Priyal Jain, et al : Synthesis Antimicrobial Activity and Chemotherapeutic Potential some Novel Benzimidazole Derivaties 633

Synthesis Antimicrobial Activity and Chemotherapeutic Potential some

Novel Benzimidazole Derivatives
Priyal Jain
1*
, Vaibhav Jain
1
, Abhishek K Jain
2
, Pradeep K Singour
3
1
Department of Pharmaceutical Chemistry, Sagar Institute of Research and Technology-Pharmacy, Bhopal, India.
2
Sagar Institute of Pharmaceutical Sciences, Sagar, India.
3
Computational and Synthetic Laboratory, VNS Institute of Pharmacy, Bhopal, India.
ABSTRACT: Benzimidazole derivates play vital role in biological field such as antimicrobial, antiviral, antidiabetic, and
anticancer activity. Therapeutic significance of these clinically useful drugs in treatment of microbial infections encouraged
the development of some more potent and significant compounds. With the purpose of finding new chemical entities with
enhanced antimicrobial activity, a series comprises 2-substituted-5-nitro benzimidazole derivatives were synthesized.
Investigation of antimicrobial activity of the compounds was done by disc diffusion method using Gram-positive (S. aureus,
S. mutans and B. subtilis), Gram-negative (E. coli, S. typhi and P. aeruginosa) bacteria. Some derivatives showed
remarkable activity comparable to that of standard against Gram-positive and Gram-negative bacteria.
KEYWORDS: Benzimidazole; antimicrobial activity; ampicillin; nalidixic acid.
Introduction
Benzimidazoles are regarded as a promising class of
bioactive heterocyclic compounds that exhibit a range of
biological activities. Specifically, this nucleus is a
constituent of vitamin-B
12
(Ansari and Lal 2009, ONiel et
al. 2001). This ring system is present in numerous
antioxidant (Ansari and Lal 2009, Ayhan-Kilcigil et al.
2007), antiparasitic (Ansari and Lal 2009, Navarrete-
Vazquez et al. 1993), antihelmintics (Ansari and Lal 2009,
Ravina et al. 1993), antiproliferative (Ansari and Lal 2009,
Garuti et al. 2000), anti-HIV (Ansari and Lal 2009, Rao et
al. 2002), anticonvulsant (Ansari and Lal 2009, Chimirri et
al. 2001), anti-inflammatory (Ansari and Lal 2009,
Thakurdesai et al. 2007), and antineoplastic (Ansari and
Lal 2009, Abdel-monem et al. 2007), activities. Varied
bioactivities exhibited by benzimidazoles, efforts have
been made from time to time to generate libraries of these
compounds and screened them for potential biological
activities.
This ring system is present in numerous antiparasitic,
fungicidal, anithelemintic and anti-inflammatory drugs
(Habib et al. 1997, Tuncbilek et al. 1997, Pedini et al. 1994
and Lackner et al. 1989). Also, some benzimidazole
nucleosides, particularly 5,6-dichlorobenzimidazole-1--
D-ribofuranoside (DRB) and its 2-substituted derivatives
show activity against human cytomegalovirus (Devivar et
al. 1994). It is also known that 5,6-dinitrobenzimidazole
can substitute 5,6-dimethylbenzimidazole in the vitamin
B12 molecule in Corynebacterium diphteriae
(Pawekiewicz et al. 1957) and 2-trifluorobenzimidazoles
are potent decouplers of oxidative phosphorylation in
mitochondria. They are also inhibitors of photosynthesis,
and some exhibit appreciable herbicidal activity (Burton et
al. 1965). Most recently, antiprotozoal activity of
substituted 2-trifluorobenzimidazoles has been reported
(Navarette-Vazquez et al. 2001), consistent with several
earlier studies on the anti-giardial activity of various
benzimidazole derivatives (Xiao et al. 1996 and Katiyar et
al. 1994). However, the general antimicrobial activity of
benzimidazole derivatives has not been extensively
investigated. The earliest report of their antibacterial
activity appeared in 1964 (Bishop et al. 1964), and more
recently we have found two groups of substituted
benzimidazoles, namely the 5, 6-dinitro and 2-
trifluoromethyl derivatives, to be promising candidates for
antimicrobial drugs (Stefaska et al. 1999). In this paper
we present new data on the antimicrobial and antiprotozoal
activities of 2-substituted-5-nitrobenzimidazoles.
Various functional groups, such as, chloro, fluoro, nitro,
methoxy, and so on, have an important significance in
medicinal chemistry. Espically the dihalogen-like dichloro,
as in antifungal azoles, provides a potent antifungal
activity for drugs such as oxiconazole, ketoconazole,
terconazole, itraconazole and so on.
International Journal of Drug Design and Discovery
Volume 2 Issue 4 October December 2011. 633-636
* For correspondence: Priyal J ain,
Mob.: 09993181881
Email: pnpharma@gmail.com
633
634 International Journal of Drug Design and Discovery Volume 2 Issue 4 October December 2011

Results and Discussion
Chemical Synthesis
In order to expand the group of benzimidazole derivatives,
we synthesized several new benzimidazole ring-containing
compounds. All synthesized compounds were
characterized by various physicochemical parameters
(TLC, melting point, solubility) and also by
spectroscopical analysis (UV, IR, MASS and NMR) for
their structure determination. The purity of all synthesized
compounds was confirmed by column chromatography.
The synthesis of target compounds was accomplished
according to the reaction sequence reported in Scheme 1.
Compounds 1A, 1B, 1C and 1D were synthesized by
condensation of corresponding aldehydes i.e., 4-
chlorobenzaldehyde, 4-fluorobenzaldehyde, pyridine-2-
carbaldehyde and 4-methylbenzaldehyde with appropriate
4-nitro substituted o-phenylenediamine, and sodium
metabisulfite in absolute ethanol (95%) (White et al.
2004).
Antimicrobial Evaluation
The in vitro antimicrobial activity was performed using the
disc diffusion method with different strains of bacteria and
fungi. Ampicillin and nalidixic acid were used as positive
control for bacteria. The results of the final compounds for
preliminary antibacterial testing are shown in table 1. The
results revealed that the majority of the synthesized
compounds showed varying degree of inhibition against
the tested microorganisms. In general, the inhibitory
activity against the Gram-positive bacteria was higher than
that of the Gram-negative bacteria. The chloro and methyl
substitutions at the 4-position of phenyl ring in the
molecule of nitro benzimidazole has the best overall
antibacterial profile. The fluoro and pyridine substituents
on phenyl ring at nitro benzimidazole displayed least
activity.
The compounds showed activity which is comparable
with control against bacterial strains in increasing order of
4- Cl >4-CH
3
>4-F >Pyridine. This shows that para
position with lipophilic group may be important to exhibit
significant activity as antimicrobial agents. It is quite
evident from the above sequence that the compound 1A
which contain nitro group (strong electron withdrawing
group) at para position and a 4-chloro substituent is highly
active. The activity decreasing as the electron withdrawing
ability of the substituent decreased. The derivatives 1A and
1D exhibited good activity in comparison to the standard.

Scheme 1 Synthesis of designed compounds. R =4-chlorophenyl, 4-fluorophenyl, 2-pyridyl, and 4-
methoxyphenyl. Reaction conditions: (a) Na
2
S
2
O
3
, absolute ethanol, reflux, 4 hours.
Table 1 Antibacterial activity of compounds (1A-1D).
Compounds
Mean Zone Inhibition (in mm)
a

Gram-Positi ve Bacteria Gram-Negati ve Bacteria
S. aureus S. mutans B. subtilis E. coli S. typhi P. aeruginosa
1A 38 20 28 18 18 14
1B 32 18 17 20 --- 10
1C 25 14 22 ---- 10 10
1D 37 18 28 16 13 12
Ampicillin
b
38 22 28 20 ---- -----
Nalidixic Acid
b
---- ---- ---- 28 20 18
a
Values are mean (n =3)
b
Ampicillin (10 g/disc) and Nalidixic acid (30 g/disc) used as positive reference; synthesized compounds (300 g/disc)
indicates no sensitivity or mean inhibition zone diameter lower than 7 mm
Priyal Jain, et al : Synthesis Antimicrobial Activity and Chemotherapeutic Potential some Novel Benzimidazole Derivaties 635

Conclusion
We have synthesized series of 2-substituted-5-nitro
benzimidazole derivatives. Among the synthesized
benzimidazoles, compounds with chloro and methyl at 4-
position of phenyl ring attached at 2-position of
nitrobenzimidazole were found to increase the antibacterial
activity potentially. Compounds with fluorophenyl and
pyridine substituents at nitrobenzimidazole showed good
antibacterial activity. More extensive study is needed to
confirm the preliminary results and mode of action studies
are required to be able to optimize the effectiveness of this
series of compounds.
Experimental
Materials and Methods
Melting points were determined in open capillaries with
the help of VEGGO melting point apparatus and are
uncorrected. IR spectra (KBr) were recorded on
SHIMADZU IR spectrophotometer. 1H NMR spectra were
recorded by Bruker WM 400 FT instrument using D2O as
solvent and tetramethylsilane (TMS) as internal reference
standard. All chemical shifts () are in ppm. The purities of
the compounds were checked by thin layer
chromatography (TLC) on silica gel-G plates. The major
chemicals were purchased from Aldrich Chemical
Corporation. All other chemicals were of analytical grade.
General procedure for Synthesis of 2-substituted-5-
nitrobenzimidazole derivatives
To a solution of 1eq of 4-nitro o-phenylenediamine and 1
eq of corresponding aldehyde in ethanol, 4 eq of Na
2
S
2
O
5

was added and the resulting mixture was reflux for 4 hours.
After reaction mixture was cooled to room temperature,
diethyl ether was added and the crude product was filtered
off. The crude product was suspended in mixture of
ethanol-diethyl ether several times until the powder was
obtained analytically pure.
Synthesis of 2-(4-Chloro-phenyl)-5-nitro-1H-
benzimidazole (1A)
To a solution of 4-nitro-o-phenylenediamine (0.001 mole,
0.15 g) and 4-chloro-benzaldehyde (0.001 mol, 0.14 g) in
ethanol, Na
2
S
2
O
5
(0.001 mole, 0.76 g) was added and the
resulting mixture was refluxed for 4 hours. After reaction
mixture was cooled to room temperature, diethyl ether was
added and the crude product was filtered off. The crude
product was suspended in mixture of ethanol-diethyl ether
several times until the powder was obtained analytically
pure. Yield 58%; melting point 275
o
C-277
o
C; IR (KBr)
(cm
-1
): 3274.3 (N-H), 3056.2 (CH str.), 1377.5 (NO
2
),
742.0 (Ar-Cl);
1
H-NMR (300 MHz, D
2
O): 7.9-8.6 (m,
3H, benzimidazole ring), 4.7 (s, H, NH), 7.2-7.5 (m, 4H,
ArH); Mass spectra m/z, 273.7
Synthesis of 2-(4-Fluoro-phenyl)-5-nitro-1H-
benzimidazole (1B)
Synthetic procedure is same as described for 1A except 4-
fluorobenzaldehyde (0.001 mole, 0.12 ml) used in place of
4-chlorobenzaldehyde. Yield 67%; melting point 221
o
C-
222
o
C; IR (KBr) (cm
-1
): 3294.7 (N-H), 3064.3 (C-H str.),
1332.8 (NO
2
), 1058.5 (Ar-F);
1
H-NMR (300 MHz, D
2
O):
7.8-8.5 (m, 3H, benzimidazole ring), 4.8 (s, H, NH), 7.2-
7.5 (m, 4H, ArH); Mass spectra m/z, 257.1.
Synthesis of 5-Nitro-2-pyridin-3-yl-1H-benzimidazole
(1C)
Synthetic procedure is same as described for 1A except
pyridine-2-carbaldehyde (0.001 mol, 0.10 mL) used in
place of 4-chlorobenzaldehyde. Yield 49%; melting point
259
o
C-261
o
C; IR (KBr) (cm
-1
): 3328.4 (N-H), 3056.9 (CH
str.), 1625.1, 1591.6 (C=N str.), 1348.5, 1517.9 (NO
2
);
1
H-
NMR (300 MHz, D
2
O): 7.8-8.6 (m, 3H, benzimidazole
ring), 4.8 (s, H, NH), 7.2-7.5 (m, 4H, ArH); Mass spectra
m/z, 240.2.
Synthesis of 2-(4-Methyl-phenyl)-5-nitro-1H-
benzimidazole (1D)
Synthetic procedure is same as described for 1A except 4-
methylbenzaldehyde (0.001 mole, 0.12 ml) used in place of
4-chlorobenzaldehyde. Yield 54%; melting point 205
o
C-
207
o
C; IR (KBr) (cm
-1
): 3258.2 (N-H), 3050.7 (C-H str.),
2915.9 (methyl -CH str.), 1366.3 (NO
2
);
1
H-NMR (300
MHz, D
2
O): 7.9-8.6 (m, 3H, benzimidazole ring), 4.7
(s, H, NH), 7.2-7.5 (m, 4H, ArH); Mass spectra m/ z,
253.3.
Antimicrobial Activity Test
The compounds (1A-1D) were tested for their in vitro
growth inhibitory activity against different microbes. The
bacterial strains used were Staphylococcus aureus ATCC
29213, Streptococcus mutans MTCC 890 and Bacillus
subtilis MTCC 741 (all Gram-positive) and Ecsherichia
coli ATCC 25922, Salmonella typhi MTCC 733 and
Pseudomonas aeruginosa MTCC 741 (all Gram-negative).
For testing the antifungal activity of the synthesized
compounds the fungal strains Candida albicans MTCC
1637, Aspergillus flavus AIIMS and Aspergillus niger
AIIMS were used. The inhibition zones of synthesized
compounds were determined using disc diffusion method
(National Comittee). In this method, paper disks (6 mm)
containing specific amounts of an antimicrobial agent (300
g for the synthesized compounds) were placed on the
surface of an agar plate inoculated with a standardized
suspension of the microorganisms tested. The plates were
incubated at 35C for 24 and 48 hours, respectively for
636 International Journal of Drug Design and Discovery Volume 2 Issue 4 October December 2011

bacteria and fungi. Ampicillin (10 g) for Gram-positive
bacteria, Nalidixic acid (30 g) for Gram-negative bacteria
and Amphotericin B (30 g) for fungi, were used as
standard drugs. Paper disks with only DMSO were utilized
as negative control. All experiments were carried out three
times. The inhibition zones produced by the various
synthesized compounds on the microbial growth were
measured (diameter in mm). The data on antimicrobial
activity of compounds (1A-1D) are shown in table 1.
Acknowledgement
The authors are thankful to IIT, Mumbai and Dhanwantri
research & analytical laboratory, Kim, Gujarat for spectral
analysis of all new compounds.
References
[1] Abdel-monem, A.; Arch. Pharm. Res., 2007, 30,678.
[2] Ansari, K. F.; Lal, C.; J . Med. Chem., 2009, 30, 1.
[3] Ayhan-Kilcigil, G.; Kus, C.; Ozdamar, E.D.; Can-Eke, B.;
Iscan, M.; Arch. Pharm. 2007, 34, 607.
[4] Bishop, B.C.; Chelton, E.T.J .; J ones, A.S.; Biochem.
Pharmacol. 1964, 13, 751.
[5] Burton, D.E.; Lambie, A.J .; Ludgate, J .C.; Newbold, G.T.;
Percival, A.; Saggers, D.T.; Nature. 1965, 208, 1166.
[6] Chimirri, A.; Sarro, A.D.; Sarro, G.D.; Gitto, R.; Zappala,
M.; Il Farmaco. 2001, 56, 821.
[7] Devivar, R.V.; Kawashima, E.; Revankar, G.R.;
Breitenbach, J .; Kreske, E.; Drach, J .; Townsend, L.; J .
Med. Chem. 1994, 37, 2942.
[8] Garuti, L.; Roberti, M.; Malagoli, M.; Rossi, T.; Castelli,
M.; Bioorg. Med. Chem. Lett. 2000, 10, 2193.
[9] Goker, H.; Kus, C.; Boykin, D.W.; Yildiz, S.; Altanlar, N.;
Bioorg. Med. Chem. 2002, 10, 2589.
[10] Habib, N.S.; Soliman, R.; Ashour, F.A.; Taiebi, M.;
Pharmazie, 1997, 52, 746.
[11] Katiyar, S.K.; Gordon, V.R.; McLaughlin, G.L.; Edlind,
T.D.; Antimicrob. Agents Chemother. 1994, 38, 2086.
[12] Kazimierczuk, Z.; Upcroft, J .A.; Upcroft, P.; Gorska, A.;
Starosciak, B.; Laudy, A.; Acta Biochim. Pol. 2002, 49,
185.
[13] Lackner, T.E.; Clissold, S.P.; Drugs, 1989, 38, 204.
[14] Navarette-Vazquez, G.; Cedillo, R.; Hernandez- Campos,
A.; Yepez, L.; Hernandez-Luis, F.; Valdez, J .; Morales, R.;
Cortes, R.; Hernandez, M.; Castillo, R.; Bioorg. Med.
Chem. Lett. 2001, 11, 187.
[15] Pawekiewicz, J .; Zodrow, K.; Acta Microbiol. Polon. 1957,
6, 9.
[16] Pedini, M.; Alunni Bistochi, G.; Ricci, A.; Bastianini, L.;
Lepri, E.; Farmaco, 1994, 49,823.
[17] Rao, A.; Chimirri, A.; Clercq, E.D.; Monforte, A.M.;
Monforte, P.; Pannecouque, C.; Zappala, M.; II Farmaco,
2002, 57, 819.
[18] Ravina, E.; Sanchez-Alonso, R.; Fueyo, J .; Baltar, M.P.;
Bos, J .; Iglesias, R.; Sanmartin, M.L.; Arzneim. Forsch,
1993, 43, 684.
[19] Stefaska, J .Z.; Gralewska, R.; Starooeciak, B.J .;
Kazimierczuk, Z.; Pharmazie, 1999, 54, 879.
[20] Thakurdesai, P.A.; Wadodkar, S.G.; Chopade, C.T.;
Pharmacology Online, 2007, 1, 314.
[21] Tuncbilek, M.; Goker, H.; Eryigit, R.; Kendi, E.; Altanlar,
E.; Arch. Pharm., 1997, 330, 372.
[22] Xiao, L.; Saeed, K.; Herd, R.P.; Vet. Parasitol., 1996, 61,
165.