Synthesis and evaluation of selected benzimidazole derivatives as potential antimicrobial agents

Article abstract of DOI:10.3390/molecules200815206

A library of 53 benzimidazole derivatives, with substituents at positions 1, 2 and 5, were synthesized and screened against a series of reference strains of bacteria and fungi of medical relevance. The SAR analyses of the most promising results showed that the antimicrobial activity of the compounds depended on the substituents attached to the bicyclic heterocycle. In particular, some compounds displayed antibacterial activity against two methicillin-resistant Staphylococcus aureus (MRSA) strains with minimum inhibitory concentrations (MICs) comparable to the widely-used drug ciprofloxacin. The compounds have some common features; three possess 5-halo substituents; two are derivatives of (S)-2-ethanaminebenzimidazole; and the others are derivatives of one 2-(chloromethyl)-1Hbenzo[ d]imidazole and (1H-benzo[d]imidazol-2-yl)methanethiol. The results from the antifungal screening were also very interesting: 23 compounds exhibited potent fungicidal activity against the selected fungal strains. They displayed equivalent or greater potency in their MIC values than amphotericin B. The 5-halobenzimidazole derivatives could be considered promising broad-spectrum antimicrobial candidates that deserve further study for potential therapeutic applications.

Full text of DOI:10.3390/molecules200815206

Molecules 2015, 20, 15206-15223; doi:10.3390/molecules200815206
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Evaluation of Selected Benzimidazole Derivatives
as Potential Antimicrobial Agents
Fatmah A. S. Alasmary 1,2,*, Anna M. Snelling 2, Mohammed E. Zain 3, Ahmed M. Alafeefy 4,
Amani S. Awaad 5 and Nazira Karodia 2,6,
*
1
Chemistry Department, College of Science, King Saud University, Riyadh 11362, Saudi Arabia
Centre for Pharmaceutical Engineering Science, Faculty of Life Sciences, University of Bradford,
Richmond Road, Bradford BD7 1DP, UK; E-Mail: a.m.snelling@bradford.ac.uk
Botany and Microbiology Department, College of Science, King Saud University, Riyadh 11362,
Saudi Arabia; E-Mail: mohamedzain@hotmail.com
2
3
4
5
6
Department of Pharmaceutical Chemistry, College of Pharmacy, Sattam bin Abdulaziz University,
Al-Kharj 11942, Saudi Arabia; E-Mail: alafeefy@hotmail.com
Pharmacognosy Department, College of Pharmacy, Salman bin Abdulaziz University,
Al-Kharj 11942, Saudi Arabia; E-Mail: amaniawaad@hotmail.com
Faculty of Science and Engineering, University of Wolverhampton; Wulfruna Street,
Wolverhampton WV1 1LY, UK
* Authors to whom correspondence should be addressed; E-Mails: fasmari@ksu.edu.sa (F.A.S.A.);
nazira.karodia@wlv.ac.uk (N.K.); Tel.: +966-506-258-414 (F.A.S.A.); +44-190-232-1920 (N.K.).
Academic Editor: Derek J. McPhee
Received: 28 June 2015 / Accepted: 10 August 2015 / Published: 20 August 2015
Abstract: A library of 53 benzimidazole derivatives, with substituents at positions 1, 2 and 5,
were synthesized and screened against a series of reference strains of bacteria and fungi of
medical relevance. The SAR analyses of the most promising results showed that the
antimicrobial activity of the compounds depended on the substituents attached to the bicyclic
heterocycle. In particular, some compounds displayed antibacterial activity against two
methicillin-resistant Staphylococcus aureus (MRSA) strains with minimum inhibitory
concentrations (MICs) comparable to the widely-used drug ciprofloxacin. The compounds
have some common features; three possess 5-halo substituents; two are derivatives of
(S)-2-ethanaminebenzimidazole; and the others are derivatives of one 2-(chloromethyl)-1H-
benzo[d]imidazole and (1H-benzo[d]imidazol-2-yl)methanethiol. The results from the
antifungal screening were also very interesting: 23 compounds exhibited potent fungicidal

Molecules 2015, 20
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activity against the selected fungal strains. They displayed equivalent or greater potency
in their MIC values than amphotericin B. The 5-halobenzimidazole derivatives could be
considered promising broad-spectrum antimicrobial candidates that deserve further study for
potential therapeutic applications.
Keywords: benzimidazole; heterocycle; antibacterial activity; antifungal activity; resistance;
Gram-negative; Gram-positive
1. Introduction
Microbial drug resistance is a serious issue, especially as increasing numbers of strains are becoming
resistant to multiple antimicrobial agents, with some bacteria now being resistant to all available antibiotics.
There is thus a critical need to develop new drugs with novel mechanisms of action. However, the
investment available for such development is frequently lower than the required level. The development
of new drug entities is hampered by several issues, notably the high cost and length of time required, as
well as the logistical and regulatory challenges of performing the necessary clinical evaluations across
multiple geographical areas. Therefore, a few new classes of antimicrobials have been developed since the
late 1980s [1–3], and much research has focused only on the chemical modification of existing drugs to
improve their potency and/or ability to overcome antibiotic resistance mechanisms. Even if this approach
does not improve antimicrobial activity directly, it may lead to derivatives that can usefully inhibit
virulence mechanisms [4].
Compounds having benzimidazole as a structural motif have been widely used in medicinal chemistry and
drug development, and researchers are actively seeking new uses and applications of this heterocycle [5].
Benzimidazole-containing compounds have numerous medical and biological activities, such as
antitumor [6] antibacterial [7–10], antifungal [11], antiviral [12–16], anticonvulsant [17], antidepressant [18],
analgesic [19], anti-inflammatory[20] and antidiabetic properties [21]. For example, derivatives, such as
thiabendazole, cambendazole, parbendazole, mebendazole, albendazole and flubendazole, are widely-used
anti-helminth drugs, used to treat people and animals with gastrointestinal worm infections [22]. Two
groups of benzimidazole derivatives, namely 5,6-dinitro- and 2-trifluromethyl derivatives, are particularly
well known for their use as antihelminth drugs [23]. 2-Methoxycarbonylamino derivatives have shown
good antiprotozoal activities against some protozoan parasites, such as Giardia lamblia and Entamoeba
histolytica, by inhibiting tubulin polymerization, and hence, making these better antiprotozoal agents than
metronidazole and albendazole [24]. Nitrogen-containing heterocyclic systems have a diverse spectrum
of pharmacological properties. Different heterocyclic motifs can be incorporated to produce molecules
with enhanced biological properties. Recent reports include benzimidazoles bearing the 1,3,4-oxadiazole
moiety, which have broad spectrum antimicrobial properties [25], and molecules containing both the
benzimidazole and indole heterocycles, which exhibit selective antibacterial activity [26].
A review of the literature thus suggests that there is the scope for the design of additional benzimidazole
derivatives with antimicrobial activity, by examining the effect of a number of different functional groups.
In this paper, we report on the synthesis of a series of benzimidazole derivatives and their antimicrobial
activity. A detailed study of the structure-activity relationship of these derivatives will pave the road to

Molecules 2015, 20
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designing more potent compounds. The compounds were tested for their ability to inhibit isolates amongst
a reference panel of 26 bacterial and 10 fungal strains, and key results are presented in Tables 1–3.
Table 1. Benzimidazole derivatives synthesized.
1–53
Compound
R
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
R1
H
R2
H
1
2
H
CH3
OCH3
Cl
3
H
4
H
5
H
Br
6
H
F
7
H
NO2
CN
H
8
H
9
CH2NH2
CH2NH2
CH2NH2
CH2NH2
CH2NH2
CH2NH2
CH(CH3)NH2
CH(CH3)NH2
CH(CH3)NH2
CH(CH3)NH2
CH(CH3)NH2
CH2CH3
CH2CH3
CH2SH
CH2SH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2Cl
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
CH3
Cl
Br
F
NO2
H
CH3
Cl
Br
F
H
NO2
H
NO2
H
CH3
OCH3
Cl
Br
F
NO2
CN
H
CH2Cl
CH3
OCH3
Cl
CH2Cl
CH2Cl

Molecules 2015, 20
15209
Table 1. Cont.
Compound
R
H
R1
R2
Br
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
CH2Cl
H
CH2Cl
F
H
CH2Cl
CH2Cl
COOH
COOH
COOH
COOH
COOH
COOH
COOH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
NO2
CN
H
H
H
H
CH3
OCH3
Cl
H
H
H
Br
H
F
H
NO2
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Cl
Br
F
OCH3
NO2
2. Results and Discussion
2.1. Chemistry
Our target molecules were based on benzimidazole derivatives. It is possible to design a wide range
of potential microbial inhibitors by replacing the hydrogen at various positions of the benzimidazole ring
with different functional groups. However, the most accessible derivatives are those with substituents at
the 1-, 2- and 5-positions. Retrosynthetic analysis of a 2,5-disubstituted benzimidazole identified two
fragments, which explains why these particular substituted benzimidazoles are easy to prepare (Figure 1).
The reactions of substituted 1,2-phenylenediamines and carboxylic acids can provide access to a library
of compounds. In this application, the design of these inhibitors focused on 5-substituted benzimidazoles
followed by the synthesis of 2,5-disubstituted analogues based on (1H-benzimidazole-2-yl)methanol
derivatives, (1H-benzimidazole-2-yl)alkylamines derivatives, (1H-benzimidazole-2-yl)-ethyl derivatives
and (1H-benzimidazole-2-yl)-methanethiol derivatives (Scheme 1). The general method for the synthesis
of these benzimidazoles 1–31 was based on the Phillips procedure [27].
N
5-position
2-position
N
H
1-position
R2
R2
NH2
NH2
N
O
R1
+
R1
N
H
HO
Figure 1. Retrosynthetic analysis of benzimidazole.

Molecules 2015, 20
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Scheme 1. 2,5-Substituted benzimidazole derivatives.
The 1H-benzimidazole-2-yl)methanol derivatives were readily reacted further (oxidation, chlorination
and N-alkylation) (Scheme 2). The hydroxyl group of (1H-benzimidazole-2-yl)methanol was converted
into the chloromethyl through a reaction with thionyl chloride to give 2-(chloromethyl)-1H-benzimidazole
Derivatives 32–39. The 2-chloromethylbenzimidazoless were required for the biological studies to provide
a direct comparison with other functional groups in the 2-position of benzimidazole [28]. Benzimidazole-
2-carboxylic Acids 40–46 were readily obtained through simple oxidation of (1H-benzimidazole-2-yl)-
methanol Derivatives 24–30 using potassium permanganate. The N-methylation of Derivatives 24–30
gave the corresponding N-methyl-5-substituted (1H-benzimidazole-2-yl)methanol derivatives 47–53.
Scheme 2. Conversion of 2-methanol benzimidazole derivatives.

Molecules 2015, 20
15211
2.2. Biological Evaluation
2.2.1. Antibacterial Activity
Firstly, a cut-off point of an inhibition zone diameter of ≥12 mm was used to define appreciable
antibacterial activity. Using this criteria, disc diffusion tests with the 53 compounds revealed that nine
compounds (9, 17, 18, 22, 32, 33, 35, 36 and 52) had some degree of antibacterial activity (Table 2); with
five exhibiting activity against representatives of at least two Staphylococcal species. Six compounds had
activity against one or more strains of Methicillin-resistant Staphylococcus aureus (MRSA), including
strains that were resistant to ciprofloxacin. Six compounds had some activity against S. epidermidis and
three against the single strain of S. haemolyticus used in this study. Regarding the Gram-negative bacteria,
only two (32 and 52) of the compounds tested were active against any of the five strains of Escherichia coli
and none against the four strains of Pseudomonas aeruginosa. Two compounds were active against the
single strain of Serratia marcescens, but six had some activity against the single strain of B. cepacia
used in the screening panel. Compounds 17, 18, 22, 35 and 36 were the most active compounds overall,
and these were selected on the basis of a broad spectrum of activity, and/or wide zones of inhibition, or
novel chemical structure. As summarised in Table 2, these five compounds were investigated further in
minimum inhibitory concentration (MIC) assays to quantify their activity against the reference isolates.
Testing confirmed that they exhibited little activity against the Gram-negative species, with the exception
of Burkholderia cepacia. However, for the Gram-positive species, there were some MICs as low as 64 and
even 32 µg/mL. Notably, Compounds 17 and 18 had MICs comparable to that of ciprofloxacin against
two of the MRSA strains, whilst 22 and 36 had consistent activity (MIC 32–64 µg/mL) against almost
all of the staphylococci. This is significant and provides lead compounds for further development.
Table 2. Antibacterial activity of selected benzimidazole derivatives against Gram-positive
and Gram-negative bacteria (series of 2-ethanamine, 2-methanthiol, 2-chlromethyl and
N-methyl benzimidazole derivatives).
Diameter of the Inhibition Zone (mm) around
Each Compound in the Agar Diffusion Test
Bacterial Species Compound
Gram-Positive
9
17
18
22
32
33 35 36
52 Ciprofloxacin
Staphylococcus aureus NCTC 6571
Staphylococcus aureus NCTC 10399
MRSA HG-1
MRSA-15 NCTC 13142
MRSA-16 NCTC 13143
MRSA BIG 0043
MRSA BIG 0044
MRSA BIG 0045
MRSA BIG 0047
MRSA BIG 0050
MRSA BIG 0052
MRSA BIG 0053
Staphylococcus epidermidis NCTC 11047
Staphylococcus epidermidis NCTC 2749
Staphylococcus haemolyticus NCTC 11042
0
0
0
0
0
0
0
0
0
0
0
0
0
17
0
10
12
10
11
10
11
9
11
12
0
11
12
11
10
13
10
12
11
13
12
13
11
11
13
10
11
16
12
12
15
11
12
14
12
11
11
13
0
13
7
0
0
0
9
10
0
0
0
0
0
10
9
11
9
7
0
0
0
10
10
12
11
9
9
10
12
9
0
0
9
9
0
13
14
14
17
13
15
13
12
12
13
13
13
12
13
12
13
15
27
33
0
28
0
0
0
0
0
0
0
0
32
35
34
10 15
9
9
9
9
9
9
9
0
11
15
11
11
15
14
14
0
0
0
0
9
13
18
10 14
11
9
0
12
0

Molecules 2015, 20
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Table 2. Cont.
Diameter of the Inhibition Zone (mm) around
Each Compound in the Agar Diffusion Test
Bacterial Species Compound
9
17
18
22
32
33 35 36
52 Ciprofloxacin
Gram-Negative
Burkholderia cepacia NCTC 10744
Escherichia coli NCTC 10418
Escherichia coli BIG 0046
12
0
0
0
0
0
0
0
0
0
0
11
0
0
0
0
0
0
0
0
0
0
10
0
0
0
0
0
0
0
0
0
0
15
0
0
0
0
0
0
0
0
0
0
0
12 15 17
12
14
9
27
33
0
13
9
9
9
9
0
9
0
0
0
0
8
9
8
8
7
7
0
0
0
0
9
9
0
8
8
0
0
0
0
0
8
Escherichia coli BIG 0048
15
11
12
10
0
11
8
0
Escherichia coli BIG 0049
0
Escherichia coli 0051
10
10
8
0
Pseudomonas aeruginosa NCTC 6749
Pseudomonas aeruginosa BIG 0039
Pseudomonas aeruginosa NCTC 10662
Pseudomonas aeruginosa BIG 0063
Serratia marcescens NCTC 1377
33
34
32
30
32
10
10
15
8
9
13
Zone diameters indicative of antibacterial activity (i.e., ≥12 mm) are highlighted in bold.
Interestingly, the MICs of Compounds 17 and 18 (2-ethanamine benzimidazole derivatives) against the
investigated bacteria were similar to each other (Table 3). This suggests that the higher electronegativity
of bromine and chlorine increased the antibacterial activity, and this was also observed by Tavman [29].
Table 3. MICs of the selected benzimidazole derivatives against the reference panel of Gram
positive and Gram negative bacteria.
MIC (µg/mL) per Compound
Bacterial Strain
17
18
22
35
36
Ciprofloxacin
Gram-Positive
Staphylococcus aureus NCTC 6571
Staphylococcus aureus NCTC 10399
MRSA HG-1
32
256
32
32
256
32
64
64
64
64
64
64
64
64
64
64
64
64
32
64
32
128
128
265
128
256
256
256
256
256
256
256
256
256
256
256
64
64
≤0.5
>32
32
64
MRSA-15 NCTC 13142
MRSA-16 NCTC 13143
MRSA BIG 0043
256
256
256
>512
256
256
32
256
256
256
>512
256
256
32
64
≤0.5
32
64
64
>32
>32
8
MRSA BIG 0044
64
MRSA BIG 0045
>512
64
MRSA BIG 0047
8
MRSA BIG 0050
64
32
MRSA BIG 0052
256
256
256
256
256
256
256
256
256
265
64
≤0.5
>32
≤0.5
≤0.5
8
MRSA BIG 0053
64
Staphylococcus epidermidis NCTC 11047
Staphylococcus epidermidis NCTC 2749
Staphylococcus haemolyticus NCTC 11042
32
64
32

Molecules 2015, 20
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Table 3. Cont.
MIC (µg/mL) per Compound
Bacterial Strain
17
18
22
35
36
Ciprofloxacin
Gram-Negative
Burkholderia cepacia NCTC 10744
Escherichia coli NCTC 10418
Escherichia coli BIG 0046
Escherichia coli BIG 0048
Escherichia coli BIG 0049
32
32
64
512
>512
512
>512
>512
>512
>512
>512
>512
>512
256
>512
>512
>512
>512
>512
512
512
512
512
>512
64
≤0.5
≤0.5
32
8
8
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
265
512
512
512
>512
Escherichia coli BIG 0051
32
Pseudomonas aeruginosa NCTC 6749
Pseudomonas aeruginosa BIG 0039
Pseudomonas aeruginosa NCTC 10662
Pseudomonas aeruginosa BIG 0063
Serratia marcescens NCTC 1377
≤0.5
≤0.5
≤0.5
≤0.5
≤0.5
Good activity, relative to the MIC of ciprofloxacin, is indicated in bold. The results are the average of three
independent readings.
For the 2-methanthiol benzimidazole derivatives (22–23, Scheme 1), the unsubstituted derivative at
position 5, Compound 22, was selectively active against all of the Gram-positive bacteria with an MIC of
32 µg/mL against S. epidermidis NCTC 11047 and S. haemolyticus NCTC 11042 and MIC 64 µg/m against
the other staphylococci plus B. cepacia (Table 3). The inhibitory activity decreases when position 5 is
methylated as in Compound 23 (>512 MIC µg/mL). This was also observed by Podunavac-Kuzmanovic
and Cvetkovic [30], who found that the unsubstituted analogue was more potent than the methyl analogues
of the 2-aminobenzimidazole against strains of Bacillus cereus, Staphylococcus aureus, Sarcina lutea
and Pseudomonas aeruginosa.
From the series of 2-chloromethyl benzimidazole derivatives (Scheme 2), 36 (5-Br) was the most active
compound with inhibition zone diameters of 13–17 mm. The largest zones of inhibition (17 mm and 15 mm
diameter) were exhibited by Compounds 35 (5-Cl) and 36, respectively, against Burkholderia cepacia
NCTC 10744. Only 35 and 36 were capable of inhibiting some strains at a concentration below 256 μg/mL.
As shown in Table 3, Compound 36, the brominated derivative, was more active than Compound 35,
which is the chlorinated derivative. This could support the hypothesis that there is a direct relationship
between biological activity and the electron withdrawing effect [29].
2.2.2. Antifungal Activity
The well diffusion tests of the 53 compounds against the fungi revealed very interesting results:
21 compounds were active against Aspergillus clavatus RCMB 2593; 28 had some activity against
Aspergillus fumigatus RCMB 02564; 26 compounds were active against Penicillium marneffei RCMB
01267; 22 compounds were active against Mucor circinelloides RCMB 07328; 20 compounds were
active against Absidia corymbifera RCMB 09635; 20 compounds were active against Syncephalastrum
racemosum RCMB 05922; 29 compounds were active against Candida albicans RCMB 05035;
29 compounds were active against Candida tropicalis RCMB 05049; 27 compounds were active against
Candida parapsilosis RCMB 05065; 13 compounds had some activity against Candida krusei RCMB
05051; 14 compounds had no detectable antifungal activity.

Molecules 2015, 20
15214
Amphotericin B was used as a positive control for the antifungal testing. The intention was not to
compare activity on a molar basis, since such a comparison does not necessarily have biological validity
when comparing compounds with very different modes of action or solubility in diffusion assays. This
allowed us to identify the compounds with the most promising activity, which could be looked at in
more detail in further studies.
Tables 4 and 5 summarise the results for the 23 compounds that were most active against the fungal
strains used. The antifungal screening of the series of 2-ethanamine benzimidazole Derivatives 9–19
showed remarkable antifungal activities against both unicellular and filamentous fungi (Table 4).
However, Compound 5 (5-Br) showed the highest antifungal activity; with a 29-mm zone of inhibition
against Candida parapsilosis RCMB 05065 and 26 mm against Candida albicans RCMB 05035,
Candida tropicalis RCMB 05049 and Aspergillus fumigatus RCMB 02564 (Table 4). Seven compounds,
2–6, 14 and 20, from the series of 5-substituted benzimidazole derivatives also showed promising
antifungal activity (Table 4).
Interestingly, all of the selected 23 compounds showed antifungal activity against both the unicellular
and filamentous fungi (Table 4). Moreover, the minimum inhibitory concentration (MIC) of some
compounds, against certain fungal species, was better (i.e., lower) than that of the reference drug (Table 5).
This suggests that when there is no substituent at position 2 and an electron withdrawing substituent at
position 5, the antifungal activity is increased (Tables 4 and 5). This confirms the hypothesis that there is
a direct relationship between the antifungal activity and the electron withdrawing effect of substituents.
Compound 5 (5-Br) was active against the selected strains with MIC equivalent to half that exhibited by
amphotericin B [31].
The antifungal screening of the series of 2-alkylamine benzimidazole derivatives (9–19, Scheme 1)
showed interesting antifungal activity for the compounds. Significant activity was observed for
Compounds 11 (5-Cl) and 14 (5-NO2), especially against Mucor circinelloides RCMB 07328 with low
MICs of 3.9 and 0.49 µg/mL, respectively. However, no activity was observed for the 5-bromo analogue
12. Both Compounds 11 and 14 showed remarkable antifungal activity that was almost similar to the
reference drug, and interestingly, Compound 14 was three-fold as potent as the amphotericin B against
Mucor circinelloides RCMB 07328. This suggests that the more electrons withdrawing the substituent
there are, the higher the antifungal activity (Table 5). Furthermore, Compounds 11 (5-Cl), 13 (5-F), 14
(5-NO2) and 15 (5-H) showed good activity against Candida parapsilosis RCMB 05065; with a MIC
range of 1.95–0.06 µg/mL, which is for Compounds 11 and 15 equivalent to the MIC of amphotericin B.
Compound 13, the 5-fluoro analogue, was three-times more potent, while 14 (5-NO2) was five-fold more
potent than the control drug against Candida parapsilosis RCMB 05065. This suggests that when the
electron withdrawing effect increases, the antifungal effect also increases.
Compound 27 (5-Br), from the series of 2-methanol benzimidazole derivatives (Scheme 2), exhibited
promising antifungal activity against Absidia corymbifera RCMB 09635, Syncephalastrum racemosum
RCMB 05922 and Candida krusei RCMB 05051 with MICs equivalent to the reference, and two-fold the
latter’s potency against Candida parapsilosis RCMB 05065. This suggests that the more electronegative
bromine increases the antifungal activity (Table 5) [29].

Molecules 2015, 20
Table 4. Antifungal activity of the benzimidazole derivatives against unicellular and filamentous fungi in the well diffusion assay.
15215
Diameter of Zone of Inhibition (mm) around Each Compound
11 13 14 15 20 22 27 28 33 37 38 42 43 44 51 52 53 Am. B
Fungal Strain
Unicellular
2
3
4
5
6
7
Candida albicans
RCMB 05035
21 21 24 26 23 21 21 20 22 19 23 17 20 18 19 17 22 17 19 20 19 21 22
17 16 19 19 18 14 14 12 17 14 20 17 19 18 21 22
22
19
18
25
Candida krusei
0
0
0
0
0
0
0
RCMB 05051
Candida parapsilosis
RCMB 05065
21 21 25 29 23 19 19 22 24 19 20 15 21 19 21 18 22 18 16 20 16 18 18
22 22 25 26 25 21 20 22 22 19 21 15 21 19 20 18 22 18 20 19 20 22 24
Candida tropicalis
RCMB 05049
Filamentous
Absidia corymbifera
RCMB 09635
21 21 22 25 23 15 14 19 21 14
0
0
19 19 19 17 20 16 18 18 20
0
0
20
22
24
18
21
20
Aspergillus clavatus
RCMB 02593
20 21 23 23 23 17 19 19 21 17 23 19 18 14 15 15 20 14 16 15 18 22 23
22 21 24 26 23 19 19 20 23 19 23 18 17 16 17 14 20 16 17 17 18 20 21
20 21 21 23 21 17 18 17 21 17 24 19 15 17 18 14 18 12 14 14 20 23 25
19 19 22 25 23 17 16 13 19 15 21 16 17 18 17 18 20 15 16 17 19 21 22
Aspergillus fumigatus
RCMB 02564
Mucor circinelloides
RCMB 07328
Penicillium marneffei
RCMB 01267
Syncephalastrum racemosum
RCMB 05922
19 19 20 20 19 13 18 18 19 17 24 21 20
0
0
0
19
0
0
0
0
23 24
Good activity, relative to the diameter of the zone of inhibition obtained with amphotericin B (i.e., a diameter ≥ amphotericin B (Am. B)), is indicated in bold.

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Table 5. MICs of the tested compounds against unicellular and filamentous fungi.
MIC (µg/mL) per Compound
Fungal Strain
2
3
4
5
6
7
11
13
14
15
20
22 27
28
33
37
38
42 43 44 51 52 53 Am. B
Unicellular
Candida albicans
RCMB 05035
0.49 0.98 0.03 0.007 0.03 0.49 0.49 0.49 0.24 1.95 0.24 31.3 0.98 3.9 1.95 7.8 0.24 7.8 0.98 0.98 0.98 0.98 0.98 0.12
7.8 15.6 1.95 0.98 0.98 31.25 62.5 31.3 7.8 62.5 1.95 15.6 0.98 >500 >500 >500 1.95 >500 >500 >500 >500 0.98 0.94 0.98
Candida krusei
RCMB 05051
Candida parapsilosis
RCMB 05065
0.24 0.12 0.03 0.003 0.06 0.98 1.95 0.24 0.06 1.95 1.95 125 0.49 1.95 0.24 1.95 0.12 1.95 0.98 0.98 15.6 7.8 7.8
1.95
Candida tropicalis
RCMB 05049
0.24 0.24 0.02 0.007 0.06 0.49 0.98 0.24 0.12 0.98 1.95 62.5 0.49 1.95 0.49 3.9 0.12 3.9 1.95 1.95 0.49 0.49 0.12 0.02
0.24 0.24 0.12 0.03 0.06 15.63 31.3 1.95 0.49 62.5 >500 >500 0.98 3.9 1.95 7.8 0.98 7.8 7.8 3.9 0.98 >500>500 0.98
Filamentous
Absidia corymbifera
RCMB 09635
Aspergillus clavatus
RCMB 02593
0.49 0.98 0.12 0.06 0.12 3.9 1.95 1.95 0.24 3.9
0.24 0.24 0.06 0.02 0.06 1.95 1.95 0.98 0.12 1.95 0.49 7.8 3.9 15.6 7.8 62.5 0.49 15.6 7.8 7.8 1.95 1.95 0.98 0.06
0.49 0.49 0.24 0.12 0.12 7.8 3.9 7.8 0.49 15.6 0.12 1.95 31.3 7.8 1.95 62.5 1.95 125 62.5 62.5 0.98 0.24 0.12 3.9
1.95 0.98 0.12 0.03 0.03 7.8 15.6 62.5 0.98 15.6 1.95 31.3 3.9 1.95 3.9 1.95 0.98 15.6 15.6 7.8 1.95 0.98 0.49 0.49
0.24 3.9 1.95 62.5 31.3 31.3 0.98 62.5 15.6 31.3 3.9 0.49 0.24 0.12
Aspergillus fumigatus
RCMB 02564
Mucor circinelloides
RCMB 07328
Penicillium marneffei
RCMB 01267
Syncephalastrum
racemosum
1.95 1.95 0.98 0.98 0.98 125 3.9 3.9 0.98 3.9
0.24 0.98 0.98 >500 >500 >500 1.95 >500 >500 >500 >500 0.24 0.12 0.98
RCMB 05922
Good activity, relative to the MIC of amphotericin B (Am. B), is indicated in bold. The results are the average of three independent readings.

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In the 2-carboxylic acid benzimidazole series (Scheme 2), good antifungal activity was obtained for
two compounds, 43 (5-Cl) and 44 (5-Br), with a MIC of 0.98 µg/mL only against Candida parapsilosis
RCMB 05065, which is twice the potency exhibited by the reference drug. However, when comparing
these results to their unsubstituted (at position 2) analogues from series of 5-substituted benzimidazoles
(5, 5-Br), the latter compounds were more active antifungals. Therefore, this result confirmed that the
activity is dependent on the halogen substituents at position 5 (Table 5) [29].
Antifungal activity for two compounds in the 2-chloromethyl benzimidazole series (Scheme 2), 33
(5-Me) and 38 (5-NO2), showed MICs of 1.95 µg/mL against Mucor circinelloides RCMB 07328, which
is one-fold as potent as amphotericin B. In addition, the MICs for these compounds were higher than the
standard drug by three- to four-fold against Candida parapsilosis RCMB 05065 (Table 5). This could
be explained, in the case of 38 (5-NO2), which was more active than the methyl derivative 33, by the
presence of the highly electron withdrawing substituent. As a result, Compound 38 (5-NO2) is almost
two times as active as Compound 33 (5-Me) (Table 5).
Compound 20 (5-H), from the 2-ethylbenzimidazole series (Scheme 1), exhibited good activity
against Mucor circinelloides RCMB 07328 (MIC of 0.12 µg/mL; five-fold the potency of amphotericin B)
and against Syncephalastrum racemosum RCMB 05922 (MIC of 0.24 µg/mL; three-fold the potency of
amphotericin B) (Table 5). Interestingly, Compound 20, which is unsubstituted at position 5, showed
promising activity, while the nitro derivative 21 was inactive (Table 5). Surprisingly, the inhibition
activity decreases when the electron withdrawing effect of the substituent at position 5 increases; this is
in contrast to previous results [32].
3. Experimental Section
3.1. Chemistry: General Methods
The melting points (m.p.) were determined using a Gallenkamp melting point apparatus. The IR
1
spectra were recorded in KBr discs on a Nicolet 140 FTIR spectrophotometer (νmax in cm−1). H- and
13C-NMR spectra were recorded using a Bruker DPX400 spectrometer. Coupling constants are given in
Hertz (Hz). Deuterated solvents were obtained from Goss Scientific Instruments. Deuterated chloroform
was stored over 4 Å molecular sieves. DMSO-d6 and D2O were stored in silica gel desiccators. The mass
spectra were obtained on electron impact using an AEI MS902 mass spectrometer. Elemental analysis
was carried out by the microanalysis service using 2 mg of the sample (MEDAC LTD, Analytical and
Chemical Consultancy Services, Surrey, UK). Spectral data (IR, NMR and mass spectra) confirmed the
structures of the compounds.
The purity of all of the compounds was established by 1H- and 13C-NMR spectroscopy. All of the known
compounds were identified by comparing their analytical and physicochemical data with previously
reported data. The data for the compounds is provided in the Supplementary Information.

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3.2. Screening for Antibacterial Activity
3.2.1. Bacterial Strains Used in This Project
All reference and clinical strains were obtained from Dr. Anna Snelling, University of Bradford.
Overall, 15 strains of Staphylococcus spp. (including 2 × S. aureus, 10 × MRSA, 2 × S. epidermidis and
1 × S. haemolyticus), 5 strains of E. coli, 4 strains of Ps. aeruginosa, 1 strain of Serratia marcescens and
1 strain of Burkholderia cepacia were used.
3.2.2. Screening for Antibacterial Activity by the Disc Diffusion Method
Disc diffusion assays for antimicrobial susceptibility testing were carried out in-line with the standard
method first described by Bauer (1966), to assess if the compounds had any appreciable antibacterial
activities. The broad spectrum antibiotic ciprofloxacin was included in these qualitative tests as a comparator
and positive control. Bacterial suspensions were prepared from fresh, overnight, agar cultures in sterile
distilled water and adjusted to a 0.5 McFarland turbidity standard (Remel, Lenexa, KS, USA). These
suspensions were used to seed Iso-sensitest agar (Oxoid, Hampshire, UK) plates evenly using a sterile
swab [33].
For test compounds, each was dissolved to 10 mg/mL in DMSO, and different amounts of each
(typically 10, 100 and 200 µg) were loaded from the working stock solution onto 6-mm diameter sterile
Whatmann paper discs (Fisher Scientific, Loughborough, UK) and allowed to dry. The loaded discs were
placed flat on the surface of the agar lawns. Each test plate was comprised of five discs: three impregnated,
one positive control (ciprofloxacin 5 µg) and one negative control (impregnated with 20 µL of 100%
DMSO). The plates were incubated at 37 °C for 24 h. At the end of incubation, the plates were examined
for zones of inhibition. Diameters of inhibition zones were measured (in mm) and recorded. If any activity
was observed, the test was repeated on a separate day, using another fresh culture.
3.2.3. Determination of Minimum Inhibitory Concentration of Compounds against Bacterial Strains
For quantitative assessment of activity, the MICs of the synthesised compounds plus ciprofloxacin
were determined by the agar dilution method using Iso-sensitest agar. Agar dilution was in the range of
0.5–512 µg/mL of each compound, in doubling dilutions. Iso-sensitest agar was sterilised in 19-mL aliquots,
using sufficient base powder for 20 mL. Once it had cooled to approximately 50 °C, an appropriate amount
of the test compound was added in a volume of 1 mL, to give the desired final concentration per plate.
Bacterial suspensions were prepared as in the previous section.
Strains were seeded (~10 µL) on to the freshly-prepared MIC agar plates using a multi-point inoculator
(Denley, Guangzhou, China). After briefly allowing the inocula to dry, the plates were incubated
overnight at 37 °C and the MIC was recorded as the lowest concentration of compound that inhibited
bacterial growth completely, as indicated by the absence of any visible growth in the inoculation spot
on the agar surface.

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3.3. Screening for Antifungal Activity
3.3.1. Fungal Strains Used in This Project
The strains were obtained from the Regional Center for Mycology and Biotechnology (RCMB),
Al-Azhar University, Cairo, Egypt, and this part of the practical work was done there, as well. Reference
isolates for the antifungal screening; included representatives of unicellular fungi, namely Candida albicans
RCMB 05035, Candida krusei RCMB 05051, Candida parapsilosis RCMB 05065 and Candida tropicalis
RCMB 05049, and filamentous fungi, namely Absidia corymbifera RCMB 09635, Aspergillus clavatus
RCMB 2593, Aspergillus fumigatus RCMB 02564, Mucor circinelloides RCMB 07328, Mucor circinelloides
RCMB 07328, Absidia corymbifera RCMB 09635, Penicillium marneffei RCMB 01267 and
Syncephalastrum racemosum RCMB 05922.
3.3.2. Screening for Antifungal Activity by the Well Diffusion Method
A qualitative assessment of antifungal activity was determined by the well diffusion method according
to the National Committee for Clinical Laboratory Standards (NCCLS) methodology (National Committee
for Clinical Laboratory Standards, 1993). Petri dishes containing 20 mL of malt extract agar (Oxoid)
were seeded with 2–3 day old cultures of fungal inoculums (suspensions of spores in sterile water). Wells
(6 mm in diameter) were cut into the agar with a sterile cork borer, and 50 μL of compound diluted in
DMSO were added at a concentration of 5 mg/mL (250 μg per well). Plates were incubated at 37 °C
(unicellular strains) or 28 °C (filamentous strains) for 3–7 days depending on the growth rate of each
strain. Antifungal activity was determined based on the measurement of the diameter of the inhibition
zone formed around each well.
3.3.3. Determination of Minimum Inhibitory Concentration of Compounds against Fungal Strains
For quantitative assessment of activity, chemical compounds showing activity in the well diffusion
tests were diluted in DMSO, and then serial doubling dilutions were made in sterile growth media.
The commonly-used antifungal drug amphotericin B (Sigma-Aldrich A9528, St. Louis, MO, USA) was
included as a comparator. Dilutions were placed in separate wells of a 96-well microtitre tray (Sarstedt).
A volume of standardized inoculum equal to the volume of the diluted compound was added to each well,
bringing the microbial concentration to approximately 5 × 105 cells per mL. The trays were incubated at a
temperature appropriate for the test species (28 °C or 37 °C), for a pre-set period (2–3 days). After
incubation, the series of dilution wells was observed for fungal growth, usually indicated by turbidity
and/or a pellet of fungi in the bottom of the well. For each test compound, the lowest concentration that
completely inhibited growth was recorded as its minimum inhibitory concentration (MIC).
4. Conclusions
A series 53 compounds were synthesized based on the 2-benzimidazole nucleus. While none of the
compounds were novel, 47 of them were herein tested against a panel of strains of bacteria and fungi for
the first time. Infections caused by MRSA variants of S. aureus are particularly difficult to treat, as the
strains are resistant to β-lactams and often manifest resistance to other classes of drug, too, such as the

Molecules 2015, 20
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fluoroquinolones. Thus, compounds active against the MRSA strains used in this study can be considered
promising lead compounds. In terms of the SAR, the key factor in this case is the presence of the chlorine
or bromine substituent at position 5 of the benzimidazole ring, and the presence of CH(CH3)NH2 or
CH2Cl at position 2. Moreover, the presence of CH2SH at position 2 (H at position 5) proved effective for
antimicrobial activity. Derivatives with CH2NH2 were not active against bacteria, but when a branched
methyl group is added (CH(CH3)NH2), the antibacterial activity is improved. The latter compounds were
prepared from S-alanine. Therefore, the product, which is also chiral, showed variable activity against the
MRSA species. Chirality is a feature for many drugs, with one of the stereoisomers active biologically and
the other inactive or even toxic. Therefore, the chirality has a significant impact on enhancing the biological
activity of the compound. However, as in the case of the antifungal assay, and according to the SAR study,
an extremely important factor is the nature of the substituent attached to the aromatic ring. The presence of
the halogen atom (Cl, Br, F) at position 5, increased the antifungal activity. For the 5-nitro derivative, the
presence of CH2NH2 at position 2 increased the activity. The N-methylated-2-methanolbenzimidazole
derivatives were more active than the unmethylated analogues, and this is observed when the substituent
at position 5 was F, OMe or NO2. To summarise, according to the SAR analysis, it can be concluded
that some derivatives of benzimidazole offer significant possibilities for the development of new
broad-spectrum antimicrobial agents. Through appropriate modification and fine-tuning of substituents
at positions 1, 2 and 5, new microbial inhibitors are possible, although thorough toxicological evaluation
will be needed to assess their utility for medicinal use. To summarise, according to the SAR analysis,
it can be concluded that some derivatives of benzimidazole offer significant possibilities for the
development of new broad-spectrum antimicrobial agents. The structures of our lead compounds are
shown in (Figures 2 and 3).
Figure 2. Promising antibacterial derivatives.
Figure 3. Promising antifungal derivatives.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/08/15206/s1.

Molecules 2015, 20
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Acknowledgments
This research project was supported by a grant from the “Research Centre of the Centre for Female
Scientific and Medical Colleges”, Deanship of Scientific Research, King Saud University.
Author Contributions
N.K. and A.M.S. designed and supervised the research; F.A.S.A. performed the research; N.K.,
A.M.S. and F.A.S.A. wrote the paper; M.E.Z., A.M.A. and A.S.A. assisted with additional tests and data
analysis. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).