Nitroimidazolyl-1,3,4-thiadiazole-based anti-leishmanial agents: Synthesis and in vitro biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2008.03.039

A series of 1-[5-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines were synthesized and evaluated in vitro against Leishmania major. Most of the target compounds exhibited good anti-leishmanial activity against the promastigote form of L. major at non-cytotoxic concentrations. The most active compound was 1-[(5-chloro-2-thienyl)carbonyl]-4-[5-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl]piperazine (5f) with an IC₅₀ value of 9.35 ± 0.67 μM against L. major promastigotes. In addition, this compound was effective against intracellular L. major and significantly decreased the infectivity index.

Full text of DOI:10.1016/j.ejmech.2008.03.039

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1758e1762
http://www.elsevier.com/locate/ejmech
Short communication
Nitroimidazolyl-1,3,4-thiadiazole-based anti-leishmanial agents:
Synthesis and in vitro biological evaluation
Fatemeh Poorrajab a, Sussan Kabudanian Ardestani a, Saeed Emami b,
Mina Behrouzi-Fardmoghadam c, Abbas Shafiee c, Alireza Foroumadi c,
*
a Institute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran, P.O. Box 13145-1384, Tehran, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
c Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14174, Iran
Received 27 November 2007; received in revised form 26 March 2008; accepted 27 March 2008
Available online 9 April 2008
Abstract
A series of 1-[5-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl]-4-aroylpiperazines were synthesized and evaluated in vitro
against Leishmania major. Most of the target compounds exhibited good anti-leishmanial activity against the promastigote form of L. major
at non-cytotoxic concentrations. The most active compound was 1-[(5-chloro-2-thienyl)carbonyl]-4-[5-(1-methyl-5-nitro-1H-imidazol-2-yl)-
1,3,4-thiadiazol-2-yl]piperazine (5f) with an IC50 value of 9.35 ꢀ 0.67 mM against L. major promastigotes. In addition, this compound was ef-
fective against intracellular L. major and significantly decreased the infectivity index.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: 1,3,4-Thiadiazole; Nitroimidazole; Leishmania major; Anti-leishmanial activity
1. Introduction
recent increase in the spread of leishmaniasis is due in part
to co-infections with the HIV/AIDS virus [5].
Leishmaniasis is a spectrum of diseases, ranging from self-
limiting cutaneous infections to more serious disseminating
diffuse cutaneous, mucocutaneous and visceral forms of the
disease caused by intracellular protozoan parasites belonging
to the genus Leishmania [1,2]. Among all leishmaniasis,
visceral leishmaniasis is a highly morbid and incapacitating
infection, which usually presents with prolonged fever, weight
loss and hepatosplenomegaly. Despite the availability of effec-
tive treatment, the disease can have a high mortality even at
referral centers [3]. The disease is endemic in 88 tropical
and subtropical countries, where 350 million people are at
risk. There is an estimate of 12 million already contaminated
people, as well as an annual incidence of 2 million cases [4].
Disease progression is dependent on both the species of leish-
mania involved (as many as 17 sub-species may infect hu-
mans) and the genetics and immune status of the host. The
There are no vaccines against leishmaniasis and, as with
other trypanosomatid diseases; treatment is dependent on
a limited range of drugs. Front line drugs include pentavalent
antimonials, amphotericin B and, in the case of visceral leish-
maniasis, the only orally administered drug, miltefosine. All
these drugs are limited to some extent by their toxicity, lack
of efficacy, requirement for hospitalisation and/or cost [6].
The problem is further aggravated by the surge of antimonial
resistance in some areas where the disease is endemic. While
a number of second line drugs including pentamidine, paromo-
mycin and the azoles are being tested or in the process of
being introduced into the clinic, there is clearly a need for
developing new, effective, cheap and safe drugs in the field
of leishmaniasis chemotherapy [6,7].
For the last decade, new potential therapies have been intro-
duced for leishmaniasis including paromomycin and sitama-
quine. The latter has completed phase II trials in India,
Kenya and Brazil [8]. Moreover, a great number of both natu-
ral and synthetic compounds have been evaluated in recent
* Corresponding author. Tel.: þ98 21 66954708; fax: þ98 21 66461178.
E-mail address: aforoumadi@yahoo.com (A. Foroumadi).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.03.039

F. Poorrajab et al. / European Journal of Medicinal Chemistry 44 (2009) 1758e1762
1759
years in anti-leishmanial assays [9e13]. In this context, the
use of nitroimidazole derivatives as antiprotozoal agents is
well established. Although there is a large amount of experi-
mental work on this heterocyclic system, it still remains an
area of active research. On the other hand, 1,3,4-thiadiazoles
have long been the subject of pharmaceutical interest as a re-
sult of their potent biological activities. Indeed, the 1,3,4-thia-
diazole derivatives have been reported to possess antiparasitic
properties [14] and their attachment with other heterocycles
often ameliorates or diminishes the bioresponses, depending
upon the type of substituent and position of attachment. As
part of our efforts to develop new compounds aimed at the
therapy of parasitic infection especially leishmaniasis
[12,13], we have synthesized and evaluated some 1-[5-(1-
methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl]-4-ar-
oylpiperazines against Leishmania major.
parasites of the digestive tract of sand flies, and amastigotes,
non-flagellated, non-motile stages that are more sensitive
and live in macrophages [17]. In this study we described the
anti-leishmanial assay of title compounds against both pro-
mastigote and amastigote forms of L. major strain (MRHO/
IR/75/ER).
Compounds 5aeg were tested for in vitro activity against
the promastigote form of the L. major strain (MRHO/IR/75/
ER) along with meglumine antimonate (GlucantimeÒ), using
MTT assay [18]. The IC50 values are presented in Table 1.
The most potent compounds against the promastigote form
of L. major were found to be N-(5-chloro-thiophen-2-yl)car-
bonyl derivative 5f and N-benzoyl analog 5a with IC50 values
of 9.35 ꢀ 0.67 and 10.39 ꢀ 0.95 mM, respectively. The
remaining compounds showed IC50 values between
15.96 ꢀ 0.77 and 42.91 ꢀ 2.38 mM, whereby the halogen sub-
stitution (chloro- or bromo-) on thiophen-2-carbonyl moiety
improves the activity against promastigotes but chloro- substi-
tution on benzoyl containing compound (5a) decreases the
overall anti-leishmanial activity. The effect of positional isom-
erism of chloro- substitution was investigated by preparing all
three possible regioisomers on benzoylpiperazine moiety. The
order of activity in chlorobenzoyl series was as follows: 3-
Cl > 2-Cl > 4-Cl. A comparison between IC50 values of the
unsubstituted benzoyl analog 5a and its unsubstituted thio-
phen-2-carbonyl counterpart 5e, against promastigotes, re-
vealed that benzoyl compound possessed better activity with
respect to corresponding thiophene derivative. In contract, in
halogenated compounds, 5-halo-thiophenes 5f,g exhibited
more potent activity than their corresponding chlorophenyl
derivatives 5bed.
2. Chemistry
The synthetic pathway to target compounds 5aeg is shown in
Scheme 1. The intermediate 2-chloro-1,3,4-thiadiazole 3 was
obtained from 1-methyl-5-nitroimidazole-5-carboxaldehyde
according to the previously described methods [15,16]. Thus,
treatment of 1 with thiosemicarbazide in the presence of HCl
afforded the corresponding thiosemicarbazone which upon
cyclization with ammonium ferric sulfate gave 2-amino-1,3,4-
thiadiazole 2. Diazotization of amine 2 in HCl solution, in the
presence of copper powder, gave 2-chloro-1,3,4-thiadiazole 3.
The reaction of compound 3 with piperazine in refluxing ethanol
gave N-piperazinyl compound 4. N-aroylation of the piperazine
analog 4 with appropriate benzoyl chlorides or thiophen-2-car-
bonyl chlorides afforded target compounds 5aeg [12,13].
The compounds 5a and f which exhibited potent activity
against promastigotes (IC50 ꢁ 10.39 mM) were also evaluated
for their activity against the amastigote form of L. major in
peritoneal macrophages (Fig. 1) [19]. As can be deducted
from Fig. 1, compounds 5a and f have significantly de-
creased the number of amastigotes per macrophage and
3. Results and discussion
The life cycle of leishmania microorganism consists of two
developmental stages: promastigotes, flagellated extracellular
N N
S
N
N
N N
S
N
i
iii
O2N
O2N
NH2
CHO
Cl
O2N
N
N
N
ii
CH3
CH3
CH3
1
2
3
O
N N
N
N N
N
NH
iv
v
N
O2N
N
O2N
N
R
N
CH3
S
N
S
CH3
5a-g
4
a: R = phenyl
e: R = thiophen-2-yl
f: R = 5-Cl-thiophen-2-yl
g: R = 5-Br-thiophen-2-yl
b: R = 2-Cl-phenyl
c: R = 3-Cl-phenyl
d: R = 4-Cl-phenyl
Scheme 1. 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.

1760
F. Poorrajab et al. / European Journal of Medicinal Chemistry 44 (2009) 1758e1762
Table 1
In vitro anti-leishmanial activity of compounds 5aeg against promastigote
form of L. major
the results are within ꢀ0.4% of the theoretical values. Merck
silica gel 60 F254 plates were used for analytical TLC.
O
4.1.1. General procedure for the synthesis of
compounds 5aeg
N N
S
N
N
O2N
N
R
N
To a mixture of compound 4 (1 mmol) in dry benzenee
pyridine (3:1 mL), appropriate thiophen-2-carbonylchloride
or benzoyl chloride (1.1 mmol) was added and the mixture
was stirred at room temperature for three days. The solvents
were removed under reduced pressure and resulting solid
was washed with methanolewater (80%) and crystallized
from ethanol to give 5aeg.
CH3
Compound
R
MW
IC50 (mM)a
5a
5b
5c
5d
5e
5f
Phenyl
399.43
433.87
433.87
433.87
405.45
439.9
10.39 ꢀ 0.95
20.26 ꢀ 0.61
17.88 ꢀ 0.72
42.91 ꢀ 2.38
25.58 ꢀ 1.03
9.35 ꢀ 0.67
2-Clephenyl
3-Clephenyl
4-Clephenyl
Thiophen-2-yl
5-Clethiophen-2-yl
5-Brethiophen-2-yl
4.1.1.1. 1-(2-Chlorobenzoyl)-4-[5-(1-methyl-5-nitro-1H-imida-
zol-2-yl)-1,3,4-thiadiazol-2-yl] piperazine (5b). Yield 80%;
mp 275e277 ꢂC; IR (KBr) 3140 (HeC imidazole), 1629
(C]O), 1537 and 1358 cmꢃ1 (NO2); 1H NMR (80 MHz,
DMSO-d6) d 8.15 (s, 1H, H-4 imidazole), 7.40e7.55 (m,
4H, phenyl), 4.35 (s, 3H, NCH3), 3.60e3.85 (m, 8H, pipera-
zine). MS (m/e, %): 435 ([Mþ þ 2], 5), 433 (Mþ, 15), 400
(45), 309 (100), 196 (58), 150 (18), 56 (48).
5g
Meglumine antimonate
484.35
15.96 ꢀ 0.77
81.97 ꢀ 3.85b
a
The values represent mean ꢀ SD.
b
IC50 in mM.
both the percentage of macrophage infectivity and infectivity
index.
The cytotoxic property of the title compounds against mac-
rophage cells were also assessed using MTT colorimetric as-
say [20]. Macrophage cells were treated with compounds in
the IC50 concentrations for 24 h, side by side the standard
drug meglumine antimonate. The results showed that these
compounds display anti-leishmanial activity at non-cytotoxic
concentrations. Whereas, the reference drug meglumine
antimonate decreased viability of macrophages up to 40%.
In conclusion, we have synthesized a series of 1-[5-(1-
methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl]-4-ar-
oylpiperazines via a versatile synthetic route and evaluated
them against L. major. Most of the target compounds exhibited
good anti-leishmanial activity against the promastigote form
of L. major at non-cytotoxic concentrations. The most active
compound was 1-[(5-chloro-2-thienyl)carbonyl]-4-[5-(1-
methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl]piper-
azine 5f which also showed good activity against intracellular
form of L. major.
4.1.1.2. 1-(3-Chlorobenzoyl)-4-[5-(1-methyl-5-nitro-1H-imida-
zol-2-yl)-1,3,4-thiadiazol-2-yl]piperazine (5c). Yield 84%;
mp 242e244 ꢂC; IR (KBr) 3140 (HeC imidazole), 1624
(C]O), 1516 and 1363 cmꢃ1 (NO2); 1H NMR (80 MHz,
DMSO-d6) d 8.2 (s, 1H, H-4 imidazole), 7.3e7.65 (m, 4H,
phenyl), 4.35 (s, 3H, NCH3), 3.69 (br s, 8H, piperazine). MS
(m/e, %): 435 ([Mþ þ 2], 6), 433 (Mþ, 15), 400 (45), 309
(100), 252 (20), 196 (58), 150 (18), 56 (48).
4.1.1.3. 1-(4-Chlorobenzoyl)-4-[5-(1-methyl-5-nitro-1H-imida-
zol-2-yl)-1,3,4-thiadiazol-2-yl]piperazine (5d). Yield 85%;
mp 245e247 ꢂC; IR (KBr) 3129 (HeC imidazole), 1639
(C]O), 1520 and 1340 cmꢃ1 (NO2); 1H NMR (80 MHz,
DMSO-d6) d 8.20 (s, 1H, H-4 imidazole), 7.50e7.53 (m,
4H, phenyl), 4.34 (s, 3H, NCH3), 3.68 (br s, 8H, piperazine).
MS (m/e, %): 435 ([Mþ þ 2], 8), 433 (Mþ, 18), 308 (20),
252 (42), 238 (30), 180 (20), 139 (100), 111 (40), 56 (54).
4.1.1.4. 4-[5-(1-Methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thia-
diazol-2-yl]-1-(2-thienyl carbonyl) piperazine (5e). Yield
79%; mp 235e237 ꢂC; IR (KBr) 3140 (HeC imidazole),
1603 (C]O), 1521 and 1358 cmꢃ1 (NO2); 1H NMR
(80 MHz, DMSO-d6) d 8.22 (s, 1H, H-4 imidazole), 7.7e7.9
(m, 1H, H-5 thiophene), 7.45e7.6 (m, 1H, H-3 thiophene),
7.15e7.25 (m, 1H, H-4 thiophene), 4.33 (s, 3H, NCH3),
3.54e3.96 (m, 8H, piperazine). MS (m/e, %): 405 (Mþ, 17),
294 (6), 278 (20), 111 (100), 55 (45).
4. Experimental
4.1. Chemistry
Chemical reagents and all solvents used in this study were
purchased from Merck AG Chemical. The key intermediate 2-
chloro-5-(1-methyl-5-nitroimidazol-2-yl)-1,3,4-thiadiazole
was prepared according to the literature method [13e16].
Melting points were determined on a Kofler hot stage appara-
tus and are uncorrected. The IR spectra were obtained on a Shi-
4.1.1.5. 1-[(5-Chloro-2-thienyl)carbonyl]-4-[5-(1-methyl-5-ni-
1
madzu 470 spectrophotometer (potassium bromide dicks). H
tro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl]piperazine
(5f).
NMR spectra were recorded on a Varian unity 80 spectrometer
and chemical shifts are reported in parts per million (d) rela-
tive to tetramethylsilane (TMS) as an internal standard. Ele-
mental analyses were carried out on a CHNeO rapid
elemental analyzer (GmbH-Germany) for C, H and N, and
Yield 85%; mp 234e235 ꢂC; IR (KBr) 3140 (HeC imidaz-
ole), 1614 (C]O), 1521 and 1362 cmꢃ1 (NO2); 1H NMR
(80 MHz, DMSO-d6) d 8.22 (s, 1H, H-4 imidazole), 7.40 (d,
1H, H-3 thiophene, J ¼ 4.0 Hz), 7.19 (d, 1H, H-4 thiophene,
J ¼ 4.0 Hz), 4.33 (s, 3H, NCH3), 3.54e3.96 (m, 8H, piperazine).

F. Poorrajab et al. / European Journal of Medicinal Chemistry 44 (2009) 1758e1762
1761
Fig. 1. In vitro activity of compounds 5a and f against intramacrophage amastigotes of L. major. (A) The mean number of amastigotes per macrophage after treat-
ment 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. The infectivity index was determined by multiplying the percentage of macrophages that had at least one intracellular parasite by the average number of
intracellular parasites per infected macrophage (60 cells were examined/well).
MS (m/e, %) 441 ([Mþ þ 2], 7), 439 (Mþ, 25), 276 (60), 251
Tehran (Iran). The infectivity of the parasites was maintained
by regular passage in susceptible BALB/c mice. The promas-
tigote form of parasite was grown in blood agar cultures at
25 ꢂC. The stationary parasite inoculation was 2 ꢄ 106 cells/
mL. For the experiments described here, the stationary phase
promastigotes were washed with phosphate buffered saline
and recultured in RPMI 1640 medium (Sigma) at 2 ꢄ 106
cells/mL density, supplemented with 10% of heat-inactivated
fetal bovine serum, 2 mM glutamine (Sigma), pH w7.2,
100 U/mL penicillin (Sigma) and 100 mg/mL streptomycin
(Sigma).
(95), 178 (76), 143 (100), 99 (45), 54 (60).
4.1.1.6. 1-[(5-Bromo-2-thienyl)carbonyl]-4-[5-(1-methyl-5-ni-
tro-1H-imidazol-2-yl)-1,3,4-thiadiazol-2-yl]piperazine
(5g).
Yield 77%; mp 260e262 ꢂC; IR (KBr) 3138 (HeC imidaz-
ole), 1608 (C]O), 1511 and 1362 cmꢃ1 (NO2); 1H NMR
(80 MHz, DMSO-d6) d 8.22 (s, 1H, H-4 imidazole), 7.34 (d,
1H, H-3 thiophene, J ¼ 3.9 Hz), 7.29 (d, 1H, H-4 thiophene,
J ¼ 3.9 Hz), 4.33 (s, 3H, NCH3), 3.58e3.96 (m, 8H, pipera-
zine) MS (m/e, %) 485 ([Mþ þ 2], 38), 483 (Mþ, 40), 403
(20), 277 (100), 251 (89), 238 (67), 189 (76), 152 (36), 99
(18), 68 (24).
4.2.2. Anti-leishmanial activity against promastigotes
form of L. major
4.2. Biological activity
The anti-leishmanial screening was performed using direct
counting and MTT assay [18]. It should be noted that at 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 ꢄ 106/mL) in the logarithmic phase were
4.2.1. Parasite isolates and culture conditions
The strain of L. major used in this study was the vaccine
strain (MRHO/IR/75/ER), obtained from Pasteur Institute,

1762
F. Poorrajab et al. / European Journal of Medicinal Chemistry 44 (2009) 1758e1762
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. 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 RPMI 1640 medium, supple-
mented with 10% of FBS, 2 mM glutamine, pH w7.2 and
antibiotics, in a volume of 200 mL. After 24 h of incubation
at 25 ꢂC, the media was renewed with 100 mg/well of MTT
(0.5 mg/mL) and plates were further incubated for 4 h at
37 ꢂC. The plates were centrifuged (2000 rpm ꢄ 5 min), the
pellets were dissolved in 200 mL of DMSO. The samples
were read using an ELISA plate reader at a wavelength of
492 nm. Two or more independent experiments in triplicate
were performed for determination of sensitivity to each
drug, the IC50 was calculated by linear regression analysis,
expressed in mean ꢀ SD. Control cells were incubated with
culture medium plus DMSO.
compound. The plates were incubated for 24 h at 37 ꢂC in
a humidified incubator with 5% CO2. Control cells were incu-
bated with culture medium plus DMSO. Cell viability was
determined by MTT colorimetric assay [20].
Acknowledgement
This work was supported by grants from the Research
Council of Tehran University of Medical sciences and Iran Na-
tional Science Foundation (INSF).
References
[1] B.L. Herwaldt, Lancet 354 (1999) 1191e1199.
[2] S.L. Croft, G.H. Coombs, Trends Parasitol. 19 (2003) 502e508.
[3] G. Malatesha, P. Mathur, K. Madan, P. Samanta, J.C. Samantaray, Trop.
Gastroenterol. 28 (2007) 28e31.
[4] P. Desjeux, Comp. Immunol. Microbiol. Infect. Dis. 27 (2004) 305e318.
[5] P.K. Sinha, K.I. Pandey, S.K. Bhattacharya, Indian J. Med. Res. 121
(2005) 407e414.
4.2.3. Anti-leishmanial activity against amastigotes
form of L. major
[6] C.R. Davies, P. Kaye, S.L. Croft, S. Sundar, BMJ 326 (2003) 377e382.
[7] T.K. Jha, Indian J. Med. Res. 123 (2006) 389e398.
[8] S.L. Croft, S. Sundar, A.H. Fairlamb, Clin. Microbiol. Rev. 19 (2006)
111e126.
Mouse peritoneal macrophages were plated in RPMI 1640
supplemented with 10% of heat-inactivated fetal bovine
serum, 2 mM glutamine, 100 U/mL penicillin (Sigma) and
100 mg/mL streptomycin. Macrophages were placed on sterile
glass cover slips in 24-well plates (1 ꢄ 106/well). After 1 h
non-adherent cells were removed by washing with RPMI,
the stationary phase promastigotes in RPMI were added
(2 ꢄ 106 parasites/well, three parasites/macrophage) to macro-
phage monolayer and the plates were kept at 37 ꢂC in a CO2
incubator for 2 h. Extracellular parasites were removed by
washing and then new media containing IC50 concentration
of the drug were added. Two sets of experiments were carried
out for each drug at 24 h. Following these procedures, cells
were fixed with methanol, stained with Giemsa stain (Sigma)
and the infectivity index was determined by multiplying the
percentage of macrophages that had at least one intracellular
parasite by the average number of intracellular parasites per
infected macrophage (60 cells were examined/well) [19].
´
´
´
[9] P. Gonzalez, C. Marın, I. Rodrıguez-Gonzalez, A. Illana, H. Mateo,
S.S. Longoni, M.J. Rosales, A. Gonzalez-Coloma, M. Reina,
´
´
´
M. Sanchez-Moreno, J.G. Dıaz, Pharmacology 76 (2006) 123e128.
´
[10] A.G. Tempone, S.E.T. Borborema, H.F. de Andrade Jr., N.C.de
ꢀ
A. Gualda, A. Yogi, C.S. Carvalho, D. Bachiega, F.N. Lupo,
S.V. Bonotto, D.C.H. Fischer, Phytomedicine 12 (2005) 382e390.
[11] K.M. Khan, M. Rasheed, Z. Ullah, S. Hayat, F.K. Kaukab,
M.I. Choudhary, Atta-ur-Rahman, S. Perveen, Bioorg. Med. Chem. 11
(2003) 1381e1387.
[12] A. Foroumadi, S. Pournourmohammadi, F. Soltani, M. Asgharian-
Rezaee, S. Dabiri, A. Kharazmi, A. Shafiee, Bioorg. Med. Chem. Lett.
15 (2005) 1983e1985.
[13] A. Foroumadi, S. Emami, S. Pournourmohammadi, A. Kharazmi,
A. Shafiee, Eur. J. Med. Chem. 40 (2005) 1346e1350.
[14] M. Behrouzi-Fardmoghadam, F. Poorrajab, S.K. Ardestani, S. Emami,
A. Shafiee, A. Foroumadi, Bioorg. Med. Chem. 16 (2008) 4509e4515.
[15] A. Foroumadi, M. Mirzaei, A. Shafiee, Il Farmaco 56 (2001) 621e623.
[16] A. Foroumadi, S. Mansouri, Z. Kiani, A. Rahmani, Eur. J. Med. Chem.
38 (2003) 851e854.
[17] S.L. Croft, V. Yardley, Curr. Pharm. Des. 8 (2002) 319e342.
[18] A. Dutta, S. Bandyopadhyay, C. Mandal, M. Chatterjee, Parasitol. Int. 54
(2005) 119e122.
4.2.4. Toxicity on peritoneal macrophages
In vitro toxicity toward mouse peritoneal macrophages was
assessed with cells plated in 96-well plates at 2 ꢄ 105 cells/
well. After cell adherence, the medium was removed and
replaced by the media containing IC50 concentration of each
[19] A.K. Tanaka, V.B. Valero, H.K. Takahashi, A.H. Straus, J. Antimicrob.
Chemother. 59 (2007) 487e492.
[20] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.