Synthesis and antimicrobial activity of new 1,2,4-triazole-3-thiol metronidazole derivatives

Article abstract of DOI:10.1007/s00706-010-0281-9

New 1,2,4-triazole-3-thiol metronidazole derivatives have been synthesized by treating 1,2,4-triazole-3-thiols with metronidazole tosylate (toluene-4-sulfonic acid 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl ester) in DMF and in the presence of potassium c

Full text of DOI:10.1007/s00706-010-0281-9

Monatsh Chem (2010) 141:471–478
DOI 10.1007/s00706-010-0281-9
ORIGINAL PAPER
Synthesis and antimicrobial activity of new 1,2,4-triazole-3-thiol
metronidazole derivatives
•
Haythem A. Saadeh Ibrahim M. Mosleh
•
•
Amal G. Al-Bakri Mohammad S. Mubarak
Received: 21 November 2009 / Accepted: 23 February 2010 / Published online: 28 April 2010
Ó Springer-Verlag 2010
Abstract New
1,2,4-triazole-3-thiol
metronidazole
importance. Derivatives of 1,2,4-triazole have been found
to have diverse pharmacological activity, for example
fungicidal, insecticidal, bactericidal, herbicidal, anti-tumor,
and anti-inflammatory, among others [1]. A large number
of 1,2,4-triazole-containing ring systems, for example
fluconazole, intraconazole, and voriconazole have been
incorporated into a wide variety of therapeutically inter-
esting drug candidates with, for example, anti-inflammatory,
CNS-stimulant, sedative, antianxiety, antimicrobial, and
antimycotic activity [2]. Moreover, there are known drugs
containing the 1,2,4-triazole group, including triazolam [3],
alprazolam [4], etizolam [5, 6], and furacylin [7, 8].
In addition, thione-substituted 1,2,4-triazoles and their
derivatives [2] have been found to have a variety of bio-
logical activity, for example antibacterial, antifungal,
antitubercular, antimycobacterial, anticancer, diuretic, and
hypoglycemic properties.
derivatives have been synthesized by treating 1,2,4-tria-
zole-3-thiols with metronidazole tosylate (toluene-4-
sulfonic acid 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl
ester) in DMF and in the presence of potassium carbonate
as a base. S-alkylated and N-alkylated products were
obtained, with the S-alkylated being the major products.
All of the newly synthesized compounds were character-
1
ized by IR, H NMR, 13C NMR, and high-resolution mass
spectrometry. The antiparasitic activity of the compounds
against Entamoeba histolytica and Giardia intestinalis was
investigated. The antibacterial and antifungal activity of
the compounds, assessed as minimal inhibitory concen-
tration, was also investigated.
Keywords Triazoles Á Metronidazole Á
Antiparasitic activity Á Antimicrobial activity
Because of their interesting and diverse biological
activity, a series of 1,2,4-triazole derivatives has been pre-
pared and their biological activity investigated. An
extensive review that deals with the synthetic procedures
employed in the preparation of mercapto and thione-
substituted 1,2,4-triazoles was published in 2006. What
follows is the most important work pertaining to triazoles
published since 2006. Amir and co-workers [9] prepared,
among others, a series of 1,2,4-triazole derivatives of
(biphenyl-4-yloxy)acetic acid; these compounds were
potential anti-inflammatory analgesic agents with minimum
ulcerogenic potential. They synthesized a series of
5-[(biphenyl-4-yloxy)methyl]-4-alkyl/aryl-3-mercapto-4H-1,2,
4-triazoles by cyclization of the corresponding thiosemic-
arbazides. Koparir and Demiradg [10] prepared a number of
4,5-substituted-4H-1,2,4-triazole-3-thiol derivatives, with
potential antibacterial and antifungul activity, from the
corresponding furan-2-carboxylic acid hydrazides and
Introduction
The chemistry of 1,2,4-triazoles has received considerable
attention because of their synthetic and biological
H. A. Saadeh Á M. S. Mubarak (&)
Chemistry Department, Faculty of Science,
The University of Jordan, Amman 11942, Jordan
e-mail: mmubarak@ju.edu.jo
I. M. Mosleh
Department of Biological Sciences, Faculty of Science,
The University of Jordan, Amman 11942, Jordan
A. G. Al-Bakri
Department of Pharmaceutics and Pharmaceutical Technology,
Faculty of Pharmacy, The University of Jordan, Amman 11942,
Jordan
123

472
H. A. Saadeh et al.
phenylacetic acid hydrazides. Deprez-Poulain and co-
workers [11] reported the synthesis of 4H-1,2,4-triazole-
3-thiols using di-2-pyridylthionocarbonate as the thiocarbonyl
transfer agent. Very recently, Kucukguzel and his team [12]
described the synthesis and wide range of antiviral activity
of some novel 5-[(4-aminophenoxy)methyl]-4-alkyl/aryl-2,
4-dihydro-3H-1,2,4-triazole-3-thiones and several related
thiourea derivatives; the anti-tuberculosis activity of the
prepared compounds was also evaluated. Karthikeyan and
Holla [13] have synthesized a series of 2,4-dichloro-
5-fluorophenyl-substituted arylidenetriazolothiazolidinones
from the corresponding 1,2,4-triazole-5-thiols and investi-
gated their anti-inflammatory and antimicrobial activity.
Interestingly, Supuran and his team [14] have very recently
synthesized novel mercapto-1,3,4-oxadiazole and 1,2,4-tri-
azole derivatives starting from 4-(4-halogeno-phenylsulfonyl)
benzoic acid hydrazides; the prepared compounds were
assayed as inhibitors of the cytosolic and tumor-associated
carbonic anhydrase isoenzymes I, II, and IX. More recently,
Rawat et al. [15] reported on the antibacterial activity of a
series of metronidazole–triazole conjugates.
addition, the antiparasitic, antibacterial, and antifungal
activity of the newly prepared compounds was investigated.
Results and discussion
Chemistry
Synthesis of compounds 3a–3e and 4a–4e was carried out
via the route shown in Scheme 1. The key starting materials,
1,2,4-triazole-3-thiones 1a–1e [26] and metronidazole tos-
ylate (2) [27] were prepared according to published
procedures. Treatment of 1,2,4-triazole-3-thiones 1a–1e
with metronidazole tosylate (2) in warm DMF in the pres-
ence of potassium iodide and potassium carbonate afforded
the desired products in moderate yields.
Under these experimental conditions, both the S-alkyl-
ated (3a–3e) and the N-alkylated (4a–4e) isomers were
obtained. Flash chromatography of the crude material
allowed the isolation of both isomers with an average yield
of 25–32% for the S-alkylated isomers and 10–14% for the
N-alkylated (ratio *3:1). This type of regioselective
alkylation reactions of thioamides is known to take place at
either the sulfur or nitrogen atom. In most cases of
nucleophilic substitution of an alkyl halide on a thioamide
system sulfur atom attack is favored [28]; very few
examples are known where attack on the nitrogen atom is
preferred [29]. The outcome of these regioselective alky-
lations of thioamide derivatives with electrophiles depends
on the character of both the thioamide derivative and the
applied electrophile [30]. Under basic conditions, thioam-
ides 1a–1e exist in two tautomeric forms (I and II) in
solution (Scheme 2); upon alkylation of I and II with 2,
compounds 4a–4e and 3a–3e, respectively, are obtained.
Because the major product was formed by S-alkylation,
the preferred tautomeric form of the thioamides is II. Sulfur
Other important drugs known for their wide range of
biological activity are metronidazole, 2-(2-methyl-5-nitro-
1H-imidazol-1-yl)ethanol (1), and its derivatives [16–19].
They are highly effective against trichomoniasis, various
forms of amoebiasis, and infections with anaerobic bacteria
and protozoa [20]; metronidazole can kill or inhibit most
anaerobic bacteria when the concentration in serum is in the
range 2 to 8 lg/cm3 [21]. In view of the wide interest in the
activity and profile of triazoles and metronidazole, and as an
extension of our ongoing research on the synthesis of new
compounds of pharmacological interest [22–25], we
describe herein the synthesis and characterization of a
number of new compounds that combine metronidazole and
1,2,4-triazole-3-thiols, which, to the best of our knowledge,
have not previously been described in the literature. In
Scheme 1
R
N
R
N
R
N
S
N
S
Ar
Ar
S
Ar
K2CO3 / KI
CH3
2N
O
+
+
N
N
N
N
N
N
NH
DMF, 75-80 °C
TsO
N
O2N
CH3
N
O2N
CH3
N
1a-1e
N
2
3a-3e
4a-4e
a
b
c
d
e
Ar
R
N
N
N
N
N
CH3
OCH3
123

Synthesis and antimicrobial activity
473
R
structures comes from mass spectral data; mass spectra of
the prepared compounds showed the correct molecular ions
(M?Á) as suggested by their molecular formulas. Each pair
of S- and N-alkylated triazoles 3 and 4 has exactly the same
molecular ion (M?Á), for example the M?Á for both 3a and
4a is 437.1. Analyses of the molecular ions were used in
the identification and characterization of these compounds.
R
R
N
_
N
S
Ar
N
S
Ar
S
Ar
K2CO3
N
NH
N
N
_
N
N
1a-1e
I
II
Scheme 2
atom alkylation is preferred to nitrogen atom alkylation
because of sterically free access, higher partial charge,
better polarizability owing to its 3p orbitals, and the aro-
maticity of the triazole ring conferred [31].
Antiamoebic, antigiardial, and cytotoxic activity
The antiamoebic and antigiardial activity of the reported
compounds was investigated by use of in-vitro bioassays.
Their bioactivity was compared with that of the standard
antiamoebic and antigiardial drug, metronidazole. The
cytotoxicity of the compounds and metronidazole on two
cell lines, Hep-2 and Vero cells, was also tested. The IC50
values of the compounds against Entamoeba histolytica,
Giardia intestinalis, and the two cell lines are given in
Table 1.
1
As reported in the literature [32, 33], analysis of the H
NMR and 13C NMR spectra of the synthesized compounds
provided proof of the structures of S- and N-alkylated tri-
azoles 3a–3e and 4a–4e, respectively. The CH2 protons
adjacent to the sulfur atom in compounds 3a–3e appear at
about 3.60 ppm and the carbon resonates at about 31 ppm.
However, CH2 protons adjacent to the nitrogen atom in
compounds 4a–4e are more downfield-shifted to about 4.70
and the carbon resonates at about 43 ppm. Finally, the 13C
NMR spectra show peaks at 169–170 ppm corresponding
to the C=S group associated with the N-alkylated deriva-
tives 4a–4e, whereas the S-alkylated derivatives 3a–3e
gave peaks at 151.3–152 ppm for the same carbon, corre-
sponding to C–S.
As shown in Table 1, all the compounds tested had bio-
logical activity against Entamoeba and Giardia. Compounds
1c, 3c, and 3d had the highest activity against Entamoeba,
with IC50 values ranging from 0.48 to 0.85 lM; other
derivatives, for example 1a, 3a, 4a, 4c, 4d, 3e, and 4e, also
had significant activity against Entamoeba with IC50 values
ranging from 1.10 to 1.53 lM, which makes them more
active than metronidazole itself. Additionally, compound 3b
was the most potent against Giardia, with an IC50 value of
0.76 lM compared with 5.03 lM for metronidazole. When
the cytotoxicity of the prepared molecules is considered
(Table 1), compound 1a seems to be the best among the
1H NMR and 13C NMR spectra of all the prepared
compounds were in total agreement with the suggested
structures. DEPT experiments were used to differentiate
secondary and quaternary carbons from primary and ter-
tiary carbons. Additional support for the proposed
Table 1 Antiamoebic,
antigiardial, and cytotoxic
activity of the tested compounds
Compound
Mean IC50 SD (lM)
E. histolytica
G. intestinalis
Hep-2
Vero
1a
1.37 0.05
1.53 0.11
1.31 0.08
2.99 0.16
3.27 0.15
3.36 0.37
0.85 0.04
0.48 0.02
1.10 0.07
2.76 0.16
0.79 0.07
1.42 0.22
2.27 0.13
1.31 0.09
1.42 0.09
4.51 0.35
1.56 0.09
1.21 0.11
2.30 0.11
3.19 0.35
0.76 0.10
3.19 0.25
2.71 0.14
1.78 0.09
3.78 0.11
2.53 0.13
1.07 0.11
1.66 0.09
2.10 0.16
1.77 0.07
1.73 0.04
5.03 0.29
531.46 11.9
469.27 11.4
472.29 6.90
249.33 11.3
297.18 8.87
403.59 12.6
205.38 10.1
204.39 9.38
142.61 7.34
140.34 8.29
92.61 4.55
68.55 3.35
548.18 9.50
413.53 11.2
471.86 8.75
1,475.5 15.0
1,122.6 9.43
614.39 3.22
605.32 9.89
237.52 13.8
317.63 9.92
287.35 8.87
303.98 8.87
150.25 8.65
117.44 12.0
141.43 8.72
114.50 6.69
97.72 7.68
720.29 11.9
565.97 7.63
216.61 7.90
1,515.2 11.0
3a
4a
1b
3b
4b
1c
3c
4c
1d
3d
4d
1e
3e
4e
Metronidazole
123

474
H. A. Saadeh et al.
derivatives of metronidazole. This derivative was around
three times more active than metronidazole against Ent-
amoeba and Giardia. Fortunately, the boosted activity of this
compound was not accompanied by any substantial increase
in cytotoxicity (Table 1). In contrast, the cytotoxicity of the
other derivatives with potent antiamoebic activity (1c, 3c,
and 3d) was higher than that of the precursor compound
metronidazole, although they remain noncytotoxic at con-
centrations much higher than the antiparasitic concentration
of the derivatives. The other potent antigiardial derivative,
3b, was also relatively nontoxic to the tested cell lines, with
an IC50 value of around 300 lM (Table 1). Interestingly,
some of the tested compounds (e.g. 3b, 3c, and 1c) had dif-
ferent patterns of activity against G. intestinalis and E.
histolytica (Table 1). In addition, the molecular modifica-
tions on our derivatives did not render any of the compounds
inactive. All other molecules had slightly higher activity than
metronidazole (Table 1). Their cytotoxicity however was
generally higher than that of metronidazole. The activity
exhibited by the derivatives, especially compounds 1a and
3b, suggest that the derivatives may be used as new lead
compounds in the development of new antiparasitic drugs.
Although drug resistance to Entamoeba and Giardia does
not, so far, appear to be a serious problem, occasional reports
of failure with metronidazole [34] and the reported variations
in drug sensitivities of isolates [35] may be alarming.
Therefore, the importance of such biologically active,
noncytotoxic metronidazole derivatives lies in their potential
contribution to overcoming the problem of resistance of
pathogens to the standard drugs. Additionally, because of the
limited number of commercially available drugs active
against anaerobic protozoan parasites and bacteria there is a
serious need for new active compounds. Molecular modifi-
cation of the original drugs therefore offers alternatives that
may bypass the already developed mechanisms adopted by
the anaerobic pathogens against the standard drugs. Our new
compounds, especially 1a and 3b, are good drug candidates
to be tested against metronidazole-resistant parasites and
bacteria.
Antibacterial and antifungal activity
The antimicrobial activity of the newly prepared compounds
and their precursor compounds, assessed as minimal inhib-
itory concentration (MIC) values, are presented in Table 2.
The spectrum of activity of the tested compounds covered
Gram-positive, Gram-negative, and fungal cultures. With
the exception of Clostridium sporogenes the reported anti-
microbial activity was significantly lower than that of the
reference tested antimicrobials. Nevertheless, it was dem-
onstrated that compounds 3c, 4d, and 3e have higher
antimicrobial activity towards the Gram-positive anaerobic
culture of C. sporogenes than the reference antimicrobial,
metronidazole (Table 2). For these compounds the MIC
Table 2 Minimum inhibitory concentrations (MIC, lM) of the tested substances and reference drugs against the tested microorganisms
Compound
MIC (lM)
P. aeruginosa
E. coli
S.aureus
Cl. sporogenes
C. albicans
Met-Ts
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
NT
ND
NT
ND
ND
514.2 273.6
ND
2,212.2 589.6
1a
ND
ND
ND
1b
ND
ND
ND
ND
1c
ND
ND
ND
ND
1d
ND
ND
ND
ND
1e
ND
ND
ND
ND
3a
ND
2,136.4 602.6
76.7 42.5
99.2 35.6
152.3 97.8
43.2 14.4
17.8 6.3
640.64 215.1
612.6 205.7
29.9 16.0
17.1 6.0
4,102.5 0.0
NT
ND
4a
ND
ND
ND
3b
ND
3,832.0 1,080.8
ND
4b
ND
ND
ND
3c
892.3 251.7
ND
446.2 125.8
ND
4c
ND
ND
3d
ND
ND
ND
4d
ND
ND
ND
3e
ND
555.8 218.8
ND
4e
ND
ND
ND
Miconazole
Ampicillin
Metronidazole
NT
NT
11.3 1.2
NT
21.7 7.3
NT
0.089 0.0
NT
NT
42.9 16.1
NT
ND Not detectable at concentrations as high as 0.5 mg/cm3, NT not tested
123

Synthesis and antimicrobial activity
475
2,4-Dihydro-4-methyl-2-[2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethyl]-5-(4-pyridinyl)-3H-[1,2,4]triazole-3-thione
(4a, C14H15N7O2S)
values were 17.8, 29.9, and 17.1 lM, respectively, making
them more active than metronidazole (MIC = 42.9 lM).
Yield 0.11 g (13%); m.p.: 138–139 °C; IR (KBr):
m = 1,528 cm
-1
(C=S); 1H NMR (CDCl3): d = 2.30
ꢀ
Experimental
(s, 3H), 3.65 (s, 3H), 4.65 (t, J = 5.7 Hz, 2H), 4.80 (t,
J = 5.8 Hz, 2H), 7.45 (d, J = 6.0 Hz, 2H), 7.95 (s, 1H),
8.80 (d, J = 6.0 Hz, 2H) ppm; 13C NMR (CDCl3): d = 14.1
(Ar-CH3), 33.4 (N–CH3), 43.9 (S-CH2), 48.5 (N–CH2),
122.1 (CH), 132.7 (C), 133.4 (CH), 138.9 (C), 148.6 (C),
150.6 (C), 150.9 (CH), 169.1 (C) ppm; HRMS (EIMS) m/z:
calcd. for C14H15N7 NaO2S [M?Na]? 368.09056, found
368.09217; m/z: calcd. for C14H16N7O2S [M?H]?
346.10862, found 346.11036.
Unless otherwise indicated, all chemicals were obtained
from commercial sources and were used as received.
Melting points were measured with a Fischer–Johns melt-
ing-point apparatus. The IR spectra were recorded, as KBr
discs, with the aid of a Thermo Nicolet Nexus 670 FT-IR
1
instrument. We obtained H and 13C NMR with a Bruker-
DPX 300 MHz spectrometer with CDCl3 or dimethyl
sulfoxide (DMSO)-d6 as solvents; chemical shifts are
reported in ppm relative to TMS as internal standard. High-
resolution mass spectral data were acquired with a Bruker
APEX (IV) mass spectrometer (Bremen, Germany). Ele-
mental analyses were obtained by use of an Eurovector
Euro EA3000, C, H, N, and S elemental analyzer and the
results obtained agreed with the calculated percentages to
within 0.4%. Compounds were checked for purity by
3-[5-[2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethylsulfanyl]-
4-phenyl-4H-[1,2,4]triazol-3-yl]pyridine
(3b, C19H17N7O2S)
Yield 0.22 g (27%); m.p.: 179–180 °C; 1H NMR (CDCl3):
d = 2.40 (s, 3H), 3.60 (t, J = 7.0 Hz, 2H), 4.65 (t,
J = 7.0 Hz, 2H), 7.35 (m, 3H), 7.55 (m, 3H), 7.65 (d,
J = 2.8 Hz, 1H), 7.95 (s, 1H), 8.48–8.55 (m, 2H) ppm; 13
C
TLC using glass plates precoated with silica gel 60 GF254
,
NMR (CDCl3): d = 14.6 (CH3), 31.5 (S–CH2), 45.4
(N–CH2), 123.3 (C), 124.1 (CH), 126.7 (C), 128.1 (CH),
130.6 (CH), 130.9 (CH), 133.5 (CH), 135.7 (CH), 138.9
(C), 148.7 (CH), 151.1 (CH), 151.9 (C), 152.3 (C), 152.8
(C) ppm; HRMS (EIMS) m/z: calcd. for C19H17NaN7O2S
[M?Na]? 430.10621, found 430.10566.
supplied by Fluka. 1,2,4-Triazole-3-thiones 1a–1e [26] and
metronidazole tosylate (2) [27] were synthesized and
purified according to published procedures.
Synthesis of compounds 3a–3e and 4a–4e
Compounds 3a–3e and 4a–4e were prepared according to
the following procedure. A mixture of 1,2,4-triazole-3-thi-
one 1a–1e (2 mmol), metronidazole tosylate (2, 2 mmol),
and K2CO3 (3 mmol) in 8 cm3 DMF was stirred at 75–80 °C
overnight. Then, 30 cm3 water was added and the resulting
mixture was extracted with CHCl3. The organic layer was
separated, washed with water (4 9 30 cm3), and dried over
Na2SO4. After solvent evaporation, the residue was purified
by flash chromatography on silica gel using CHCl3–CH3OH
(95:5) to afford 3a–3e and 4a–4e.
2,4-Dihydro-2-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)-
ethyl]-4-phenyl-5-(3-pyridinyl)-3H-[1,2,4]-
triazole-3-thione (4b, C19H17N7O2S)
Yield 0.08 g (10%); m.p.: 203–204 °C; IR (KBr):
-1
1
ꢀ
m = 1,529 cm (C=S); H NMR (CDCl3): d = 2.40 (s,
3H), 4.75 (t, J = 5.8 Hz, 2H), 4.85 (t, J = 5.6 Hz, 2H),
7.20 (m, 3H), 7.50 (m, 4H), 8.00 (s, 1H), 8.45 (d,
J = 2.1 Hz, 1H), 8.60 (dd, J1 = 2.1, J2 = 5.8 Hz, 1H)
ppm; 13C NMR (CDCl3): d = 14.2 (CH3), 43.9 (S–CH2),
48.6 (N–CH2), 121.2 (C), 123.4 (CH), 126.5 (C), 128.0
(CH), 130.2 (CH), 130.5 (CH), 133.4 (CH), 134.3 (C),
135.4 (CH), 138.9 (C), 148.8 (CH), 150.7 (C), 151.7 (CH)
169.5 (C) ppm; HRMS (EIMS) m/z: calcd. for
C19H17N7NaO2S [M?Na]? 430.10621, found 430.11119.
4-[4-Methyl-5-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)-
ethylsulfanyl]-4H-[1,2,4]triazol-3-yl]pyridine
(3a, C14H15N7O2S)
Yield 0.25 g (29%); m.p.: 134–135 °C; 1H NMR
(CDCl3): d = 2.60 (s, 3H), 3.65 (m, 5H), 4.80 (t,
J = 6.9 Hz, 2H), 7.58 (d, J = 5.8 Hz, 2H), 7.95 (s, 1H),
8.80 (d, J = 5.8 Hz, 2H) ppm; 13C NMR (CDCl3):
d = 14.6 (CH3), 31.5 (S–CH2), 31.9 (N–CH3), 45.3 (N–
CH2), 122.2 (CH), 133.5 (CH), 134.1 (C), 137.5 (C),
150.7 (CH), 151.6 (C), 152.3 (C), 153.9 (C) ppm; HRMS
(EIMS) m/z: calcd. for C14H15N7NaO2S [M?Na]?
368.09056, found 368.09512.
4-[4-(4-Methoxyphenyl)-5-[2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)ethylsulfanyl]-4H-[1,2,4]triazol-3-yl]pyridine
(3c, C20H19N7O3S)
Yield: 0.28 g (32%); m.p.: 164–165 °C; 1H NMR (CDCl3):
d = 2.60 (s, 3H), 3.55 (t, J = 7.0 Hz, 2H), 3.85 (s, 3H),
4.80 (t, J = 7.0 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 7.13 (d,
J = 8.5 Hz, 2H), 7.32 (d, J = 5.2 Hz, 2H), 7.92 (s, 1H),
123

476
H. A. Saadeh et al.
8.55 (d, J = 5.2 Hz, 2H) ppm; 13C NMR (CDCl3):
d = 14.6 (CH3), 31.0 (S–CH2), 45.2 (N–CH2), 55.7 (O–
CH3), 115.7 (CH), 121.3 (CH), 125.4 (C), 128.3 (CH),
133.4 (CH), 133.8 (C), 138.4 (C), 150.3 (CH), 151.4 (C),
153.1 (C), 153.9 (C), 161.2 (C) ppm; HRMS (EIMS) m/z:
calcd. for C20H19N7NaO3S [M?Na]? 460.11678, found
460.11623.
(C), 170.5 (C) ppm; HRMS (EIMS) m/z: calcd. for
C23H19N7NaO2S [M?Na]? 480.12186, found 480.12708.
3-[5-[2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethylsulfanyl]-
4-(1-naphthalenyl)-4H-[1,2,4]triazol-3-yl]pyridine
(3e, C23H19N7O2S)
Yield 0.24 g (26%); m.p.: 160–161 °C; 1H NMR (CDCl3):
d = 2.60 (s, 3H), 3.60 (t, J = 6.4 Hz, 2H), 4.80 (t,
J = 6.4 Hz, 2H), 7.13 (m, 1H), 7.20 (d, J = 5.4 Hz, 1H),
7.42 (d, J = 7.9 Hz, 1H), 7.50-7.58 (m, 3H), 7.72 (d,
J = 7.9 Hz, 1H), 7.95 (s, 1H), 7.95 (d, J = 7.9 Hz, 1H),
8.05 (d, J = 8.1 Hz, 1H), 8.45-8.55 (m, 2H) ppm; 13C
NMR (CDCl3): d = 14.7 (CH3), 31.1 (CH2), 45.3 (CH2),
121.2 (CH), 121.4 (C), 122.8 (C), 123.5 (CH), 125.4 (CH),
125.6 (CH), 126.4 (CH), 127.8 (CH), 128.8 (CH), 129.1
(CH), 129.3 (C), 131.1 (C), 131.7 (CH), 133.5 (CH), 134.6
(CH), 146.3 (C), 147.8 (C), 150.9 (CH), 151.5 (CH) ppm;
HRMS (EIMS) m/z: calcd. for C23H19N7NaO2S [M?Na]?
480.12186, found 480.12503.
2,4-Dihydro-4-(4-methoxyphenyl)-2-[2-(2-methyl-5-nitro-
1H-imidazol-1-yl)ethyl]-5-(4-pyridinyl)-3H-[1,2,4]tria-
zole-3-thione (4c, C20H19N7O3S)
Yield 0.12 g (14%); m.p.: 175–176 °C; IR (KBr):
-1
(C=S); 1H NMR (CDCl3): d = 2.40
ꢀ
m = 1,531 cm
(s, 3H), 3.85 (s, 3H), 4.75 (t, J = 5.4 Hz, 2H), 4.85
(t, J = 5.4 Hz, 2H), 7.01 (d, J = 8.9 Hz, 2H), 7.08 (d,
J = 6.1 Hz, 2H), 7.13 (d, J = 8.9 Hz, 2H), 7.97 (s, 1H),
8.55 (d, J = 6.1 Hz, 2H) ppm; 13C NMR (CDCl3): d = 14.2
(CH3), 43.8 (S–CH2), 48.7 (N–CH2), 55.7 (O–CH3), 115.4
(CH), 121.5 (CH), 126.6 (C), 129.1 (CH), 132.3 (C), 133.4
(CH), 139.2 (C), 147.7 (C), 150.5 (CH), 150.7 (C), 160.8
(C), 170.2 (C) ppm; HRMS (EIMS) m/z: calcd. for
C20H19N7NaO3S [M?Na]? 460.11678, found 460.12292.
2,4-Dihydro-2-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)-
ethyl]-4-(1-naphthalenyl)-5-(3-pyridinyl)-3H-[1,2,4]tria-
zole-3-thione (4e, C23H19N7O2S)
4-[5-[2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethylsulfanyl]-
4-(1-naphthalenyl)-4H-[1,2,4]triazol-3-yl]pyridine
(3d, C23H19N7O2S)
Yield: 0.12 g (13%); m.p.: 173–174 °C; IR (KBr):
-1
1
ꢀ
m = 1,525 cm (C=S); H NMR (CDCl3): d = 2.50 (s,
3H), 4.80 (t, J = 5.4 Hz, 2H), 4.90 (t, J = 5.4 Hz, 2H), 7.05
(dd, J1 = 8.1, J2 = 4.9 Hz, 1H), 7.35 (m, 3H), 7.55 (m, 3H),
7.93 (m, 1H), 7.98 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H), 8.34 (d,
J = 2.1 Hz, 1H), 8.48 (dd, J1 = 4.8, J2 = 1.4 Hz, 1H) ppm;
13C NMR (CDCl3): d = 14.5 (CH3), 44.0 (S–CH2), 48.6
(N–CH2), 121.3 (C), 121.7 (CH), 123.4 (CH), 125.6 (CH),
127.3 (CH), 127.4 (CH), 128.5 (CH), 129.1 (CH), 129.4
(C), 130.7 (C), 131.5 (CH), 133.4 (CH), 134.5 (C), 134.8
(CH), 139.2 (C), 148.2 (CH), 148.4 (C), 150.8 (C), 151.6
(CH), 170.2 (C) ppm; HRMS (EIMS) m/z: calcd. for
C23H19N7NaO2S [M?Na]? 480.12186, found 480.12131.
Yield 0.25 g (28%); m.p.: 197–198 °C; 1H NMR (CDCl3):
d = 2.60 (s, 3H), 3.60 (t, J = 6.8 Hz, 2H), 4.80 (t,
J = 6.8 Hz, 2H), 6.95–7.22 (m, 3H), 7.45 (d, J = 7.4 Hz,
1H), 7.52 (t, J = 7.0 Hz, 1H), 7.60 (m, 2H), 7.90 (s, 1H),
8.01 (d, J = 8.2 Hz, 1H), 8.10 (d, J = 8.4 Hz, 1H) 8.42 (d,
J = 6.2 Hz, 2H) ppm; 13C NMR (CDCl3): d = 14.7 (CH3),
31.1 (S–CH2), 45.3 (N–CH2), 120.7 (CH), 121.2 (CH), 125.6
(CH), 126.2 (CH), 127.8 (CH), 128.9 (CH), 129.0 (CH),
129.1 (C), 129.4 (C), 131.7 (CH), 133.4 (CH), 133.6 (C),
134.5 (C), 138.4 (C), 150.4 (CH), 151.4 (C), 153.7 (C), 154.5
(C) ppm; HRMS (EIMS) m/z: calcd. for C23H19N7NaO2S
[M?Na]? 480.12186, found 480.12305.
Antiamoebic and antigiardial activity
To test the antiamoebic and antigiardial activity of the
compounds E. histolytica HK-9 strain (ATCC number
30015) cultured in LYI-S-2 medium and G. intestinalis
WB strain (ATCC number 30957) grown in a modified YI-
S medium were used in all assays. The antiamoebic and
antigiardial activity of the prepared compounds and met-
ronidazole as the standard antiamoebic and antigiardial
drug were tested as described elsewhere [25]. Briefly, the
tested compounds and metronidazole were dissolved in
DMSO then in medium and filter-sterilized. In a 15-cm3
glass tube, twofold dilutions starting at 15 lg/cm3 were
prepared in a final volume of 15 cm3 to exclude air from
the tube. Each tube was inoculated with 20,000 cells of the
parasite being tested (Entamoeba or Giardia). Each
2,4-Dihydro-2-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)-
ethyl]-4-(1-naphthalenyl)-5-(4-pyridinyl)-3H-[1,2,4]tria-
zole-3-thione (4d, C23H19N7O2S)
Yield 0.13 g (14%); m.p.: 209–210 °C; IR (KBr):
-1
1
ꢀ
m = 1,530 cm (C=S); H NMR (CDCl3): d = 2.50 (s,
3H), 4.85 (t, J = 5.3 Hz, 2H), 4.95 (t, J = 5.3 Hz, 2H),
6.95 (d, J = 6.2 Hz, 2H), 7.30 (d, J = 6.8 Hz, 1H), 7.35
(d, J = 7.3 Hz, 1H), 7.55 (m, 3H), 7.95 (d, J = 8.2 Hz,
1H), 8.00 (s, 1H), 8.05 (d, J = 8.42 Hz, 1H), 8.40 (d,
J = 7.2 Hz, 2H) ppm; 13C NMR (CDCl3): d = 14.5 (CH3),
43.9 (S–CH2), 48.6 (N–CH2), 120.9 (CH), 121.6 (CH),
125.7 (CH), 127.2 (CH), 127.5 (CH), 128.5 (CH), 129.1
(CH), 129.3 (C), 130.7(C), 131.5 (CH), 132.1 (C), 133.4
(CH), 134.4 (C), 139.1 (C), 148.3 (C), 150.4 (CH), 150.7
123

Synthesis and antimicrobial activity
477
compound was assayed in duplicate in each of three
independent experiments. In each assay, the appropriate
controls were performed, including one without any com-
pound and another with metronidazole as the positive
control. The biological activity of the compounds was
evaluated by counting the parasites in each tube by use of a
standard hemacytometer. In each count, trypan blue was
used to distinguish live from dead parasites.
substance stock solution (10 mm3) was added to the first well.
Twofold serial dilution was then carried out across the plate.
Overnight batch culture (10 mm3) was used to inoculate the
wells to achieve a final inoculum size of 1 9 106 cfu/cm3 and
the plate was incubated for 24 h at 37 °C. MIC was expressed
as the mean concentration between the well showing growth
and the well showing no growth. Growth was detected as
turbidity (630 nm) relative to an un-inoculated well, by use of
a microtitre plate reader (Biotek, USA). MIC for C. sporog-
enes ATCC 19404 was determined in micro centrifuge tubes
(Bio Basic). Negative controls were performed using only
sterile broth and positive controls were performed with only
overnight culture and 10 mm3 DMSO. Each MIC determi-
nation was carried out in triplicate.
The cytotoxicity of the reported compounds and the
reference drug, metronidazole, was investigated on Hep-2
and Vero cells using the standard cytotoxicity assay and the
trypan blue exclusion method [25]. Briefly, 100 mm3
portions of each cell suspension were added to the wells of
96-well plates, incubated for 24 h, and the medium in each
well was then replaced with 150 mm3 fresh medium.
Solutions of the compounds or the reference drug were
dissolved in DMSO, prepared in medium, and filter ster-
ilized. Then, 150 mm3 twofold serial dilutions of each of
the compounds and the reference drug starting at a con-
centration of 1,000 lg/cm3 in culture medium were
prepared on the plates. After 48 h incubation, the number
of cells in each well was determined by use of a hemacy-
tometer. Each compound was assayed in duplicate in each
of three independent experiments. In each assay the neg-
ative controls (without any compound or reference drug)
were included in duplicates.
Acknowledgments This work was financially supported by the
Deanship of Scientific Research at the University of Jordan; this
support is highly acknowledged.
References
1. Swamy SN, Basappa, Priya BS, Prabhuswamy B, Doreswamy
BH, Prasad JS, Rangappa KS (2006) Eur J Med Chem 41:531
2. Shaker RM (2006) Arkivoc ix:59
3. Brucato A, Coppola A, Gianguzza S, Provenzano PM (1978) Boll
Soc Ital Biol Sper 54:1051
4. Coffen DL, Fryer RL (1974) US Pat 3849 434
5. Shiroki M, Tahara T, Araki K (1975) Jap Pat 75100096
6. Shiroki M, Tahara T, Araki K (1975) Chem Abstr 84:59588k
7. Povelista FD, Gural AG (1973) Antibiotiki (Moscow) 18:71
8. Povelista FD, Gural AG (1973) Chem Abstr 78:93044
9. Kumar H, Javed SA, Khan SA, Amir M (2008) Eur J Med Chem
43:2688
10. Cansiz A, Koparir M, Demiradg A (2004) Molecules 9:204
11. Deprez-Poulain RF, Charton J, Leroux V, Deprez BP (2007)
Tetrahedron Lett 48:8157
Antimicrobial activity
The antimicrobial activity of the compounds, ampicillin,
miconazole (both obtained as a gift from Dar Al Dawa
Pharmaceutical Company, Naour-Jordan), and metronida-
zole, determined as MIC, was assessed using the broth
microdilution method recommended by CLSI with some
modifications [36]. Overnight cultures of Pseudomonas
aeruginosa ATCC 9027, Escherichia coli ATCC 8739,
Staphylococcus aureus ATCC 6538P, C. sporogenes
ATCC 19404, and Candida albicans ATCC 10231 were
used. Aerobic bacteria were grown in nutrient broth med-
ium (Oxoid, UK). C. sporogenes ATCC 19404 was grown
in thioglycollate medium (Oxoid, UK), and C. albicans
was grown in Sabaroud medium (Hi Media, India). Batches
of medium (20 cm3) were inoculated from fresh culture
slopes and incubated overnight at 37 °C. Anaerobic growth
conditions for C. sporogenes were achieved by incubating
the cultures in an anaerobic jar (Oxoid, UK) and using gas-
generating sachets of CO2 Gen. (Oxoid, UK).
12. Kuecuekguezel I, Tatar E, Kuecuekguezel SG, Rollas S, De
Clercq E (2008) Eur J Med Chem 43:381
13. Karthikeyan MS, Holla BS (2008) Monatsh Chem 139:691
14. Almajan GL, Barbuceanu S-F, Innocenti A, Scozzafava A,
Supuran CT (2008) J Enzyme Inhib Med Chem 23:101
15. Beena K, Nitin R, Rajesh K, Roy N, Rawat DS (2009) Bioorg
Med Chem Lett 19:1396
16. Bertinaria M, Galli U, Sorba G, Fruttero R, Gasco A, Brenciaglia
MI, Scaltrito MM, Dubini F (2003) Drug Dev Res 60:225
17. Martino GD, Regina GL, Pasquali AD, Ragno R, Bergamini A,
Ciaprini C, Sinistro A, Maga G, Crespan E, Artico M, Silvestri R
(2005) J Med Chem 48:4378
18. Hay MP, Anderson RF, Ferry DM, Wilson WR, Denny WA
(2003) J Med Chem 46:5533
¨
19. Gu¨nay NS, C¸ apan G, Ulusoy N, Ergenc¸ N, Otu¨k G, Kaya D
(1999) Farmaco 54:826
20. Goldman P, Wuest JD (1981) J Am Chem Soc 103:6224
21. Salimi A, Izadi M, Hallaj R, Rashidi M (1997) Electroanalysis
19:1668
22. Al-Zghoul KHA, Salih KSM, Ayoub MT, Mubarak MS (2005)
Heterocycles 65:2937
MIC was determined by using the twofold broth dilution
method in 96 well microtitre plates (CellstarÒ, Greinerbio-
one, Germany). Stock solution of each substrate (10 mg/cm3)
was prepared in DMSO (Tedia, USA) under aseptic condi-
tions. The first experimental well was filled with 190 mm3
media and the other wells were filled with 100 mm3. Each
23. Salih KSM, Al-Zghoul KHA, Mubarak MS, Ayoub MT (2006)
J Saudi Chem Soc 9:623
24. Abu Shairah EA, Saadeh HA, Mosleh IM, Ayoub MT, Mubarak
MS (2009) Eur Pat Appl EP 2985394 A2
123

478
H. A. Saadeh et al.
25. Saadeh HA, Mosleh IM, Mubarak MS (2009) Molecules 14:1483
26. Zamani K, Faghihi K, Tofighi T, Shariatzadeh M (2004) Turk J
Chem 28:95
33. Sarva MC, Romeo G, Guerrera F, Siracusa M, Salerno L, Russo F,
Cagnotto A, Goeganb M, Menninib T (2002) Bioorg Med Chem
10:313
27. Clayton R, Ramsden C (2005) Synthesis 16:2695
28. Ibrahim IA (1995) M.Sc. degree thesis. Organic Chemistry
Department, Faculty of Science, Suez Canal University
29. Kricheldorf HR (1973) Justus Liebigs Ann Chem 5:772
34. Knight R (1980) J Antimicrob Chemother 6:577
35. Majewska AC, Kasprzak W, De Jonckheere JF, Kaczmarek E
(1991) Trans R Soc Trop Med Hyg 85:67
36. National Committee for Clinical Laboratory Standards (2003)
Performance standards for antimicrobial susceptibility testing.
NCCLS, Wayne
ˇ
30. Fathalla W, Cajan M, Pazdera P (2004) Molecules 5:1210
ˇ
31. Fathalla W, Cajan M, Pazdera P (2001) Molecules 6:557
`
32. Salerno L, Siracusa M, Guerrera F, Romeo G, Pittala V, Modica M,
Mennini T, Russo F (2004) Arkivoc v:312
123