Synthesis and anti-helicobacter pylori activity of (4-nitro-1- imidazolylmethyl)-1,2,4-triazoles, 1,3,4-thiadiazoles, and 1,3,4-oxadiazoles

Article abstract of DOI:10.3906/kim-1004-522

A series of [(4-nitro-1 H-imidazol-1-yl) methyl]-1,2,4-triazoles and 1,3,4-thiadiazoles were prepared and evaluated for their activity against sensitive and resistant H. pylori strains. Study of the structure-activity relationship of these series of compounds indicated that the type of nitroimidazole moiety and the pendent group on the heteroaryl analog dramatically impact the anti-H. pylori activity. In triazole series, compound 5d, containing a 4-methyl phenyl group on the triazole ring, was the most potent compound tested against both metronidazole-sensitive and-resistant strains at a concentration of 8 μg. TUeBITAK.

Full text of DOI:10.3906/kim-1004-522

Turk J Chem
35 (2011) , 307 – 316.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1004-522
Synthesis and anti-Helicobacter pylori activity of
(4-nitro-1-imidazolylmethyl)-1,2,4-triazoles,
1,3,4-thiadiazoles, and 1,3,4-oxadiazoles
Asal FALLAH TAFTI1, Tahmineh AKBARZADEH1, Parastoo SANIEE2,
Farideh SIAVOSHI2, Abbas SHAFIEE1, Alireza FOROUMADI1,∗
1Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran, 14176, IRAN
2Microbiology Department, Faculty of Sciences, University of Tehran,
Tehran-IRAN
e-mail: aforoumadi@yahoo.com
Received 07.04.2010
A series of [(4-nitro-1H -imidazol-1-yl)methyl]-1,2,4-triazoles and 1,3,4-thiadiazoles were prepared and
evaluated for their activity against sensitive and resistant H. pylori strains. Study of the structure-activity
relationship of these series of compounds indicated that the type of nitroimidazole moiety and the pendent
group on the heteroaryl analog dramatically impact the anti-H. pylori activity. In triazole series, compound
5d, containing a 4-methyl phenyl group on the triazole ring, was the most potent compound tested against
both metronidazole-sensitive and -resistant strains at a concentration of 8 μg.
Key Words: 4-Nitroimidazole, 1,2,4-triazoles, 1,3,4-thiadiazoles, 1,3,4-oxadiazoles, anti-H. pylori activity
Introduction
It is well known that Helicobacter pylori, an s-shaped spiral microaerophilic gram-negative bacterium first
isolated in human gastric mucosa in 1982,1−3 is considered the major causative agent of several gastric
pathologies, such as chronic active gastritis, peptic ulcer disease, and gastric cancer.4−6
Hence, the World Health Organization (WHO) has classified H. pylori as a Class 1 carcinogen in humans;
the eradication of H. pylori can significantly reduce the risk of ulcer relapse and may help prevent corresponding
∗Corresponding author
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Synthesis and anti-Helicobacter pylori activity of..., A. FALLAH TAFTI, et al.,
gastric disorders.7 Since oral administration of metronidazole, PPI, clarithromycin, and amoxicillin was put to
use, infection can be cured in up to 80%-90% of cases. However, eradication is not always successful and a few
problems have been observed in the use of these drugs, such as the emergence of drug resistance, especially
against metronidazole, clarithromycin, and amoxicillin,8 and low compliance9 related to the occurrence of a
number of harmful side effects.10−11 Nitroimidazole compounds, especially metronidazole, have been frequently
used in treatment regimens for H. pylori infection; moreover, the antimicrobial properties of 1,2,4-triazole, 1,3,4-
oxadiazole, and 1,3,4-thiadiazole derivatives are well documented. Their attachment with other heterocycles
often ameliorates the bioresponses, depending on the type of substituent and the position of attachment.12−16
In continuation of our research on nitroheterocyclic compounds as anti-H. pylori agents,17−20 we decided
to synthesize a new series of (4-nitroimidazol-1-ylmethyl)-1,2,4-triazoles (4a-e and 5a-l), 1,3,4-thiadiazoles (6a-
h), and 1,3,4-oxadiazoles (7a-d) and evaluate their activity against clinical isolates of H. pylori strains.
Experimental
Melting points were determined on a Kofler hot-stage microscope and were corrected. The 1 H-NMR spectra
were obtained using Bruker FT-80 or Varian Unity Plus 400 instruments with tetramethylsilane as the internal
standard. The solvents used were CDCl3 and DMSO-d6. IR spectra were recorded on a Nicolet Magna FTIR
550 spectrometer (KBr disks). Mass spectra were taken using a Finnigan TSQ-70 at 70 eV. Elemental analyses
were carried out on a CHN-O-rapid elemental analyzer (Heraeus GmbH, Hanau, Germany) for C, H, and N,
and the results were within 0.4% of the theoretical values. Liarre Starsonic ultrasonic cleaner (Italy) was used
as an ultrasonic device with a generator variable frequency of 28-34 KHz and 100 W of power line heating.
2-(4-Nitro-1H-imidazol-1-yl)acetohydrazide (2a)
Hydrazine hydrate (15 mL) was added dropwise to a stirred solution of 1a (12 g, 0.06 mol) in methanol (40 mL)
in an ice-water bath. The mixture was stirred at the same temperature for 2 h. The precipitate was filtered
and recrystallized from methanol to give 10.4 g (83%) of 2a; mp 122-123 ◦ C. IR (KBr): ν = 3345, 3300, 3124
(NH-NH2), 1660 cm−1 (C=O). 1 H-NMR (CDCl3): δ 8.27 (d, J = 1.2 Hz, H-5 imidazole), 7.77 (d, J = 1.2
Hz, 1H, H-2 imidazole), 4.76 ppm (s, 2H, N-CH2). MS m/z (%): 185 (M+ , 78), 154 (44), 139 (75), 127 (100),
114(18), 110 (12). Anal. Calcd. for C5 H7 N5 O3 ; C, 32.44; H, 3.81; N, 37.83. Found C, 32.49; H, 3.79; N,
37.56.
2-(2-Methyl-4-nitro-1H-imidazol-1-yl)acetohydrazide (2b)
This compound was prepared in a similar manner to 2a with 87% yield. Mp 190-192 ◦ C (methanol). IR (KBr):
ν = 3343, 3269, 3151 (NH-NH2), 1695 cm−1 (C=O). 1 H-NMR (CDCl3): δ 8.24 (s, 1H, H-5 imidazole), 4.70
(s, 2H, N-CH2), 2.33 ppm (s, 3H, CH3). MS m/z (%): 199 (M+ , 90), 168 (85), 152 (37), 141 (100), 124 (18),
81 (9). Anal. Calcd. for C6 H9 N5 O3 ; C, 36.18; H, 4.55; N, 35.16. Found C, 36.50; H, 4.77; N, 35.07.
308

Synthesis and anti-Helicobacter pylori activity of..., A. FALLAH TAFTI, et al.,
General procedure for the synthesis of 4-alkyl(aryl)-5-[(4-nitro-1H-imidazol-1-yl)
methyl]-4H-1,2,4-triazole-3-thiol (4a-d)
To a stirred solution of compound 2 (0.01 mol) in ethanol (20 mL), NaOH (2 N, 5 mL) and alkyl(aryl)isothiocya-
nate (0.01 mol) were added. The mixture was stirred for 2 h at room temperature and then acidified with an
aqueous solution of HCl in an ice bath. The mixture was kept in a refrigerator for 3 h to complete the
precipitation. The precipitate was filtered and crystallized from ethanol to give compounds 3a-m, which were
pure enough to be used in the next reaction. Compound 3 (0.01 mol) was dissolved in 10 mL of an aqueous
solution of sodium bicarbonate (5%) while being heated and stirred. The mixture was refluxed for 2.5 h, cooled
to room temperature, and acidified with an aqueous solution of HCl in an ice bath. The precipitate was filtered
and crystallized from ethanol to give 4a-d.
The chemical and physical properties of compounds 4a-d are given in Table 1.
General procedure for the synthesis of 4-alkyl(aryl)-5-[(4-nitro-1H-imidazol-1-yl)
methyl]-3-methylthio-1,2,4-triazoles (5b-i)
To a mixture of compound 4 (1.6 mmol) in ethanol (1 mL), 0.83 mL of an aqueous solution of sodium hydroxide
(10%) was added; it was kept in ultrasonic conditions for 1 min. Methyl iodide (0.15 mL, 2.4 mmol) was added
to the solution and the reaction was continued for 20 min. The precipitate was filtered and crystallized from
ethanol to give compounds 5b-i.
The chemical and physical properties of compounds 5b-i are presented in Table 1.
General procedure for the synthesis of 5-alkyl(aryl)amino-2-[(4-nitro-1H-imidazol-
1-yl)methyl]1,3,4-thiadiazoles (6a-g)
A mixture of compound 3 (0.6 mmol) and concentrated H2 SO4 (1 mL) was stirred overnight at room temper-
ature, poured into ice-cold water, and neutralized with liquid ammonia, and the resulting solid was crystallized
from methanol to give compounds 6a-g.
The chemical and physical properties of compounds 6a-g are shown in Table 1.
The synthesis and physicochemical properties of compounds 4b, 4c, 4e, 5a, 5f, 5g, 5j, 5k, 5l, 6d, 6h,
and 7a-d were reported previously.17
Biological activity
Bacterial isolates and culture conditions
Clinical H. pylori isolates from gastric biopsy specimens were obtained from Shariati Hospital (Tehran, Iran).
Primary isolation was performed on a selective blood agar base (Oxoid, Basingstoke, UK) supplemented with
horse blood (5%, v/v) and 1 Selectatab tablet (500 mg; Mast Diagnostics, Merseyside, UK). Following primary
selective isolation, H. pylori bacteria cells were identified according to colony morphology, Gram staining,
microaerophilic growth at 37 ◦ C, oxidase +, catalase +, urease +, nitrate -, H2 S, and hippurate hydrolysis
309

Synthesis and anti-Helicobacter pylori activity of..., A. FALLAH TAFTI, et al.,
310

Synthesis and anti-Helicobacter pylori activity of..., A. FALLAH TAFTI, et al.,
311

Synthesis and anti-Helicobacter pylori activity of..., A. FALLAH TAFTI, et al.,
Y
Y
Y
N
O2N
O
N
O
H
N
N
H
N
O
ii
i
Et
N
N
NH2
N
O
N
H
R1
N
H
O2N
O2N
S
3 a-m
1 a-b
2 a-b
Y
N
N
N
O2N
iv , v
N
Me
S
Y
N
N
N
N
iii
R1
5 a-k
N
SH
N
O2N
Y
N
N
R1
iv , vi
N
O2N
4 a-e
N
Et
3
S
N
5l
Y
R1
N
N
N
N
vii
NHR1
S
O2N
6 a-h
Y
N
N
N
N
viii , ix or x
2
R2
O
O2N
7 a-d
Scheme. Reagents: i) NH2 NH2 .H2 O, ii) R1 N=C=S, iii) Na2 CO3 , iv) NaOH, v) MeI, vi) EtI, vii) H2 SO4 , viii)
CS2 , ix) KOH, x) BrCN; Y=H, CH3 ; R1 = Et, n-Bu, C6 H11 , C6 H5 , 4-CH3 -C6 H4 , 4-OCH3 -C6 H4 , 4-F-C6 H4 ,
4-NO2 -C6 H4 . 7a-b: R2 = SH; 7c-d: R2 = NH2 . 3a: R1 = C2 H5 , Y=H; 3b: R1 = n-Bu, Y=H; 3c: R1 = n-Bu,
Y=CH3 ; 3d: R1 = C6 H11 , Y=H. 3e: R1 = C6 H5 , Y=H; 3f: R1 = 4-CH3 -C6 H4 , Y=H; 3g: R1 = 4-CH3 -C6 H4 ,
Y=CH3 . 3h: R1 = 4-OCH3 -C6 H4 , Y=H; 3i: R1 = 4-OCH3 -C6 H4 , Y=CH3 ; 3j: R1 = 4-F-C6 H4 , Y=H. 3k: R1
=
4-F-C6 H4 , Y=CH3 ; 3l: R1 = 4-NO2 -C6 H4 , Y=H; 3m: R1 = 4-NO2 -C6 H4 , Y=CH3 .
-. Growth of H. pylori was maintained at 37 ◦ C for 3-5 days in an atmosphere of 5% O2 , 15% CO2 , and
80% N2 in an anaerobic chamber (Hirayama, Tokyo, Japan). To maintain a moist atmosphere, a moist paper
towel was placed in the chamber. Bacterial strains were stored at -70 ◦ C in brain heart infusion broth (BHIB)
(Difco, East Molesey, UK) containing 10% (v/v) fetal calf serum (FCS) and 15% (v/v) glycerol. Frozen clinical
isolates were thawed and inoculated on Mueller-Hinton agar (MHA) plates (Oxoid), supplemented with 10%
312

Synthesis and anti-Helicobacter pylori activity of..., A. FALLAH TAFTI, et al.,
horse blood and incubated under microaerophilic conditions. Given the importance of inoculum homogeneity,
cellular viability was controlled microscopically by morphological observation with Gram staining. In order to
control the proportions of coccoid cells in the cultures, cultures were always used after 48 h of incubation, when
they generally did not present coccoid forms. Bacterial growth was taken from the plates and resuspended
in sterile saline. The inoculum was prepared to contain 5 × 107 CFU/mL by adjusting the turbidity of the
suspension to match the McFarland No. 2 standard.
Bacterial growth inhibition assay (disk diffusion method)
Growth inhibition was performed by the filter paper disk diffusion method on selective Brucella agar with
7% defibrinated horse blood under microaerophilic conditions at 37 ◦ C. The samples were evaluated for
their anti-Helicobacter activity, dissolved in dimethylsulfoxide (DMSO). All compounds were assayed against
metronidazole-sensitive and metronidazole-resistant H. pylori strains at 3 concentrations (8, 16, and 32 μg/disk);
the surfaces of the Brucella blood agar plates were inoculated with 100 μL of bacterial suspensions. Blank stan-
dard disks (6 mm in diameter) were deposited on the plates and impregnated with 10 μL of different dilutions
of test compounds. Following incubation for 3-5 days at 37 ◦ C, the inhibition zone around each disk (aver-
age diameter), if any, was recorded. The control disks received 10 μL of DMSO. All tests were performed in
triplicate and the antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by
the tested compounds. The antibacterial activity was classified as follows: strong response, zone diameter >20
mm; moderate response, zone diameter 16-20 mm; weak response, zone diameter 11-15 mm; and little or no
response, zone diameter <10 mm.21
Result and discussion
Chemistry
The title compounds were synthesized according to the sequence shown in the Scheme. As we have reported
previously, compounds 1a-b were prepared by a known procedure.17 Reaction of the ester group of compounds
1a-b with hydrazine hydrate gave corresponding acetic acid hydrazides 2a-b. Treatment of compounds 2a-
b with different isothiocyanates gave thiosemicarbazide 3, which, on cyclization with aqueous Na2 CO3 or
concentrated H2 SO4 , afforded 1,2,4-triazole-5-thiones 4a-e or 5-amino-l,3,4-thiadiazoles 6a-h, respectively.
The reaction of compounds 4a-e with alkyl iodide in alkaline media under ultrasonic conditions produced
alkylthio derivatives 5a-l.
Anti-Helicobacter pylori activity
The preliminary evaluation of compounds 4-7 against 2 metronidazole-sensitive and metronidazole-resistant
H. pylori strains are summarized in Table 2. Some of the synthesized triazole analogs (5d, 5g), one of the
thiadiazole analogs (6g), and one of the oxadiazole derivatives (7d) exhibited strong antimicrobial activity
against both metronidazole-sensitive and metronidazole-resistant H. pylori strains at concentrations of 16 and
32 μg/disk (inhibition zone diameter: >20 mm). Compound 5d was the most potent compound tested,
displaying strong activity at 8 μg/disk (inhibition zone diameter: 23 mm) against both metronidazole-sensitive
and metronidazole-resistant strains.
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Synthesis and anti-Helicobacter pylori activity of..., A. FALLAH TAFTI, et al.,
314

Synthesis and anti-Helicobacter pylori activity of..., A. FALLAH TAFTI, et al.,
Generally, the type of nitroimidazole moiety and the pendent group on the heteroaryl analog impact
the anti-H. pylori activity. In triazole series, substitution of an aromatic ring at the N4 position of the 1,2,4-
triazole ring with phenyl (5c), 4-methylphenyl (5d), 4-methoxyphenyl (5g), and 4-flurorophenyl (5h) resulted
in compounds with potent anti-H. pylori activity. Replacement of the aryl group at the N4 position with
aliphatic moiety eliminated the antibacterial activity, except for compound 4a having an ethyl group in that
position.
Methylation of the thiol group in compound 4e, having 4-nitrophenyl moiety at the N4 position, resulted
in compound 5j with activity against metronidazole-resistant strains. Replacement of methyl with an ethyl
group (compound 5l) increased anti-H. pylori activity against both metronidazole-sensitive and metronidazole-
resistant strains. The same result was obtained when the thiol analog 4d was methylated (compound 5h).
In 1,3,4-thiadiazole series, substitution of the amino group at the 5 position with alkyl moiety resulted
in compounds without anti-H. pylori activity. However, substitution with 4-methylphenyl moiety resulted in
compound 6g, which showed a potent inhibitory effect against both metronidazole-sensitive and metronidazole-
resistant strains.
Compound 7d, with a 5-amino group on the oxadiazole ring, was the most potent compound in this
series, which may show the superiority of the amino group to the thiol group in this position.
As can be seen in Table 2, compounds 5j, 6g, and 7d showed better activity against resistant strains of
H. pylori compared to sensitive strains, which possibly reveals the interference of the mentioned compounds in
the resistance mechanism.
In conclusion, we identified a series of (4-nitro-1-imidazolylmethyl)-1,2,4-triazoles, 1,3,4-thiadiazoles, and
1,3,4-oxadiazole with in vitro anti-H. pylori activity. Biological data indicated that triazole analog 5d, contain-
ing 4-methylphenyl and methylthiol moiety, was the most potent compound tested, which makes this compound
a promising lead for the development of an effective therapeutic agent for anti-Helicobacter chemotherapy.
Acknowledgments
This work was supported by grants from the Research Council of Tehran University of Medical Sciences and
the Iran National Science Foundation (INSF).
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