Synthesis and in vitro antimicrobial evaluation of 5-(1-methyl-5-nitro-2-imidazolyl)-4H-1,2,4-triazoles

Article abstract of DOI:10.1002/ardp.200290004

The increasing clinical importance of drug-resistant pathogens has lent additional urgency to antimicrobial research. Various 5-(1-methyl-5-nitro-2-imidazolyl)-4H-1,2,4-triazoles (4a-6f) were synthesized and tested in vitro for their antibacterial and antifungal activities. Compounds 4a and 4b exhibited significant effects against Bacillus subtilis at MIC ranges of 0.5-1 μg/mL and moderate effects against Staphylococcus aureus.

Full text of DOI:10.1002/ardp.200290004

Arch. Pharm. Pharm. Med. Chem. 2002, 10, 495–499
5-(1-Methyl-5-nitro-2-imidazolyl)-4H-1,2,4-triazoles 495
Abbas Shafiee,
Abolfazl Sayadi,
Synthesis and in vitro Antimicrobial Evaluation of
5-(1-Methyl-5-nitro-2-imidazolyl)-4H-1,2,4-triazoles
Marjan Hosseini Roozbahani,
Alireza Foroumadi,
Fatemeh Kamal
Department of Medicinal
Chemistry,
Faculty of Pharmacy,
Tehran University of Medical
Sciences, Tehran, Iran
The increasing clinical importance of drug-resistant pathogens has lent additional
urgency to antimicrobial research. Various 5-(1-methyl-5-nitro-2-imidazolyl)-4H-
1,2,4-triazoles (4a–6f) were synthesized and tested in vitro for their antibacterial
and antifungal activities.Compounds 4a and 4b exhibited significant effects against
Bacillus subtilis at MIC ranges of 0.5–1 µg/mL and moderate effects against
Staphylococcus aureus.
Keywords: 1,2,4-Triazoles; 5-Nitroimidazoles; Antimicrobial activity
Received: March 25, 2001 [FP686]
Considering these two small active heterocycles having
potential antimicrobial effects, in continuation of our pre-
Introduction
The considerable biological importance of imidazoles
and triazoles has stimulated much work on these hetero-
cycles [1–5]. Traditionally, small molecules have been a
reliable source for discovering novel biologically active
compounds. Compared to their natural counterparts
with complex structures, these molecules are easily syn-
thesized and their structural optimization would usually
lead to a feasible candidate compound. Here we consid-
er two heterocycles 5-nitroimidazole and 4H-1,2,4-tri-
azole as the small effective molecules.
vious research on the synthesis and biological evalua-
tion of small bioactive heterocyclic molecules [13–14] a
new series of 5-(1-methyl-5-nitro-2-imidazolyl)-4H-
1,2,4-triazoles were synthesized and their antimicrobial
properties were evaluated.
Chemistry
5-(1-Methyl-5-nitro-2-imidazolyl)-4H-1,2,4-triazoles
were synthesized for their possible antimicrobial effect.
The introduction of nitroheterocyclic drugs in the late
1950s and the 1960s heralded a new era in the treatment
of infections caused by gram-negative and gram-positive
bacteria and a range of pathogenic protozoan parasites.
The antibiotic Azomycin (a 2-nitroimidazole) isolated in
Japan from a streptomycete, was the first active nitroim-
idazole to be discovered and acted as the main impetus
for the systematic search for drugs with activity against
anaerobic protozoa [6]. Now nitroimidazoles are impor-
tant drugs because of their antibacterial, antifungal, anti-
protozea, antihelmitic, and anticancer effects [7, 8]. Pre-
vious studies showed that the best and simplest struc-
ture for this heterocycle is 1-alkyl-5-nitroimidazole offer-
ing no steric hindrance for the nitro group [9]. Substitu-
tion in 1 and 2 position increases the effect of com-
pounds [10].
1-Methyl-5-nitroimidazole-2-carboxylic acid hydrazide
(2) was prepared from the reaction of hydrazine hydrate
with methyl 1-methyl-5-nitroimidazole-2-carboxylate (1)
in ethanol. In order to prepare different thiosemicar-
bazides, compound 2 was allowed to react with various
isothiocyanates in THF. The aromatic substituted isothio-
cyanate needed more reflux time to complete the reac-
tion (R1 = methyl, ethyl: 2 h, R1 = phenyl: 4 h).The cycli-
zation of compounds 3 was accomplished in aqueous
media in the presence of sodium bicarbonate.The sub-
stitution on the thiosemicarbazide played an important
role in the cyclization step. Namely, the effort to cyclize
the semicarbazide with an aromatic group (compound
3c) was unsuccessful. Reaction of compounds 4 with
alkyl halides afforded compounds 5. In the alkyation re-
actions, the steric hindrance of alkyl group played an im-
portant role in affecting the time of completion of the re-
action (R2 = methyl, ethyl: 12 h, R1 = propyl: 40 h, R2 =
isopropyl:72 h).Oxidation of substituted thio derivatives
using MCPBA in dichloromethane afforded the corre-
sponding sulfone 6.
In addition, the triazole nucleus has a broad-spectrum of
antimicrobial activity [11].Many alkylthio- or alkylsulfonyl
substitued triazole analogues have been reported to exi-
hibit excellent efficacy in in vitro animal models of candi-
diasis and aspergillosis [12].
Correspondence: Abbas Shafiee, Department of Medicinal
Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences. P.O.Box: 14155-6451, Tehran, Iran. Phone:
+98 21 6406757, Fax: +98 21 6461178, e-mail:
ashafiee@ams.ac.ir.
Results and discussion
In order to obtain novel chemotherapeutic heterocyclic
cores, we synthesized new 5-(1-methyl-5-nitro-2-imid-

496 Shafiee et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 495–499
Acknowledgments
This work was partially supported by a grant from the re-
search council of the Tehran University of Medical Sci-
ences.
Experimental part
Materials
Most chemicals were purchased from Aldrich Chemical Com-
pany, Deisenhofen Germany. Methyl 1-methyl-5-nitroimid-
azole-2-carboxylate was prepared from 2-hydroxymethyl-1-
methyl-5-nitroimidazole according to a previously reported
method [15].
The 1H-NMR spectra were obtained using a Bruker FT-80 or a
Varian unity plus 400 MHz instrument, with tetramethylsilane
as the internal standard. The solvents used were CDCl3 and
CF3COOD. IR spectra were taken on a Perkin-Elmer 781 (KBr
disks). UV spectra were obtained using a Perkin-Elmer model
550 SE. Mass Spectra were taken using a Finnigan TSQ 70 at
70 eV. Melting points were determined on a Kofler hot stage
microscope. Elemental microanalyses were within ±0.4% of
theoretical value for C, H, and N.
1-Methyl-5-nitroimidazole-2-carboxylic acid hydrazide (2)
To a stirred mixture of 2.25 g (50 mmole) methyl 1-methyl-5-ni-
troimidazole-2-carboxylate in ethanol (130 mL), 3.21 g (3 mL)
hydrazine hydrate was added dropwise. After a few minutes,
the crystals obtained were filtered off and washed with hexane
to give 8.7 g (47 mmole) of the hydrazide 2, pure enough to be
1
used in the next reaction; 25%, mp = 144–145°C. – H NMR
(CDCl3, CF3COOD): δ 8.08 (s, 1H, H4 imidazole) and 4.44 (s,
3H, N1-CH3).
1-(l-Methyl-5-nitroimidazole-2-carbonyl)-4-methylthiosemi-
carbazide (3a)
Compound 2 (4 g, 22 mmole), was added to a stirred solution of
1.6 g (22 mmole) methylisothiocyanate (1.6 g, 22 mmole) in
anhydrous THF (100 mL).The mixture was refluxed for 2 hours
and stirred overnight at room temperature.The mixture was fil-
tered and hexane was added to the filtrate to obtain more pre-
cipitate. Total precipitate was crystallized from THF-hexane to
Scheme 1. Synthesis of substituted 5-(1-methyl-5-nitro-
2-imidazolyl)triazoles.
1
give 4.86 g (18.8 mmole) of 3a: 85%, mp = 196–198°C. – H
NMR (CDCl3, CF3COOD): δ 8.07 (s, 1H, H4 imidazole), 4.40 (s,
3H, N4-CH3 imidazole), 3.15 (s, 3H, CH3).
azolyl)-1,2,4-triazoles that showed antibacterial activi-
ties on some pathogenic bacterial organisms. Com-
pounds 4a and 4b had a significant inhibitory effect on
Bacillus subtilis in MIC ranges of 0.5–1 µg/mL (com-
pared to MIC 0.5 µg/mL for gentamycin), whereas their
activities were moderate against Staphylococcus au-
reus and weak against the other bacteria tested.The oth-
er compounds exhibited weak effects against the bacte-
ria tested. Compared to miconazole none of the com-
pounds exerted a significant effect against the fungi
Candida albicans and Aspergillus niger.The detailed da-
ta are tabulated in Table1. It seems that in these com-
pounds, alkylation and subsequent oxidation of the mer-
capto group, in general, decreases the antimicrobial
properties.
1-(l-Methyl-5-nitroimidazole-2-carbonyl)-4-ethylthiosemi-
carbazide (3b)
This compound was prepared similar to 3a in 88% yield, mp =
129–131°C. – IR (KBr): ν cm–1 3360, 3250 (N-H), 3120 (C4-H
imidazole), 1710 (C=O), 1550, 1370 (NO2), 1225 (C=S). – 1H-
NMR (CDCl3, CF3COOD): δ 8.04 (s, 1H, H4 imidazole), 4.42 (s,
3H, N-CH3), 3.63 (q, 2H, J = 7.3 Hz, N-CH2), 1.27 (t, 3H, J =
7.3 Hz, CH3).
1-(1-Methyl-5-nitro-2-imidazolyl)-4-phenylthiosemicarbazide
(3c)
This compound was prepared in a similar manner to 3a. The
reflux time was 4 hours and after stirring overnight the solution
was concentrated at reduced pressure and the residue was
crystallized from ethanol to give 3c in 90% yield, mp = 150–
152°C. – 1H NMR (CDCl3, CF3COOD): δ 8.13 (s, 1H, H4 imida-
zole), 7.55–7.29 (m, 5H, phenyl), 4.43 (s, 3H, N-CH3).

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 495–499
5-(1-Methyl-5-nitro-2-imidazolyl)-4H-1,2,4-triazoles 497

498 Shafiee et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 495–499
Table 2. Physical constants for compounds 5 a–5 f.
No.
R1
R2
Time Yield
Mpa (°C)
1H NMR (CDCl3)
(h)
12
12
40
(%)
5 a
5 b
5 c
CH3
CH3
CH3
CH3
67
183–6
140–2
115–7
8.13 (s, 1 H, H4 imidazole), 4.35 (s, 1 H, N-CH3 imid-
azole), 4.01 (s, 1 H, N-CH3 triazole), 2.92 (2, 3 H, S-CH3).
8.09 (s, 1 H, H4 imidazole), 4.47 (s, 3 H, N-CH3 imid-
azole), 3.38 (q, 2 H, S-CH2), 1.59 (t, 3 H, CH3).
8.09 (s, 1 H, H4 imidazole), 4.47 (s, 3 H, N-CH3 imid-
azole), 3.92 (s, 3 H, N-CH3 triazole), 3.35 (t, 2 H, S-CH2),
1.86 (m, 2 H, CH2), 1.08 (t, 3 H, CH3).
C2H5
C3H7
52
69.3
5 d
5 e
5 f
CH3
CH3
C2H5
i-C3H7
C6H5CH2
CH3
72
12
12
63
55
82
118–20 8.09 (s, 1 H, H4 imidazole), 4.47 (s, 3 H, NCH3 imid-
azole), 4.00 (heptet, 1 H, S-CH), 3.93 (s, 3 H, NCH3 tria-
zole), 1.50 (d, 6 H, CH3).
161–3
8.23 (s, 1 H, H4 imidazole), 7.36 (s, 5 H, phenyl), 4.68 (s,
2 H, S-CH2), 4.29 (s, 3 H, N-CH3 imidazole), 3.91 (s, 3 H,
N-CH3 triazole).
166–8
8.08 (s, 1 H, H4 imidazole), 4.47 (s, 3 H, N-CH3 imida-
zole), 4.43 (q, 2 H, N-CH2), 2.83 (s, 3 H, S-CH3), 1.40 (t,
3 H, CH3).
a
All compounds were crystallized from ethyl acetate.
Table 3. Physical constants for compounds 6 a–6 f.
No.
R1
R2
Yield Mpa (°C)
(%)
1H NMR (CDCl3)
6 a
6 b
CH3
CH3
CH3
92
66
200–2
127–9
8.17 (s, 1 H, H4 imidazole), 4.39 (s, 3 H, N-CH3 imidazole), 4.27 (s,
3 H, N-CH3 triazole), 3.59 (s, 3 H, SO2CH3).
8.13 (s, 1 H, H4 imidazole), 4.46 (s, 3 H, N-CH3 imidazole), 4.32 (s,
3 H, N-CH3 triazole), 3.75 (q, 2 H, J = 7.3 Hz, SO2-CH2), 1.58 (t,
3 H, J = 7.3 Hz, CH3).
C2H5
6 c
CH3
C3H7
94
150–2
8.12 (s, 1 H, H4 imidazole), 4.46 (s, 3 H, N-CH3 imidazole) 4.31 (s,
3 H, N-CH3 triazole), 3.70 (t, 2 H, J = 7.2 Hz, SO2-CH2), 2.04 (m,
2 H, CH2), 1.16 (t, 3 H, J = 7.2 Hz, CH3).
6 d
6 e
6 f
CH3
CH3
C2H5
i-C3H7
C6H5CH2
CH3
65
80
85
166–9
156–8
185–7
8.13 (s, 1 H, H4 imidazole), 4.46 (s, 3 H, NCH3 imidazole), 4.32 (s,
3 H, NCH3 triazole), 4.05 (heptet, 1 H, SO2CH), 1.56 (d, 6 H, CH3).
8.08 (s, 1 H, H4 imidazole), 7.35 (s, 5 H, phenyl), 4.86 (s, 2 H, SO2-
CH2), 4.33 (s, 3 H, N-CH3 imidazole), 3.84 (s, 3 H, N-CH3-triazole).
8.19 (s, 1 H, H4 imidazole), 4.76 (q, 2 H, SO2-CH2), 4.33 (s, 3 H,
N-CH3 imidazole), 3.60 (s, 3 H, N-CH3 triazole), 1.51 (t, 3 H, CH3).
a
All compounds were crystallized from ethanol.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 495–499
5-(1-Methyl-5-nitro-2-imidazolyl)-4H-1,2,4-triazoles 499
Antibacterial and antifungal assay
5-(1-Methyl-5-nitro-2-imidazolyl)-4-methyl-2,4-dihydro-3H-
1,2,4-triazole-3-thione (4 a)
Agar dilution method was used to determine the antimicrobial
potency of the compounds [16].The test was done as duplicate
and the lowest concentration of compound that allowed no
more growth compared to solvent control was the MIC.The mi-
croorganisms used were Staphylococcus aureus ATCC 6538
and ATCC 29737; Staphylococcus epidermidis ATCC 12228,
Streptococcus fecalis ATCC 29212, Bacillus subtilis ATCC
6633, Escherichia coli ATCC 35218, Klebsiella pneumonia
ATCC 33495, Pseudomonas aeruginosa ATCC 27853, Salmo-
nella paratiphi B (Local) and Proteus vulgaris ATCC 8427, Fun-
gi used were Candida albicans ATCC 10231, and Aspergillus
niger ATCC 16404.
Compound 3 a (4.65 g, 18 mmole) was dissolved in 80 mL of
1.5 M aqueous solution of sodium bicarbonate while heating and
stirring. The mixture was refluxed 5 h and then cooled to room
temperature and filtered. The precipitate was filtered and the fil-
trate was acidified with concentrated hydrochloric acid.The pre-
cipitate was filtered. The total precipitate was crystallized from
ethanol to give 3.48 g (14.5 mmole) of 4 a: 81%, mp = 234–
237 °C, UV: λmax = 256 nm (log ε = 4.12). – IR (KBr): ν cm–1 3100
(C4-H imidazole), 2920 (C-H aliph.), 1540 and 1365 cm–1 (NO2).–
1H NMR (CDCl3, CF3COOD):δ = 8.18 (s, 1 H, H4 imidazole), 4.32
(s, 3 H, N-CH3 imidazole) 3.96 ppm (s, 3 H, N-CH3 triazole).– MS:
m/e (%) = 240 (M+, 100), 210 (75), 207 (18), 195 (20), 180 (31),
168 (29), 153 (89), 141 (40), 140 (28), 120 (16), 107 (38), 81 (18),
80 (46), 68 (34), 60 (43), 55 (61) and 42 (50).
Compounds 4 a–6 f were dissolved in DMSO to make a con-
centration of 2560 µg/mL and diluted in a two-fold manner to
give less concentrations.The plates were incubated at 35 °C for
16–20 h.
5-(1-Methyl-5-nitro-2-imidazolyl)-4-ethyl-2,4-dihydro-3H-1,2,4-
triazole-3-thione (4 b)
This compound was prepared in a similar manner to 4 a in 80%
yield. The reflux time was 3 h, mp = 203–205 °C (ethanol), UV
(C2H5OH): λmax = 269 nm (log ε = 3.84). – 1H NMR (CDCl3,
CF3COOD):δ8.12 (s, 1 H, H4 imidazole), 4.61 (q, 2 H, J = 7 Hz, N-
CH2), 4.33 (s, 3 H, N-CH3) and 1.42 (t, 3 H, J = 7 Hz, CH3). – MS:
m/e (%) = 254 (M+, 100), 239 (30), 227 (48), 225 (79), 221 (85),
208 (62), 198 (35), 197 (75), 180 (74), 167 (79), 166 (35), 153
(86), 141 (69), 139 (75), 128 (60), 109 (57), 107 (67), 97 (36), 85
(31), 82 (50), 69 (79), 60 (46), 55 (71), and 42 (82).
References
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4-Methyl-5-(1-methyl-5-nitro-2-imidazolyl)-3-methylthio-4H-
1,2,4-triazole (5 a)
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To a stirred mixture of 4 a (1.56 g, 6.5 mmole) in one molar aque-
ous solution of sodium bicarbonate (25 mL), methyl iodide (3.7 g,
26 mmole) in ethanol 2 mL was added. After stirring overnight at
room temperature, the reaction mixture was concentrated at re-
duced pressure and the residue was crystallized from ethyl ace-
tate to give 1.1 g (4.33 mmol) of 5 a: 67%, mp = 183–186 °C, UV
(C2H5OH) λmax = 338 nm (log ε = 3.94). – IR (KBr): ν cm–1 3120
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1
[7] J. Heyes, N. Ward, Ger. Offen. 1972, 2, 142, 832, Chem.
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(C4-H imidazole), 1530 and 1350 (NO2). – H NMR (CDCl3): δ
8.13 (s, 1 H, H4 imidazole), 4.35 (s, 1 H, N-CH3 imidazole), 4.01
(s, 3 H, N-CH3 triazole), 2.92 (s, 3 H, S-CH3). – MS: m/e (%) 254
(M+, 100), 225 (46), 224 (98), 209 (35) 208 (46), 207 (19), 194
(62), 167 (95), 155 (83), 153 (81), 110 (39), 107 (41), 91 (70), 88
(58), 82 (50), 80 (75), 67 (83), 54 (76) and 42 (80).
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Compounds 5 b–5 f were prepared similarly (Table 2).
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Tchelitcheff, R. Vaupre, Arzneim. Forsch. 1966, 16,
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4-Methyl-5-(1-methyl-5-nitro-2-imidazolyl)-3-methylsulfonyl-4H-
1,2,4-triazole (6 a)
m-Chloroperbenzoic acid (1.45 g, 8.4 mmole, MCPBA) was ad-
ded in one portion to a stirring mixture of 5 a (0.53 g, 2.1 mmol) in
dichloromethane (16 mL). The stirring was continued overnight.
Dichloromethane (32 mL) was added. The organic layer was
washed with saturated sodium bicarbonate solution (2 times),
saturated saline solution (one time) and dried over anhydrous so-
dium sulfate.The organic layer was then concentrated in vacuum
and the residue was crystallized from ethanol to give 0.556 g
(1.94 mmole) of 6 a: 92%, mp = 200–202 °C, UV (C2H5OH): λmax
= 311 nm (log ε = 3.85).– IR (KBr):ν cm–1 3120 (C4-H imidazole),
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1
1530, 1370 (NO2) 1320 and 1150 (SO2). – H NMR (CDCl3): δ
8.17 (s, 1 H, H4 imidazole), 4.39 (s, 3 H, N-CH3 imidazole), 4.27
(s, 3 H, N-CH3 triazole), 3.59 (s, 3 H, SO2CH3).– MS:m/e (%) 286
(M+, 41), 256 (100), 200 (65), 187 (71), 153 (54), 107 (25), 106
(27), 91 (13), 80 (46), 79 (57), 69 (33), 68 (49), 55 (41) and 42
(45).
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Compounds 6 b–6 f were prepared similarly (Table 3).