SAR study of clubbed [1,2,4]-triazolyl with fluorobenzimidazoles as antimicrobial and antituberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2008.12.001

In the present study, a series of novel 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-substituted-4,6-difluoro-1H-benz o[d]imidazole derivatives are synthesized by the alkylation of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-4,6-difluoro-1H-benzo[d]imidazole with substituted alkyl and aryl halides. The compounds were evaluated for their preliminary in-vitro antibacterial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Salmonella typhosa and then were screened for their antitubercular activity against Mycobacterium tuberculosis H37Rv strain by broth microdilution assay method. The antibacterial data suggested that the analogs with electronegative substituents emerged as promising antimicrobials. It was also observed that the promising antimicrobials have proved to be better antimycobacterials. Few of selected analogs are under further evaluation for secondary antitubercular screening, as they have shown better activity compared to rifampin.

Full text of DOI:10.1016/j.ejmech.2008.12.001

European Journal of Medicinal Chemistry 44 (2009) 2930–2935
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
SAR study of clubbed [1,2,4]-triazolyl with fluorobenzimidazoles
as antimicrobial and antituberculosis agents
,
Ganesh Rajaram Jadhav a b, Mohammad Usman Shaikh b, Rajesh Prabhakar Kale b,
,
Mahendra Ramesh Shiradkar c, Charansingh Harnamsingh Gill a
*
a Department of chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431 004, Maharashtra, India
b Wockhardt Research Centre, D-4, Chikathana, M.I.D.C. Area, Aurangabad 431 210, Maharashtra, India
c Chemical Biology Program, Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, OR 97239, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2008
Received in revised form
10 November 2008
Accepted 8 December 2008
Available online 16 December 2008
In the present study, a series of novel 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-substituted-4,6-difluoro-
1H-benzo[d]imidazole derivatives are synthesized by the alkylation of 2-[4-(1H-[1,2,4]-triazol-1-
yl)phenyl]-4,6-difluoro-1H-benzo[d]imidazole with substituted alkyl and aryl halides. The compounds
were evaluated for their preliminary in-vitro antibacterial activity against Pseudomonas aeruginosa,
Escherichia coli, Staphylococcus aureus, and Salmonella typhosa and then were screened for their antitu-
bercular activity against Mycobacterium tuberculosis H37Rv strain by broth microdilution assay method.
The antibacterial data suggested that the analogs with electronegative substituents emerged as prom-
ising antimicrobials. It was also observed that the promising antimicrobials have proved to be better
antimycobacterials. Few of selected analogs are under further evaluation for secondary antitubercular
screening, as they have shown better activity compared to rifampin.
Keywords:
Antimycobacterial
Antibacterial activity
Air oxidation
Difluorobenzimidazole
[1,2,4]-Triazole
Alkylation
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
Heterocyclic species like triazole and fluorobenzimidazole
derivatives [7] represent a novel emerging major chemical entity as
Despite the availability of highly potential antitubercular agents,
tuberculosis remains primary cause of comparatively high
mortality worldwide. The statistics shows that around three million
people throughout the world die annually from tuberculosis [1,2]
and today more people die from tuberculosis than ever before [3].
Therefore, the development of new drugs with activity against
multi drug-resistant (MDR) TB, extensively drug-resistant (XDR)
TB, and latent TB is a priority task. Although new agents that will
shorten the duration of current chemotherapy are also needed. A
special interest has been focused on five membered heterocyclic
compounds like imidazole, pyrrole, oxadiazole, specially triazoles
and other heterocyclic system derivatives have been reported [4].
The azole antitubercular may be regarded as a new class providing
truly effective drugs, which is reported to inhibit bacteria by
blocking the biosynthesis of certain bacterial lipids and/or by
additional mechanisms [5,6].
antimicrobial agent. Triazoles, in particular, [1,2,4]-triazole coun-
terparts of fluorobenzimidazoles, are among the various hetero-
cycles that have received much attention as potential antimicrobial
agents, antifungal, anti-inflammatory, CNS stimulants, sedatives,
antianxiety [8,9]. Benzimidazole moiety has already been reported
for its antimicrobial activity [10,11] along with antiviral, antiulcer,
antihypertension [12], cardiotonic [13] Literature survey shows that
2-substituted benzimidazoles are found to be more potent and
hence the design and synthesis of 2-substituted benzimidazole are
the potential area of research [14]. After extensive literature search,
it was observed that, till date enough efforts have not been made to
combine these two moieties as a single molecular scaffold and to
study its biological activity. In continuation with our earlier work
[15] to establish probable pharmacological activities of [1,2,4]-tri-
azoles [16–22] and benzimidazole [23], we herein report the
synthesis of new [1,2,4]-triazole derivatives clubbed with fluo-
robenzimidazole moiety.
In recent years, environmentally benign synthetic methods
have received considerable attention [24–33]. A fast, highly effi-
cient and eco-friendly, catalyst-free, chemical transformation by air
* Corresponding author.
E-mail address: chgill50@yahoo.com (C.H. Gill).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.12.001

G.R. Jadhav et al. / European Journal of Medicinal Chemistry 44 (2009) 2930–2935
2931
oxidation, for the synthesis of title compounds is designed. We
now wish to disclose our results towards the synthesis of anti-
bacterial and antimycobacterial activity of 2-[4-(1H-[1,2,4]-triazol-
1-yl)phenyl]-1-substituted-4,6-difluoro-1H-benzo[d]imidazole
derivatives (9) Schemes 1–3.
their antitubercular activity. The antitubercular activity data are
mentioned in Tables 1 and 2
At the commencement of this study in the preliminary
screening, compound (9) displayed better activity and showed
>95% inhibition at 6.25 mg concentration. This result encouraged
us to consider compound (9) as our lead molecule. Subsequent
structural modifications were carried out for lead optimization.
The first step towards lead optimization was incorporation of
simple alkyl groups like CH3, CH2CH3, and CH2CH2CH3 at NH of
imidazole ring system. The antituberculosis study data of these
compounds showed <90% of inhibition at 6.25 mg concentration.
The next structural modification was introduction of more elec-
tronegative groups viz. CH2CH]CH2, CH2CCH, CH2CN, and
CH2C6H5. This change resulted in substantial improvement in
activity. More than 96% inhibition at 6.25 mg concentration was
noticed. Enhancement in activity proves that this modification is
step up towards synthesis of a pharmacophore. On the basis of
biological data, improvement in biological activity was observed
with increase in electronegativity of molecule. Hence we planned
to introduce electron withdrawing and donating groups at different
positions to study its influence on activity. Different analogs with
electron withdrawing groups e.g. fluorine and chlorine and
donating groups like methyl and methoxy were synthesized. These
analogs were evaluated for antimicrobial activity. In majority of the
cases, compounds having electronegative atoms like fluorine and
chlorines (10h, 10i and 10l) exhibited good activity i.e. >96% inhi-
bition at 6.25 mg concentration. In exceptional case, 3-methoxy
substituted analog was also found better in activity. The same result
was applicable only in primary screening while in secondary level
screening same response was not produced. Therefore we have
introduced trifluoromethyl substitution (10k) to increase electro-
negativity. This change resulted in substantial loss in activity. This
loss in activity may be due to retardation in intracellular transport
because of high electronegativity.
2. Results and discussion
2.1. Chemistry
The synthesis of title compounds was started with conden-
sation of 3,5-difluorobenzene-1,2-diamine (8) [35] condensed
with 4-(1H-[1,2,4]-triazol-1-yl) benzaldehyde (3) [34] in refluxing
toluene for 1 h by air oxidation without using any oxidizing agent
or catalyst, to furnish 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-4,6-
difluoro-1H-benzo[d]imidazole (9). The chemical transformation
of (9) with alkyl and substituted aryl halides was carried out
using NaH as a base proceeded at room temperature to furnish 2-
[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-substituted-4,6-difluoro-1H-
benzo[d]imidazole derivatives. The physical and spectral data of
compounds (10a–10o) are presented in Section 3.3.
2.2. Structure–activity relationship study
In an on-going research work, we have synthesized benzimid-
azoles clubbed with different heteroaromatic nucleus like [1,2,3]-
triazole, [1,2,4]-oxadiazole, [1,2,4]-triazole etc. These newly
synthesized scaffolds were screened for antimicrobial activities
against various gram þve and gram ꢀve microorganisms.
Compound 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-4,6-difluoro-1H-
benzo[d]imidazole (9) exhibited better antimicrobial activity as
compared to other scaffolds.
Having confirmed the activity of compound (9), we embarked
on a hit-to-lead exploration program focusing on N1–H of imidazole
nucleus. The NH was alkylated with different alkyl and substituted
aryl halides. Thus our aim was to explore SAR trends and to find out
the lead for further optimization.
All those compounds which were found to be active in first level
of screening viz. 10e, 10f, 10h, 10i 10l and 10n then tested to
determine actual minimum inhibitory concentration. We have
drawn the inference that compounds 10h and 10i have proved to be
the best and most active with MIC value ranging from 0.36 to 0.58.
2.2.1. Antimicrobial activity
2.2.3. Conclusion
The newly synthesized compounds (10a–10o) were evaluated
for in-vitro antimicrobial activities. They were assessed against two
representative of each gram þve and gram ꢀve strains like Staph-
ylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmo-
nella typhosa. Gentamycin was used as a reference drug molecule.
The data generated from preliminary screening showed that
compounds displayed moderate to better activity. SAR study
reveals that compounds like 10a, 10e, 10f, 10h, 10i, and 10l dis-
played maximum activity, while compound 10n was moderately
active. The other compounds viz. 10b, 10c, 10d, 10g, 10j, 10k, 10m,
and 10o showed least activity. This in-vitro antimicrobial activity
data are tabulated in Table 3.
A series of novel analogs of fluorobenzimidazole clubbed with
[1,2,4]-triazoles were synthesized and assessed for their antibac-
terial and antituberculosis H37Rv strain activity. The antibacterial
data indicates that the analogs with electronegative substituents
emerged as promising antimicrobials showing better to moderate
activity. It was also observed that the promising antimicrobials
have proved to be better antimycobacterials. Specially, compounds
10h and 10i, due to their better activity against the H37Rv strain,
are the best choice for the preparation of new derivatives in order
to improve antimycobacterial activity in future. Also, we have
developed an efficient methodology for the synthesis of 10h and
10i benzimidazole derivatives towards the development of new
pharmacophore. Such activity and their ease of preparation make
them attractive candidate for further assessment in antitubercu-
losis agents.
2.2.2. Antitubercular activity
The encouraging results from the antibacterial activity promp-
ted us to opt for preliminary screening of the titled compounds for
3. Experimental section
O
H
O
H
a
N
N
The melting points were estimated by Veggo programmable
(microprocessor based) melting point apparatus and are uncor-
rected. 1H NMR spectra were recorded on a Varian 400 MHz spec-
trometer MHz instrument using CDCl3 as solvent using TMS as
F
N
HN
+
N
N
1
2
3
a: K2CO3, DMF, 120°C
internal standard; the chemical shifts (d) are reported in ppm.
Scheme 1.
Signal multiplicities are represented by s (singlet), d (doublet),

2932
G.R. Jadhav et al. / European Journal of Medicinal Chemistry 44 (2009) 2930–2935
F
F
F
F
F
H
H
NH2
NO2
NH2
NH2
N
NH2
N
d
a
b
c
O
O
F
F
NO2
F
F
F
7
8
6
5
4
a: Acetic anhydride, reflux; b: HNO3, H2SO4, RT; C: 2N HCl, reflux; d: Pd/C, H2, EtOH
Scheme 2.
t (triplet), ds (double singlet), dd (double doublet), m (multiplet)
and br s (broad singlet). IR spectra were recorded on KBr disc using
a FTIR bruker Vector 22 Spectrophotometer. Elemental analyses
were determined on Elementor Vario instrument. EI-MS spectra
recorded on micromass-quatro-II. The purity of the compounds
was checked on Merck precoated silica gel 60 F-254. All the
reagents, solvents used were of commercial grade only.
Step II: nitration of N-(2,4-difluorophenyl)acetamide (5) was
carried out (12 g, 70.2 mmol) in nitric acid (25 mL), by the addition
of conc. sulfuric acid (25 mL) slowly at 5–10 ꢁC. Then gradually
warming up the reaction mixture at room temperature and stirring
was continued for 2–3 h. Monitored the progress of the reaction by
TLC system (ethylacetate:hexane, 2:8). Quenched the reaction in ice
water, a pale yellow colour solid precipitates out, filtered and suck
dried to give as N-(2,4-difluoro-6-nitrophenyl)acetamide (6)
(12.0 g, 80% yield). 1H NMR (CDCl3)
d (ppm): 2.23 (3H, s, CH3), 7.26
(1H, m, ArH), 7.62 (1H, m, ArH), 8.01 (1H, s, –NH). m/z 215 (M)ꢀ.
Step III: N-(2,4-difluoro-6-nitrophenyl)acetamide (6) (12 g,
55.5 mmol) was suspended in 2 N HCl (50 mL) and refluxed for 3–
4 h. The reaction mixture was cooled gradually to room tempera-
ture, neutralized with aq. NaHCO3 solution and where upon
a yellow colour needle type crystal separates out. It was filtered,
suck dried to give as 2,4-difluoro-6-nitrobenzenamine. (7) (7.0 g,
3.1. General experimental procedure for the synthesis of
4-(1H-[1,2,4]-triazol-1-yl)benzaldehyde (3)
To a solution of 4-fluorobenzaldehyde (1) (5.0 g, 40.32 mmol)
and 1H-[1,2,4]-triazole (2) (3.3 g, 47.82 mmol) in DMF (50 mL) was
added powdered K2CO3 (6.67 g, 48.38 mmol). The mixture was
stirred at 120 ꢁC for 12 h. Reaction mixture was warmed up at room
temperature and quenched with water. It was extracted with eth-
ylacetate (2 ꢂ 150 mL). The combined organic fractions were
washed with water (100 mL) followed to brine solution (100 mL)
and dried over anhydrous Na2SO4. Solvent was removed under
reduced pressure and purification was done by flash chromatog-
raphy (hexane:ethylacetate, 3:1 elution) to give 4-(1H-[1,2,4]-tri-
azol-1-yl)benzaldehyde as a off-white solid (3) (5.0 g, 72% yield). 1H
73% yield). 1H NMR (CDCl3)
d (ppm): 6.05 (2H, s, NH2), 7.12 (1H, m,
ArH), 7.73 (1H, s, ArH). m/z 173 (M)ꢀ.
Step IV: 2,4-difluoro-6-nitrobenzenamine. (7) (7.0 g, 40.2 mmol)
was hydrogenated in ethanol solution by using 10% Pd/C (700 mg)
under 60 psi H2 pressure. Monitored the progress of the reaction on
TLC system (ethyl acetate:hexane, 1:1). After completion, catalyst
was filtered. Solvent was removed under reduced pressure The final
product 3,5-difluorobenzene-1,2-diamine (8) was purified by
NMR (CDCl3)
d (ppm): 8.01–8.10 (4H, m), 8.15 (1H, s, triazole-H),
9.43 (1H, s, ArH), 10.10 (1H, s, CHO). m/z 173 (Mþ).
sublimation under vacuum,
obtained (4.9 g, 85% yield).
a cotton like off-white material
3.2. General experimental procedure for the synthesis of
3,5-difluorobenzene-1,2-diamine (8)
3.3. General experimental procedure for the synthesis of 2-[4-(1H-
[1,2,4]-triazol-1-yl)phenyl]-4,6-difluoro-1H-benzo[d]imidazole (9)
Step I: acetic anhydride (7.9 g, 77.5 mmol) was added slowly to
2,4-difluorobenzenamine (4) (10 g, 77.5 mmol) at 0–5 ꢁC, where
upon a solid material precipitates out after 15–20 min. Reaction
mixture was quenched in ice water and stirred for 30 min. A off-
violet colour solid material separates out, filtered and suck dried to
obtained as N-(2,4-difluorophenyl)acetamide. (5) (12.0 g, 91%
yield). m/z 172 (M).
A mixture of 4-(1H-[1,2,4]-triazol-1-yl)benzaldehyde (3) (5.0 g,
28.9 mmol) and 3,5-difluorobenzene-1,2-diamine (8) (6.0 g,
41.7 mmol) in toluene (25 mL) was refluxed for 2–3 h. The reaction
mixture was then cooled to 5 ꢁC gradually, stirred for 15–20 min.
The pale yellow solid material formed was filtered through buchner
funnel and washed with ice-cold toluene. The obtained pale yellow
F
F
NH2
N
N
O
a
N
N
N
N
N
N
H
NH2
H
F
F
8
3
9
F
F
b
N
N
N
N
N
N
N
RX
N
N
N
H
F
F
R
9
10a-10o
a: 4-(1H- [1,2,4]-triazol-1-yl)benzaldehyde, toluene,110 °C; b: NaH, RX, DMF, RT
Scheme 3.

G.R. Jadhav et al. / European Journal of Medicinal Chemistry 44 (2009) 2930–2935
2933
Table 1
First antituberculosis activity of compounds 10a–10o.
Table 3
First antibacterial screening of compounds 10a–10o.
Compound
R
MICa
GI (%)b
Compound
R
Organisms
Sa
10a
10b
10c
10d
10e
–CH3
–CH2CH3
–CH2CH2CH3
–CH2CH]CH2
–CH2CCH
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
0.015–0.125
–
–
–
–
96
96
–
96
96
–
–
96
–
Pa
Ec
St
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
–CH3
–CH2CH3
–CH2CH2CH3
–CH2CH]CH2
–CH2CCH
28
23
21
20
25
27
19
29
26
14
22
25
21
24
19
34
26
18
23
22
23
26
20
26
22
12
21
23
18
22
21
35
21
16
18
23
21
24
15
22
19
16
20
19
18
22
18
31
19
14
19
23
24
20
13
24
18
16
18
21
16
21
14
30
10f
–CH2CN
–CH2C6H5
10g
10h
10i
10j
10k
10l
10m
10n
10o
Rifampin
–CH2CN
–CH2C6H5
–CH2(3-FC6H5)
–CH2(3-ClC6H5)
–SO2C6H5CH3
–CH2(3-CF3C6H5)
–CH2(4-FC6H5)
–CH2(3-MeC6H5)
–CH2(3-OMe C6H5)
–CH2(2-MeC6H5)
–
–CH2(3-FC6H5)
–CH2(3-ClC6H5)
–SO2C6H5CH3
–CH2(3-CF3C6H5)
–CH2(4-FC6H5)
–CH2(3-MeC6H5)
–CH2(3-OMe C6H5)
–CH2(2-MeC6H5)
–
96
–
97
a
MIC in 1
m
g/mlꢀ1
.
Gentamycin
b
Growth inhibition of virulent H37Rv strain of M. tuberculosis.
Sa: Staphylococcus aureus, Ec: Escherichia coli, Pa: Pseudomonas aeruginosa, St:
Salmonella typhosa.
colour solid (9) was triturated with diethyl ether and filtered (7.5 g,
61% yield), m.p. 220–222 ꢁC. 1H NMR (CDCl3, 400 MHz),
(ppm):
3.4.2. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-ethyl
4,6-difluoro-1H-benzo[d]imidazole (10b)
d
7.07 (1H, m, ArH), 7.26 (1H, m, ArH), 8.07 (2H, d, J ¼ 8.17 Hz, ArH),
8.27 (1H, s, triazole-H), 8.32 (2H, d, J ¼ 8.21 Hz, ArH), 9.39 (1H, s,
triazole-H) 12.18 (1H, s, NH). m/z 298.1 (M). Anal. calcd for
C15H9F2N5: C, 60.61; H, 3.05; N, 23.56; found C, 60.75; H, 3.10;
N, 23.64.
The compound obtained as a pale yellow colour crystalline solid
by using iodoethane (yield 78%), m.p. 148–150 ꢁC. IR (KBr cmꢀ1):
3334, 3099, 1636, 1521, 1435, 1348, 1279, 1216, 1101, 981, 842, 673.
1H NMR (CDCl3, 400 MHz):
d (ppm) 1.46 (3H, t, CH3), 4.28 (2H, q,
CH2), 7.06 (1H, m, ArH), 7.27 (1H, m, ArH), 7.80 (2H, d, J ¼ 7.92 Hz,
ArH), 8.07 (2H, d, J ¼ 7.94 Hz, ArH), 8.52 (1H, s, triazole-H), 9.18 (1H,
s, triazole-H), m/z: (Mþ) 326.2; Anal. calcd. for C17H13F2N5: C, 62.76;
H, 4.03; N, 21.53; found C, 62.77; H, 4.27; N, 21.55.
3.4. General experimental procedure for the 2-[4-(1H-[1,2,4]-
triazol-1-yl) phenyl]-1-substituted-4,6-difluoro-1H-benzo[d]
imidazole derivatives (10a–10o)
3.4.3. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-4,
6-difluoro-1-propyl-1H-benzo[d]imidazole (10c)
To a solution of sodium hydride (1.1 eq.) in dimethyl formamide
was added 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-4,6-difluoro-1H-
benzo[d]imidazole (9) (1.0 eq.), stirred the reaction mixture at
room temperature for 30 min. To this reaction mixture, charge
substituted aryl or alkyl halide (1.2 eq.) and continue stirring for
30 min. After completion of reaction, the reaction mixture was
quenched in the ice water, a solid precipitates out, was filtered, with
bed wash of water (100 mL) and suck dried (yield ¼ 69–81%).
The compound obtained as a off-white colour crystalline solid
by using 1-bromopropane (yield 79%), m.p. 138–140 ꢁC. IR (KBr
cmꢀ1): 3091, 2985, 1639, 1539, 1413, 1280, 1110, 981, 846, 669. 1H
NMR (CDCl3, 400 MHz) d (ppm): 0.71 (3H, t, CH3), 1.69 (2H, m, CH2),
4.29 (2H, q, CH2), 7.14 (1H, m, ArH), 7.41 (1H, m, ArH), 7.94 (2H, d,
J ¼ 8.0 Hz, ArH), 8.10 (1H, s, triazole-H), 8.29 (2H, d, J ¼ 8.01 Hz,
ArH), 9.40 (1H, s, triazole-H), m/z: (Mþ) 340.1. Anal. calcd. for
C18H15F2N5: C, 63.71; H, 4.46; N, 20.64; found C, 63.76; H, 4.55;
N, 20.79.
3.4.1. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-4,
6-difluoro-1-methyl-1H-benzo[d]imidazole (10a)
The compound obtained as a pale green colour crystalline solid
by using iodomethane (yield 72%), m.p. 100–102 ꢁC. IR (KBr, cmꢀ1):
3328, 3104, 1602, 1502, 1279, 1214, 1149, 980, 838, 672. 1H NMR
3.4.4. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-allyl-4,
6-difluoro-1H-benzo[d]imidazole (10d)
The compound obtained as a yellow colour crystalline solid by
using 3-bromoprop-1-ene (yield 68%). m.p. 156–158 ꢁC. IR (KBr
cmꢀ1): 3263, 3129, 1640, 1539, 1436, 1366, 1275, 982, 844, 796, 674.
(CDCl3, 400 MHz): d (ppm): 3.81 (3H, s, CH3), 7.13 (1H, m, ArH), 7.29
(1H, m, ArH), 8.14 (2H, d, J ¼ 8.06 Hz, ArH), 8.26 (1H, s, triazole-H),
8.28 (2H, d, J ¼ 8.08 Hz, ArH), 9.29 (1H, s, triazole-H), m/z: 312.2
(Mþ). Anal. calcd. for C16H11F2N5: C, 61.73; H, 3.56; N, 22.50; found
C, 61.74; H, 3.56; N, 22.55.
1H NMR (CDCl3, 400 MHz)
d (ppm): 4.18 (2H, s, CH2), 6.76 (2H, m,
ArH), 6.71 (1H, m, allylic), 7.48 (1H, m, ArH), 7.66 (1H, m, ArH), 7.81
(2H, d, J ¼ 7.92 Hz, ArH), 8.08 (2H, d, J ¼ 7.95 Hz, ArH), 8.14(1H, s,
triazole-H), 8.65 (1H, s, triazole-H), m/z: (Mþ) 338.1. Anal. calcd.
C18H13F2N5 for: C, 64.09; H, 3.88; N, 20.76; found C, 64.10; H, 3.91;
N, 20.81.
Table 2
Second level antituberculosis screening.
3.4.5. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-4,
6-difluoro-1-(prop-2-ynyl)-1H-benzo[d]imidazole (10e)
SN
MICa g/mlꢀ1
m
10e
10f
10h
10i
10l
10n
Rifampin
3.13
1.56
0.36
0.58
3.13
3.13
The compound obtained as a buff colour crystalline solid by
using 3-bromoprop-1-yne (yield 70%). m.p. 180–182 ꢁC. IR (KBr
cmꢀ1): 3193, 3125, 1690,1599,1505,1430,1388,1275, 982, 844, 674.
1H NMR (CDCl3, 400 MHz):
d (ppm) 3.58 (1H, s, prop-2-ynyl-H),
5.22 (2H, d, CH2), 7.21 (1H, m, ArH), 7.48 (1H, m, ArH), 8.06 (2H, d,
J ¼ 8.07 Hz, ArH), 8.15 (2H, d, J ¼ 8.08 Hz, ArH), 8.30 (1H, s, triazole-
H), 9.43 (1H, s, triazole-H), m/z: (Mþ) 336.1. Anal. calcd. for
0.015–0.125
a
Actual minimum inhibitory concentration (MABA assay).

2934
G.R. Jadhav et al. / European Journal of Medicinal Chemistry 44 (2009) 2930–2935
C18H11F2N5: C, 64.48; H, 3.31; N, 20.89; found C, 64.49; H, 3.41;
N, 20.98.
77%). m.p. 133–135 ꢁC. IR (KBr cmꢀ1): 3142, 2363, 1658, 1581, 1498,
1457, 1326, 1166, 1048, 985, 846, 718, 672. 1H NMR (CDCl3,
400 MHz): d (ppm): 5.48 (2H, s, CH2), 6.72 (2H, d, ArH), 6.88 (1H, m,
3.4.6. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-4,
6-difluoro-1H-benzo [d]imidazole-1-acetonitrile (10f)
ArH), 7.18 (3H, m, ArH), 7.68 (2H, d, J ¼ 8.02 Hz, ArH), 7.98 (2H, d,
J ¼ 8.05 Hz, ArH), 8.14 (1H, s, triazole-H), 9.39 (1H, s, triazole-H), m/
z: 456.1 (Mþ). Anal. calcd. for C23H14F5N5: C, 60.66; H, 3.10; N,15.38;
found C, 60.72; H, 3.45; N, 15.39.
The compound obtained as a buff colour crystalline solid by
using bromoacetonitrile (yield 72%). m.p. 216–218 ꢁC. IR (KBr
cmꢀ1): 3112, 2932, 1641, 1604, 1503, 1431, 1226, 1122, 981, 845, 674.
1H NMR (CDCl3, 400 MHz):
d
(ppm) 5.42 (2H, s, CH2), 7.23 (1H, t,
3.4.12. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-(4-
fluorobenzyl)-4,6-difluoro-1H-benzo[d]imidazole (10l)
ArH), 7.48 (1H, dd, ArH), 8.09 (2H, d, J ¼ 7.98 Hz, ArH), 8.21 (2H, d,
J ¼ 7.97 Hz, ArH), 8.41 (1H, s, triazole-H), 9.45 (1H, s, triazole-H),
m/z: 337.2 (Mþ); Anal. calcd. for C17H10F2N6: C, 59.63; H, 2.50;
N, 26.08; found C, 59.81; H, 2.71; N, 25.99.
The compound obtained as a cremish colour crystalline solid by
using 1-(bromomethyl)-4-fluorobenzene (yield 69%). m.p. 128–
130 ꢁC. IR (KBr cmꢀ1): 3138, 2361, 1665, 1574,1497,1460,1326,1154,
1055, 987, 844, 718, 672. 1H NMR (CDCl3, 400 MHz):
d (ppm): 5.51
3.4.7. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-benzyl-
4,6-difluoro-1H-benzo[d]imidazole (10g)
(2H, s, CH2), 6.90 (2H, d, ArH), 7.13 (2H, m, ArH), 7.31 (2H, m, ArH),
7.75 (2H, d, J ¼ 8.01 Hz, ArH), 8.10 (2H, d, J ¼ 8.05, Hz, ArH), 8.27 (1H,
s, triazole-H), 9.48 (1H, s, triazole-H), m/z: 406.1 (Mþ). Anal. calcd.
for C22H14F3N5: C, 65.18; H, 3.48; N,17.28; found C, 65.42; H, 3.64; N,
17.44.
The compound obtained as a off-white colour crystalline solid
by using 1-(bromomethyl)benzene (yield 81%). m.p. 172–174 ꢁC. IR
(KBr cmꢀ1): 3113, 2361, 1643, 1597, 1502, 1455, 1358, 1173, 1078,
982, 847, 724, 672. 1H NMR (CDCl3, 400 MHz):
d (ppm) 5.62 (2H, s,
CH2), 6.96 (2H, d, ArH), 7.12 (2H, m, ArH), 7.25–7.32 (3H, m, ArH),
7.95 (2H, d, J ¼ 7.97 Hz, ArH), 8.05 (2H, d, J ¼ 7.98 Hz, ArH), 8.27 (1H,
s, triazole-H), 9.37 (1H, s, triazole-H), m/z: 388.2 (Mþ). Anal. calcd.
for C22H15F2N5: C, 68.21; H, 3.90; N, 18.08; found C, 68.23; H, 3.92;
N, 18.11.
3.4.13. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-(3-
methylbenzyl)-4,6-difluoro-1H-benzo[d]imidazole (10m)
The compound obtained as a buff colour crystalline solid by
using 1-(bromomethyl)-3-methylbenzene (yield 72%). m.p. 166–
163 ꢁC. IR (KBr cmꢀ1): 3098, 2363,1663,1574,1511,1464,1338,1188,
1044, 980, 847, 718, 671. 1H NMR (CDCl3, 400 MHz):
d (ppm): 2.78
3.4.8. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-(3-
fluorobenzyl)-4,6-difluoro-1H-benzo[d]imidazole (10h)
(3H, s, CH3) 5.49 (2H, s, CH2), 6.84 (2H, d, ArH), 7.11 (1H, m, ArH),
7.27 (3H, m, ArH), 7.85 (2H, d, J ¼ 7.98 Hz, ArH), 8.09 (2H, d,
J ¼ 8.0 Hz, ArH), 8.22 (1H, s, triazole-H), 9.48 (1H, s, triazole-H), m/z:
402.1 (Mþ). Anal. calcd. for C23H17F2N5: C, 68.82; H, 4.27; N, 17.45;
found C, 68.89; H, 4.51; N, 17.66.
The compound obtained as a off-white colour crystalline solid by
using 1-(bromomethyl)-3-fluorobenzene (yield 74%). m.p. 148–
152 ꢁC. IR (KBr cmꢀ1): 3133, 2359, 1651, 1577, 1498, 1456, 1328, 1168,
1058, 986, 846, 718, 672. 1H NMR (CDCl3, 400 MHz):
d (ppm): 5.51
(2H, s, CH2), 6.88(2H, d, ArH), 7.09 (1H, m, ArH), 7.27 (3H, m, ArH), 7.85
(2H, d, J ¼ 7.98 Hz, ArH), 8.11 (2H, d, J ¼ 8.0 Hz, ArH), 8.24 (1H, s, tri-
azole-H), 9.42 (1H, s, triazole-H), m/z: 406.1 (Mþ). Anal. calcd. for
C22H14F3N5: C, 65.18;H, 3.48;N,17.28;foundC, 65.21;H, 3.51;N,17.36.
3.4.14. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-(3-
methoxybenzyl)-4,6-difluoro-1H-benzo[d]imidazole (10n)
The compound obtained as a off-white colour crystalline solid
by using 1-(bromomethyl)-3-methoxybenzene (yield 78%). m.p.
150–152 ꢁC. IR (KBr cmꢀ1): 3141, 2362, 1648, 1581, 1511, 1461, 1332,
3.4.9. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-1-(3-
chlorobenzyl)-4,6-difluoro-1H-benzo[d]imidazole (10i)
1172, 1066, 984, 847, 719, 673. 1H NMR (CDCl3, 400 MHz):
d (ppm):
3.98 (3H, s, OCH3), 5.48 (2H, s, CH2), 6.79 (2H, d, ArH), 7.10 (1H, m,
ArH), 7.24 (3H, m, ArH), 7.79 (2H, d, J ¼ 7.92 Hz, ArH), 8.10 (2H, d,
J ¼ 7.96 Hz, ArH), 8.22 (1H, s, triazole-H), 9.39 (1H, s, triazole-H),
m/z: 418.1 (Mþ). Anal. calcd for C23H17F2N5O: C, 66.18; H, 4.11;
N, 16.78; found C, 66.23; H, 4.51; N, 16.90.
The compound obtained as a pale yellow colour crystalline solid
by using 1-(bromomethyl)-3-chlorobenzene (yield 71%). m.p. 127–
129 ꢁC. IR (KBr cmꢀ1): 3153, 2361, 1647, 1554, 1489, 1444, 1331, 1178,
1048, 991, 847, 722, 672. 1H NMR (CDCl3, 400 MHz):
d (ppm): 5.49
(2H, s, CH2), 6.93 (2H, d, ArH), 7.12 (1H, m, ArH), 7.35 (3H, m, ArH),
7.77 (2H, d, J ¼ 7.96 Hz, ArH), 8.10 (2H, d, J ¼ 7.98 Hz, ArH), 8.26 (1H,
s, triazole-H), 9.36 (1H, s, triazole-H), m/z: 422.1(Mþ). Anal. calcd.
for C22H14ClF2N5: C, 62.64; H, 3.35; N,16.60; found C, 62.68; H, 3.51;
N, 16.66.
3.4.15. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl) phenyl]-1-(2-
methylbenzyl)-4,6-difluoro-1H-benzo[d]imidazole (10o)
The compound obtained as a buff colour crystalline solid by
using 1-(bromomethyl)-2-methylbenzene (yield 79%). m.p. 134–
136 ꢁC. IR (KBr cmꢀ1): 3121, 2359, 1656, 1578, 1510, 1438, 1351, 1179,
3.4.10. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl)phenyl]-4,
6-difluoro-1-(tosyl-methyl)-1H-benzo[d]imidazole (10j)
1055, 981, 846, 717, 672. 1H NMR (CDCl3, 400 MHz):
d (ppm): 2.68
(3H, s, CH3) 5.51 (2H, s, CH2), 6.87 (2H, d, ArH), 7.11 (2H, m, ArH),
7.24 (2H, m, ArH), 7.78 (2H, d, J ¼ 7.92 Hz, ArH), 8.01 (2H, d,
J ¼ 7.93 Hz, ArH), 8.22 (1H, s, triazole-H), 9.42 (1H, s, triazole-H).
m/z: 402.1 (Mþ). Anal. calcd. for C23H17F2N5: C, 68.82; H, 4.27;
N, 17.45; found C, 68.89; H, 4.51; N, 17.66.
The compound obtained as a pale yellow colour crystalline solid
by using 4-methylbenzene-1-sulfonyl chloride (yield 73%); m.p.
182–184 ꢁC. IR (KBr cmꢀ1): 3324, 3125, 2960,1638,1516,1386,1283,
1190, 1114, 948, 847, 669. 1H NMR (CDCl3, 400 MHz)
d (ppm): 2.64
(3H, s, CH3), 7.12 (1H, m, ArH), 7.32 (1H, m, ArH), 7.48 (4H, d, ArH),
7.7 (2H, d, J ¼ 8.01 Hz, ArH), 7.95 (2H, d, J ¼ 8.0 Hz, ArH), 8.26 (1H, s,
triazole-H), 9.36 (1H, s, triazole-H), m/z: 452.1 (Mþ). Anal. calcd for
C22H15F2N5O2S: C, 58.53; H, 3.35; N, 15.51; found C, 58.34; H, 3.32;
N, 15.50.
3.5. Pharmacological activity
3.5.1. Antitubercular activity
Primary screening was conducted at 6.25 m
g/mLꢀ1 against
Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B
medium using a broth microdilution assay, the microplate Alamar
Blue assay (MABA) [36]. Compounds exhibited fluorescence were
tested in the BACTEC 460 radiometric system [37]. Compounds
showing more than 95% inhibition in the primary screening were
3.4.11. Synthesis of 2-[4-(1H-[1,2,4]-triazol-1-yl) phenyl]-1-[3-
(trifluoromethyl)-benzyl]-4,6-difluoro-1H-benzo[d]imidazole (10k)
The compound obtained as a pale yellow colour crystalline solid
by using 1-(bromomethyl)-3-(trifluoromethyl)-benzene (yield

G.R. Jadhav et al. / European Journal of Medicinal Chemistry 44 (2009) 2930–2935
2935
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considered active and again retested at lower concentration against
M. tuberculosis H37Rv in order to determine the actual MIC, using
MABA. The MIC is defined as the lowest concentration effecting
a reduction in fluorescence of 95% with respect to the controls.
Rifampin (RMP) was used as the reference compound (MIC ¼ 0.015–
0.125 mg/mLꢀ1). We also have done cytotoxicity analysis of the
above-synthesized compounds, using neutral red uptake by using
Vero-C-1008 cell line at various concentrations (6.25–50 mg/mL),
none of them were found toxic. Hence the activities of the above-
synthesized compounds were not due to cytotoxicity of compounds.
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The compounds listed in were screened for the antimicrobial
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Acknowledgement
The authors are thankful to The Head, Department of Chemistry,
Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-
431004 (MS), India for providing laboratory facility and Wockhardt
Research Centre, Aurangabad, Maharashtra, India for their valuable
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