3.57–4.0 (m, 8H, piperazine).
7.85 (d, 1H, H4-furan, J = 4.0 Hz), 7.40
(d, 2H, H3-furan, H3-thiophene,
421 ([M++2], 4), 419 (M+, 10),
376 (25), 307 (76), 373 (25), 196
(43), 139 (91), 91 (100), 56 (73).
391 (M+, 17), 280 (10), 166 (20),
111 (100).
10a
1608 (C@O), 1539 and 1352 cmÀ1 (NO2)
11a
12a
1602 (C@O), 1533 and 1356 cmÀ1 (NO2)
1607 (C@O), 1540, 1361 cmÀ1 (NO2)
426 ([M++2], 7), 424 (M+, 18),
262 (34), 237 (19), 164 (24), 145
(100), 143 (95), 81 (37), 55 (40).
J = 4.0 Hz), 7.19 (d, 1H, H4-thiophene,
J = 4.0 Hz), 3.57–4.0 (m, 8H, piperazine).
7.85 (d, 1H, H4-furan, J = 4.0 Hz), 7.40
(d, 1H, H3-thiophene, J = 4.0 Hz), 7.34
(d, 1H, H3-furan, J = 3.9 Hz), 7.29 (d,
1H, H4- thiophene, J = 3.9 Hz), 3.59–3.96
(m, 8H, piperazine).
471 ([M++2], 40), 469 (M+, 40),
390 (20), 281 (24), 264 (90), 239
(64), 187 (100), 167 (60), 80 (75),
54 (98).
7b
8b
9b
1634 (C@O), 1530, 1337 cmÀ1 (NO2)
1644 (C@O), 1536, 1347 cmÀ1 (NO2)
1634 (C@O), 1527, 1347 cmÀ1 (NO2)
8.17 (d, 1H, H4-thiophene, J = 4.0 Hz),
7.35–7.65 (m, 5H, H3-thiophene and
phenyl), 3.65–3.95 (m, 8H, piperazine).
8.18 (d, 1H, H4-thiophene, J = 4.2 Hz),
7.40–7.60 (m, 5H, H3-thiophene and
phenyl), 3.7–4.0 (m, 8H, piperazine).
8.12 (d, 1H, H4-thiophene, J = 4.2 Hz),
7.57 (d, 1H, H3-thiophene, J = 4.2 Hz),
7.51 (s, 4H, phenyl ), 3.66 (br s, 8H,
piperazine).
437 ([M++2], 7), 435 (M+, 18),
308 (13), 223 (18), 139 (100), 111
(56), 56 (40).
437 ([M++2], 5), 435 (M+, 13),
139 (100), 111 (50), 55 (48).
437 ([M++2], 7), 435 (M+, 10),
280 (18), 254 (25), 182 (25), 139
(100), 111 (50), 56 (46).
10b
11b
12b
1608 (C@O), 1521, 1342 cmÀ1 (NO2)
1605 (C@O), 1522, 1368 cmÀ1 (NO2)
1609 (C@O), 1519, 1346 cmÀ1 (NO2)
8.13 (d, 1H, H4-nitrothiophene,
J = 4.2 Hz), 7.7–7.9 (m, 1H, H5-
407 (M+, 10), 279 (15), 152 (18),
111 (60), 109 (100).
thiophene, J = 4.2 Hz), 7.65 (d, 1H, H3-
nitrothiophene, J = 4.2 Hz), 7.45–7.6 (m,
1H, H3-thiophene), 7.15–7.25 (m, 1H, H4-
thiophene), 3.52–4.0 (m, 8H, piperazine).
8.15 (d, 1H, H4-nitrothiophene,
443 ([M++2], 4), 441 (M+, 10),
280 (15), 240 (15), 181 (22), 144
(100),69 (18), 56 (51).
J = 4.2 Hz), 7.59 (d, 1H, H3-
nitrothiophene, J = 4.2 Hz), 7.39 (d, 1H,
H3-thiophene, J = 4.0 Hz), 7.19 (d, 1H,
H4-thiophene, J = 4.0 Hz), 3.52–4.05 (m,
8H, piperazine).
8.13 (d, 1H, H4-nitrothiophene,
J = 4.2 Hz), 7.61 (d, 1H, H3-
488 ([M++2], 15), 486 (M+, 16),
281 (37), 278 (45), 187 (100), 183
(65), 162 (24), 125 (40), 101 (76),
80 (87), 52 (98).
nitrothiophene, J = 4.2 Hz), 7.35 (d, 1H,
H3-thiophene, J = 4.0 Hz), 7.27 (d, 1H,
H4-thiophene, J = 4.0 Hz), 3.50–3.96 (m,
8H, piperazine).
The results obtained are given in Table 3. The calculated
values of logP for derivatives of nitrothiophene were
about 0.35 higher than for the corresponding compounds
with a nitrofuran moiety. It could be assumed that the in-
crease in lipophilicity (log P) of the compounds within the
series 6–12b results in the decrease of anti-leishmanial
activity. On the other hand, the observed differences in
anti-leishmanial activities of nitrofurans and nitrothioph-
enes may be due to the reduction potential of the single-
electron transfer ArNO2/ArNOꢀ2À. Nitroheterocylic com-
pounds are generally believed to exert their cytotoxic ef-
fects only after activation by single-electron reduction
of their corresponding nitro anion radicals.20–22 Under
aerobic conditions, the nitro radical anion reacts with
oxygen to form superoxide anion and hydroxyl radical.
The resulting oxygen-derived free radicals would damage
the enzyme, DNA or important structures in the sur-
rounding cell, and result in a cytotoxic action. Under
anaerobic conditions, the radical anion can be trans-
formed into the corresponding nitroso-derivative. This

M. Behrouzi-Fardmoghadam et al. / Bioorg. Med. Chem. 16 (2008) 4509–4515
4513
Table 3. In vitro activities of compounds 6–12a,b against promastigote form of L. major and peritoneal macrophages
N N
N
R
S
N
X
O2N
LogPa
O
Compound
X
O
R
Toxicity on L. major [IC50 (lM)]b
Toxicity on macrophages [Cell death (%)]c
6a
2.17
11.69 1.3
7.1 0.08
Cl
7a
O
1.95
10.73 0.69
8.7 0.06
Cl
8a
O
O
O
O
O
S
1.95
1.95
0.78
1.92
2.22
2.52
2.29
13.19 0.96
26.22 0.69
19.18 1.95
11.75 1.1
13.11 0.86
25.00 1.25
34.44 1.14
9.7 0.07
9.7 0.02
2.4 0.03
7.6 0.09
7.7 0.13
8.9 0.02
6.1 0.10
9a
Cl
10a
11a
12a
6b
S
Cl
Br
S
S
Cl
7b
S
Cl
8b
S
S
S
S
S
2.29
2.29
1.13
2.27
2.57
>50
5.5 0.12
7.2 0.06
8.7 0.07
9.8 0.07
9b
24.11 1.14
28.26 0.61
23.78 0.65
Cl
10b
11b
S
Cl
Br
S
12b
24.69 1.03
30 1.41d
9.2 0.02
S
Glucantime
40.4 0.09
a LogP, that is, the logarithm of the partition coefficient for n-octanol/water, was calculated using the program HyperChem ver. 7.0.
b The values represent means SD.
c Macrophage cells were treated with compounds in the IC50 concentrations (observed against promastigotes) for 24 h.
d IC50 in mg/mL.
nitroso form has been put forward as an efficient scaven-
ger of essential thiols in the cell.23,24
2-yl]-4-aroylpiperazines, with in vitro anti-leishmanial
activity. From our biological results, it is evident that
most of title compounds exhibited potent anti-leishman-
ial activity against both promastigote and amastigote
forms of L. major at non-cytotoxic concentrations. How-
ever, the 5-nitrofurans were evidently more active than
the corresponding 5-nitrothiophene analogues.
In conclusion, we have described a series of nitrohetero-
aryl-1,3,4-thiadiazole-based compounds including 1-[5-
(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpipera-
zines and 1-[5-(5-nitrothiophen-2-yl)-1,3,4-thiadiazol-

4514
M. Behrouzi-Fardmoghadam et al. / Bioorg. Med. Chem. 16 (2008) 4509–4515
Figure 2. In vitro activity of selected 5-(5-nitrofuran-2-yl)-1,3,4-thiadiazoles against intramacrophage amastigotes of L. major: (A) The mean number
of amastigotes per macrophage after treatment with drug for 24 h. (B) The percentage of infected macrophages after treatment. (C) Infectivity index
of macrophages cultured 24 h in the presence of selected drugs.
4. Experimental
4.2. Biological activity
Chemicals and all solvents used in this study were
purchased from Merck AG and Aldrich Chemical.
Intermediates 5a,b were prepared according to the lit-
erature.14–16 Melting points were determined on a
Kofler hot stage apparatus and are uncorrected.
The IR spectra were obtained on a Shimadzu 470
spectrophotometer (potassium bromide disks). 1H
NMR spectra were measured using a Bruker FT-80
spectrometer, and chemical shifts are expressed as d
(ppm) with tetramethylsilane as internal standard.
The mass spectra were run on a Finigan TSQ-70
spectrometer (Finigan, USA) at 70 eV. Elemental
analyses were carried out on a CHN–O rapid elemen-
tal analyzer (GmbH-Germany) for C, H and N, and
the results are within 0.4% of the theoretical values.
Merck silica gel 60 F254 plates were used for analyt-
ical TLC. Yields are of purified product and were
not optimized.
The strain of L. major used in this study was the vac-
cine strain (MRHO/IR/75/ER), obtained from Pasteur
Institute, Tehran (Iran). The infectivity of the parasites
was maintained by regular passage in susceptible
BALB/C mice. The promastigote form of parasite
was grown in blood agar cultures at 25 ꢂC. The station-
ary parasite inoculation was 2 · 106 cells/mL. For the
experiments described here, the stationary phase prom-
astigotes were washed with phosphate buffered saline
and recultured in RPMI 1640 medium (Sigma) at
2 · 106 cells/mL density, supplemented with 10% of
heat-inactivated fetal bovine serum, 2 mM glutamine
(Sigma), pH ꢀ 7.2, 100 U/mL penicillin (Sigma) and
100 lg/mL streptomycin (Sigma).
4.2.1. Anti-leishmanial activity against promastigotes
form of L. major. The anti-leishmanial screening was
performed using direct counting and MTT assay. It is
noted that at the first, the growth curve of the L. major
strain was determined daily under light microscope and
counting in a Neubauer’s chamber. Then, parasites
(2 · 106/mL) in the logarithmic phase were incubated
with a serial range of drug concentrations for 24 h at
25 ꢂC. To determine 50% inhibitory concentrations
(IC50), the tetrazolium bromide salt (MTT) assay was
used.17 Briefly, promastigotes, from early log phase of
growth, were seeded in 96-well plastic cell culture trays,
containing serial dilution of drug and phenol red free
4.1. General procedure for synthesis of compounds 6–12
To a mixture of compound 5a,b (2.0 mmol) in dry ben-
zene (5 mL), pyridine (1 mL) and appropriate aroyl
chloride (2.0 mmol) were added, respectively. The mix-
ture was stirred at room temperature overnight. The sol-
vents were removed under reduced pressure and the
resulting solid was washed with water and crystallized
from ethanol to give compounds 6–12.

M. Behrouzi-Fardmoghadam et al. / Bioorg. Med. Chem. 16 (2008) 4509–4515
4515
RPMI 1640 medium, supplemented with 10% of FBS,
References and notes
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This work was supported by a grant from INSF (Iran
National Sciences Foundation).