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Synthesis and Microbial Activities of 2, 5, 6-Substituted
Benzimidazole Derivatives
Dhondiba Vishwanath
1
, Devaraju
2
, Vijayanand Vithalrao
3
1
GWFGC Kalaburagi, Karnataka, India
2
Yuvaraja college Mysore, Karnataka, India
3
GC(Autonomous) kalaburagi, Karnataka, India
DOI: https://dx.doi.org/10.51584/IJRIAS.2025.101100038
Received: 24 November 2025; Accepted: 30 November 2025; Published: 09 December 2025
ABSTRACT
Benzimidazole derivatives are the analogues of purine nucleosides which are found in human body. These are
an important heterocyclic organic compounds containing phenyl ring (six membered ring) fused with imidazole
(five membered ring) which possesses wide range of starting material for various compounds and exhibit clinical
applications such as anti-inflammatory, antibacterial, antifungal, antiviral, analgesic, proton pump inhibitors,
antihistamines, anticancer, etc. The presence of electron-donating groups (OH, -CH
3
, -OCH
3
) causes significant
increase in the biological activity, while the electron-withdrawing groups (-Cl, -Br,–NO
2
) decreases the
biological activity of synthesized of benzimidazole derivatives.
Keywords: Benzimidazole; 4,5-substituted phenylene-1,2-diamine, trimethoxy benzaldehyde; magnetic stir;
Na
2
S
2
O
5
; microbial activity.
INTRODUCTION:
Benzimidazole derivatives are an important benzfused heterocyclic organic compounds which contains benzene
ring and imidazole ring [1-2]. They are analogue of nucleosides which are found in living organism. They possess
an extensive range of clinical applications such as anti-inflammatory, antibacterial, antifungal, antiviral,
analgesic, and anticancer [3-6]. Benzimidazole compounds can be further improved by changing substituent
groups on the core structure. This method is the most familiar to improve their biological activities. Many
commercially available drugs based on the benzimidazole skeleton such as antifungal (carbendazole),
anthelmintics (albendazole, mebendazole, fenbendazole, and oxibendazole), proton pump inhibitors
(omeprazole), antihypertensives (candesartan and telmisartan), anticancer (bendamustine, nocodazole, and
abemaciclib), antiviral(enviradene) and antihistamines(emedastine and clemizole). In this article we planned to
modify the benzimidazole nucleus at 2, 4 and 5 –positions to prepare better biological active compounds. The
synthesis of benzimidazole derivatives typically involved in the reaction between 4,5-substituted phenylene 1,2
dimine and 4,5,6-trimethoxy benzaldehyde using oxidizing agent sodium metabisuphite in ethanol[7-10].
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Experimental: Material and Methods
All the reagents and chemicals were purchased from Sigma-Aldrich and used without further purification.
Melting points were found using thiel’s tube and paraffin oil with open capillary tubes and are uncorrected. TLC
is performed with E. Merck pre-coated silica gel plates (60F-254) with ninhydrin as a spot developing agent.
Acme, India silica gel, 60–120 mesh is used for column chromatography. IR spectra in KBr were recorded on
Perkin-Elmer model 683 spectrometers.
1
H NMR (300 MHz) and
13
C NMR (100 MHz) spectra were recorded
CDCl
3
solvent containing tetra methyl silane (TMS) as internal references were recorded on Bruker
spectrometer; Elemental analyses were performed on a PerkinElmer 2400. Mass spectra were obtained by Water-
QTOF ultima spectrometer. Micro analytical data were obtained by elemental-Vario EL-III [10-13].
General methods of preparation of 2, 5, 6-substituted derivatives benzimidazoles: A mixture of substituted
phenyl 1,2-dimine 1(a-f) 0.02 mole, 3, 4, 5-trimethoxybenzaldehyde 0.02 mole and 0.04 mole sodium
metabisulphite was added in 30cm
3
of ethanol. The resulting reaction mixture was stirred using magnetic stirrer
at room temperature for about 2-3 hours. The progress of reaction was checked by TLC. After complete
completion of reaction, the product was concentrated with vacuum distillation. The solid product 2(a-f) was
washed with water and then n-hexane and dried in anhydrous P
2
O
5
for overnight. Product was again dried at
room temperature and determined the physical constant.
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Scheme
Compound
R
1
R
2
1-2(a)
Cl
Cl
1-2(b)
Br
Br
1-2(c)
CH
3
CH
3
1-2(d)
OCH
3
OCH
3
1-2(e)
OH
H
1-2(f)
NO
2
H
5, 6-dichloro-2-(3, 4, 5-trimethoxyphenyl)-1H-benzimidazole 2(a): Colour: Light yellow solid. M. P.
145149
0
C, Yield 75.5%. IR (KBr): 3250cm
-1
(N-H), 1500cm
-1
(C=N), 750cm
-1
(Ar-Cl) 1387cm
-1
(Ar-OCH
3
).
1
H-
NMR (CDCl
3
); δ (ppm): 3.71-3.83 (bs, 9H, OCH
3
), 6.97 – 8.30 (m, 4H, Ar.-H), 12.5(s, 1H, NH).
13
C-NMR
(CDCl
3
); δ (ppm):56.1, 60.1, 60.8, 139.2, 153.1, 104.6, 124.9, 152.9, 130.9, 117.2, 128.6. Mass (m/z): 354.20.
Elemental Analysis (%): For C
16
H
14
O
3
Cl
2
N
2
, Calculated: C, 54.41; H, 4.00; O, 13.59; Cl, 20.07; N, 7.93. Found:
C, 54.45; H, 3.91; O, 20.00; Cl, 13.52; N, 7.89.
5, 6-dibromo-2-(3, 4, 5-trimethoxyphenyl)-1H-benzimidazole 2(b): Colour: Yellow solid. M. P. 155-159
0
C,
Yield 83.7%. IR (KBr): 3250cm
-1
(N-H), 1500cm
-1
(C=N), 650cm
-1
(Ar-Br) 1387cm
-1
(Ar-OCH
3
).
1
H- NMR
(CDCl
3
); δ (ppm): 3.71-3.83 (bs, 9H, OCH
3
), 6.97 – 7.76 (m, 4H, Ar.-H), 12.56(s, 1H, NH).
13
C-NMR (CDCl
3
);
δ (ppm):56.1, 60.1, 139.2, 153.1, 104.6, 124.9, 152.9, 140.1, 120.9, 120.8. Mass (m/z): 442.11. Elemental
Analysis (%): For C
16
H
14
O
3
Br
2
N
2
, Calculated: C, 43.47; H, 3.19; O, 10.85; Br, 36.15; N, 6.34. Found: C, 43.35;
H, 3.25; O, 10.91; Br, 36.10; N, 6.39.
5, 6-dimethyl-2-(3, 4, 5-trimethoxyphenyl)-1H-benzimidazole 2(c): Colour: Colourless solid. M. P.
107109
0
C, Yield 71.8%. IR (KBr): 3250cm
-1
(N-H), 1500cm
-1
(C=N), 1387cm
-1
(Ar-OCH
3
).
1
H- NMR (CDCl
3
);
δ (ppm): 2.47(s, 6H, CH
3
), 3.71-3.83 (bs, 9H, OCH
3
), 6.97 – 7.35 (m, 4H, Ar.-H), 12.56(s, 1H, NH).
13
C-NMR
(CDCl
3
); δ (ppm):18.8, 56.1, 60.8, 139.2, 153.1, 104.6, 124.9, 132.6, 115.2, 130.3. Mass (m/z): 312.37. Elemental
Analysis (%): For C
18
H
20
O
3
N
2
, Calculated: C, 69.21; H, 6.45; O, 15.37; N, 8.97. Found: C, 69.25; H, 6.41; O,
15.42; N, 8.92.
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5, 6-dimethoxy-2-(3, 4, 5-trimethoxyphenyl)-1H-benzimidazole 2(d): Colour: Colourless solid. M. P.
127129
0
C, Yield 79.4%. IR (KBr): 3250cm
-1
(N-H), 1500cm
-1
(C=N), 1387cm
-1
(Ar-OCH
3
).
1
H- NMR (CDCl
3
);
δ (ppm): 3.71-3.83 (bs, 15H, OCH
3
), 6.97 – 7.14 (m, 4H, Ar.-H), 12.56(s, 1H, NH).
13
C-NMR (CDCl
3
); δ (ppm):
56.1, 60.8, 139.2, 153.1, 104.6, 152.9, 132.2, 101.8, 142.0. Mass (m/z): 344.37. Elemental Analysis (%): For
C
18
H
20
O
5
N
2
, Calculated: C, 62.78; H, 5.85; O, 23.23; N, 8.13. Found: C, 62.70; H, 5.88; O, 23.28; N, 8.14.
5-hydroxy-2-(3, 4, 5-trimethoxyphenyl)-1H-benzimidazole 2(e): Colour: Orange solid. M. P. 162-165
0
C,
Yield 84.1%. IR (KBr): 3250cm
-1
(N-H), 1500cm
-1
(C=N), 3450cm
-1
(Ar-OH) 1387cm
-1
(Ar-OCH
3
).
1
H- NMR
(CDCl
3
); δ (ppm): 3.71-3.83 (bs, 9H, OCH
3
), 6.97 – 7.39 (m, 5H, Ar.-H), 9.45(s, 1H, OH), 12.56(s, 1H, NH).
13
C-NMR (CDCl
3
); δ (ppm): 56.1, 60.8, 139.2, 153.1, 104.6, 124.9, 152.9, 134.3, 140.3, 102.4, 116.6, 111.4,
151.7. Mass (m/z): 300.31. Elemental Analysis (%): For C
16
H
16
O
4
N
2
, Calculated: C, 63.99; H, 5.37; O, 21.31;
N, 9.33. Found: C, 63.91; H, 5.35; O, 21.35; N, 9.39.
5-nitro-2-(3, 4, 5-trimethoxyphenyl)-1H-benzimidazole 2(f): Colour: Red solid. M. P. 187-199
0
C, Yield
85.0%. IR (KBr): 3250cm
-1
(N-H), 1500cm
-1
(C=N), 1450cm
-1
(Ar-NO
2
) 1387cm
-1
(Ar-OCH
3
).
1
H- NMR (CDCl
3
);
δ (ppm): 3.71-3.83 (bs, 9H, OCH
3
), 6.97 – 8.09 (m, 5H, Ar.-H), 12.56(s, 1H, NH).
13
C-NMR (CDCl
3
); δ (ppm):
56.1, 60.8, 139.2, 153.1, 104.6, 124.9, 152.9, 139.8, 147.8, 112.9, 116.1, 118.6, 144.3. Mass (m/z): 329.31,
Elemental Analysis (%): For C
16
H
15
O
5
N
3
, Calculated: C, 58.36; H, 4.59; O, 24.29; N, 12.76. Found: C, 58.31;
H, 4.62; O, 24.33; N, 12.74.
Antimicrobial Activity:
1. Antibacterial Activity
The pure compounds 2(a-f) were carried out for their antibacterial activity by using disc diffusion method. The
nutrient agar broth prepared by the usual method, was inoculated aseptically with 0.5 cm
3
of 24 hours old
subculture of Staphylococcus aureus(SA) and Escherichia coli(EC) in separate conical flask at 35
o
C-45
o
C and
mixed well by shaking. About 30 cm
3
of the contents of the flask were poured and evenly spread in Petridis (90
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mm in diameter) and allowed to set for three hours. The cups (8 mm in diameter) were formed by the help of
borer in agar medium and filled with 0.1 cm
3
(1mg/ cm
3
) solution of sample in propanone. The plates were
incubated at 25-30
o
C for 7 days. After the completion of incubation period, the zones of inhibition growth is in
the form of diameter in mm was measured. Along the test solution in each Petridis one cup was filled up with
ciprofloxacin as standard and another cup was filled up with distilled water (DW) which acts as negative control
all over the experiment of 50 μg concentration [11-13].
Table-1: Antibacterial activity of the synthesized compounds 2(a-f)
Sl.
No.
Compounds
Zone of inhibition in mm
Staphylococcus aureus
Escherichia coli
1
2a
12
14
2
2b
15
11
3
2c
23
20
4
2d
24
24
5
2e
21
23
6
2f
16
15
7
Ciprofloxacin
20
18
8
Distilled water
14
12
Graph-1: Inhibition zone against bacteria of the synthesized compounds 2(a-f)
Antifungal Activity
The pure compounds 2(a-f) were screened for their antifungal activity by using disc diffusion method. The fungi
like Trichoderma harzianum(TH), Aspergillus niger(AN), Colletotrichum capsici(CC), Aspergillus tamari(AT),
Aspergillus flavus(AF), Alternaria solani(AS), and Penicillium oxalicum(PO) were employed for testing. The
culture was maintained on sabouraud dextrose agar slants. Sterilized Sabouraud dextrose agar medium was
inoculated with 72 hr old 0.5 cm
3
suspension of fungal spores in a separate flask. About 25 cm
3
of the inoculated
medium was evenly spreader in a sterilized Petridis and allowed to set for 5 hours. The cups (8 mm in diameter)
were punched in Petridis and loaded with 0.1 cm
3
(2 mg/ cm
3
) of solution of sample in propanone. The plates
were incubated at 25-30
o
C for 7 days. After the completion of incubation period, the zones of inhibition growth
is in the form of diameter in mm was measured. Along the test solution in each Petridis one cup was filled up
with nystatin as standard and another cup was filled up with distilled water (DW) which acts as negative control
all over the experiment of 50 μg concentration [14-16].
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Table-2: Antifungal activity of the synthesized compounds 2(a-f)
Sl.
No.
Compounds
Zone of inhibition in mm
TH
AN
CC
AT
AF
AS
PO
1
2a
16
9
13
15
17
15
14
2
2b
14
11
15
15
15
14
14
3
2c
19
17
21
20
23
20
19
4
2d
19
20
21
19
21
22
21
5
2e
22
18
16
17
22
19
22
6
2f
13
15
10
12
13
15
12
7
Nystatin
17
16
18
17
18
18
15
8
Distilled water
13
11
11
10
13
14
10
Graph-2: Inhibition zone against fungi of the synthesized compounds 2(a-f)
RESULTS AND DISCUSSION
2,5,6 -substituted benzimidazole derivatives were prepared in good yields by oxidation reaction of 4,5-
substituted phenyl 1,2-dimine 1(a-f) with 3, 4, 5-trimethoxybenzaldehyde using sodium metabisulphite in
ethanol. The resulting reaction mixture was stirred using magnetic stirrer at room temperature for about 2-3
hours. The progress of reaction was checked by TLC. After complete completion of reaction, the product was
concentrated with vacuum distillation. The solid product 2(a-f) was washed with water and then n-hexane and
dried in anhydrous P
2
O
5
for overnight. Product was again dried at room temperature and determined the physical
constant. The products were recrystallized using hot water. The structure of 2,5,6- substituted benzimidazole
derivatives of was confirmed by IR,
1
H- NMR,
13
C-NMR and mass spectral data.
The synthesized compounds were conducted for antibacterial activity and antifungal activity and the results were
summarized in table-1 & graph-1 and table-2 & graph-2 respectively. From the table-1 & graph-1, the
synthesized compounds 2c and 2d shows very active and 2e moderately active against both bacteria due to the
presence of electron releasing groups in their structures where as 2a, 2b and 2f compounds less active due to the
presence of electron withdrawing group against the ciprofloxacin as standard. Similarly from the table-2 &
graph-2, the synthesized compounds 2c, 2d and 2e shows very active against all the fungi due to the presence of
electron releasing groups in their structures where as 2a, 2b and 2f compounds less or moderately active against
all the fungi due to the presence of electron withdrawing group against the nystatin as standard.
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CONCLUSION
We succeeded for the synthesizing of 2, 5, 6- substituted benzimidazole derivatives and their microbial activities.
Some synthesized compounds were very active against fungi and bacteria, others are remains inactive compared
to the standard Ciprofloxacin and nystatin which are already available in the market.
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