One-pot synthesis of novel benzimidazoles with a naphthalene moiety as antimicrobial agents and molecular docking studies

Article abstract of DOI:10.1002/jccs.202000125

In an attempt to design a greener approach for the synthesis of a potent class of antimicrobials, 1,2-phenylenediamine derivatives were reacted with various 1/2-carboxylic acid-substituted naphthalene derivatives to generate a series of naphthyl-substituted benzimidazole derivatives (11–19) using polyphosphoric acid as catalyst under microwave irradiation and conventional synthesis method. This is an eco-friendly and swift reaction method for a synthetic approach to diverse benzimidazoles. Structures of the synthesized compounds were established on the basis of spectral data and they were screened for their antimicrobial activity. Compound 18 showed maximum potency against all Gram-positive and Gram-negative bacterial strains with a minimum inhibitory concentration (MIC) value in the range of 7.81–62.50 μg/ml. Only compound 17 was found to be the most active against all fungal strains with a MIC value of 15.62 μg/ml. In this study, we performed molecular docking experiments to understand the interactions between compounds 17 and 18 and E. coli topoisomerase I, and we compared the results obtained with that of 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin?1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole (DMA). Compounds 17 and 18 demonstrated strong interactions with important active site residues, similar to DMA. As a result, the compounds obtained from this study can be used in designing new potent inhibitors of E. coli topoisomerase I.

Full text of DOI:10.1002/jccs.202000125

Received: 21 April 2020
Revised: 1 September 2020
Accepted: 3 September 2020
DOI: 10.1002/jccs.202000125
A R T I C L E
One-pot synthesis of novel benzimidazoles with a
naphthalene moiety as antimicrobial agents and molecular
docking studies
1
1
2
3
Ronak Haj Ersan | Ahmet Yuksel | Tugba Ertan-Bolelli | Aylin Dogen
|
4
1
Serdar Burmaoglu | Oztekin Algul
1
Department of Pharmaceutical
Abstract
Chemistry, Faculty of Pharmacy, Mersin
University, Mersin, Turkey
In an attempt to design a greener approach for the synthesis of a potent class of
antimicrobials, 1,2-phenylenediamine derivatives were reacted with various
2
Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Ankara
University, Ankara, Turkey
1/2-carboxylic acid-substituted naphthalene derivatives to generate a series of
3
naphthyl-substituted benzimidazole derivatives (11–19) using polyphosphoric acid
as catalyst under microwave irradiation and conventional synthesis method. This
is an eco-friendly and swift reaction method for a synthetic approach to diverse
benzimidazoles. Structures of the synthesized compounds were established on the
basis of spectral data and they were screened for their antimicrobial activity. Com-
pound 18 showed maximum potency against all Gram-positive and Gram-
negative bacterial strains with a minimum inhibitory concentration (MIC) value
in the range of 7.81–62.50 μg/ml. Only compound 17 was found to be the most
active against all fungal strains with a MIC value of 15.62 μg/ml. In this study, we
performed molecular docking experiments to understand the interactions between
compounds 17 and 18 and E. coli topoisomerase I, and we compared the results
obtained with that of 2-(3,4-dimethoxyphenyl)-5-[5-(4-methylpiperazin−1-yl)-1H-
benzimidazol-2-yl]-1H-benzimidazole (DMA). Compounds 17 and 18 demon-
strated strong interactions with important active site residues, similar to DMA. As
a result, the compounds obtained from this study can be used in designing new
potent inhibitors of E. coli topoisomerase I.
Department of Pharmaceutical
Microbiology, Faculty of Pharmacy,
Mersin University, Mersin, Turkey
4
Department of Chemistry, Faculty of
Science, Atatürk University, Erzurum,
Turkey
Correspondence
Oztekin Algul, Department of
Pharmaceutical Chemistry, Faculty of
Pharmacy, Mersin University, Mersin,
Turkey.
Email: oztekinalgul@mersin.edu.tr
Funding information
Mersin Üniversitesi, Grant/Award
Number: 2017-1-TP2-1981
K E Y W O R D S
antirobial, benzimidazole, MIC, molecular docking, naphthalene
1
| INTRODUCTION
addition of o-phenylenediamine to different aldehydes in
[6]
the presence of a catalytic amount of indium triflate,
[7]
Benzimidazoles and their derivatives have a wide range
of applications in medicine,
zinc triflate in ethanol solvent at reflux temperature,
[1,2]
[8]
[9]
and several methods
sodium dodecyl sulfate or nickel acetate, and the reac-
have been developed for their synthesis. Condensation of
tion of o-phenylenediamines with acid anhydrides, urea,
[
10]
o-phenylene-diamine and different acids in the presence of
and esters
are some of the methods reported so far.
[3]
concentrated hydrochloric acid, ammonium chloride as
However, the methodologies mentioned above have some
disadvantages regarding their applicability for different
benzimidazole derivatives. Therefore, comparative studies
[4]
a catalyst, microwave irradiation under solvent-free con-
[
5]
ditions which are catalyzed by alumina and silica gel,
J Chin Chem Soc. 2020;1–10.
http://www.jccs.wiley-vch.de
© 2020 The Chemical Society Located in Taipei & Wiley-VCH GmbH
1

2
ERSAN ET AL.
of these methods are required. Thus, more effective benz-
imidazole synthesis methods should be developed for each
substituted benzimidazole derivatives.
benzimidazole derivatives with or without a methyl linker
to investigate the role of the methyl linker to investigate the
role of the methyl linker on antimicrobial activity. Second,
we substituted the naphthalene moiety of the most active
compounds with a hydroxyl group and substituted an R
In addition, recent observations suggest that
substituted benzimidazoles and their derivatives possess
an extensive range of biological activities including ant-
iviral, antifungal, antimicrobial, antiprotozoal, anti-
inflammatory, anticancer, antioxidant, anticoagulant,
group (CH , Cl, and di-Cl) in the C-5/−6 position of the
3
most active benzimidazole derivative for the evaluation of
the most active compounds (Figure 2).
Also, we performed molecular docking experiments
to exhibit strong interactions between compounds and
E. coli topoisomerase I active site residues.
[11–19]
antidiabetic, and antihypertensive activities.
It has
been reported that benzimidazole derivatives with a func-
tional group at the C-1, −2, and/or − 5/−6 positions
[2]
show increased biological activity.
Recent develop-
ments regarding 2-substituted benzimidazoles have rev-
ealed that aromatics varied at the C-2 position give rise to
potent antimicrobial agents that are effective against dif-
2 | RESULTS AND DISCUSSION
[20,21]
ferent bacteria and fungi.
Furthermore, 2-aromatic-
We carried out the best reaction methods, and we com-
pared five different synthesis methods on the first group
compounds (11 and 12) (Table 1). The use of microwave
irradiation and conventional method with PPA simplified
and improved organic reactions. The microwave irradiation
method with PPA demonstrated high yield, clean reactions,
and short reaction time (Table 2). Thereby all compounds
were synthesized for comparison by using PPA by micro-
substituted benzimidazoles with a functional group at the
[22–25]
C-5 position are potent antimicrobial agents.
Similarly, naphthalene derivatives also have a variety
[26–28]
of biological activities.
Many studies have supported
the antimicrobial effect of a naphthalene ring on bacte-
[29–31]
rial and fungal strains.
In fact, several naphthalene-
containing drugs such as nafcillin, naftifine, tolnaftate,
and terbinafine are currently available to treat microbial
infections (Figure 1). Naphthalene derivatives bearing a
functional group, especially an -OH group, on α and β
[39,40]
wave irradiation and conventional methods.
The syn-
thetic route of the compounds is shown in Scheme 1. The
synthesized compounds were classified into three groups.
The compounds in the first group (11–14) were synthesized
by reacting 1,2-phenylenediamine (1) with 1/2-naphthoic
acid (5 and 7) and 2-(naphthalen-1/2-yl)acetic acid (6 and
8), whereas those in the second set of compounds (15 and
16) were synthesized by reacting 1,2-phenylenediamine (1)
with 1-hydroxy-2-naphthoic acid (10) and 2-hydroxy-
1-naphthoic acid (9). Finally, the compounds in the third
[
32,33]
positions possess very good antimicrobial properties.
It has recently reported that 2-(3,4-dimethoxyphenyl)-
5
-[5-(4methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-
benzimidazole (DMA), a bisbenzimidazole, has no
effect on cell viability in mammalian cells but effi-
ciently inhibited E. coli topoisomerase I by enhancing
DNA cleavage products. DMA has no significant inhibi-
tory effect on human topoisomerase I, which suggests
that DMA acts as a potential antibacterial agent, selec-
group
(17–19)
were
synthesized
by
reacting
3,4-diaminotoluene (2), 4-chloro-o-phenylenediamine (3)
and 4,5-dichloro-o-phenylenediamine (4) with 1-hydroxy-
2-naphthoic acid (10) (Scheme 1).
[
34–36]
tively targeting E. coli topoisomerase I enzyme.
On the basis of aforesaid facts and in continuation of
our research to develop the synthesis of benzimidazoles as
[37,38]
potent, selective, and less toxic therapeutic agents
,
herein we report a facile and an effective methodology for
the synthesis of a variety of benzimidazoles in the presence
of polyphosphoric acid (PPA) as a catalyst under microwave
irradiation or conventional conditions. In this study, we
2.1 | Antimicrobial activity
Minimum inhibitory concentration (MIC) values for all
synthesized compounds revealed good to potent
demonstrated
a
series
of
naphthyl-substituted
antibacterial
activity
against
Escherichia
coli,
CH
3
CH
CH3
3
3
CH3
O
N
S
CH
H
N
S
N
HCl
N
H C
O
3
CH3
O
HCl
OH
O
O
Nafcillin
Naftifine
Tolnaftate
Terbinafine
FIGURE 1 Structures of some naphthalene containing drugs

ERSAN ET AL.
3
Pseudomonas aeruginosa, Enterococcus faecalis, Staphylo-
coccus aureus, Streptococcus pneumoniae, Bacillus subtilis,
Candida albicans, and Candida parapsilosis.
In the first group, nonsubstituted naphthyl-
benzimidazole derivatives (11–14) were screened for antimi-
crobial activity. The compounds without the methyl linker
14). In particular, compound 11 demonstrated good inhibi-
tory activity against Candida glabrata, with a MIC value of
15.62 μg/ml, and compound 13 demonstrated good inhibi-
tory activity against, S. pneumoniae, with a MIC value of
62.50 μg/ml (Table 3).
A plausible explanation could be the increased
lipophilicity of these compounds compared with the stan-
dards, which should improve drug uptake via the lipid-
mediated pathway. Therefore, the naphthalene moiety of
the active compounds (11 and 13) was substituted on the
α/β positions by an -OH group to generate second com-
pounds (15 and 16). Compared to compound 15, com-
pound 16 showed increased growth inhibitory activity
against E. faecalis, S. aureus, and B. subtilis, with a MIC
value of 15.62 μg/ml (Table 4).
Third group of compounds (17–19) were designed to
enhance antimicrobial activity by substituting the benzimid-
azole ring of the most active compound from the second
group (16) on C-5/−6 positions. We found that chloro-
substituted naphthyl-benzimidazole (18) demonstrated the
most potent antibacterial activity against S. AUREUS and
P. AERUGINOSA, with a MIC value of 7.81 μg/ml, which
was four-fold more than the standard drug ampicillin (MIC
(11 and 13) demonstrated increased microbial growth inhi-
bition compared to those with the methyl linker (12 and
N
H
N
NH
N
N
H
N
N
N
H
HO
H
N
HO
31.25 μg/ml). Additionally, compound 18 had a MIC value
N
of 15.62 μg/ml against E. coli, E. faecalis, and B. subtilis. On
the other hand, methyl-substituted naphthyl-benzimidazole
N
N
H
(
17) was the most active against all fungal strains, with a
MIC value of 15.62 μg/ml. In addition, the activity
decreased with dichloro-substituted naphthyl-
benzimidazole (19), which had a log p value higher than
monochloro-substituted compounds (Table 5)
HO
R
N
N
H
2.2 | Molecular docking studies
R= CH , Cl, di-Cl
3
We conducted molecular docking experiments to under-
FIGURE 2 Synthetic strategy pathway
stand the interactions between compounds 17 and 18 and
TABLE 1 Optimization of conventional method for the synthesis of compounds 11 and 12
ꢀ
Compound no
Catalyst/mol%
Solvent
HCl
Temperature ( C)
Time
Yield (%)
Ref.
1
1
—
—
—
—
Reflux
180–200
120
48
18
18
42
50
49
20
20
40
50
8
[3]
PPA
PPA
30
65
15
14
6
[39]
Ethylene glycol
Toluene
HCl
Reflux
Reflux
Reflux
180–200
120
[57]
[58]
[3]
B(oh)
3
1
2
—
—
PPA
35
69
17
19
[39]
—
PPA
—
-
Ethylene glycol
Toluene
Reflux
Reflux
[57]
[58]
B(oh)
3

4
ERSAN ET AL.
TABLE 2 Conventional method and effect of MWI in watts on the synthesis of compounds
Microwave heating
Conventional heating PPA
1
2
3
ꢀ
Compound no
R
R
n
0
1
0
1
0
0
0
0
0
R
Time (min)
Power (watt)
100–150
100–150
100–150
100–150
100–150
100–150
100–150
100–150
100–150
Yield (%)
Temp. ( C)
120
Time (h)
Yield (%)
11
12
13
14
15
16
17
18
19
H
H
H
H
H
H
H
H
H
Cl
H
12
10
10
12
8
65
81
83
90
85
82
90
80
83
18
16
20
17
21
21
20
22
22
45
57
59
65
59
55
62
52
58
H
H
120
H
H
120
H
H
120
H
OH
OH
OH
OH
OH
120
H
10
10
16
14
120
CH
Cl
Cl
3
120
120
120
E. coli topoisomerase 1, and we compared the obtained
results with that of DMA, which is a known inhibitor of
E. coli topoisomerase 1. Compounds 17 and 18 showed
strong interactions with the important active site resi-
dues, similar to DMA. Compound 17 formed H-bonds
with Arg114, Glu547, and Asp551 and salt bridges with
Glu547 and Asp551 (Figure 3c), whereas compound 18
formed H-bonds with Asp323 and salt bridges with
Asp113 and Asp323 (Figure 3d). DMA formed H-bonds
with Arg493, Glu547, and Asp551 and salt bridges with
Asp113, Asp323, Glu547, and Asp551. The results from
the docking experiments and the interactions of the
tested compounds are shown in Table 6 and Figure 3.
First group
H
NH2
NH2
N
HOOC
n
n
PPA
+
N
n
0
1
n
1
5
6
0
1
11
12
H
HOOC
NH2
NH2
N
n
PPA
n
+
N
n
0
1
1
n
0
1
13
14
7
8
3
| EXPERIMENTAL
Second group
HO
H
N
COOH
The reagents and solvents used were obtained from Sigma–
Aldrich chemicals. Reagents and solvents are used directly
without further purification. Reaction progress and product
mixtures were monitored by thin layer chromatography
(TLC) using E-Merck 0.25 silica gel plates. Colorless prod-
ucts were detected by UV light (254 nm). Melting points
were determined by using one end open capillary tube on
HO
N
NH2
1
5
PPA
+
9
NH2
1
OH
HO
HOOC
N
N
H
1
0
1
6
Third group
R1
1
an uncorrected Analab melting point apparatus. H- and
OH
HO
NH2
NH2
13
R1
R2
N
C-NMR spectra were recorded on Bruker Avance 400 spec-
HOOC
PPA
+
R2
trometers, operated at 400 MHz for 1H and 100 MHz for
13C nuclei, relative to TMS as the internal standard on the
δ (ppm) scale with deuterated dimethylsulfoxide (DMSO-
d ) and chloroform (CDCl ) as the solvents. The IR spectra
N
H
1
0
R1
CH
Cl
R1
H
H
R
CH
Cl
1
R
1
H
H
3
2
3
4
3
17
18
19
Cl
Cl
Cl
Cl
6
3
were obtained on a Perkin Elmer Spectrum One FT-IR
spectrometer. The spectra of all of the investigated com-
pounds are provided as Supporting Information.
SCHEME 1 Synthesis of benzimidazoles with naphthalene
moiety compounds (11–19)

ERSAN ET AL.
5
TABLE 3 MICs of first group compounds 11–14 against the selected microbial strains
MIC (mg/ml)
cLog
P
E.
coli
P.
E.
facealis
S.
aureus
S.
B.
subtilis
C.
C.
a
No
aeuroginosa
pneumoniae
albicans
glabrata
1
1
1
1
1
2
3
4
4,837
4,806
4,837
4,576
-1,2045
125,00 125,00
250,00 250,00
125,00 125,00
125,00 125,00
125,00
250,00
62,50
125,00
*
250,00
250,00
62,50
250,00
*
250,00
250,00
62,50
250,00
*
125,00
250,00
62,50
125,00
*
31,25
125,00
125,00
125,00
—
15,62
125,00
62,50
62,50
—
Ampicillin
32,25
32,25
Fluconazole −0,44
—
—
—
—
—
—
*
*
a
cLogP value of the synthesized compounds (calculated from ChemBioDrawUltra 12.0.3).
TABLE 4 MICs of second group compounds 15, 16 against the selected microbial strains
MIC (mg/ml)
cLog
P
E.
coli
P.
E.
facealis
S.
aureus
S.
B.
subtilis
C.
C.
a
No
aeuroginosa
pneumoniae
albicans
glabrata
1
5
6
4,060
4,060
250,00 250,00
125,00 125,00
250,00
15,62
*
125,00
15,62
*
125,00
62,50
*
125,00
15,62
*
125,00
62,50
—
62,50
62,50
—
1
Ampicillin
−1,2045 32,25
32,25
Fluconazole −0,44
—
—
—
—
—
—
*
*
a
cLogP value of the synthesized compounds (calculated from ChemBioDrawUltra 12.0.3).
TABLE 5 MICs of third group compounds 17–19 against the selected microbial strains
MIC (mg/ml)
cLog
P
E.
coli
P.
E.
facealis
S.
aureus
S.
B.
subtilis
C.
C.
a
No
aeuroginosa
pneumoniae
albicans
glabrata
1
1
1
7
8
9
4,560
15,62
15,62
62,50
32,25
—
15,62
7,81
62,50
32,25
—
31,25
15,62
31,25
*
7,81
7,81
31,25
*
62,50
62,50
125,00
*
15,62
15,62
31,25
*
15,62
62,50
62,50
—
15,62
62,50
62,50
—
4,851
5,471
Ampicillin
−1,2045
Fluconazole −0,44
—
—
—
—
*
*
a
cLogP value of the synthesized compounds (calculated from ChemBioDrawUltra 12.0.3).
ꢀ
3
.1 | General procedure for the synthesis
The reaction mixture was heated at 180 C for 13–15 hr.
After the reaction was monitored by TLC, the mixture
was poured into ice water and neutralized by 5 M NaOH
until it reached a slightly basic pH (8–9) to obtain a pre-
cipitate. The resulting precipitate was filtered off, washed
with cold water, and recrystallized with a suitable solvent
(H O, EtOH, EtOH-H O). The resulting crystalline com-
of 2-naphthelen substituted benzimidazole
derivatives (11–19)
3
.1.1 | Conventional heating method
o-Phenylenediamine derivatives (1–4) (1 eq) and
corresponding naphthalene carboxylic acid derivatives
2
2
pounds were filtered, and the vacuumed product was
dried.
(5–10) (1.1 eq) in PPA (5 ml) were placed in a 25 ml flask.

6
ERSAN ET AL.
FIGURE 3 (a) Crystal structure of E. coli Topoisomerase I enzyme (PDB ID: 3PX7). (b) Superposition of DMA (orange), compound
7 (pink) and compound 18 (blue). (c) Docked position of compound 17: revealed H-bonds with Arg114, Glu547, Asp551; salt bridges with
Glu547 and Asp551. (d) Docked position of compound 18: revealed H-bond with Asp323 and salt bridges with Asp113 and Asp323. *H-bond
1
(
(
green arrow), Salt-bridge (red-blue arrow), negative charge (orange), positive charge (purple), Hydrophobic (green), Polar interactions
turquoise)
TABLE 6 Docking results of the tested compounds
No
Docking score
Glide score
Interactions
a
b
b,e
e
1
7
8
−7.243
−7.243
Asp113 , Arg114 , Arg161, Ser361 , Gln362 ,
a
d
a,c
a,c
Glu363 , Ala554 , Glu547 , Asp551
Asn555
,
e
e
a,c
b
b
1
−7.158
−7.158
Asn111 , Asp113 , Arg114 , Ptr319, Arg321 ,
e
a,c
e
e
a
Thr322 , Asp323 , Ser361 , Gln362 , Glu363 ,
Ala364 , Hie365 , Arg493 , Pro494 , Ser495 ,
DT8
d
e
b
d
e
e
a,c
b
a
DMA
−10.640
−10.797
Asn111 , Asp113 , Arg114 , Glu115 , Ptr319,
b
e
a,c
e
e
Arg321 , Thr322 , Asp323 , Ser361 , Gln362 ,
Glu363 , Ala364 , Hie365 , Arg493 , Pro494 ,
Ser495 , Ala544 , Glu547 , Asp551 , DT8
a
d
e
b
d
e
a
a,c
a,c
Note: Bold: H-bond, a: negative charge, b: positive charge, c: Salt-bridge, d: Hydrophobic, e: Polar interactions.

ERSAN ET AL.
7
3.1.2 | Microwave irradiation method
111.3. Anal. calcd for C17H12N2: C, 83.58; H, 4.95; N,
[45,46]
11.47; Found: C, 83.63; H, 4.83; N, 11.55.
In the presence of PPA, the mixture of o-
phenylenediamine derivatives (1–4) (1 eq) and
corresponding
naphthalene
carboxylic
acid
3.1.6 | 2-(naphthalen-1-ylmethyl)-1H-
benzo[d]imidazole (14)
derivatives (5–10) (1.1 eq) was stirred and irradiated in
MW (7–12 min, 100–150 W). After monitoring of the
reaction with TLC, the mixture was poured into ice water
and neutralized with 5 M NaOH until it reached a
slightly basic pH (8–9) to obtain the precipitate. The
resulting precipitate was filtered off, washed with cold
ꢀ
−1
m.p.: 210–213 C; IR (KBr, cm ) 3,087 (aromatic = C-H),
2,900 (aliphatic –C-H), 1,541 (C=N), 1,493 (aromatic C=C);
1
H NMR (400 MHz, CDCl3): 8.11–8.01 (m, 2H, Ar-H),
7.89–7.69 (m, 3H, Ar-H), 7.56–7.42 (m, 5H, Ar-H), 7.32–7.25
(m, 1H, Ar-H), 4.90 (bs, 2H, -CH2); C NMR (100 MHz,
CDCl3): δ 137.0, 133.3, 132.6, 127.9, 126.9, 125.5, 125.4,
125.3, 125.0, 44.3. Anal. calcd for C18H14N2: C, 83.69; H,
13
water, and recrystallized with a suitable solvent (H O,
2
EtOH, EtOH-H O). The resulting crystalline compounds
2
were filtered, and the vacuumed product was dried.
[47,48]
5.46; N, 10.84; Found: C, 83.58; H, 5.49; N, 10.77.
3
.1.3 | 2-(Naphthalen-2-yl)-1H-benzo[d]
imidazole (11)
3.1.7 | 1-(1H-benzo[d]imidazol-2-yl)
naphthalen-2-ol (15)
ꢀ
−1
m.p.: 188–191 C; IR (KBr, cm ) 3,268 (aromatic = C-H),
1
ꢀ
−1
1
,733 (C=N), 1,510–1,419 (aromatic C=C); H NMR
m.p.: 186–189 C; IR (KBR, cm ) 3,045 (aromatic = C-
1
(400 MHz, d6-DMSO): δ 13.12 (s, 1H, -NH), 8.76 (bs, 1H,
H), 1,532 (C=N), 1,488 (aromatic C=C); H NMR
Ar-H), 8.34 (dd, J = 1.62, 8.76 Hz, 1H, Ar-H), 8.09 (d,
J = 8.76 Hz, 1H, Ar-H), 8.08–7.98 (m, 2H, Ar-H), 7.70 (bs,
(400 MHZ, CDCL3): δ 8.93 (d, J = 8.51 Hz, 1H, AR-H),
8.20 (d, J = 8.15 Hz, 1H, AR-H), 8.00–7.92 (m, 4H, AR-
13
1
H, Ar-H), 7.63–7.58 (m, 3H, Ar-H), 7.25–7.24 (m, 2H,
H), 7.64–7.43 (m, 5H, AR-H); C NMR (100 MHZ,
CDCL3): δ 151.4, 143.9, 133.6, 130.5, 130.1, 128.4, 127.0,
126.3, 125.2, 122.6, 121.6, 111.3. Anal. calcd for
C17H12N2O: C, 78.44; H, 4.65; N, 10.76; Found: C,
13
Ar-H); C NMR (100 MHz, d6-DMSO):δ 151.2, 133.4,
32.8, 128.5, 128.4, 128.0,127.7, 127.1, 126.9, 125.8, 123.9.
Anal. calcd for C17H12N2: C, 83.58; H, 4.95; N, 11.47;
1
[41,42]
[49,50]
Found: C, 83.47; H, 4.77; N, 11.38.
78.29; H, 4.77; N, 10.59.
3
.1.4 | 2-(naphthalen-2-ylmethyl)-1H-
3.1.8 | 2-(1H-benzo[d]imidazol-2-yl)
naphthalen-1-ol (16)
benzo[d]imidazole (12)
ꢀ
−1
ꢀ
−1
m.p.: 196–199 C; IR (KBr, cm ) 3,049 (aromatic = C-H),
m.p.: 198–201 C; IR (KBr, cm ) 3,057 (aromatic = C-H),
1
1
1
,576(C=N), 1,567(aromatic C = C); H NMR (400 MHz,
1,657 (C=N), 1,584 (aromatic C=C); H NMR (400 MHz,
d6-DMSO): δ 7.97–7.84 (m, 5H, Ar-H), 7.80 (bs, 1H, Ar-H),
d6-DMSO): δ 8.37–8.35 (m, 1H, Ar-H), 8.12–8.10 (m, 1H, Ar-
H), 7.92–7.90 (m, 1H, Ar-H), 7.71 (bs, 2H, Ar-H), 7.63–7.53
13
7
.59–7.42 (m, 5H, Ar-H), 3.72 (s, 2H, CH2); C NMR
100 MHz, d6-DMSO):δ 155.6, 152.4, 134.7, 128.0, 127.6,
25.8, 124.7, 122.8, 122.3, 118.5, 105.4. Anal. calcd for
C18H14N2: C, 83.69; H, 5.46; N, 10.84; Found: C, 83.75; H,
13
(
1
(m, 3H, Ar-H), 7.33–7.29 (m, 2H, Ar-H); C NMR (100 MHz,
d6-DMSO):δ 133.0, 131.6, 128.3, 127.4, 127.3, 127.1, 125.8,
125.2, 121.1, 14.6. Anal. calcd for C17H12N2O: C, 78.44; H,
[43,44]
[51,52]
5.33; N, 10.71.
4.65; N, 10.76; Found: C, 78.39; H, 4.61; N, 10.65.
3
.1.5 | 2-(naphthalen-1-yl)-1H-benzo[d]
3.1.9 | 2-(5-METHYL-1H-benzo[d]
imidazol-2-yl)naphthalen-1-ol (17)
imidazole (13)
ꢀ
−1
ꢀ
−1
m.p.: 186–189 C; IR (KBr, cm ) 3,045 (aromatic = C-H),
m.p.: 202–205 C; IR (KBr, cm ) 3,057 (aromatic = C-H),
1
1
1
,532 (C=N), 1,488 (aromatic C=C); H NMR (400 MHz,
2,853 (aliphatic–C-H), 1,657 (aromatic C=C); H NMR
CDCl3): δ 8.93 (d, J = 8.51 Hz, 1H, Ar-H), 8.20 (d,
J = 8.15 Hz, 1H, Ar-H), 8.00–7.92 (m, 4H, Ar-H), 7.64–7.43
(400 MHz, d6-DMSO): δ 14.64 (s, 1H, -OH), 13.22 (s, 1H,
-NH), 8.40–8.88 (m, 4H, Ar-H), 7.68–7.40 (m, 4H, Ar-H),
13
13
(
m, 5H, Ar-H); C NMR (100 MHz, CDCl3):δ 151.4, 143.9,
7.15 (d, J = 7.15 Hz, 1H, Ar-H), 2.49 (s, 3H, -CH3); C NMR
133.6, 130.5, 130.1, 128.4, 127.0, 126.3, 125.2, 122.6, 121.6,
(100 MHz, d6-DMSO):δ 155.5, 135.9, 134.6, 129.9, 128.3,

8
ERSAN ET AL.
1
27.8, 127.7, 127.6, 125.7, 124.8, 124.7, 123.7, 122.8, 122.3,
tested chemical solution was added to the first well, and
two-fold dilutions were prepared. Then, 5 μl of fungal sus-
pension was added to each tube except the last one, which
acted as the control well. For antibacterial activity testing,
120.6, 118.4, 105.5,21.3. Anal. calcd for C18H14N2O: C,
78.81; H, 5.14; N, 10.21; Found: C, 78.73; H, 5.25; N, 10.33.
100 μl Mueller–Hinton broth (MHB) was added to each of
3
2
.1.10 | 2-(5-chloro-1H-benzo[d]imidazol-
-yl)naphthalen-1-ol (18)
the 11 wells. A 100 μl aliquot of the chemical derivative
solution was added to the first tube, and two-fold dilutions
were prepared. Then, 5 μl of the bacterial suspension was
added to each tube, except the last control well. A control
tube containing 5 μl of the fungal and bacterial suspen-
sions alone without the tested compounds was also pre-
ꢀ
−1
m.p.: 194–197 C; IR (KBr, cm ) 3,050 (aromatic = C-H),
1
1
8
,515 (aromatic C=C); H NMR (400 MHz, d6-DMSO): δ
.44–8.08 (m, 2H, Ar-H), 8.06–7.49 (m, 6H, Ar-H), 7.34
13
ꢀ
(
d, J = 1.92 Hz, 1H, Ar-H);
d6-DMSO): δ 155.7, 153.7, 134.9, 128.2, 127.6, 125.9,
24.7, 123.1, 122.9, 122.3, 118.7, 105.1. Anal. calcd for
C17H11ClN2O: C, 69.28; H, 3.76; N, 9.50; Found: C,
9.14; H, 3.88; N, 9.41.
C NMR (100 MHz,
pared. All plates were incubated at 28 C (for fungi) and at
ꢀ
37 C (for bacteria) for 24 hr. After incubation, the MICs
1
(Tables 3–5) were obtained by noting the growth inhibi-
tion. The concentration resulting in a 50% reduction in
the optical density (OD) values was compared to a repli-
cate plate control at 450 nm by spectrophotometric evalu-
ation and defined as the MIC value. Fluconazole and
ampicillin were used as reference drugs. The results were
read visually and by measuring OD for 24 hr.
6
3
.1.11 | 2-(5,6-dichloro-1H-benzo[d]
imidazol-2-yl)naphthalen-1-ol (19)
ꢀ
−1
m.p.: 207–210 C; IR (KBr, cm ) 3,057 (aromatic = C-H),
1
1
,657 (C=N), 1,520 (aromatic C=C); H NMR (400 MHz,
3.3 | Molecular docking methodology
d6-DMSO): δ 8.87–8.24 (m, 2H, Ar-H), 8.15–7.92 (m, 3H,
Ar-H), 7.85–7.48 (m, 3H, Ar-H); C NMR (100 MHz,
d6-DMSO): δ 155.8, 154.9, 134.9, 128.21, 127.8, 127.8,
13
Molecular docking studies were performed by using
[55]
Schrödinger molecular modeling software. Ligands were
prepared by using the LigPrep module and the 2D struc-
tures of the ligands converted to the full 3D structure by
assigning the OPLS-2005 force field. LigPrep can generate
the expected ionized forms at significant concentrations
corresponding to the pH 7.0 ± 3.0, generate variations, per-
form verification, and optimize structures. It generates a
maximum of 32 stereochemical structures per ligand. Bind-
ing of ligands to the receptors adopts more than one con-
formation, and the lowest energy conformer is important
for docking studies. The crystal structure of the E. coli topo-
isomerase I enzyme was extracted from the Protein Data
1
25.9, 125.0, 124.6, 122.9, 122.5, 118.7, 105.0. Anal. calcd
for C17H10Cl2N2O: C, 62.03; H, 3.06; N, 8.51; Found: C,
1.95; H, 3.17; N, 8.44.
6
3.2 | Antimicrobial assay
Antimicrobial susceptibility testing was performed by a
[53,54]
modification of literature methods.
We used the
microbial strains Escherichia coli (ATCC 25922), Pseudo-
monas aeruginosa (ATCC 25853), Enterococcus faecalis
[56]
(ATTC 29212), Staphylococcus aureus (ATCC 25923),
Bank (PDB ID: 3PX7)
(Figure 3a). Prior to docking the
Streptococcus pneumoniae (ATCC 10353), Bacillus subtilis
ATCC 6633, Candida albicans (ATCC 4322), and Can-
dida parapsilosis (ATTC 22019).
ligands onto the protein's active site, the protein was pre-
pared using the protein preparation wizard in the
Schrödinger software. During protein preparation all het-
ero atoms and water molecules were removed. Hydrogen
atoms were added, and the active site of the protein was
defined to generate the grid. The grid box was limited to a
size of 20 Å at the active site. Finally, docking studies were
carried out using the Grid-based Ligand Docking with
Energetics module of the Schrödinger Software, the ligands
were docked into the prepared grid by using “Extra preci-
sion mode”, and no constraints were defined. To determine
the spatial fit into the active site of the receptor, favorable
ligand poses were generated, and the best-fit conformations
of the ligands were evaluated and minimized to generate
glide scores. To predict the binding affinities and best
The fungal and bacterial cell inoculum were prepared
ꢀ
from a stock culture grown in tryptic soy agar at 28 C for
ꢀ
2
2
4 hr, and Mueller–Hinton agar (MHA) at 37 C for
4 hr, respectively. The concentrations of microorganism
suspension were adjusted according to MCFARLAND 0.5
turbidity tubes using sterilized saline. Stock solutions of
the title compounds were prepared in DMSO at
1
000 μg/ml. A modified microdilution test was applied
for antimicrobial activity, and the experiments were run
in duplicate independently
For antifungal activity testing, 100 μl Tryptic Soy Broth
was added to each of the 11 wells. A 100 μl aliquot of the

ERSAN ET AL.
9
alignment of the compounds at the active site of the
enzyme, hydrogen bonds and other interactions formed
with the surrounding amino acids and glide scores were
used. All the results are shown in Table 6.
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series of 2-naphthyl-substituted benzimidazole deriva-
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incorporation of methyl, one chlorine, and two chlorine
atoms at the fifth and sixth positions of benzimidazole,
mono chloro-substituted compound 18 showed 8- and
[
[
[15] Y. F. Li, G. F. Wang, P. L. He, W. G. Huang, F. H. Zhu, H. Y.
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6-fold increased activity against E. coli and
[
P. aeuroginosa, respectively, compared to unsubstituted
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53, 1076.
In conclusion, we have demonstrated that mono
chloro substitution of the benzimidazole moiety at the
C-5 position and the presence of an -OH group on the
C-1 position of the naphthalene moiety are crucial ele-
ments of antimicrobial activity. Studies suggest that DMA
is a selective E. coli topoisomerase I inhibitor and acts as
a potential antibacterial agent. Therefore, we assessed the
most active compounds against E. coli (17 and 18) using
molecular modeling studies. According to the docking
results, compound 18 and, especially, compound 17
showed strong interactions with important active site res-
idues, similar to DMA. Therefore, the compounds gener-
ated in this study can be useful in designing new potent
inhibitors of E. coli topoisomerase I, as lead compounds.
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This study was supported by 2017-1-TP2-1981 BAP Pro-
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One-pot synthesis of novel benzimidazoles with a
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