Synthesis, molecular docking and ADME prediction of some new benzimidazole carboxamidines derivatives as antimicrobial agents

Article abstract of DOI:10.1007/s00044-020-02621-5

In this study, 15 new 1H-benzimidazole-5-carboxamidine derivative compounds that could be new antimicrobial agents were synthesized and their antimicrobial activities were determined using the microdilution method. When the activity results were examined,

Full text of DOI:10.1007/s00044-020-02621-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02621-5
ORIGINAL RESEARCH
Synthesis, molecular docking and ADME prediction of some new
benzimidazole carboxamidines derivatives as antimicrobial agents
1
Ismail Celik1,2 Ozlem Temiz-Arpaci2 Hakan Goker2 Fatma Kaynak-Onurdag3 Suzan Okten3
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Meryem Erol
Received: 9 May 2020 / Accepted: 26 August 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
In this study, 15 new 1H-benzimidazole-5-carboxamidine derivative compounds that could be new antimicrobial agents
were synthesized and their antimicrobial activities were determined using the microdilution method. When the activity
results were examined, it was observed that the antibacterial effects of the new benzimidazole derivatives were weaker than
standard drugs, but some derivatives showed significant efficacy against MRSA and VREF with the value of MIC: 8 µg/ml
compared to reference drugs. The antifungal effects of the compounds were found to be weaker compared to the reference
drugs. Molecular docking studies of compounds and reference drugs used were performed against PBP4 and the active and
allosteric site of PBP2a, and estimated ADME profiles were calculated. In addition, 2D and 3D interactions of N10, one of
the most effective antimicrobial compounds compared to reference drugs, were demonstrated in both sites.
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Keywords Benzimidazole Carboxamidine Antimicrobial activity ADME prediction Molecular docking
Introduction
increased disability. Without effective antimicrobials for the
prevention and treatment of infections, many medical pro-
cedures such as organ transplantation, cancer treatment,
diabetes and surgical operations become a high risk
(Spellberg et al. 2013; Ventola 2015; Peters et al. 2019). In
recent years, there has been an alarming increase in the
incidence of Gram (+) infection. Among these, methicillin-
resistant Staphylococcus aureus (MRSA) and vancomycin-
resistant Enterococcus faecalis (VREF) infections are an
increasingly important treatment problem in hospital
patients (Boucher et al. 2009).
In S. aureus, methicillin resistance is realized by “Peni-
cillin Binding Protein” (PBP), which is a different
mechanism than penicillinase (beta-lactamase) production.
PBPs are responsible for carrying and attaching their pep-
tidoglycan precursors to the cell wall being constructed, and
a different PBP called PBP2 or PBP2a is synthesized in
MRSAs (Hackbarth and Chambers 1993). Unlike other
PBPs, PBP2/2a shows low affinity for antibiotics in beta-
lactam structure (Gordon and Lowy 2008). In E. faecalis,
decreased sensitivity to B-lactam antibiotics is due to the
expression of PBP4 (Moon et al. 2018; Rice et al. 2018).
Benzimidazole ring system has an important role to play
in many pharmacological activities such as antimicrobial,
antiviral, antiparasitic, anthelmintic, antihistamine, antiulcer,
antihypertensive, anticancer, antioxidant, spasmolytic,
anticonvulsant, antitumor, anti-inflammatory, antiastmatic,
Infectious diseases seriously threaten public health, but the
biggest problem in combating infectious diseases is the
rapid development of resistance to existing drugs (Blair
et al. 2015). Antimicrobial resistance is currently respon-
sible for more than 700,000 deaths annually worldwide, and
by 2050 it is estimated that it will reach an estimated eco-
nomic cost of $100 million and more than 10 million deaths
per year (Jansen et al. 2018). Resistance to antimicrobials
(antibiotics, antifungals, antivirals, anthelmintics, anti-
malarials) leads to higher medical costs, treatment failure,
increased mortality and morbidity, longer hospital stay, and
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02621-5) contains supplementary
material, which is available to authorized users.
* Meryem Erol
eczacimeryem@gmail.com
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Erciyes University, 38280 Talas/Kayseri, Turkey
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ankara University, 06560 Yenimahalle/Ankara, Turkey
3
Department of Pharmaceutical Microbiology, Faculty of
Pharmacy, 22130 İskender/Edirne, Turkey

Medicinal Chemistry Research
1
analgesic, and neuroleptic (Salahuddin et al. 2017). Since the
benzimidazole ring system is structural analogs of hetero-
cyclic bases in the structure of nucleic acids, it is thought
that they can show their microbiological activities in this
way (Oehlers et al. 2004). Therefore, studies on these deri-
vatives have been increased in recent years. Researches to
date reveal that the benzimidazole ring system is mostly
substituted from the 1st, 2nd, and 5th positions.
Aromatic amidine and diamidine group compounds
show strong activity against many bacteria, ameba, protozoa
and viruses. Although the mechanism of action of the
amidine group compounds has not been adequately illu-
minated, it has revealed that most of the compounds of this
class show antimicrobial effects by binding to the minor
cavity in the AT rich region of DNA (Huang et al. 2001).
Another mechanism is that it can show its activities by
inhibiting Topoisomerase I and/or II enzyme by reversible
interaction with DNA (Bell et al. 1991; Cory et al. 1992;
Fairley et al. 1993; Tidwell et al. 1993).
point device and results were given without correction. H
(400 MHz) and 13C (100 MHz) nuclear magnetic resonance
spectroscopy was taken with Varian Mercury 400 MHz
High Performance Digital FT-NMR spectrometer (Palo
Alto, CA, USA). Dimethyl sulfoxide-d6 (DMSO-d6) was
used as the solvent, chemical shifts were expressed in ppm
according to TMS and coupling constants (J) were given as
Hertz. The molecular weights of the synthesized com-
pounds were taken on the Waters 2695 Alliance Micromass
ZQ LC/MS spectrometer (Milford, MA, USA) using the
ESI (+) method.
General procedures for preparation of compounds
(N1–N15)
It was obtained by reacting 1a, 4-chloro-3-nitrobenzonitrile
and cyclohexylamine in DMF. Dry HCl gas was passed
through absolute EtOH cooled in an ice water bath for
30 min and mixed with 1 in a sealed flask for 3 days to
obtain the imidate ester (1b). All imidate esters was con-
tinued their reactions without any purification due to their
unstable structure. Imidate esters were left to stir overnight
with absolute ethanol passed NH3 gas to obtain 1c. Then, the
amine group (1d) was formed by reducing the nitro group in
the compound with H2/Pd-C. The target compounds N1–
N15 were prepared by condensation of 1d with Na2S2O5
adduct of related aldehydes in DMF. A portion of the
resulting crude product was purified by column chromato-
graphy using a chloromethane:methanol:ammonia (10:1:0.1)
solvation mixture, while the other was crystallized from
methanol. The HCl salts of the compounds were obtained in
a mixture of HCl passed EtOH. List of synthesized com-
pounds (Table S1) and their physical and spectral data were
reported below (see also Supplementary Information).
1-cyclohexyl-2-(2,4-dimethylphenyl)-1H-benzimida-
zole-5-carboxamidine HCI (N1): Yield: 35%, M.p.
195–200 °C. 1H-NMR (400 MHz, DMSO-d6): δ =
1.12–1.20 (m, 4H, –CH2), 1.28–1.34 (t, H, –CH), 1.56 (d,
H, –CH), 1.78 (d, 2H, –CH2), 2.09 (s, 3H, –CH3), 2.17 (d,
2H, –CH2), 2.36 (s, 3H, –CH3), 3.95–4.04 (t, H, N–CH),
7.17 (d, H, Jo = 8.4 Hz, H-3′), 7.25 (d, 2H, Jo = 7.6 Hz, H-
5′, 6′), 7.68 (dd, H, Jo = 8.4 Hz, Jm = 1.6 Hz, H-6), 8.05 (d,
H, Jo = 8.4 Hz, H-7), 8.20 (d, H, Jm = 1.2 Hz, H-4). 13C-
NMR (100 MHz, DMSO-d6): δ = 15.22, 24.18, 25.49,
28.02, 30.33, 56.25, 113.18, 120.15, 120.53, 121.87,
123.58, 126.41, 129.19, 136.65, 139.78, 143.75, 145.17,
155.51, 166.41. MS (ESI+) m/z: 347.3 (M + H, 100%),
265.2 (100%). Anal. Calcd for C24H26N4·HCl·C2H6O: C,
67.19; H, 7.75; N, 13.06. Found: C, 67.35; H, 8.05;
N, 13.06.
In the light of this information, it was aimed in this study
to synthesize some new mono cationic benzimidazole car-
boxamidine derivatives (Fig. 1), to clarify the structures of
1
the compounds using H-NMR, 13C-NMR, mass spectro-
scopy, elemental analysis methods and to investigate
in vitro antimicrobial activities compared to clinical refer-
ence drugs. Estimation of ADME profiles of all compounds,
molecular docking studies and molecular mechanics gen-
eralized born surface area (MM-GBSA) calculations were
performed with Schrödinger software.
Materials and methods
Chemistry
Chemicals and solvents were purchased from Sigma-
Aldrich, Across Organics, Merck, Riedel de Haen and used
without further purification. Silica gel 60 HF254 chroma-
toplates (Merck) for TLC and chloroform:methanol
(10:0.5) were used as the mobile phase. UV light of 254
and 366 nm wavelength was used to detect stains. Melting
points were determined with Büchi B-540 capillary melting
1-cyclohexyl-2-(2,4-dimethoxyphenyl)-1H-benzimida-
zole-5-carboxamidine HCI (N2): Yield 15%, M.p.
182–185 °C. 1H-NMR (400 MHz, DMSO-d6): δ = 1.22–1.40
Fig. 1 Designed 1H-benzimidazole-5-carboxamidine derivatives

Medicinal Chemistry Research
(m, 4H, –CH2), 1.60 (d, H, –CH), 1.83 (d, 3H, –CH2 and
–CH), 2.20 (m, 2H, –CH2), 3.79 (s, 3H, OCH3), 3.86 (s, 4H,
OCH3 and N–CH), 6.70 (dd, H, Jo = 8.4, Jm = 2, H-5′), 6.76
(d, H, Jm = 2, H-3′), 7.35 (d, H, Jo = 8.4, H-6′), 7.70 (d, H,
Jo = 8.8, H-6), 8.03 (d, H, Jo = 9.2, H-7), 8.21 (s, H, H-4).
13C-NMR (100 MHz, DMSO-d6): δ = 15.15, 24.14, 25.98,
28.05, 30.34, 56.80, 113.27, 119.24, 120.47, 121.00, 122.68,
127.14, 129.19, 135.89, 136.66, 139.79, 142.76, 155.49,
166.04. MS (ESI+) m/z: 379.5 (M + H, 100%), 297.4
(100%). Anal. Calcd for C20H20F2N4·0.5HCl·0.5C2H6O: C,
66.82; H, 7.08; N, 13.35. Found: C, 66.81; H, 6.89; N, 13.56.
1-cyclohexyl-2-(2,4-difluorophenyl)-1H-benzimida-
zole-5-carboxamidine HCI (N3): Yield 8%, M.p.
196–200 °C. 1H-NMR (400 MHz, DMSO-d6): δ =
1.17–1.40 (m, 3H, –CH2 and –CH), 1.61 (s, H, –CH), 1.86
(d, 4H, –CH2), 2.20 (d, 2H, –CH2), 4.01 (t, H, N–CH),
7.33–7.37 (m, 2H, H-3′,5′), 7.53–7.58 (m, H, H-6′),
7.71–7.77 (m, H, H-6), 8.40 (d, H, Jo = 8.4, H-7), 8.29 (s,
H, H-4), 9.04 (s, 2H), 9.34 (s, 2H). 13C-NMR (100 MHz,
DMSO-d6): δ = 24.85, 25.12, 31.87, 55.12, 113.60, 116.57,
119.80, 121.53, 122.37, 125.60, 131.01, 136.84, 142.65,
154.15, 160.71, 163.15, 166.01. MS (ESI+) m/z: 355.1 (M
+ H, 75%), 273.4 (100%). Anal. Calcd for C20H20F2N4: C;
59.24, H; 6.16, N; 12.56. Found: C; 58.95, H; 6.40,
N; 12.34.
1-cyclohexyl-2-(2-chlorophenyl)-1H-benzimidazole-5-
carboxamidine HCI (N4): Yield: 13%, M.p. 200–202 °C.
1H-NMR (400 MHz, DMSO-d6): δ = 1.17–1.25 (m, 3H,
–CH2 and –CH), 1.34–1.37 (m, H, –CH), 1.67 (d, H,
–CH), 1.82 (3H, –CH2 and –CH), 2.19 (m, 2H, –CH2),
3.86–3.90 (t, H, N–CH), 7.15–7.19 (t, H, H-3′), 7.27 (d,
H, Jo = 8.4 Hz, H-5′), 7.45 (dd, H, Jo = 7.6 Hz, Jm =
1.6 Hz, H-4′), 7.61–7.64 (m, H, H-6′), 7.77 (dd, H, Jo =
8.8 Hz, Jm =2 Hz, H-6), 8.04 (d, H, Jo = 8.8 Hz, H-4),
8.29 (s, H, H-7). 13C-NMR (100 MHz, DMSO-d6): δ =
24.71, 25.32, 30.86, 57.08, 113.62, 115.78, 119.81,
121.67, 122.73, 125.65, 131.10, 132.91, 1 36.46, 142.90,
154.15, 160.72, 163.51, 166.01. MS (ESI+) m/z: 353.4
(M + H, 90%), 271.3 (100%). Anal. Calcd for
C20H21ClN4·1.25HCl·2C2H6O·0.5H2O: C, 68.08; H, 6.00;
N, 15.88. Found: C, 68.41; H, 6.29; N:15.45.
for C20H21ClN4·HCl·0.5C2H6O·0.25 H2O: C, 58.97; H,
5.94; N, 13.82. Found: C, 58.65; H, 5.67; N, 13.82.
1-cyclohexyl-2-(3,4-dichlorophenyl)-1H-benzimida-
zole-5-carboxamidine HCI (N6): Yield 23%, M.p.
178–182 °C. 1H-NMR (400 MHz, DMSO-d6): δ =
1.12–1.20 (m, 3H, –CH2 and –CH), 1.28–1.34 (t, H, –CH),
1.56 (d, H, –CH), 1.78 (d, 3H, –CH2 and –CH), 2.17 (d, 2H,
–CH2), 3.84–3.90 (t, H, N–CH), 7.17 (d, 2H, Jo = 8.4 Hz,
H-2′, 5′), 7.24 (d, H, Jm = 1.6 Hz, H-6′), 7.68 (dd, H, Jo =
8.4 Hz, Jm = 1.6 Hz, H-6), 8.05 (d, H, Jo = 8.4 Hz, H-7),
8.20 (d, H, Jm = 1.2 Hz, H-4). 13C-NMR (100 MHz,
DMSO-d6): δ = 24.80, 25.23, 31.86, 56.85, 113.27, 119.24,
121.00, 122.68, 127.14, 129.19, 132.25, 136.66, 139.79,
142.76, 146.87, 155.49, 160.78, 166.04. MS (ESI+) m/z:
387.27 (M + H, 100%). Anal. Calcd for C20H20Cl2N4·3HCl:
C, 48.36; H, 4.67; N, 11.28. Found: C, 48.52; H, 5.05;
N, 11.02.
1-cyclohexyl-2-(2,4-dichlorophenyl)-1H-benzimida-
zole-5-carboxamidine HCI (N7): Yield: 23%, M.p.
186–188 °C. 1H-NMR (400 MHz, DMSO-d6): δ =
1.12–1.20 (m, 3H, –CH2 and –CH), 1.28–1.34 (t, H, –CH),
1.56 (d, H, –CH), 1.78 (d, 3H, –CH2 and –CH), 2.17 (d, 2H,
–CH2), 3.75–3.78 (t, H, N–CH), 7.14 (d, 2H, Jo = 8.4 Hz,
H-5′, H-6′), 7.24 (d, H, Jm = 1.6 Hz, H-3′), 7.65 (dd, H, Jo
= 8.4 Hz, Jm = 1.6 Hz, H-6), 8.14 (d, H, Jo = 8.4 Hz, H-7),
8.25 (d, H, Jm = 1.2 Hz, H-4). 13C-NMR (100 MHz,
DMSO-d6): δ = 24.18, 25.02, 30.25, 55.80, 113.58, 119.63,
121.02, 122.84, 127.41, 129.15, 130.45, 136.67, 139.92,
142.77, 145.82, 155.87, 166.04. MS (ESI+) m/z: 387.4
(M + H, 100%), 305.4 (100%). Anal. Calcd for
C20H20Cl2N4·1.2HCl·0.8C2H6O·1.5H2O C, 55.45, H, 5.60;
N, 11.97. Found: C, 55.82; H, 5.31; N, 11.58.
1-cyclohexyl-2-(2,6-dichlorophenyl)-1H-benzimida-
zole-5-carboxamidine HCI (N8): Yield: 17%, M.p.
200–202 °C. 1H-NMR (400 MHz, DMSO-d6): δ =
1.28–1.36 (m, 3H, –CH2 and –CH), 1.70 (d, H, –CH), 1.75
(d, 2H, –CH2), 1.83 (d, 2H, –CH2), 2.29–2.37 (m, 2H,
–CH2), 4.24–4.30 (m, H, N–CH), 7.64 (d, 2H, Jo = 8.4 Hz,
H-3′,5′), 7.80 (dd, H, Jo = 8.4 Hz, Jm = 1.6 Hz, H-6), 7.85
(d, H, Jo = 8 Hz, H-4′), 8.17 (d, H, Jo = 8.4 Hz, H-7), 8.31
(d, H, Jm = 1.2 Hz, H-4). 13C-NMR (100 MHz, DMSO-d6):
δ = 24.72, 25.41, 30.87, 57.11, 113.69, 120.31, 121.45,
123.92, 128.17, 131.39, 131.73, 137.28, 142.22, 142.79,
154.89, 166.78. MS (ESI+) m/z: 387.24 (M + H, 100%),
305.35 (48%). Anal. Calcd for C20H21N4Cl2·HCI·1.5-
C2H6O·2H2O: C, 52.24; H, 5.48; N, 12.19. Found: C,
51.98; H, 5.65; N, 12.52.
1-cyclohexyl-2-(3-nitro-4-chloro)-1H-benzimidazole-
5-carboxamidine HCI (N9): Yield 7%, M.p. 174–176 °C.
1H-NMR (400 MHz, DMSO-d6): δ = 1.12–1.21 (m, 3H,
–CH2 and –CH), 1.25–1.32 (t, H, –CH), 1.67 (d, H, –CH),
1.79 (d, 3H, –CH2 and –CH), 2.21 (d, 2H, –CH2), 3.86–3.92
(t, H, N–CH), 7.22 (d, 2H, Jo = 8.4 Hz, H-5′, 6′), 7.25 (d,
1-cyclohexyl-2-(3-chlorophenyl)-1H-benzimidazole-5-
carboxamidine HCI (N5): Yield 18%, M.p. 188–190 °C.
1H-NMR (400 MHz, DMSO-d6): δ = 1.15–1.41 (m, 3H,
–CH2 and –CH), 1.62 (d, H, –CH), 1.87 (d, 2H, –CH2),
1.92 (d, 2H, –CH2), 2.24–2.33 (m, 2H, –CH2), 4.24–4.29
(m, H, N–CH), 7.48–7.69 (m, 4H, H-2′,4′,5′,6′), 7.73 (dd,
H, Jo = 8.4 Hz, Jm = 2 Hz, H-6), 8.11 (d, H, Jo = 8.8 Hz,
H-7), 8.27 (d, H, Jm = 1.6 Hz, H-4). 13C-NMR (100 MHz,
DMSO-d6): δ = 24.15, 25.34, 30.86, 57.05, 113.64,
116.59, 119.94, 121.35, 122.48, 125.62, 131.11, 132.51,
136.46, 143.49, 154.51, 160.72, 163.45, 165.01. MS (ESI
+) m/z: 353.2 (M + H, 100%), 271.1 (100%). Anal. Calcd

Medicinal Chemistry Research
H, Jm = 1.6 Hz, H-2′), 7.86 (dd, H, Jo = 8.4 Hz,
Jm=1.6 Hz, H-6), 8.11 (d, H, Jo=8.4 Hz, H-7), 8.24 (d, H,
Jm = 1.2 Hz, H-4). 13C-NMR (100 MHz, DMSO-d6): δ =
24.82, 25.25, 31.89, 57.24, 112.68, 121.20, 121.34, 121.89,
123.92, 129.17, 131.56, 131.87, 132.58, 137.29, 143.68,
145.41, 156.68, 165.76. MS (ESI+) m/z: 398.3 (M + H,
100%) 316.3 (50%). Anal. Calcd for C20H20ClN5O2·2.2H-
CI·0.75C2H6O: C, 50.25; H, 4.68; N, 14.65. Found: C,
49.91; H, 4.87; N, 13.98.
1-cyclohexyl-2-(3,4-dibenzyloxyphenyl)-1H-benzimi-
dazole-5-carboxamidine HCI (N10): Yield: 28%, M.p.
104–106 °C. 1H-NMR (400 MHz, DMSO-d6): δ =
1.20–1.27 (m, 3H, –CH2 and –CH), 1.31–1.39 (m, H, –CH),
1.60 (d, H, –CH), 1.79–1.85 (t, 3H, –CH2 and –CH),
2.18–2.27 (q, 2H, –CH2), 4.24–4.30 (t, H, N–CH), 5.20 (s,
2H, –OCH2), 5.23 (s, 2H, –OCH2), 7.18–7.47 (m, 13H, 2′,
5′, 6′, 2″,3″, 4″, 5″, 6″, 2‴, 3‴, 4‴, 5‴, 6‴), 7.70 (d, H,
Jo=8.4, H-6), 8.05 (d, H, Jo = 8.4, H-7), 8.25 (d, H, Jo =
1.6, H-4), 9.19 (br, 4H, NHamidine). 13C-NMR (100 MHz,
DMSO-d6): δ = 24.14, 25.37, 30.34, 56.80, 69.92, 70.09,
113.36, 113.94, 115.07, 119.75, 120.90, 121.35, 122.41,
122.57, 127.31, 127.53, 127.75, 127.82, 128.34, 128.36,
136.80, 136.88, 137.17, 142.61, 147.92, 149.71, 155.53,
165.72. MS (ESI+) m/z: 531.7 (M + H, 75%), 449.5
(100%). Anal. Calcd for C34H34N4O2·2HCI·1.4C2H6O·H2O:
C, 62.60; H, 7.25; N, 14.13. Found: C, 62.51; H, 7.58;
N, 13.85.
1-cyclohexyl-2-(naphthalen-2-yl)-1H-benzimidazole-5-
carboxamidine HCI (N11): Yield 18%, M.p. 206–210 °C.
1H-NMR (400 MHz, DMSO-d6): δ = 1.17–1.42 (m, 3H,
–CH2 and –CH), 1.60–1.74 (d, H, –CH), 1.83 (d, 2H, –CH),
1.98 (d, 2H, –CH2), 2.28–2.36 (m, 2H, –CH2), 4.35–4.41 (m,
H, N–CH), 7.62–7.68 (m, 2H, H-6′-7′), 7.84 (m, 2H, H-5′-
8′), 8.05 (dd, 2H, Jo = 8.8, Jm = 2.4, H-3′,4′), 8.11 (dd, 2H,
Jo = 8.0, Jm = 1.2, H-1′-6), 8.19 (d, H, Jm = 1.2, H-7), 8.27
(s, H, H-4). 13C-NMR (100 MHz, DMSO-d6): δ = 24.01,
25.26, 30.50, 56.97, 113.28, 120.15, 121.06, 121.73, 124.68,
125.19, 126.59, 127.21, 127.59, 128.38, 130.37, 131.60,
132.88, 136.75, 142.77, 165.79. MS (ESI+) m/z: 369.4 (M
+ H, 100%), 287.2 (100%). Anal. Calcd for
C24H24N4·2.25HCl·C2H6O·1.8H2O: C, 59.03; H, 6.83; N,
10.59. Found: C, 59.48; H, 6.58; N, 11.00.
1-cyclohexyl-2-(naphthalen-1-yl)-1H-benzimidazole-
5-carboxamidine HCI (N12): Yield: 18%, M.p.
268–272 °C. 1H-NMR (400 MHz, DMSO-d6): δ =
1.20–1.40 (m, 3H, –CH2 and –CH), 1.59 (d, H, –CH), 1.82
(d, 2H, –CH2), 1.95–1.98 (t, 2H, –CH2), 2.26–2.34 (m, 2H,
–CH2), 4.32–4.39 (m, H, N–CH), 7.61–7.66 (m, 2H, H-6′,
7′), 7.71–7.76 (m, 2H, H-2′, 3′), 8.00–8.11 (m, 4H, H-4′, 5′,
8′, 6), 8.19 (s, H, H-7), 8.25 (s, H, 4), 9.08 (br, 4H, NHa-
midine). 13C-NMR (100 MHz, DMSO-d6): δ = 24.26, 25.47,
30.55, 56.98, 113.11, 118.92, 121.31, 125.26, 126.21,
127.63, 128.34, 128.53, 129.30, 132.45, 133.17,
136.13,142.94, 155.00, 164.85, 176.88. MS (ESI+) m/z:
369.2 (M + H, 70%), 287.2 (100%). Anal. Calcd for
C24H24N4·0.8HCl·C2H6: C, 70.38; H, 7.42; N, 12.63.
Found: C, 70.54; H, 7.58; N, 13.02.
1-cyclohexyl-2-(1H-indol-3-yl)-1H-benzimidazole-5-
carboxamidine HCI (N13): Yield 30%, M.p. 222–225 °C.
1H-NMR (400 MHz, DMSO-d6): δ = 1.26–1.46 (m, 3H,
–CH2 and –CH), 1.66 (d, H, –CH), 1.87 (d, 2H, –CH2), 1.95
(d, 2H, –CH2), 2.30–2.51 (m, 2H, –CH2), 4.61–4.67 (m, H,
N–CH), 7.17 (dd, H, Jo = 7.6 Hz, Jm = 2 Hz, H-5′),
7.22–7.26 (m, H, H-4′), 7.55 (d, H, Jo = 8, H-7′), 7.66 (dd,
H, Jm = 8, Jm = 2, H-6′), 7.84 (s, H, H-2′), 7.98 (d, H, Jo
= 7.6, H-6), 8.04 (d, H, Jo = 8.8, H-7), 8.22 (d, H, Jm =
1.6, H-4). 13C-NMR (100 MHz, DMSO-d6): δ = 24.34,
25.57, 30.55, 56.56, 104.27, 112.05, 112.91, 118.66,
120.22, 121.77, 122.31, 126.57, 127.09, 136.14, 136.94,
143.60, 151.74, 166.29, 175.85. MS (ESI+) m/z: 358.5
(M + H, 100%), 276.3 (100%). Anal. Calcd for
C22H23N5·2HCl·0.3C2H6O·1.2H2O: C, 58.27; H, 6.32; N,
15.03. Found: C, 58.51; H, 6.54; N, 15.38.
1-cyclohexyl-2-(1-methyl-1H-indol-3-yl)-1H-benzimi-
dazole-5-carboxamidine HCI (N14): Yield: 25%, M.p.
225–230 °C. 1H-NMR (400 MHz, DMSO-d6): δ =
1.15–1.23 (m, 3H, –CH2 and –CH), 1.87 (d, 2H, –CH2),
1.94(d, 2H, –CH2), 2.31–2.39 (m, 3H, –CH2 and –CH),
3.95 (s, 3H, –N–CH3), 4.62–4.69 (m, H, N–CH),
7.20–7.23 (t, H, H-5′), 7.29–7.33 (m, H, H-6′), 7.59 (d, H,
Jo = 8.4, H-4′), 7.66 (dd, H, Jo = 8, Jm = 1.6, H-7′), 7.85
(s, H, H-2′), 8.00–8.04 (m, 2H, H-6, 7), 8.21 (d, H, Jm =
1.6, H-4). 13C-NMR (100 MHz, DMSO-d6): δ = 24.32,
25.50, 30.53, 32.96, 56.48, 103.34, 110.40, 112.98,
118.70, 120.48, 120.54, 120.61, 121.72, 122.43, 126.88,
130.91, 136.65, 143.60, 151.37, 166.26, 175.65. MS (ESI
+) m/z: 372.5 (M + H, 100%), 290.2 (100%). Anal. Calcd
for C23H25N5·2HCl: C, 61.49; H, 7.27; N, 14.64. Found:
C, 61.82; H, 7.58; N, 14.31.
1-cyclohexyl-2-(benzofuran-2-yl)-1H-benzimidazole-
5-carboxamidine HCI (N15): Yield 12% M.p.
256–258 °C. 1H-NMR (400 MHz, DMSO-d6): δ =
1.43–1.52 (m, 3H, –CH2 and –CH), 1.73 (s, H, –CH), 1.92
(d, 2H, –CH2), 2.01 (d, 2H, –CH2), 2.28–2.36 (q, 2H,
–CH2), 4.88–4.94 (m, H, N–CH), 7.37–7.41 (m, H, H-5′),
7.46–7.51 (m, H, H-6′), 7.65 (s, H, H-3′), 7.73–7.77 (m, 2H,
H-4′, 7′), 7.83 (d, H, Jo=8, H-6), 8.10 (d, H, Jo = 8.8, H-7),
8.25 (d, H, Jm = 1.6, H-4). 13C-NMR (100 MHz, DMSO-
d6): δ = 24.19, 25.44, 30.50, 57.35, 104.27, 112.05, 112.91,
118.66, 120.22, 121.77, 122.31, 126.57, 127.09, 136.14,
136.94, 143.60, 151.74, 166.29, 175.85. MS (ESI+) m/z:
359.5 (M + H, 85%), 277.4 (100%). Anal. Calcd for
C22H22N4O: C, 59.30; H, 6.89; N, 11.53. Found: H, 59.58;
H, 6.45; N, 11.18.

Medicinal Chemistry Research
Antimicrobial evaluation
Quinazolinone (PDB ID: 4CJN at 1.95 Å resolution) and
crystal structure of E. faecalis PBP4 in the ceftaroline-
bound ((PDB ID: 6MKI at 2.98 Å resolution) was impor-
ted from protein data bank (https://www.rcsb.org/).
Hydrogens were added, non-bonding command with
metals, formation of disulfide bonds, deletion of water at
5 Å distance from het groups and preprocess by creating
pH: 7.00 2.00 het states using Epik. In the second step,
the appropriate chain was selected. Water molecules and
other structures other than ligands were deleted. pH:
7.00 2.00 regenerate state was created with S2. In the
third step, the Hydrogen bond determination was opti-
mized using PROKA pH: 7.00 with water sample orien-
tation. Finally, protein was prepared by minimizing
OPLS3 field forces. Receptor Grid Generation was used to
binding site: for the 4CJN allosteric site x: 45.05, y: 44.42,
and z: 7.36 coordinates, for the 4CJN active site x: -35.89,
y: −12.57, and z: −23.65 coordinates and for the 6MKI
active site x: 35.24, y: 1.34, and z: 10.06 coordinates was
created in 20 × 20 × 20 Å. Docking with XP (extra preci-
sion) was performed using a previously prepared, ligand,
protein, and grid box by using Glide Ligand Docking
module. Ligands were flexible specials and added Epik
states penalties to docking score. Glide emodel and XP
Gscore values of all compounds were calculated and two-
and three-dimensional ligand–protein interactions were
determined (Erol et al. 2020).
In microbiological studies, P. aeruginosa ATCC 27853,
S. aureus ATCC 29213, E. coli ATCC 25922, E. faecalis
ATCC 29212, C. albicans ATCC 10231 standard strains
and clinical isolates of these microorganisms known to be
resistant to various antibiotics were used. Resistance states
of isolates were investigated by Kirby Bauer Disk Diffusion
method according to CLSI guidelines and resistant strains
were studied (Wayne 2018). The synthesized result was
prepared by DMSO for the stock solutions of the com-
pounds. First, microorganisms were passaged by single
colony cultivation method for purity and viability control.
Bacteria were incubated on SDA plates at 24 °C for 24 h
and mushrooms at 35 °C for 24–48 h at 37 °C. The resulting
synthesized compounds were diluted in 96-well microplates
in liquid medium (MHB or RPMI-1640) to give con-
centrations of 1024, 512, 256, 128, 64, 32, 16, 8 μg/ml.
Reference antimicrobial drugs were diluted in liquid media
(MHB or RPMI-1640) in 96-well microplates to achieve
concentrations of 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μg/ml.
A bacterial susceptibility test was performed at the Mueller
Hinton Broth (MHB) feeder. Bacterial suspensions to be
used in the susceptibility test were prepared at a density of
106 cfu/ml, diluted with fresh broth from overnight culture
at a density of 0.5 McFarland (108 cfu/ml). The bacterial
suspension at a concentration of 106 cfu/ml was inoculated
in 10 μl of wells containing 0.1 ml of diluted compounds.
After inoculation, there are 105 cfu/ml bacteria in the wells.
The microplates were incubated at 35 °C for 24 h. Fungal
susceptibility testing was performed in RPMI-1640 medium
buffered with pH 7 MOPS containing L-glutamine. For
inoculation, the yeast suspension adjusted to a McFarland
0.5 concentration (106 cfu/ml) was diluted 1/100, followed
by 1/20 dilution and inoculated to 0.9 ml in wells containing
1 ml of diluted compounds. After inoculation, there are 5 ×
102 cfu/ml yeast in the wells. Microplates were incubated at
35 °C for 24–48 h.
MM-GBSA free energy calculations
MM-GBSA is a calculation method used to estimate
protein–ligand free energies. Calculations were made
using the Schrödinger Prime module. It is calculated by the
formula ΔGbind = Ecomplex (minimized) − [Eligand
(unbound, minimized) + Ereceptor (unbound, mini-
mized)]. The ligands and protein construct were selected
from the XP implantation performed earlier. VSGB was
used as the solvent model and OPLS3 was used as the
Force Field. MM-GBSA (dG Bind) value of all com-
pounds was calculated (Genheden and Ryde 2015).
Molecular docking studies
All computerized studies were performed using Maestro
10.5 suit software (Schrödinger 2018). Molecular docking
studies were carried out in four stages: ligand preparation,
protein preparation, grid creation, and docking. The struc-
ture of the compounds was plotted using ChemDraw 17.1.
LigPrep module was used to prepare ligands. OPLS3 was
preferred for field forces. Ionization was carried out using
Epik in the range of possible pH: 7.00 2.00, desalted and
tautomer was formed. Protein preparation was carried out in
three stages using Protein Preparation Wizard module.
Import and Process, Review and Modify, and Refine.
Crystal structure of S. aureus PBP2a in complex with
Theoretical ADME prediction
The Schrodinger QikPprop module (Release 2018) was
used to determine the “absorption, distribution, metabo-
lism, and excretion” profiles of the compounds, and some
important parameters were selected. Molecular weight,
number of recipient and donor hydrogen bonds, estimated
octanol/water distribution coefficient, predicted human
oral absorption, Van der Waals surface area, number of
violations of Lipinski’s five rules, and number of viola-
tions of Jorgensen’s three defining feature rules were
calculated.

Medicinal Chemistry Research
Scheme 1 Synthesis pathways of the target compounds
Results and discussion
controls of the synthesized compounds (TLC studies) and
their melting points were determined, spectral analyzes
1
Chemistry
were performed to prove the structures. H-NMR, 13C-
NMR, mass spectral analysis, and elemental analysis were
1
Target compounds were synthesized in 5 steps according
to the method specified in the literature (Alp et al. 2009;
Alp et al. 2014; Ates-Alagoz et al. 2006; Goker et al.
2005; Karataş et al. 2012). Compounds N1–N15 were
prepared using the methods outlined in Scheme 1. 1a was
prepared by the reaction of 4-chloro-3-nitrobenzonitrile
and cyclohexylamine in the presence of DMF. Then,
imidate esters (1b) from the cyano group were formed by
the Pinner reaction method. The obtained and unstable
imidate esters were rapidly converted to the amidine
group (1c) with NH3/EtOH, and the nitro group was
reduced by Pd/C catalyzed hydrogen gas to obtain the
amine group (1d). The targeted result products (N1–N15)
were obtained by reaction of 1d with Na2S2O5 additive
products of aldehydes in DMF. Some of the derivatives
were purified by column chromatography (chlorofom:
methanol:ammonia (10:1:0.1) solvent mixture), while
some were re-crystallized from methanol. After the purity
found to prove the expected structures. The H-NMR and
13C-NMR spectra of the compounds were taken by dis-
solving in DMSO-d6. Since these solvents are not 100%
deuterated and contain 98–99.8% deuterium, there are
also peaks of non-deuterated forms of solvents in the
spectra. The peak observed in the range of 3.25 to
3.65 ppm of the compounds dissolved with DMSO-d6 is
due to the non-deuterated DMSO in the solvent. In addi-
tion, the peaks observed in the range of 2.20–2.40 ppm in
the analysis made with DMSO-d6 are the peaks from the
water in the solvent. 1-Cyclohexylamino-2-substitue-5-
carboxamidine compounds have aromatic protons
between 6.85 and 8.35 ppm, and –CH2 protons in cyclo-
hexyl are between 1.14 and 1.52 ppm (protons
overlapped-net unallocated), and the cyclohexyl –CH of
benzimidazole bound to the N atom is 3.84–4.94 ppm was
also observed. The methyl protons attached to the phenyl
ring at the 2nd position of the benzimidazole are between

Medicinal Chemistry Research
Table 1 In vitro antimicrobial
MIC values (μg/mL) of
Compound
A
B
C
D
E
F
G
H
I
synthesized compounds
(N1–N15) and reference drugs
N1
256
128
128
32
128
128
128
32
64
64
64
64
16
64
16
128
512
32
16
8
16
128
64
32
32
16
8
16
16
N2
128 128
N3
128 128 512
128 128 32
64
32
16
16
8
512
128
32
N4
N5
128
32
128
32
64
64
8
N6
128 128
128 128
128 128
8
32
N7
32
32
8
8
>512
64
N8
32
32
8
16
16
8
32
16
8
32
16
8
N9
128
128
32
128
128
32
64
64
64
64
16
8
64
N10
16
N11
128 128
8
8
8
8
64
N12
128
128
128
128
–
128
128
128
128
–
64
64
512 512
512
8
512
8
512 512
N13
64
8
8
8
8
64
64
16
–
N14
128 128
8
32
16
32
16
N15
64
–
64
–
8
Vancomycin
Ampicillin
Meropenem
Ciprofloxacin
Gentamicin
Amphotericin B
<0.0625 <0.0625 <0.0625 >8
2
2
–
–
2
>8
2
–
2
–
–
>8
–
–
<0.0625 <0.0625 0.5
0.5
2
<0.0625 >8
–
<0.0625 <0.0625
1
0.5
1
0.5
2
2
–
0.25
0.25
0.5
–
>8
–
–
–
–
–
–
–
–
0.5
Note: A: E. coli ATCC 25922, B: E. coli isolate, C: P. aeruginosa ATCC 27853, D: P. aeruginosa isolate, E:
S. aureus ATCC 29213, F: S. aureus isolate (MRSA), G: E. faecalis ATCC 29212, H: E. faecalis isolate
(VREF), I: C. albicans ATCC 10231. E. coli isolate is susceptible to the tested antimicrobial agents. S.
aureus isolate is a methicillin-resistance (MRSA) isolate. Antibacterial drugs are not tested against fungi,
while antifungal drugs are not tested against bacteria. P. aeruginosa is naturally resistant to ampicillin. Gram
(−) bacteria used in this study are resistant to vancomycin
Bold values indicates the most effective compounds compared to standard drugs
2.09 and 2.36 ppm, the methoxy protons are singlets,
3.79–3.86 ppm. Among the protons belonging to benzyl-
CH2, the singlet was observed between 5.20 and
5.23 ppm. While the protons belonging to the amidine
group were observed as 9.04–9.34 ppm in some spectra, in
the form of a large singlet 4H, in some spectra as 2H and
2H singlet peaks, in some spectra it could not be observed.
MASS spectral analysis, electrospray ionization (ESI)
method and molecular ion peak was observed as M + H. M
+ H + 2 was also observed in compounds containing
chlorine atoms. The C, H, and N percentages of the syn-
thesized compounds were found, and these findings were
compared with the theoretical calculations to determine the
purity of the compounds and the amount of water, ethanol
and HCl contained in them, and the amount of C, H, and N
elements in all compounds were within the limits of 0.4%.
and amphotericin B against fungi. The antimicrobial effects
of compounds and reference drugs in the form of MIC
values were determined and are given in Table 1. According
to the microbiological results, when the effects of deriva-
tives against E. coli and E. coli isolate are examined; it was
determined that it is sensitive to ampicillin, meropenem,
ciprofloxacin, and gentamicin, and reference drugs have a
much better effect than benzimidazole derivatives.
Compounds generally had a weak antimicrobial effect
against Gram-negative P. aeruginosa ATCC 27853 and
P. aeruginosa isolate (MIC: 64–128 µg/ml—except N12).
More than half of the compounds against S. aureus ATCC
29123 showed good activity with MIC: 8 µg/ml, while
showing weaker efficacy compared to reference drugs.
The results of the N6, N7, N10, N11, N13, N14, and N15
derivatives against MRSA (MIC: 8 µg/ml) are quite
satisfactory compared to ampicillin and meropenem. The
compounds showed a broad range of activity against the
Gram-positive bacterium E. faecalis ATCC 29212 (MIC:
8–512 µg/ml) compared to the reference drugs. Against
VREF; N7, N10, N11, and N13 derivatives against VREF
showed better efficacy than vancomycin and ampicillin
with MIC: 8 µg/ml. The antifungal activities of the
In vitro antimicrobial activities
Antimicrobial effect determinations of all synthesized
compounds (N1–N15) were determined in vitro by micro-
dilution technique. Vancomycin, ampicillin, meropenem,
ciprofloxacin, and gentamicin were used as reference drugs,

Medicinal Chemistry Research
compounds against C. albicans were in the range of 8–
>512 μg/ml and much less effective than amphotericin B.
When the microbiology findings were evaluated in
general, it was found that the tested derivatives showed
more important antimicrobial activity against Gram-
positives than Gram-negatives, and the best antibacterial
activity was observed against MRSA and VREF. The
methyl substituent of the benzimidazole in the 2nd posi-
tion, which gave electrons to the ring on the phenyl ring,
had no positive effect on activity. In addition, it has been
observed that the fluorine substituent has low activity.
Instead, it has been found that substitution with electron
withdrawal groups such as 4-chlorine and 3,4-dibenzy-
loxy increases antimicrobial activity. The fact that the
chlorine group becomes ortho or meta decreases the
activity. Also, the presence of heterocyclic rings at 2nd
position of the benzimidazole contributed positively to
activity. While 2-naphthyl showed significant anti-
microbial activity, 1-naphthyl group did not. While the
indol-3-yl substituent is effective against both MRSA and
VREF, the anti-VREF activity of the 1-methylindol-3-yl
substituent is weaker.
RMSD values indicates that the docking process predicts
fairly accurate binding exposure. According to the results of
the docking studies performed in the 4CJN allosteric site, it
was concluded that the target compounds may act by
binding to the 4CJN allosteric site, since the Glide emodel
scores of most compounds were calculated lower than the
active site. The calculated Glide emodel, MM-GBSA (dG
Bind) and XP Gscore scores of all compounds were given
in Table 2. N10 gave the lowest Glide emodel scores
against both MRSA (Glide emodel: −57.901) and VREF
(Glide emodel: −82.278).
According to antimicrobial activity results, 2D and 3D
interactions of N10, one of the most effective compounds
(also, Glide emodel score is lowest in both sites), were
shown in both sites. In the PBP2a allosteric site, it formed a
1-Å long hydrogen bond between -NH2 and GLN613 in the
amidine group. It also showed hydrophobic interactions
with MET641, ALA642, ALA646, TYR446, VAL578,
VAL579, TYR588, and PHE617. 2D and 3D interactions of
N10 in this site were shown in Fig. 2.
In the PBP4 binding site, it formed two separate hydro-
gen bonds between the length of 1.83 and 2.08 Å between
the NH2 and GLN542 in the amidine group, and a hydrogen
bond between the 4th position benzyloxy oxygen and
THR620. N10; it also showed hydrophobic interactions
with TYR605, ALA664, PHE638, TYR540, and VAL467
in this region. Figure 3 was showed 2D and 3D interactions
of N10 in the PBP4 active site.
Results of molecular docking
Molecular docking techniques are often used to investigate
how drug or drug candidates and enzyme, nucleic acid, and
receptor proteins fit together in computer-aided rational drug
design. In these docking studies, binding energies to the
receptor with a clear three-dimensional structure can be
determined and the position of the ligand in the binding site
of the receptor can be revived. This can be useful for
understanding the type of binding and designing molecule
and more compatible ligands that target proteins (Celik et al.
2020). Also, MM-GBSA is a method used to estimate free
binding energies, requires less calculation than free energy
methods, and is often reported to give more accurate results
than the molecular docking scoring function. MM-GBSA is
widely used in biomolecular studies such as protein–ligand
binding and protein–protein interaction (Wang et al. 2019).
In this study, some of the synthesized compounds
exhibited higher activity against MRSA and VREF than
reference drugs. As mentioned in the “Introduction”,
methicillin resistance in S. aureus results from a different
synthesis of PBP called PBP2 or PBP2a, while decreased
sensitivity to β-lactam antibiotics in E. faecalis was due to
expression of PBP4. So we focused on PBP2a and PBP4
resistance proteins (PDB: 4CJN and 6MKI). In order to
ensure the validation of the docking process, the ligands
were removed from the protein–ligand complex crystal
structure and re-docking was performed on these regions. In
4CJN allosteric site gave RMSD = 0.068 and in the 6MKI
active site gave RMSD = 0.053 value. The low of these
Theoretical ADME prediction
In the process of drug discovery and development, molecules
are asked to show high biological activity with low toxicity.
ADME parameters can be estimated by in silico methods
using the molecular structure. Therefore, in silico studies
allow us to learn about the possibility of a compound being a
potentially good drug. Access to the therapeutic goal and
concentration in the organism is equally important. Early
estimation of ADME parameters greatly reduces the rate of
pharmacokinetic failure in clinical phases during the dis-
covery phase. It is increasingly accepted to predict ADME
parameters in cases where numerous compounds are
screened for ADME or access to physical samples is limited.
Estimates of ADME properties of all synthesized compounds
(N1–N15) were made with QikProp. According to Lipinski’s
five rules and Jorgensen’s three rules, drug-like compounds
should not have more than one violation. Data of the com-
pounds such as molecular weight, logP, % oral absorption,
polar surface area, hydrogen donor number, hydrogen
acceptor number, and volume are given in Table 3 together
with the violation of the third and fifth rules. From the table,
it can be seen that N10 violates the Lipinski rule, but all other
compounds comply with these rules.

Medicinal Chemistry Research
Table 2 Calculated Glide emodel, MM-GBSA (dG Bind) and XP Gscore values of compounds and reference drugs
4CJN
6MKI
Comp.
Allosteric site
Active site
Active site
Glide emodel MM-GBSA XP Gscore Glide emodel MM-GBSA XP Gscore Glide emodel MM-GBSA XP Gscore
(dG Bind)
(dG Bind)
(dG Bind)
N1
−31.147
−37.561
−36.411
−40.164
−38.393
−39.582
−37.376
−39.803
−39.653
−57.901
−39.190
−35.178
−40.638
−33.487
−40.814
−44.73
−33.84
−34.14
−41.08
−39.58
−44.66
−38.92
−44.70
−40.74
−36.41
−39.20
−44.68
−28.90
−38.34
−38.95
−57.85
−27.08
−40.88
−44.19
−37.05
−4.251
−3.637
−4.130
−3.863
−3.960
−3.974
−3.143
−3.599
−3.329
−2.931
−3.344
−4.411
−4.004
−1.843
−3.695
−6.233
−3.589
−3.410
−4.556
−2.678
−28.268
−33.081
−35.354
−40.964
−41.035
−34.156
−36.517
−42.160
−40.385
−50.826
−23.204
−34.027
−29.064
−27.547
−21.147
−70.037
−32.240
28.645
−32.19
−46.76
−34.69
−42.24
−47.17
−52.56
−33.04
−44.08
−40.30
−48.09
−34.47
−28.56
−21.04
−22.87
−10.82
−30.43
−32.61
−17.92
−32.54
−54.78
2.316
−2.483
−2.628
−2.624
−3.048
−2.347
−0.439
−2.925
−3.155
−3.615
−2.978
−1.940
−4.275
−4.352
−3.615
−5.487
−3.719
−2.183
−6.472
−5.998
−46.007
−45.627
−45.832
−52.641
−50.017
−54.093
−50.499
−57.377
−53.660
−82.278
−54.775
−53.463
−51.264
−46.410
−49.778
−70.737
−43.251
−77.755
−48.571
−46.662
−65.42
−56.69
−54.97
−70.82
−62.54
−66.58
−64.67
−72.92
−58.48
−70.96
−63.50
−59.91
−51.49
−59.43
−52.98
−95.36
−25.30
−68.84
−46.11
−52.33
−7.025
−7.251
−4.544
−7.697
−5.382
−7.504
−5.033
−6.663
−5.185
−8.093
−7.678
−5.437
−5.435
−5.541
−5.017
−6.770
−4.936
−2.082
−5.184
−5.216
N2
N3
N4
N5
N6
N7
N8
N9
N10
N11
N12
N13
N14
N15
Vancomycin −75.607
Ampicillin −36.564
Gentamycin −31.879
Meropenem −40.453
Ciprofloxacin −32.610
−37.238
−47.965
Fig. 2 2D and 3D interaction between N10 with PBP2a (4CJN) at allosteric site
Conclusions
N13 derivatives were observed more effectively compared to
reference drugs against MRSA and VREF with MIC: 8 µg/
ml. Derivatives showed better antibacterial activity against
Gram-positives than Gram-negatives; It was observed that
especially the binding of heterocyclic rings to the 2 position
of benzimidazole or groups that attract electrons to the
In this study, a series of 1H-benzimidazole-5-carboxamidine
derivative compounds that we hope might be a new anti-
microbial agent were designed, synthesized and their anti-
microbial activities were determined. N7, N10, N11, and

Medicinal Chemistry Research
Fig. 3 2D and 3D interaction between N10 with PBP4 (6MKI) at active site
Table 3 Calculated ADME parameters (N1–N15)
Comp.
MW
DonorHB
AccptHB
QPlogPo/w
QPlogS
% HOA
PSA
63.589
RuleOfFive
RuleOfThree
N1
336.411
378.473
354.402
352.866
352.866
387.311
387.311
387.311
397.863
530.668
368.480
368.480
357.457
371.484
358.442
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
3.000
4.000
3.000
3.000
3.000
4.500
3.000
3.000
3.000
3.000
3.000
3.000
4.000
4.500
3.000
3.000
3.000
3.000
3.500
3.954
3.954
4.132
4.071
4.196
4.636
4.564
4.452
3.565
7.602
4.643
4.567
3.864
4.684
4.038
−5.654
−5.631
−5.844
−5.624
−5.968
−6.638
−6.362
−6.073
−6.129
−9.954
−6.424
−6.136
−5.744
−6.621
−5.870
100.000
100.000
100.000
100.000
100.000
100.000
100.000
100.000
82.570
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
1
0
1
1
1
1
1
1
1
1
1
1
1
N2
78.638
63.707
63.836
63.561
63.608
63.838
62.805
107.732
76.852
63.511
63.597
76.216
64.809
72.567
N3
N4
N5
N6
N7
N8
N9
N10
N11
N12
N13
N14
N15
95.039
100.000
100.000
95.364
100.000
100.000
Donor HB: Estimated number of hydrogen bonds that would be donated by the solute to water molecules in an aqueous solution (recommended
value: 0–6); acceptor HB: Estimated number of hydrogen bonds that would be accepted by the solute from water molecules in an aqueous solution
(recommended value: 2–20); QPlogPo/w: Predicted octanol/water partition coefficient (recommended value: −2–6.5); QPlogS: Predicted aqueous
solubility (recommended value: −6.5–0.5); % HOA: percent of human oral absorption; PSA: Van der Waals surface area of polar nitrogen and
oxygen atoms and carbonyl carbon atoms (recommended value: 7–200); RuleOfFive: Number of violations of Lipinski’s rule of five. The rules are:
mol_MW < 500, QPlogPo/w < 5, donorHB ≤ 5, and accptHB ≤ 10. Compounds that satisfy these rules are considered druglike (the “five” refers to
the limits), RuleOfThree: Number of violations of Jorgensen’s rule of three. The three rules are: QPlogS > −5.7, QP PCaco > 22 nm/s, and
# Primary Metabolites < 7
Bold values indicates the compound that violated the Lipinski rule
phenyl ring significantly increased antimicrobial activity.
Molecular docking studies of all compounds and reference
drugs were conducted and when the scores of all compounds
were examined, a good relationship was found between
Glide emodel scores and antimicrobial effect. 2D/3D inter-
actions of N10, which is one of the most effective anti-
microbial compounds and also has the highest Glide emodel
score, were demonstrated in the PBP2a allosteric site and
PBP4 active site. According to all these results, the synthe-
sized compounds showed promising antimicrobial activity.
Acknowledgements NMR, mass spectra and elemental analysis of the
compounds were performed by Ankara University Faculty of Phar-
macy Central Laboratory and Erciyes University Technology and
Research Center (TAUM). This work was supported by The Scientific
and Technological Research Council of Turkey (TUBITAK) Grant
[315S333].

Medicinal Chemistry Research
Compliance with ethical standards
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