Synthesis and antileishmanial activity of novel 5-(5-nitrofuran-2-y1)-1,3, 4-thiadiazoles with piperazinyl-linked benzamidine substituents

Article abstract of DOI:10.1016/j.ejmech.2011.03.053

In order to optimize the antileishmanial activity of piperazinyl-linked 5-(5-nitrofuran-2-yl)-1,3,4-thiadiazoles, we synthesized a series of 5-(5-nitrofuran-2-y1)-1,3,4-thiadiazoles with piperazinyl-linked benzamidine substituent as scaffold found in pentamidine related antiprotozoals. The structure of target compounds was confirmed by IR, ¹H NMR, ¹³C NMR and Mass spectral data. All compounds were tested for in vitro activity against the promastigote and amastigote forms of Leishmania major. From the results, we found that the substitution on amidine nitrogen has profound role in the biological activity of these compounds. The 5-nitrofuran-2-yl-1,3,4-thiadiazoles having n-propyl, n-butyl and benzyl side chain on benzamidine (as in compounds 2d, 2e and 2g, respectively) showed very good activity in both forms of promastigote and amastigote. The most active compound was N-propyl-4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl) piperazin-1-yl) benzamidine hydrochloride (2d) with IC₅₀ value of 0.08 μM in promastigote model. This compound showed a very low level of toxicity against macrophages (CC₅₀ = 785 μM), with the highest selectivity index (SI = 78.5) among the tested compounds.

Full text of DOI:10.1016/j.ejmech.2011.03.053

European Journal of Medicinal Chemistry 46 (2011) 2602e2608
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antileishmanial activity of novel 5-(5-nitrofuran-2-y1)-1,3,
4-thiadiazoles with piperazinyl-linked benzamidine substituents
Azar Tahghighi a, Farzane Rezazade Marznaki b, Farzad Kobarfard c, Siavoush Dastmalchi a,
Javid Shahbazi Mojarrad a, Sepideh Razmi b, Sussan Kabudanian Ardestani b, Saeed Emami d,
,
Abbas Shafiee e, Alireza Foroumadi e
*
a Department of Medicinal Chemistry, School of Pharmacy and Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
b Institute of Biochemistry and Biophysics, Department of Biochemistry, University of Tehran, Tehran, Iran
c Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
d Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
e Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14174, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to optimize the antileishmanial activity of piperazinyl-linked 5-(5-nitrofuran-2-yl)-1,3,4-thia-
diazoles, we synthesized a series of 5-(5-nitrofuran-2-y1)-1,3,4-thiadiazoles with piperazinyl-linked
benzamidine substituent as scaffold found in pentamidine related antiprotozoals. The structure of target
compounds was confirmed by IR, 1H NMR, 13C NMR and Mass spectral data. All compounds were tested
for in vitro activity against the promastigote and amastigote forms of Leishmania major. From the results,
we found that the substitution on amidine nitrogen has profound role in the biological activity of these
compounds. The 5-nitrofuran-2-yl-1,3,4-thiadiazoles having n-propyl, n-butyl and benzyl side chain on
benzamidine (as in compounds 2d, 2e and 2g, respectively) showed very good activity in both forms of
promastigote and amastigote. The most active compound was N-propyl-4-(4-(5-(5-nitrofuran-2-yl)-
Received 1 December 2010
Received in revised form
16 March 2011
Accepted 24 March 2011
Available online 31 March 2011
Keywords:
In vitro antileishmanial activity
Nitrofuran, 1,3,4-thiadiazole
Benzamidine
1,3,4-thiadiazol-2-yl)piperazin-1-yl) benzamidine hydrochloride (2d) with IC50 value of 0.08
promastigote model. This compound showed very low level of toxicity against macrophages
(CC50 ¼ 785
mM in
a
m
M), with the highest selectivity index (SI ¼ 78.5) among the tested compounds.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
leishmaniasis AIDS, in some countries, have worried the authorities
[5,6]. In addition, most of the drugs currently in use are expensive
and require long-term treatment [7]. Thus, the development of
new, cheap, efficient, and safe drugs for the treatment of this
disease is imperative.
Leishmaniasis is caused by protozoan parasites belonging to the
genus Leishmania. Official data show that there are 12 million
infected people around the world, 350 million at risk of acquiring
the disease, and 1.5e2 million that will be infected annually [1].
Leishmaniasis showed a complex and diverse clinical manifesta-
tions and epidemiology [2]. In spite of the socioeconomic impor-
tance of this tropical infection, efforts directed toward the
discovery of new drugs and/or vaccines against it are underdevel-
oped [3,4]. Drugs currently in use as the antimony derivative glu-
cantine, the bis-amidines, stilbamidine or the glycomacrolide
amphotericin B are quite toxic and cause severe side effects such as
pancreatitis and cardiac toxicity. In addition, the development of
the clinical resistance and the increase of co-infections
Aromatic diamidine compounds based on pentamidine (Fig. 1)
have shown excellent activity against Leishmania parasites. The
antileishmanial activity of several such diamidines was reported in
1990 [8]. Since that time, many new aromatic diamidines have been
synthesized and improved assay systems have been developed for
the in vitro testing of candidate drugs as antiprotozoal agents. For
example, diminazene (Fig. 1), is an aromatic diamidine anti-
protozoal related to pentamidine that has been used in veterinary
practice in the treatment of trypanosomiasis and babesiosis.
Furthermore, imidocarb bearing cyclic diamidine (imidazoline) has
antiprotozoal and antibacterial activity [9e11]. On the other hand,
the antiparasitic property of 5-(5-nitrofuran-2-yl)-1,3,4-thiadia-
zoles is well established and their linkage with other heterocycles
often modulates their biological activities, depending upon the
* Corresponding author. Tel.: þ98 21 66954708; fax: þ98 21 66461178.
E-mail address: aforoumadi@yahoo.com (A. Foroumadi).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.053

A. Tahghighi et al. / European Journal of Medicinal Chemistry 46 (2011) 2602e2608
2603
NH2
NH
NH
NH
NH2
HN
NH2
H2N
N
O
O
N
H
N
Pentamidine
Diminazene
O
N N
NH
NH
N
X
N
S
O
O2N
N
H
HN
1
N
Imidocarb
X= CH2, O, NH, N-R
.HCl
NH
N N
S
N
N
O2N
O
NR1R2
2a-j
a: R1 = H, R2 = H
f: R1 = H, R2 = sec-C4H9
g: R1 = H, R2 = PhCH2
b: R1 = H, R2 = CH3
c: R1 = H, R2 = C2H5
d: R1 = H, R2 = n-C3H7
e: R1 = H, R2 = n-C4H9
h: R1 = H, R2 = 4-MeOPhCH2
i: R1 = H, R2 = (Tetrahydrofuran-2-yl)methyl
j: NR1R2 = Morpholine
N N
N
.HCl
n
N
N
O2N
S
O
N
H
2k-l
k: n = 1
l: n = 2
Fig. 1. Structures of diamidine antiprotozoal agents (pentamidine, diminazene and imidocarb), 2-(5-nitrofuran-2-yl)-5-substituted-1,3,4-thiadiazoles (1) and designed compounds
(2ael).
type of substituent and position of attachment [12e15]. In our
previous papers [16e18], 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), which several of them showed
promising antileishmanial properties (Fig. 1). In view of the bio-
logical importance of pentamidine analogs [19e21], it was of our
interest to combine the structural features of 2-(5-nitrofuran-2-yl)-
5-substituted-1,3,4-thiadiazoles (1) with the amidines, we
synthesized and evaluated twelve analogs of 1-[5-(5-nitrofuran-2-
y1)-1,3,4-thiadiazol-2-y1] piperazineelinked benzamidines 2aej
and related derivatives 2k,l (Fig. 1) as potential new antileishmanial
agents.
dichloromethane with ethanol and gaseous hydrochloric acid
afforded the intermediate benzeneimidate 8 which was subse-
quently reacted with ammonia or appropriate amines to afford
benzamidines 2aej. Reaction of diamine (ethylenediamine or pro-
pylenediamine) with imidate 8 enabled to prepare compounds 2k,l
bearing an imidazoline or a tetrahydropyrimidine system as cyclic
amidine groups (Scheme 1).
3. Pharmacology
3.1. Parasite and culture
The strain of Leishmania major used in this study was the vaccine
strain (MRHO/IR/75/ER), obtained from Pasteur Institute, Tehran
(Iran) [24]. 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 ꢀC. The stationary
parasite inoculation was 2 ꢁ 106 cells/mL. For the experiments
described here, the stationary phase of promastigotes 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 glutamine (Sigma), pH
2. Chemistry
The pathway for the synthesis of compounds 2ael is shown in
Scheme 1. 4-(Piperazin-1-yl)benzonitrile (4) was obtained from the
reaction of excess piperazine with 4-fluorobenzonitrile (3). The
intermediate 2-chloro-1,3,4-thiadiazole 6 was obtained from 5-
nitrofurfurylidine diacetate according to the previously described
method [16]. The reaction of chloro-thiadiazole 6 with piperazine 4
in refluxing ethylmethylketone gave compound 7. Conversion of
the nitrile 7 into the amidines 2ael was effected by the Pinner
reaction [22,23]. Treatment of a solution of compound 7 in
w7.2, 100 U/mL penicillin (Sigma) and 100
mg/mL streptomycin
(Sigma).

2604
A. Tahghighi et al. / European Journal of Medicinal Chemistry 46 (2011) 2602e2608
F
CN
CH(OCOCH3)2
O2N
O
5
3
b, c, d
a
N N
S
HN
N
CN
Cl
O2N
O
6
4
e
N N
S
N
N
CN
O2N
O
7
f
N N
NH .HCl
OEt
N
N
O2N
S
O
8
g
h
N N
N N
N
N
S
S
N
NH
N
O
O
O2N
O2N
N
NR1R2
.HCl
n
HN
.HCl
2a-j
2k-l
Scheme 1. Synthesis of compounds 2ael. Reagents and conditions: (a) piperazine, ethylmethylketone, K2CO3 reflux; (b) thiosemicarbazide, EtOH, reflux; (c) NH4Fe(SO4)2$12H2O,
H2O, reflux; (d) NaNO2, HCl, Cu, 0 ꢀC / r.t, (e) ethylmethylketone, K2CO3; (f) CH2Cl2, EtOH, gaseous HCl, r.t; (g) appropriate amine, EtOH, reflux; (h) appropriate diamine, EtOH,
reflux.
3.2. Antileishmanial activity against promastigotes form of L. major
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 ꢂ SD. Control cells were incubated with culture
medium plus DMSO.
The antileishmanial screening of compounds 2ael, 7 and 8 was
performed using direct counting and MTT assay [25]. 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 Neu-
bauer’s chamber. Then, parasites (2 ꢁ 106/mL) in the logarithmic
phase were incubated with a serial range of drug concentrations for
24 h, 48 h and 72 h at 25 ꢀC. 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 dilu-
tion of drug and phenol red free RPMI 1640 medium, supplemented
with 10% of FBS, 2 mM glutamine, pH w7.2 and antibiotics, in
3.3. Antileishmanial activity against amastigotes form of L. major
Mouse peritoneal macrophages were plated in RPMI 1640 sup-
plemented with 10% of heat-inactivated fetal bovine serum, 2 mM
glutamine, 100 U/mL penicillin (Sigma) and 100
mg/mL strepto-
mycin. Macrophages were placed on sterile glass cover slips in 24-
well plates (1 ꢁ 106/well). After 1 h, non-adherent cells were
removed by washing with RPMI, the stationary phase promasti-
gotes in RPMI were added (2 ꢁ 106 parasites/well, three parasites/
macrophage) to macrophage monolayer and the plates were kept at
37 ꢀC in a CO2 incubator for 2 h. Extracellular parasites were
a volume of 200 m
L. After 24 h, 48 h or 72 h of incubation at 25 ꢀC,
the media was renewed with 100 mg/well of MTT (0.5 mg/mL) and
plates were further incubated for 4 h at 37 ꢀC. The plates were
centrifuged (2000 rpm ꢁ 5 min), the pellets were dissolved in

A. Tahghighi et al. / European Journal of Medicinal Chemistry 46 (2011) 2602e2608
2605
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 (100 cells were examined/well) [26].
introduction of an alkyl group on a nitrogen atom of the amidine
function. Indeed, longer alkyl chains such as n-propyl and n-butyl
(compounds 2d and 2e) increased the anti-promastigote activity,
whereas a smaller alkyl group such as methyl or ethyl produced
moderate activity. The benzyl derivative (compound 2g) with IC50
value of 0.4 ꢂ 0.2
m
M has better activity respect to the 4-methox-
ybenzyl derivative 2h (IC50 ¼ 2 ꢂ 0.2
mM). It should be noted that
compounds 2k and 2l which substituted by a five-membered ring,
namely the imidazoline and six-membered ring, namely tetrahy-
dropyrimidine were devoid of activity (IC50 values >200 mM). The
3.4. Toxicity against macrophages
lack of activity may be due to the steric hindrance around the
nitrogen atoms or to the absence of ]NH function.
The toxicity of compounds 7 and 2aej was assessed against
mouse peritoneal macrophages plated in 96-well plates at
2 ꢁ 105 cells/well. After cell adherence, the medium was removed
and replaced by the media containing different concentrations of
each compounds. The plates were incubated for 24 h at 37 ꢀC in
a humidified incubator with 5% CO2. Cell viability was determined
by MTT colorimetric assay [27]. Two independent experiments in
triplicate were performed for determination of toxicity of each
compound, the CC50 (cytotoxic concentration for 50% inhibition)
were calculated by linear regression analysis.
Some of the compounds were also evaluated for their activity
against the amastigote form of L. major in peritoneal macrophages
(Fig. 2) [25,26]. As can be deducted from Fig. 2, all tested
compounds have significantly decreased the number of amasti-
gotes per macrophage and both the percentage of macrophage
infectivity and infectivity index. It is notable that non-amidine
compounds 7 and 8, N-methylamidine 2b, sec-butyl analog 2f
which showed moderate activity against promastigotes being
relatively effective compounds against amastigotes as showed in
the terms of amastigote number per macrophage, the percentage of
macrophage infectivity and infectivity index.
4. Result and discussion
The in vitro cytotoxic activity of compounds 7 and 2aej against
mouse peritoneal macrophages was also determined using
MTT assay (Table 1). The CC50 values for tested compounds
demonstrated that, most of them had low toxicity for macrophages
In the present study, we describe the evaluation of anti-
leishmanial activity of target compounds 2ael, 7 and 8 on both
developmental forms of Leishmania consisting promastigotes
(extracellular parasites) and amastigotes (that live in macrophages
of mammalian hosts). The test compounds were evaluated for anti-
promastigote activity along with meglumine antimonate
(CC50 > 100
showed very low level of toxicity against macrophages
(CC50 ¼ 785 M) with the highest selectivity index (SI ¼ 78.5).
mM). The most active antileishmanial compound 2d,
m
(GlucantimeÒ), using MTT assay [25]. The IC50 values (in
mM)
5. Conclusion
against promastigotes after 24, 48 or 72 h incubation, in compar-
ison with Glucantime, are listed in Table 1.
In our earlier studies we have shown that 2-(5-nitrofuran-2-yl)-
5-substituted-1,3,4-thiadiazoles showed pronounced in vitro anti-
leishmanial profile in the terms of activity against promastigote
and amastigote forms of L. major [16e18]. In view to further
optimization of the activity profile, we synthesized a series of 5-(5-
nitrofuran-2-y1)-1,3,4-thiadiazoles with piperazinyl-linked ben-
zamidine substituent as scaffold fond in pentamidine related
antiprotozoals. From the results we found that the substitution on
amidine nitrogen has profound role in the biological activity of
these compounds. The 5-nitrofuran-2-yl-1,3,4-thiadiazoles having
n-propyl, n-butyl and benzyl side chain on benzamidine (as in
compounds 2d, 2e and 2g, respectively) showed very good activity
in promastigote as well as amastigote model. However, benzonitrile
substitution as in compound 7 and O-ethyl benzimidate substitu-
tion as in compound 8 showed lower activity in promastigote
model but was found effective in the amastigote model. In
summary, we have identified N-substituted benzamidine deriva-
tives of 5-(5-nitrofuran-2-y1)-1,3,4-thiadiazoles as a promising
new hit for the antileishmanial chemotherapy.
The most potent compounds against the promastigote form of
L. major were found to be compound 2d followed by compounds 2e,
2g and 2h with IC50 values of 0.08 ꢂ 0.01, 0.2 ꢂ 0.1, 0.4 ꢂ 0.2 and
2 ꢂ 0.2
mM recorded at 72 h, respectively. The remaining
compounds showed IC50 values between 4 and 32 mM after 72 h
incubation. These results indicate that the propyl, butyl and benzyl
substitutions on the amidine residue improve the activity against
promastigotes. The parent amidine 2a, exhibits low inhibition and
it appears that the inhibitory activity can be modulated by the
Table 1
In vitro antileishmanial activity of compounds 2ael, 7 and 8 against promastigote
form of L. major.
Compound
Anti-promastigote activity IC50
24 h 48 h 72 h
(m
M) Cytotoxicitya Selectivity
CC50
(
m
M)
index
(SI) CC50
/
IC50ꢃ24 h
7
8
104 ꢂ 0.7 86 ꢂ 1.3
48 ꢂ 0.42 46 ꢂ 0.7
32 ꢂ 0.45
26 ꢂ 0.31
159
e
1.53
e
2a
2b
2c
2d
2e
2f
2g
2h
2i
111 ꢂ 0.8 111 ꢂ 1.4 26 ꢂ 0.3
342
223
337
3.08
9.69
10.21
78.50
11.18
23.33
60.77
0.86
9.69
0.59
e
23 ꢂ 0.25 22 ꢂ 0.5
33 ꢂ 0.62 26 ꢂ 0.4
10 ꢂ 0.65 5 ꢂ 0.5
21 ꢂ 0.14
16 ꢂ 0.7
6. Experimental protocols
0.08 ꢂ 0.01 785
6.1. Chemistry
11 ꢂ 1.4
4 ꢂ 1.4
0.2 ꢂ 0.1
13 ꢂ 0.21
123
770
546
69
921
55
33 ꢂ 0.21 18 ꢂ 1.5
9 ꢂ 1.8
80 ꢂ 1.6
95 ꢂ 0.8
93 ꢂ 0.9
>200
1.9 ꢂ 0.7 0.4 ꢂ 0.2
Chemical reagents and all solvents used in this study were
purchased from Merck AG Chemical. The key intermediate 2-
chloro-5-(5-nitrofuran-2-yl)-1,3,4-thiadiazole (6) was prepared
according to the literature method [16e18]. Melting points were
determined on a Kofler hot stage apparatus and are uncorrected.
The IR spectra were obtained on a Shimadzu 470 spectrophotom-
eter (potassium bromide dicks). 1H NMR spectra were recorded on
76 ꢂ 1.2
2 ꢂ 0.2
88 ꢂ 0.14 5 ꢂ 0.3
88 ꢂ 1.02 4 ꢂ 0.2
2j
2k
2l
e
e
e
e
e
>200
e
e
Glucantimeb 35 ꢂ 0.2
30 ꢂ 0.18 30 ꢂ 0.167
e
e
a
Cytotoxicity was evaluated against mouse peritoneal macrophages.
The IC50 of Glucantime was in mg/mL.
b
a Varian unity 500 spectrometer and chemical shifts (d) are

2606
A. Tahghighi et al. / European Journal of Medicinal Chemistry 46 (2011) 2602e2608
6.1.1. 4-(Piperazin-1-yl)benzonitrile (4)
A mixture of 4-fluorobenzonitrile (3, 0.36 g, 3 mmol), piperazine
(0.64 g, 7.4 mmol) and K2CO3 (0.85 g, 6.1 mmol) in ethyl-
methylketone (50 mL) was refluxed for 4 days. The completion of
reaction was detected by TLC. The solvent was evaporated under
reduced pressure and then chloroform and water was added. The
organic layer was extracted. After evaporation of chloroform, the
residue was crystallized from chloroform to give compound 4 as
a cream solid. Yield: 95%; m.p 82e85 ꢀC; IR (KBr, cmꢃ1): 3325, 2215,
1606. 1H NMR (500 MHz, CDCl3): 7.50 (d, 2H, J ¼ 9.2 Hz, phenyl),
6.86 (d, 2H, J ¼ 9.2 Hz, phenyl), 3.29 (t, 4H, J ¼ 5.2 Hz, piperazine),
3.02 (t, 4H, J ¼ 5.2 Hz, piperazine). MS (ESI): 188 [M þ Hþ]. Anal.
Calcd for C11H13N3: C, 70.56; H, 7.0; N, 22.44. Found: C, 70.35; H,
6.82; N, 22.73.
6.1.2. 4-(4-(5-(5-Nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl)piperazin-
1-yl)benzonitrile (7)
A mixture of compound 4 (0.13 g, 0.7 mmol), compound 6
(0.16 g, 0.7 mmol) and K2CO3 (0.145 g, 1.05 mmol) in ethyl-
methylketone (30 mL) was refluxed for 8 h. After completion of the
reaction, the solvent was removed under reduced pressure to
obtain a yellow solid (yield 90%). The crude product was purified by
column chromatography (silica gel, chloroform). Yield: 78%;
m.p > 300 ꢀC; IR (KBr, cmꢃ1): 2217, 1608, 1355, 1254. 1H NMR
(500 MHz, CDCl3): 7.50 (d, 2H, J ¼ 8.4 Hz, phenyl), 7.44 (d, 1H,
J ¼ 4 Hz, furan), 7.19 (d, 1H, J ¼ 4 Hz, furan), 6.92 (d, 2H, J ¼ 8.4 Hz,
phenyl), 3.82 (m, 4H, piperazine), 3.54 (m, 4H, piperazine). MS
(ESI): 382.9 [M þ Hþ]. Anal. Calcd for C17H14N6O3S: C, 53.40; H,
3.69; N, 21.98. Found: C, 53.54; H, 3.82; N, 21.76.
6.1.3. O-Ethyl-4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl)
piperazin-1-yl)benzimidate hydrochloride (8)
A mixture of compound 7 (0.3 g, 0.78 mmol) in dry dichloro-
methane (90 mL) and dry ethanol (3.5 mL) was saturated with HCl
gas and the reaction mixture was left at room temperature for 5
days. The precipitate 8 was filtered and washed with acetone. Yield:
75%; m.p > 300 ꢀC; IR (KBr, cmꢃ1): 3448, 1601, 1357, 1242; 1H NMR
(500 MHz, DMSO-d6): 7.85 (d, 2H, J ¼ 9 Hz, phenyl), 7.87 (d, 1H,
J ¼ 4 Hz, furan), 7.41 (d, 1H, J ¼ 4 Hz, furan), 7.10 (d, 2H, J ¼ 9 Hz,
phenyl), 4.48 (q, 2H, J ¼ 7 Hz, eCH2e), 3.67 (m, 8H, piperazine), 1.43
(t, 3H, J ¼ 7 Hz, methyl). 13C NMR (62.5 MHz, DMSO-d6)
d 13.7
(eCH3), 45.3 (C-8, piperazine), 46.4 (eCH2e), 48.7 (C-7, piperazine),
112.1 (C-9, phenyl), 113.3 (C-10, phenyl), 114 (C-3, furan), 115.1 (C-2,
furan), 130.4 (C-11, phenyl), 145.5 (C-12, phenyl), 147.2 (C-4, furan),
151.5 (C-1, furan),153.8 (C-6, thiadiazole),168.6 (eC]N),172.3 (C-5,
thiadiazole). MS (ESI): 429 [M þ Hþ]. Anal. Calcd for C19H21ClN6O4S:
C, 49.08; H, 4.55; N, 18.08. Found: C, 48.93; H, 4.84; N, 17.77.
6.1.4. Synthesis of compounds 2ael
6.1.4.1. 4-(4-(5-(5-Nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl)piperazin-
1-yl)benzamidine hydrochloride (2a) [28]. This compound was
prepared according to the literature method. The crude product
was washed with propanol and crystallized from meth-
anolediethylether. Yield: 35%; m.p > 300 ꢀC; IR (KBr, cmꢃ1): 3456,
3354, 1608, 1355, 1234; 1H NMR (500 MHz, DMSO-d6): 9.01 (br s,
2H), 8.64 (br s, 2H), 7.87 (d, 1H, J ¼ 4 Hz, furan), 7.78 (d, 2H,
J ¼ 9.2 Hz, phenyl), 7.41 (d, 1H, J ¼ 4 Hz, furan), 7.13 (d, 2H,
J ¼ 9.2 Hz, phenyl),3.74 (br s, 4H, piperazine), 3.64 (br s, 4H,
piperazine). MS (ESI): 400 [M þ Hþ]. Anal. Calcd for C17H18ClN7O3S:
C, 46.84; H, 4.16; N, 22.49. Found: C, 47.10; H, 3.97; N, 22.18.
Fig. 2. In vitro activity of selected compounds 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) Infec-
tivity 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 (100 cells were examined/well).
reported in parts per million (ppm) relative to tetramethylsilane
(TMS) as an internal standard. The mass spectra were run on an
Agilent 6410. Merck silica gel 60 F254 plates were used for
analytical TLC. Compound 2a [28] has been described in the
literature.
6.1.4.2. N-Methyl-4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl)
piperazin-1-yl)benzamidine hydrochloride (2b). The compound was
obtained by treatment of the crude imidate 8 (0.1 g, 0.21 mmol)
with methylamine 40% (0.31 mL, 4 mmol) in ethanol (5 mL) at

A. Tahghighi et al. / European Journal of Medicinal Chemistry 46 (2011) 2602e2608
2607
reflux for 90 min. The reaction mixture was concentrated under
reduced pressure and the residue was washed with acetone and
crystallized from ethanoledioxan .Yield: 30%; m.p > 300 ꢀC; IR
(KBr, cmꢃ1): 3487, 1610, 1351, 1242; 1H NMR (500 MHz, DMSO-d6):
9.14 (br s, 3H), 7.85 (d, 1H, J ¼ 3.8 Hz, furan), 7.71 (d, 2H, J ¼ 8.6 Hz,
phenyl), 7.39 (d, 1H, J ¼ 3.8 Hz, furan), 7.13 (d, 2H, J ¼ 8.6 Hz,
phenyl), 3.74 (br s, 4H, piperazine), 3.60 (br s, 4H, piperazine), 2.96
eCH2e), 0.94 (t, 3H, J ¼ 7.1 Hz, methyl). 13C NMR (62.5 MHz, DMSO-
d6)
d 13.6 (eCH3), 19.5 (eCH2e), 29.5 (eCH2e), 42.1 (eCH2e), 45.7
(C-8, piperazine), 48.8 (C-7, piperazine), 112.1 (C-9, phenyl), 113.8
(C-10, phenyl), 115.1 (C-3, furan), 116.9 (C-2, furan), 129.5 (C-11,
phenyl), 145.5 (C-12, phenyl), 147.2 (C-4, furan), 151.5 (C-1, furan),
153.4 (C-6, thiadiazole), 161.9 (eC]N), 172.4 (C-5, thiadiazole). MS
(ESI): 456 [M þ Hþ]. Anal. Calcd for C21H26ClN7O3S: C, 51.27; H,
5.33; N, 19.93. Found: C, 51.49; H, 5.22; N, 19.57.
(s, 3H, methyl). 13C NMR (62.5 MHz, DMSO-d6)
d 29.4 (eCH3), 45.7
(C-8, piperazine), 48.9 (C-7, piperazine), 112.1 (C-9, phenyl), 113.8
(C-10, phenyl), 115.1 (C-3, furan), 116.6 (C-2, furan), 129.3 (C-11,
phenyl), 145.5 (C-12, phenyl), 147.2 (C-4, furan), 151.5 (C-1, furan),
153.3 (C-6, thiadiazole), 162.3 (eC]N), 172.3 (C-5, thiadiazole). MS
(ESI): 414 [M þ Hþ]. Anal. Calcd for C18H20ClN7O3S: C, 48.05; H,
4.48; N, 21.79. Found: C, 47.86; H, 4.38; N, 22.13.
6.1.4.6. N-(2-Butyl)-4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-
yl)piperazin-1-yl)benzamidine hydrochloride (2f). This compound
was prepared by treatment of the crude imidate 8 with 2-butyl-
amine in ethanol at reflux for 6 h and resulting solid was crystal-
lized from ethanolediethylether and then from methanoledioxan.
Yield: 25%; m.p > 300 ꢀC; IR (KBr, cmꢃ1): 3433, 1606, 1353,1238; 1H
NMR (500 MHz, DMSO-d6): 7.89 (d, 1H, J ¼ 3.85 Hz, furan), 7.68 (d,
2H, J ¼ 8.4 Hz, phenyl), 7.43 (d, 1H, J ¼ 3.85 Hz, furan), 7.15 (d, 2H,
J ¼ 8.4 Hz, phenyl), 3.85 (m, 1H); 3.76 (br s, 4H, piperazine), 3.59 (br
s, 4H, piperazine), 1.65 (m, 1H, J ¼ 7.1 Hz, eCHe), 1.59 (m, 1H,
J ¼ 6.5 Hz, eCHe), 1.22 (d, 3H, J ¼ 6.25 Hz, methyl), 0.92 (t, 3H,
6.1.4.3. N-Ethyl-4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl)
piperazin-1-yl)benzamidine hydrochloride (2c). The compound was
obtained by treatment of the crude imidate 8 (0.1 g, 0.21 mmol)
with ethylamine 70% (0.17 mL, 3 mmol) in ethanol (5 mL) at reflux
for 60 min. The reaction mixture was concentrated under reduced
pressure and the residue was crystallized from meth-
anolediethylether. Yield: 25%; m.p > 300 ꢀC; IR (KBr, cmꢃ1): 3406,
1606, 1360, 1231; 1H NMR (500 MHz, DMSO-d6): 7.87 (d, 1H,
J ¼ 4 Hz, furan), 7.67 (d, 2H, J ¼ 8.8 Hz, phenyl), 7.41 (d, 1H, J ¼ 4 Hz,
furan), 7.07 (d, 2H, J ¼ 8.8 Hz, phenyl), 3.74 (t, 4H, piperazine), 3.53
(t, 4H, piperazine), 3.3 (q, 2H, J ¼ 7.2 Hz, CH2), 1.19 (t, 3H, J ¼ 7.2 Hz,
J ¼ 7.2, methyl). 13C NMR (62.5 MHz, DMSO-d6)
d 10.4 (eCH3), 19.3
(eCH2e), 28.2 (eCH2e), 30.9 (eCH2e), 45.9 (C-8, piperazine), 48.9
(C-7, piperazine), 50.2 (eCH2e), 112.1 (C-9, phenyl), 113.8 (C-10,
phenyl), 115.1 (C-3, furan), 116.9 (C-2, furan), 129.6 (C-11, phenyl),
145.5 (C-12, phenyl), 147.2 (C-4, furan), 151.5 (C-1, furan),153.3 (C-6,
thiadiazole), 161.3 (eC]N), 172.4 (C-5, thiadiazole). MS (ESI): 456
[M þ Hþ]. Anal. Calcd for C21H26ClN7O3S: C, 51.27; H, 5.33; N, 19.93.
Found: C, 51.56; H, 5.45; N, 19.74.
methyl). 13C NMR (62.5 MHz, DMSO-d6)
d 14.0 (eCH3), 37.7
(eCH2e), 46.2 (C-8, piperazine), 49 (C-7, piperazine), 112.1 (C-9,
phenyl), 114.1 (C-10, phenyl), 115.1 (C-3, furan), (C-3, furan), 116.8,
128.7 (C-11, phenyl), 145.5 (C-12, phenyl), 147.2 (C-4, furan), 151.5
(C-1, furan), 152.5 (C-6, thiadiazole), 160.2 (eC]N), 172.4 (C-5,
thiadiazole). MS (ESI): 428 [M þ Hþ]. Anal. Calcd for C19H22ClN7O3S:
C, 49.19; H, 4.78; N, 21.13. Found: C, 48.87; H, 4.92; N, 21.22.
6.1.4.7. N-Benzyl-4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl)
piperazin-1-yl)benzamidine hydrochloride (2g). This compound was
prepared by treatment of the crude imidate 8 with benzylamine in
ethanol at reflux for 3 h and resulting solid was washed with THF
and crystallized from methanolediethylether. Yield: 25%;
m.p > 300 ꢀC; IR (KBr, cmꢃ1): 3434, 1606, 1350, 1236; 1H NMR
(500 MHz, DMSO-d6): 7.88 (d, 1H, J ¼ 4 Hz, furan), 7.75 (d, 2H,
J ¼ 8 Hz, phenyl), 7.41 (m, 5H, phenyl), 7.34 (d, 1H, J ¼ 4 Hz, furan),
7.15 (d, 2H, J ¼ 8 Hz, phenyl), 4.7 (s, 2H, eCH2e), 3.75 (br s, 4H,
piperazine), 3.61 (br s, 4H, piperazine). MS (ESI): 490 [M þ Hþ].
Anal. Calcd for C24H24ClN7O3S: C, 54.80; H, 4.60; N, 18.64. Found: C,
54.47; H, 4.83; N, 18.83.
6.1.4.4. N-(1-Propyl)-4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-
yl)piperazin-1-yl) benzamidine hydrochloride (2d). The compound
was obtained by treatment of the crude imidate 8 (0.1 g, 0.21 mmol)
with propylamine (0.16 mL, 2 mmol) in ethanol (5 mL) at reflux for
60 min. The reaction mixture was concentrated under reduced
pressure and the residue was washed with diethylether and crys-
tallized from 2-propanoledioxan. Yield: 38%; m.p > 300 ꢀC; IR (KBr,
cmꢃ1): 3487,1607,1359,1238; 1H NMR (500 MHz, DMSO-d6): 9.39 (s,
1H); 9.11 (s,1H), 8.67 (s,1H), 7.87 (d,1H, J ¼ 3.9 Hz, furan), 7.68 (d, 2H,
J ¼ 8.7 Hz, phenyl), 7.41 (d,1H, J ¼ 3.9 Hz, furan), 7.15 (d, 2H, J ¼ 8.7 Hz,
phenyl), 3.76 (t, 4H, piperazine), 3.61 (t, 4H, piperazine),1.66 (m, 2H,
J ¼ 7.2 Hz, eCH2e), 0.96 (t, 3H, J ¼ 7.2 Hz, methyl). 13C NMR
6.1.4.8. N-(4-Methoxybenzyl)-4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thia-
diazol-2-yl)piperazin-1-yl)benzamidine hydrochloride (2h). This
compound was prepared by treatment of the crude imidate 8 with
p-methoxybenzylamine in ethanol at reflux for 60 min. The
resulting solid was washed with diethylether and acetone and
crystallized from ethanolediethylether. Yield: 40%; m.p > 300 ꢀC;
IR (KBr, cmꢃ1): 3440, 1608, 1351, 1237; 1H NMR (500 MHz, DMSO-
d6): 8.07 (br s, 3H), 7.88 (d, 1H, J ¼ 3.8 Hz, furan), 7.72 (d, 2H,
J ¼ 8.7 Hz, phenyl), 7.42 (d, 1H, J ¼ 3.8 Hz, furan), 7.36 (d, 2H,
J ¼ 8.35 Hz, phenyl), 7.15 (d, 2H, J ¼ 8.7 Hz, phenyl), 6.98 (d, 2H,
J ¼ 8.35), 3.94 (s, 2H, eCH2e), 3.76 (br s, 8H, piperazine), 3.62 (s, 3H,
methoxy). MS (ESI): 519.9 [M þ Hþ]. Anal. Calcd for C25H26ClN7O4S:
C, 54.00; H, 4.71; N, 17.63. Found: C, 54.19; H, 4.57; N, 17.92.
(62.5 MHz, DMSO-d6) d 11.2 (eCH3), 20.9 (eCH2e), 44 (eCH2e), 45.8
(C-8, piperazine), 48.9 (C-7, piperazine),112.1 (C-9, phenyl),113.8 (C-
10, phenyl),115.1 (C-3, furan),116.9 (C-2, furan),129.5 (C-11, phenyl),
145.5 (C-12, phenyl),147.2 (C-4, furan), 151.5 (C-1, furan), 153.4 (C-6,
thiadiazole), 161.9 (eC]N), 172.4 (C-5, thiadiazole). MS (ESI): 442
[M þ Hþ]. Anal. Calcd for C20H24ClN7O3S: C, 50.26; H, 5.06; N, 20.51.
Found: C, 49.95; H, 4.87; N, 20.78.
6.1.4.5. N-(1-Butyl)-4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-
yl)piperazin-1-yl)benzamidine hydrochloride (2e). This compound
was prepared by treatment of the crude imidate 8 with butylamine
in ethanol at reflux for 45 min and resulting solid was washed with
acetone and crystallized from methanolediethylether. Yield: 40%;
m.p > 300 ꢀC; IR (KBr, cmꢃ1): 3432, 1610, 1351, 1233; 1H NMR
(500 MHz, DMSO-d6): 9.39 (s, 1H), 9.12 (s, 1H); 8.68 (s, 1H), 7.89 (d,
1H, J ¼ 3.35 Hz, furan), 7.68 (d, 2H, J ¼ 8.4 Hz, phenyl), 7.42 (d, 1H,
J ¼ 3.35 Hz, furan), 7.15 (d, 2H, J ¼ 8.4 Hz, phenyl), 3.76 (br s, 4H,
piperazine), 3.61 (br s, 4H, piperazine), 3.57 (t, 2H, J ¼ 7.1 Hz,
eCH2e), 1.61 (m, 2H, J ¼ 7.1 Hz, eCH2e), 1.39 (m, 2H, J ¼ 7.1 Hz,
6.1.4.9. N-((Tetrahydrofuran-2-yl)methyl)-4-(4-(5-(5-nitrofuran-2-
yl)-1,3,4-thiadiazol-2-yl)piperazin-1-yl)benzamidine hydrochloride
(2i). This compound was prepared by treatment of the crude imi-
date 8 with 2-tetrahydrofurfurylamine in ethanol at reflux for
60 min. The resulting solid was washed with hot acetone, dieth-
ylether and chloroform. Yield: 25%; m.p > 300 ꢀC; IR (KBr, cmꢃ1):
3430, 1607, 1351, 1234; 1H NMR (500 MHz, DMSO-d6): 7.85 (d, 1H,
J ¼ 4 Hz, furan), 7.66 (d, 2H, J ¼ 8.8 Hz, phenyl), 7.39 (d, 1H, J ¼ 4 Hz,
furan), 7.14 (d, 2H, J ¼ 8.8 Hz, phenyl), 4.09 (m, 1H, OCHe), 3.8 (m,

2608
A. Tahghighi et al. / European Journal of Medicinal Chemistry 46 (2011) 2602e2608
1H, eCHe); 3.75 (m, 4H, piperazine), 3.68 (m, 1H, eCHe), 3.60 (m,
1H, eCHe), 3.47 (m, 5H, piperazine and eCHe), 2.02 (m, 1H,
eCHe), 1.87 (m, 2H, eCH2e); 1.59 (m, 1H, eCHe). MS (ESI): 484
[M þ Hþ]. Anal. Calcd for C22H26ClN7O4S: C, 50.81; H, 5.04; N, 18.86.
Found: C, 51.16; H, 5.13; N, 18.61.
Acknowledgment
This work was supported by grants from the Research Council of
Tehran University of Medical Sciences and Iran National Science
Foundation (INSF).
6.1.4.10. Morpholino(4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-
yl)piperazin-1-yl)phenyl)methanimine
hydrochloride
(2j). This
References
compound was prepared by treatment of the crude imidate 8 with
morpholine in ethanol at reflux for 60 min. The resulting solid was
washed with 2-propanol and acetone and crystallized from meth-
anol and dioxan. Yield: 35%; m.p > 300 ꢀC; IR (KBr, cmꢃ1): 3448,
1604, 1353, 1229; 1H NMR (500 MHz, DMSO-d6): 7.88 (d, 1H,
J ¼ 3.75 Hz, furan), 7.47 (d, 2H, J ¼ 8.5 Hz, phenyl), 7.42 (d, 1H,
J ¼ 3.75, furan), 7.16 (d, 2H, J ¼ 8.5 Hz, phenyl), 3.75 (br s, 8H,
piperazine and morpholine), 3.57 (br s, 8H, piperazine and mor-
pholine). MS (ESI): 469.9 [M þ Hþ]. Anal. Calcd for C21H24ClN7O4S:
C, 49.85; H, 4.78; N, 19.38. Found: C, 49.72; H, 4.57; N, 19.57.
[1] http://www.who.int/tdr/diseases/leish/diseaseinfo.htm.
[2] P. Desjeux, Comp. Immunol. Microbiol. Infect. Dis. 27 (2004) 305e318.
[3] S.L. Croft, Parasitology 114 (1997) S3eS15.
[4] H.D. Engers, R. Bergquist, F. Moddaber, Dev. Biol. Stand. 87 (1996) 73e84.
[5] M. Ouellette, J. Drummelsmith, B. Papadopoulou, Drug Resist. Update 7 (2004)
257e266.
[6] S.L. Croft, K. Seifert, V. Yardley, Indian J. Med. Res. 123 (2006) 399e410.
[7] H.W. Murray, Am. J. Trop. Med. Hyg. 71 (2004) 787e794.
[8] C. Bell, J. Hall, D. Kyle, M. Grogl, K. Ohemeng, M. Allen, R. Tidwell, Antimicrob.
Agents Chemother. 34 (1990) 1381e1386.
[9] S.C. Sweetman (Ed.), Martindale: The Complete Drug Reference, thirty fifth ed.
Pharmaceutical Press, London (UK), 2007.
[10] K. Werbovetz, Antimicrob. Agents Chemother. 52 (2008) 4098e4114.
[11] D.A. Patrick, S.A. Bakunov, S.M. Bakunova, E.V.K. Suresh Kumar, H. Chen,
S.K. Jones, T. Wenzler, T. Barzcz, K.A. Werbovetz, R. Brun, R.R. Tidwell, Eur. J.
Med. Chem. 44 (2009) 3543e3551.
[12] R.F. Rodrigues, E.F. da Silva, A. Echevarria, R. Fajardo-Bonin, V.F. Amaral,
L.L. Leon, M.M. Canto-Cavalheiro, Eur. J. Med. Chem. 42 (2007) 1039e1043.
[13] A. Khalaj, M. Nakhjiri, A.S. Negahbani, M. Samadizadeh, L. Firoozpour,
S. Rajabalian, N. Samadi, M.A. Faramarzi, N. Adibpour, A. Shafiee,
A. Foroumadi, Eur. J. Med. Chem. 46 (2011) 65e70.
[14] E. Alipour, S. Emami, A. Yahya-Meymandi, M. Nakhjiri, F. Johari, S.K. Ardestani,
F. Poorrajab, M. Hosseini, A. Shafiee, A. Foroumadi, J. Enzyme Inhib. Med.
Chem. 26 (2011) 123e128.
[15] M.H. Moshafi, A. Yahya-Meymandi, S.E. Sadat-Ebrahimi, S. Emami, M. Nakhjiri,
F. Siavoshi, M. Omrani, M. Vosooghi, E. Alipour, A. Shafiee, A. Foroumadi,
J. Serb, Chem. Soc. 76 (2011) 201e210.
[16] A. Foroumadi, S. Pournourmohammadi, F. Soltani, M.A. Rezaee, S. Dabiri,
A. Kharazmi, A. Shafiee, Bioorg. Med. Chem. Lett. 15 (2005) 1983e1985.
[17] F. Poorrajab, S.K. Ardestani, A. Foroumadi, S. Emami, A. Kariminia,
M. Behrouzi-Fardmoghadam, A. Shafiee, Exp. Parasitol. 121 (2009) 323e330.
[18] M. Behrouzi-Fardmoghadam, F. Poorrajab, S.K. Ardestani, S. Emami, A. Shafiee,
A. Foroumadi, Bioorg. Med. Chem. Lett. 16 (2008) 4509e4515.
[19] V.J. Ram, M. Nath, Curr. Med. Chem. 3 (1996) 303e316.
[20] S.L. Croft, V. Yardley, Curr. Pharm. Des. 8 (2002) 319e342.
[21] A. Mayence, J.J. Vanden Eynde, L. LeCour Jr., L.A. Walker, B.L. Tekwani,
T.L. Huang, Eur. J. Med. Chem. 39 (2004) 547e553.
[22] R.L. Shriner, F.W. Neumann, Chem. Rev. 35 (1944) 351e425.
[23] R. Roger, D.G. Neilson, Chem. Rev. 61 (1961) 179e211.
[24] Z. Nazarian, S. Emami, S. Heydari, S.K. Ardestani, M. Nakhjiri, F. Poorrajab,
A. Shafiee, A. Foroumadi, Eur. J. Med. Chem. 44 (2010) 1424e1429.
[25] A. Dutta, S. Bandyopadhyay, C. Mandal, M. Chatterjee, Parasitol. Int. 54 (2005)
119e122.
6.1.4.11. 1-(4-(4,5-Dihydro-1H-imidazol-2-yl)phenyl)-4-(5-(5-nitro-
furan-2-yl)-1,3,4-thiadiazol-2-yl)piperazine hydrochloride (2k). This
compound was prepared by treatment of the crude imidate 8 with
1,2-ethylene diamine in ethanol at reflux for 60 min. The resulting
solid was washed with acetone and crystallized from etha-
nolediethylether. Yield: 40%; m.p > 300 ꢀC; IR (KBr, cmꢃ1): 3444,
1606, 1357, 1235; 1H NMR (500 MHz, DMSO-d6): 7.87 (d, 1H,
J ¼ 3.55Hz,furan), 7.41(d, 2H,J ¼ 8.35 Hz,phenyl), 7.42 (d,1H, J ¼ 3.55,
furan), 7.02 (d, 2H, J ¼ 8.35 Hz, phenyl), 3.74 (br s, 4H, imidazoline),
3.57 (br s, 4H, piperazine), 3.41 (br s, 4H, piperazine). MS (ESI): 426
[M þ Hþ]. Anal. Calcd for C19H20ClN7O3S: C, 49.40; H, 4.36; N, 21.23.
Found: C, 49.11; H, 4.55; N, 21.51.
6.1.4.12. 1,4,5,6-Tetrahydro-2-(4-(4-(5-(5-nitrofuran-2-yl)-1,3,4-
thiadiazol-2-yl)piperazin-1-yl)phenyl)pyrimidine
hydrochloride
(2l). This compound was prepared by treatment of the crude imi-
date 8 with 1,3-diaminopropane in ethanol at reflux for 60 min. The
resulting solid was washed with acetone and crystallized from
ethanolediethylether. Yield: 32%; m.p > 300 ꢀC; IR (KBr cmꢃ1):
3461, 1605, 1350, 1229; 1H NMR (500 MHz, DMSO-d6): 9.75 (s, 1H),
8.11 (br s, 2H), 7.87 (br s, 1H, furan), 7.69 (d, 2H, J ¼ 7.6 Hz, phenyl),
7.41 (br s, 1H, furan), 7.15 (d, 2H, J ¼ 7.6 Hz, phenyl), 3.75 (br s, 4H,
piperazine), 3.60 (br s, 4H, piperazine), 3.45 (br s, 2H, 6-CH2 tet-
rahydropyrimidine), 2.88 (br s, 2H, 4-CH2 tetrahydropyrimidine),
1.95 (m, 2H, 5-CH2 tetrahydropyrimidine). MS (ESI): 440 [M þ Hþ].
Anal. Calcd for C19H20ClN7O3S: C, 50.47; H, 4.66; N, 20.60. Found: C,
50.11; H, 4.53; N, 20.85.
[26] A.K. Tanaka, V.B. Valero, H.K. Takahashi, A.H. Straus, J. Antimicrob. Chemother.
59 (2007) 487e492.
[27] A.F. Kiderlen, P.M. Kaye, J. Immunol. Methods 127 (1990) 11e18.
[28] B. Tao, T.L. Huang, Q. Zhang, L. Jackson, S.F. Queener, I.O. Donkor, Eur. J. Med.
Chem. 34 (1999) 531e538.