Synthesis and in vitro leishmanicidal activity of 2-(1-methyl-5-nitro-1H- imidazol-2-yl)-5-substituted-1,3,4-thiadiazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2005.07.002

A series of 2-(1-methyl-5-nitroimidazol-2-yl)-5-(1-piperazinyl, 1-piperidinyl and 1-morpholinyl)-1,3,4-thiadiazoles (3a-g) were synthesized and evaluated for in vitro leishmanicidal activity against Leishmania major promastigotes. The leishmanicidal data

Full text of DOI:10.1016/j.ejmech.2005.07.002

European Journal of Medicinal Chemistry 40 (2005) 1346–1350
www.elsevier.com/locate/ejmech
Short communication
Synthesis and in vitro leishmanicidal activity
of 2-(1-methyl-5-nitro-1H-imidazol-2-yl)-5-substituted-1,3,4-thiadiazole
derivatives
Alireza Foroumadi a,b,*, Saeed Emami c, Shirin Pournourmohammadi b,
Arsalan Kharazmi d, Abbas Shafiee a
a Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Tehran University of Medical Science, Tehran 14174, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Kerman University of Medical Sciences, Haft Bagh Street, Kerman 76351, Iran
c Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, P.O. Box 48175-861, Sari, Iran
d LICA Pharmaceuticals, Symbion Sciences Park, Copenhagen DK 2100, Denmark
Received 13 February 2005; received in revised form 28 June 2005; accepted 1 July 2005
Available online 10 August 2005
Abstract
A series of 2-(1-methyl-5-nitroimidazol-2-yl)-5-(1-piperazinyl, 1-piperidinyl and 1-morpholinyl)-1,3,4–thiadiazoles (3a–g) were synthe-
sized and evaluated for in vitro leishmanicidal activity against Leishmania major promastigotes. The leishmanicidal data revealed that com-
pounds 3a–g had strong and much better leishmanicidal activity than the reference drug pentostam. Compound 3c (piperazine analog) was the
most active compound (IC50 = 0.19 µM).
© 2005 Elsevier SAS. All rights reserved.
Keywords: 5-Nitro-1H-imidazole; 1,3,4-Thiadiazole; Leishmania major; Anti-promastigote activity
1. Introduction
The pharmacological interest of the imidazole ring has been
established, nitroimidazoles being extensively used in therapy
against amoebic, trichomonal, giardial and anaerobic infec-
tions, or as hypoxic cell radiosensitizers [8]. Metronidazole
and N-substituted imidazoles (ketoconazole, fluconazole and
itraconazole) are well-tolerated drugs that are potentially
active against Leishmania, but their use in the treatment of
cutaneous and visceral leishmaniasis has produced conflict-
ing results [9]. On the other hand the antiparasitic property of
1,3,4-thiadiazoles is well documented and their attachment
with other heterocycles often ameliorates or diminishes the
bioresponses, depending upon the type of substituent and posi-
tion of attachment [10].
In our previous paper [11], we described the synthesis and
in vitro antileishmanial activity of a series of 2-(5-nitrofuran-
2-yl)-5-substituted-1,3,4-thiadiazoles (1), and 2-(5-nitro-2-
thienyl)-5-substituted-1,3,4-thiadiazoles (2), which several of
them showed promising antileishmanial properties. In view
of the biological importance of nitroimidazoles [12–14], it
was of our interest to prepare 2-(1-methyl-5-nitro-1H-
The World Health Organization (WHO) has identified
leishmaniasis as major and increasing public health problem
[1]. In spite of the socioeconomic importance of this tropical
infectious, efforts directed towards the discovery of new drugs
and/or vaccines against it are underdeveloped [2,3]. Drugs
currently in use as the antimony derivative glucantine, the
bis-amidines, pentamidine and stilbamidine or the glycomac-
rolide amphotericin B, display high liver and heart toxicities,
develop clinical resistance after a few weeks of treatment and
currently contribute to increase co-infections leishmaniasis-
AIDS in some countries [4,5]. In addition, most of the drugs
currently in use are expensive and require long-term treat-
ment [6]. Given the prospect that antileishmanial vaccines
may not become available in the near future [7], the develop-
ment new effective, cheap, and safe drugs for the treatment
of leishmaniasis is there fore an urgent task.
* Corresponding author. Tel.: +98 21 6640 6757; fax: +98 21 6646 1178.
E-mail address: aforoumadi@yahoo.com (A. Foroumadi).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.07.002

A. Foroumadi et al. / European Journal of Medicinal Chemistry 40 (2005) 1346–1350
1347
imidazol-2-yl)-1,3,4-thiadiazoles (3), as potential new anti-
leishmanial agents.
pound with concentrated nitric acid at 70 °C afforded the cor-
responding 5-nithroimidazole (5). Oxidation of 5 by hydrogen
peroxide in trifluoroacetic acid leads to the sulfone (6) where
a nucleophilic substation by cyanide anion produces the cor-
responding carbonitrile (7) in good yield. Compound 8 was
obtained by treatment of 7 with thiosemicarbazide in an acid
catalyzed reaction followed by a ring closure process.
Diazotization of amine (8) in hydrochloric acid in the pres-
ence of copper powder [16] gave 2-chloro-5-(1-methyl-5-
nitro-1H-2-imidazolyl)-1,3,4-thiadiazole (9) [17]. Reaction
of compound 9 with piperidine or morpholine in refluxing
ethanol gave compounds 3a or 3b, respectively. Similarly,
the reaction of compound
9
with piperazine,
N-methylpiperazine and N-phenylpiperazine gave the corre-
sponding compounds 3c–e, respectively. Acetylation of the
piperazine analogue 3c with acetic anhydride gave acety-
lated compound 3f. The reaction of compound 3c with ben-
zoyl chloride yielded benzoylated analogue 3g in high yield
(Fig. 1).
2. Chemistry
The synthesis of 2-(1-methyl-5-nitro-1H-2-imidazolyl)-5-
substituted-1,3,4-thiadiazoles (3a–g) was achieved with a ver-
satile and efficient synthetic route outline in Fig. 1. The
required 2-amino-5-(1-methyl-5-nitro-1H-2-imidazolyl)-
1,3,4-thiadiazole (8) [15] was prepared according to the newly
synthetic route suitable for multigrams scale [13]. The reac-
tion of 1-methylimidazole (4) with n-butyllitium at –30 °C
and subsequent addition of dimethyl disulfide gave 1-methyl-
2-(methylthio)-1H-imidazole. Nitration of the latter com-
3. Pharmacology
The in vitro leishmanicidal activity of the compounds
(3a–g) on promastigotes of Leishmania major (ATCC J 774,
HB-197) was assessed by a previously described method [18].
Promastigotes (3 × 106) were cultured in medium 199 con-
taining 10% heat-inactivated fetal calf serum. Incubation and
growth of the parasite were carried out at 26 °C. Promastig-
Fig. 1. Synthetic pathway to compounds 3a–g.

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A. Foroumadi et al. / European Journal of Medicinal Chemistry 40 (2005) 1346–1350
otes were harvested on day 4 of the culture and used. The
culture of parasite was diluted with the fresh medium to a
final concentration of 5 × 106 parasites per ml. The com-
pounds to be checked were dissolved in DMSO (15 mM) and
further diluted to appropriate concentrations. In a 96-well
microtiter plates, 160 µl of the promastigotes suspension was
added to 40 µl of various concentrations of each compound,
medium alone, or pentavalent antimonial Pentostam as posi-
tive control. After 2 h, 1 µCi of 3H-thymidine was added to
with respect to the Pentostam (IC50 = 243.3 µM). The overall
activity profile of compounds 3a–g demonstrated that there
is a small difference in their IC50 values. Thus, the biological
activity was slightly influenced by the type of cyclic amine
attachment at the 5-position of the 1,3,4-thiadiazole nucleus
and a structure–activity relationship study was not crucial.
However, it is notable to observe that unsubstituted pipera-
zine derivative (3c) (IC50 = 0.19 0.08 µM) proved to be sta-
tistically the most effective in this series (P values < 0.05),
followed by piperidine and morpholine derivatives (3a and
3b) with IC50 values of 0.42–0.44 µM, and this level of activ-
ity could be maintained with a N-benzoylated piperazine
group (compound 3g). Comparison of the IC50 values of 3c
with N4-substituted piperazine derivatives (3d–g) reveals that
certain substituents (such as, methyl, phenyl, acetyl and ben-
zoyl) at N4-position of piperazine ring are permitted and well-
tolerated but, not improved the leishmanicidal activity.
3
each well. The cultures were incubated for 18 h and H-
thymidine incorporation was measured. To ensure that the
solvent had no effect on parasite growth a control test was
performed with test medium and DMSO at the same dilu-
tions as used in the experiment. The results are from three
experiments and are given as IC50 (mean S.D.) as mea-
sured by 3H-thymidine incorporation (Table 1).
From our biological results, it is evident that
nitroimidazolyl-1,3,4-thiadiazoles (3) exhibited very potent
antileishmanial activity. It is worthwhile observing that, in
comparison with nitrofuran derivatives (1), previously
described by us [11], both of them have a better antileishma-
nial activity than nitrothiophene derivatives (2). However, the
nitroimidazoles (3a–d) were evidently more active than the
corresponding nitrofuran analogs [11]. These results brought
to our attention that this pharmacomodulation (replacement
of furan with imidazole) in many cases exerted a positive
effect.
4. Results and discussion
The in vitro leishmanicidal activity of the synthesized com-
pounds was determined by measuring the 3H-thymidine incor-
poration in promastigotes of L. major, as reported in Table 1.
The IC50 values of the test derivatives indicate that all com-
pounds exhibit high activity against L. major (IC50 ≤ 1.77 µM)
Table 1
Effect of compounds 3a–g on the growth of L. major promastigotes
The antileishmanial activity of these new nitroimidazolyl-
1,3,4-thiadiazole derivatives (3) may be due to the reduction
potential of the single-electron transfer ArNO2/ArNO2•–.
Nitroheterocylic drugs (e.g. nifurtimox, metronidazole, ben-
znidazole and megazole) are generally believed to exert their
cytotoxic effects only after activation by single-electron reduc-
tion of their corresponding nitro anion radicals [19–21]. Under
anaerobic conditions, the radical anion can be transformed
into the corresponding nitroso derivative. This nitroso form
has been put forward as an efficient scavenger of essential
thiols in the cell. 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 surrounding cell, and result in a cytotoxic action [22–25].
Thus, it seems that the antileishmanial activity of the new
nitroimidazolyl-1,3,4-thiadiazole derivatives (3) (structur-
ally related to megazole and other nitroimidazoles) are asso-
ciated with their interference with oxygen metabolism as well
as their role as thiol scavenger.
Compound
R
IC50 (µM)a
0.44 0.14
3a
3b
0.42 0.11
0.19 0.08
1.38 0.27
1.63 0.14
1.77 0.32
0.48 0.09
243.3 3.8
3c
3d
3e
3f
In conclusion, the high in vitro leishmanicidal activity of
compounds 3 makes these compounds a promising lead for
the development of an effective therapeutic agent. However,
this study will be completed by additional tests in
amastigote/macrophage in vitro models or in vivo mouse mod-
els.
3g
Pentostam
a Values are expressed as mean S.D. for three independent experiments.

A. Foroumadi et al. / European Journal of Medicinal Chemistry 40 (2005) 1346–1350
1349
5. Experimental
5.1.5. 1-[5-(1-Methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thia-
diazol-2-yl]-4-phenylpiperazine (3e)
This compound was prepared by the same method used
5.1. Chemistry
for 3a from the reaction of compound
9 with
N-phenylpiperazine. The reaction time was 2 h and the result-
ing product was purified using silica gel column chromatog-
raphy eluting with CH2Cl2 containing 2% ethanol.Yield 88%.
M.p. 257–259 °C (EtOH). IR (KBr) mmax: 3135 (H-C4 imida-
Melting points were determined on a Kofler hot stage appa-
ratus and are uncorrected. The IR spectra were obtained on a
Shimadzu 470 spectrophotometer (potassium bromide disks).
1H-NMR spectra were recorded on a Varian unity 400 spec-
trometer and chemical shifts are reported in parts per million
(d) relative to tetramethylsilane (TMS) as an internal stan-
dard. Elemental analyses were carried out on a CHN-O rapid
elemental 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 analytical TLC; col-
umn chromatography was performed on Merck silica gel (70–
230 mesh).
1
zole), 1487 and 1360 cm−1 (NO2). H-NMR (CDCl3) d:
8.04(s, 1H, H4-imidazole), 7.32–6.90 (m, 5H, phenyl), 4.50
(s, 3H, CH3), 3.87 (m, 4H, piperazine), and 3.35 ppm (m, 4H,
piperazine).
5.1.6. 4-Acetyl-1-[5-(1-methyl-5-nitro-1H-imidazol-2-yl)-
1,3,4-thiadiazol-2-yl]piperazine (3f)
A mixture of compound 3c (885 mg, 3.0 mmol), acetic
acid (5 ml) and acetic anhydride (0.5 ml) was refluxed for
20 min. After cooling, the reaction mixture was added to a
mixture of ice and water and the precipitated product was
filtered and washed with water and crystallized from ethanol
5.1.1. 1-[5-(1-Methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thia-
diazol-2-yl]piperidine (3a)
A mixture of compound 9 [17] (491 mg, 2 mmol) and pip-
eridine (340 mg, 2 mmol) in ethanol (5 ml) was refluxed for
30 min. After cooling, water was added and the precipitated
solid was washed with water and crystallized from
ethanol/water to give 3a (81%). M.p. 220–222 °C. IR (KBr)
mmax: 3135 (CH-imidazole), 1520 and 1363 cm−1 (NO2).
1H-NMR (CDCl3) d: 8.03 (s, 1H, H4-imidazole), 4.48 (s, 3H,
CH3), 3.62 (brs, 4H, CH2NCH2), 1.74-1.71 ppm (m, 6H, pip-
eridine).
to give 890 mg (88%) of 3f. M.p. 269–270 °C. IR (KBr) mmax
:
3120 (H-C4 imidazole), 1645 (C=O), 1525 and 1355 cm−1
(NO2). 1H-NMR (CDCl3) d: 8.03 (s, 1H, H4-imidazole), 4.49
(s, 3H, NCH3), 3.72–3.67 (m, 8H, piperazine) and 2.16 ppm
(s, 3H, CH3CO).
5.1.7. 4-Benzoyl-1-[5-(1-methyl-5-nitro-1H-imidazol-2-yl)-
1,3,4-thiadiazol-2-yl]piperazine (3g)
To a mixture of compound 3c (590 mg, 2.0 mmol), in dry
benzene (5 ml) and pyridine (1 ml) was added benzoyl chlo-
ride (281 mg, 2.0 mmol) and the mixture was stirred at room
temperature overnight. The solvents were removed under
reduced pressure and the resulting solid was washed with
water and crystallized from ethanol to give 638 mg (80%) of
3g. M.p. 225–227 °C. IR (KBr) mmax: 3140 (H-C4 imidazole),
1640 (C=O), 1520 and 1356 cm−1 (NO2). 1H-NMR (CDCl3)
d: 8.02 (s, 1H, H4-imidazole), 7.45 (brs, 5H, phenyl), 4.49 (s,
3H, CH3) and 3.75–3.71 ppm (m, 8H, piperazine).
5.1.2. 4-[5-(1-Methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thia-
diazol-2-yl]morpholine (3b)
This compound was prepared by the same method used
for 3a in 80% yield (the reaction time was 1.5 h). M.p. 231–
232 °C (EtOH/H2O). IR (KBr) mmax : 3152 (H-C4 imidazole),
1
1510 and 1356 cm−1 (NO2). H-NMR (CDCl3) d: 8.03 (s,
1H, H4-imidazole), 4.49 (s, 3H, CH3), 3.82-3.79 (m, 4H,
CH2OCH2) and 3.70–3.66 ppm (m, 4H, CH2NCH2).
5.1.3. 1-[5-(1-Methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thia-
diazol-2-yl]piperazine (3c)
Acknowledgments
Compound 3c was obtained by the same method used for
3a in 85% yield. M.p. 238–240 °C. IR (KBr) mmax: 3140 (H-C4
imidazole) 1520 and 1340 cm−1 (NO2). 1H-NMR (CDCl3) d:
8.05 (s, 1H, H4-imidazole), 4.50 (s, 3H, NCH3), 3.62–3.58
(m, 4H, piperazine), 2.97–2.93 ppm (m, 4H, piperazine).
This work was supported by TheAcademy of Sciences for
the Developing world (TWAS).
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