Discovery of novel bacterial FabH inhibitors (Pyrazol-Benzimidazole amide derivatives): Design, synthesis, bioassay, molecular docking and crystal structure determination

Article abstract of DOI:10.1016/j.ejmech.2019.03.026

The enzyme FabH catalyzes the initial step of fatty acid biosynthesis that is essential for bacterial survival. Therefore, FabH has been identified as an attractive target for the development of new antibacterial agents. We present here the discovery of a

Full text of DOI:10.1016/j.ejmech.2019.03.026

Accepted Manuscript
Discovery of novel bacterial FabH inhibitors (Pyrazol-Benzimidazole amide
derivatives): Design, synthesis, bioassay, molecular docking and crystal structure
determination
Yan-Ting Wang, Tian-Qi Shi, Jie Fu, Hai-Liang Zhu
PII:
S0223-5234(19)30244-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.026
EJMECH 11196
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 December 2018
Revised Date: 11 March 2019
Accepted Date: 11 March 2019
Please cite this article as: Y.-T. Wang, T.-Q. Shi, J. Fu, H.-L. Zhu, Discovery of novel bacterial FabH
inhibitors (Pyrazol-Benzimidazole amide derivatives): Design, synthesis, bioassay, molecular docking
and crystal structure determination, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.03.026.
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ACCEPTED MANUSCRIPT
Discovery of Novel Bacterial FabH Inhibitors (Pyrazol-Benzimidazole amide
derivatives): Design, Synthesis, Bioassay, Molecular Docking and Crystal
Structure Determination
Yan-Ting Wang a,b,*, Tian-Qi Shi b, Jie-Fu a,*, Hai-Liang Zhu b,*
a
Department of Environmental Science & Engineering, Fudan University, Shanghai
200433, P. R. China
b
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, P. R. China
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Abstract
The enzyme FabH catalyzes the initial step of fatty acid biosynthesis that is
essential for bacterial survival. Therefore, FabH has been identified as an attractive
target for the development of new antibacterial agents. We present here the discovery
of a promising new series of Pyrazol-Benzimidazole amides with low toxicity and
potent FabH inhibitory. Twenty-seven novel compounds have been synthesized, and
1
13
all the compounds were characterized by H-NMR, C-NMR and MS. Afterwards
they were evaluated for in-vitro antibacterial activities against E. coli, P. aeruginosa,
B. subtilis and S. aureus, along with E. coli FabH inhibition and cytotoxicity test.
Some compounds proved to be of low toxicity and potent, especially compound 31
exhibited the most potential to be a new drug with MIC of 0.49-0.98 µg/mL against
the tested bacterial strains and IC50 of 1.22 µM against E. coli FabH. Eight analogues
16, 28, 30, 31, 33, 34, 35 and 36 with low range MIC against wild type Xanthomonas
Campestris exhibited no inhibition against FabH-deficient mutant strain, which firmly
proved the class of compounds arrived at antibacterial activity via interacting with
FabH. In silico ADMET (Absorption, Distribution, Metabolism, Excretion and
Toxicity) evaluation also pointed out that these compounds are potential for
druggability. Further, effective overall docking scores of all the compounds have been
recorded, and docking simulation of compound 31 into E. coli FabH binding pocket
has been conducted, where solid binding interactions has been identified.
Keywords: FabH, Inhibitor, Pyrazole, Antibacterial, Molecule docking
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1. Introduction
Although many kinds of antibacterial agents were discovered and used for
clinical treatment, the incidences of drug resistance of microorganisms to antibacterial
agents were constantly reported [1, 2]. Therefore, the development of new types of
antibacterial agents is a very vital task and much of the research effort is oriented to
the design of new antibacterial agents with high efficiency [3]. Recently, the research
has been focused toward development of new antibacterial agents with novel target. A
promising target is the fatty acid synthase (FAS) pathway in bacteria.
Bacterial FAS (type II) has been proved to be indispensable for bacteria cell
survival [4, 5], which provides essential fatty acids for use in the assembly of key
cellular components such as cell envelope, phospholipids, lipoproteins,
lipopolysaccharides, and mycolic acids.
A key enzyme in this pathway is β-ketoacyl-acyl carrier protein synthase III
(FabH) that initiates the fatty acid elongation cycles [6] and participates in the
feedback regulation of FAS via product inhibition [7-9]. As shown in Fig. 1., FabH
catalyzes the condensation reaction between a CoA-attached acetyl group and an
ACP-attached malonyl group, yielding acetoacetyl-ACP as its final product [10]. In
addition, FabH proteins are highly conserved at the sequence and structural level in
both Gram-positive and -negative bacteria, while there are no significantly
homologous proteins in humans. These attributes suggest that small molecule
inhibitors of FabH enzymatic activity could be potential development candidates
leading to selective, nontoxic, and broad-spectrum antibacterial [6].
The natural products platensimycin and platecin (Fig. 2.), discovered by
employing a novel antisense differential sensitivity screening strategy, were reported
recently from soil bacterial strains of Streptomyces platensis [11-13]. Platencin is
considered a dual FabH-FabF inhibitior. The most potent of the natural products
targeting the condensing enzymes is platensimycin, a nanomolar inhibitor of bacterial
elongation condensing enzymes, which has a MIC of between 0.5-1.0 µg/ml for
several important pathogens, including S. aureus, Enterococcus faecium, and
Streptococcus pneumonia [14, 15]. Compound 1 is a representative benzoic acid
FabH inhibitor [16], which exhibits potent inhibitory activity against Enterococcus
faecalis FabH and Streptococcus pyogenes FabH, IC50 = 0.004umol/L.）FAS20013 is
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an anti-tuberculosis drug developed by FASgen Company [17], which was originally
designed with FabH as its target. It exerts potent inhibition against multi-drug
resistant Mycobacterium tuberculosis, MIC = 0.75-1.5 mg/L.) as well as compounds 2
and 3, discovered by McKinney et al. [18].
According to the structure analysis of these patent FabH inhibitiors reported in
the literatures, phenylamide is an important structural fragment, which can be used as
the skeleton structure of new FabH inhibitors. Besides, with the aid of computer-aided
drug design, Li screened a large number of compounds with different structures, and
results also revealed that phenylamide fragment in drug was beneficial to FabH
inhibition [19]. In addition, Subramani et al. exactly right exploited benzoyl amino
benzoic acid (compound 4) as the skeleton structure to design and develop novel
drugs targeting FabH which exhibited potent activity [20].
Moreover, Polycyclic compounds containing benzimidazole ring units exhibit
multiple excellent biological activities, including antibacterial, antifungal, anticancer,
analgesic, anti-inflammatory, antioxidant antidiabetic, antiparasitic, antihypertensive
ones and so on [21-26].
Further, Pyrazole possesses low molecular weight and steric hindrance, and is
often contributed as an active structural fragment to antimicrobial design [27].
Yet, the effectiveness of fragment hybrid of phenyl amide, benzimidazole and
pyrazole has not been studied till now. To address this knowledge gap, a series of
novel Pyrazol-Benzimidazole amides targeting FabH was synthesized by fragment
hybrid, then modification.
In this paper, some work has been done: discuss the synthetic method of this
series of novel compounds, depict the results of reactivity studies; evaluate their
antibacterial and anti-FabH activities; detect cytotoxicity and hemolytic activities;
appraise in silico ADMET (Absorption, Distribution, Metabolism, Excretion,
Toxicity). Additionally, molecular docking provided more information that could
elucidate antimicrobial mechanisms.
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Fig. 1. Fatty acid biosynthesis pathyway (FabH catalyzes the initiation reaction) [10]
Fig. 2. Reported antibacterial agents targeting FabH and fragment hybrid of seclected
segments for the design of novel compounds binding to FabH.
2. Results and discussion:
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2.1. Chemistry
The synthesis of compounds 13-39 followed the general pathway was outlined in
Scheme 1. The target compounds were obtained in four steps as described in
experimental section. All of the synthetic compounds are being reported for the first
1
13
time (Table 1) and gave satisfactory analysis and spectroscopic data. H NMR, C
NMR, melting test and mass spectroscopy, and analysis results were in full
accordance with their depicted structures.
Scheme 1a General synthesis of compounds 13-39.
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a Reagents and conditions: (i) sodium acetate, phenylhydrazine hydrochloride, H2O, rt,
3 h; (ii) POCl3, DMF, 58 ℃, 5 h, reflux; (iii) o-diaminobenzene, sodium metabisulfite,
DMF, 110 ℃, 4 h, reflux; (iv) benzoic acid, DMAP, EDC·HCl, DCM, rt, 4 h; (v)
nicotinic acid, DMAP, EDC·HCl, DCM, rt, 4 h; (vi) isonicotinic acid, DMAP,
EDC·HCl, DCM, rt, 4 h.
Table 1. Structures of compounds 13-39
Compound
R1
CH3
CH3
CH3
CH3
CH3
F
R2
Compound
R1
CH3
CH3
CH3
F
R3
H
R4
-
H
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
2-CH3
3-CH3
4-CH3
4-Cl
5-CH3
6-CH3
H
-
-
-
F
5-CH3
6-CH3
H
-
H
F
-
F
2-CH3
3-CH3
4-CH3
4-Cl
CF3
CF3
CF3
CH3
F
-
F
5-CH3
6-CH3
-
-
F
-
F
H
H
H
CF3
CF3
CF3
CF3
CF3
H
-
2-CH3
3-CH3
4-CH3
4-Cl
CF3
-
2.2. Crystal structure determination
The crystal structures of compound 17 were determined by X-ray diffraction
analysis. The crystal data presented in Table 2 and Fig. 3 gave the perspective views
of 17 with the atomic labeling system. The crystallographic data have been deposited
at the Cambridge Crystallographic Data Centre (CCDC) and the deposition number of
17 is 1494813.
Table 2. Crystallographic and experimental Data for compound 17
Compound
17
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Formula
Formula weight
Crystal system
Space group
a (Å)
C30H21ClN4O
488.98
Z
4
1.339
Dc(g•cm-3)
Monoclinic
P-1
µ(mm-1)
0.189
F(000)
1016
10.062(3)
10.864(3)
23.171(6)
89.852(7)
89.696(7)
73.268(7)
2425.6(11)
θrang(o)
2.11- 28.76
10275
b (Å)
Reflns collected
Reflns unique
Goodness-of-fit on F2
R1, wR2[I>2σ(I)]
R1, wR2[all data]
Max, min∆ρ(e Å-3)
c (Å)
651
α(o)
1.048
β(o)
0.0777, 0.2074
0.0989, 0.2275
0.539, -0.538
γ(o)
V(Å3)
Fig. 3. ORTEP drawing of the crystal structure of 17
2.3. Biological activity
2.3.1. Antibacterial activity
Two Gram-negative bacterial strains: E. coli and P. aeruginosa and two
Gram-positive bacterial strains: B. subtilis and S. aureus were exploited in the
antimicrobial assay. All the synthesized compounds 13-39 were evaluated for their
antibacterial activity with TTC double dilution method, with DDCP and kanamycin B
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as controls. The results were listed in Table 3, and as shown, the MIC (minimum
inhibitory concentration) value indicated that these novel compounds showed better
inhibitory activity against B. subtilis than other bacteria. Some of them possessed
potent activity, for example, compounds 16, 21, 26, 28, 30, 31, 33, 34, 35 and 36
exerted the similar inhibitory activity with the controls. Notably, compound 31
exhibited the most potent activity and broad-spectrum antibacterial activity against all
the four bacteria strains, with MIC of 0.98, 0.49, 0.98, 0.98 µg/mL, respectively for E.
coli, P. aeruginosa, B. subtilis and S. aureus, which was better than the positive
controls DDCP (3.9, 0.98, 3.9, 1.95 µg/mL) and kanamycin B (1.95, 3.9, 0.98, 0.98
µg/mL).
According to the data listed in Table 3, we could arrive at the conclusion that the
activity of the tested compounds may be correlated to the variation and modification
of the structure. Structure-activity relationship (SAR) analysis demonstrated that type
and location of the substituent have important influences on the antibacterial activity.
We drew that nicotinic groups have better antibacterial effects than benzoic acid
groups according to the activity data: 28-39 overall exhibited better antibacterial
activities than 13-27. At the position of R1 of the benzene ring attached to the pyrazole,
the order of activity of the group introduced is 4-F > 4-CF3 > 4-CH3, indicating that
introduction of a halogen group at R1 enhances the antibacterial activity.
Among compounds 13-27, different substituents were confirmed. With R1 fixed,
4-CH3 or 4-Cl was introduced at the R2 position, and data showed that the activity
sequence was 4-CH3 > 4-Cl. The results showed that the introduction of an
electron-donating group at R2 can enhance the antibacterial activity. In addition,
substituents at different positions are evaluated as well. Also with R1 fixed, 2-CH3 /
3-CH3 / 4-CH3 is introduced at the R2 position, and the order of activity from the
antibacterial data is para- > meta- > ortho- (5d>5j>5m, 5c>5k>5n, 5f>5l>5o).
Furthermore, among compounds 28-39, when the R1 position was fixed and -H,
5-CH3, 6-CH3 was introduced at the R3 position, the activity sequence can be obtained
from the antibacterial data -H < 6-CH3 < 5-CH3 (35 < 36 < 34, 32 < 33 < 31),
indicating that H at R3 enhances antibacterial activity. Compounds 37, 38, 39 have
lower antibacterial activity, demonstrating that isonicotinic acid groups can not
enhance antibacterial activity.
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Then, we drew two conclusions as follows: (1) F substituent in R1 could enhance
antibacterial activity. (2) Nicotinic acid group substitution linked to amide bonds
could also increase the activity. (3) Electron-donating groups and para-substituent at
R2 can enhance the antibacterial activity.
Table 3. MIC of synthetic compounds against bacteria and IC50 against FabH
MIC (µg/mL)
E. coli FabH bIC50
±
Compounds
Gram-negative
Gram-positive
S. aureus
SD (µM)
E. coli P. aeruginosa B. subtilis
>62.5
62.5
62.5
3.9
62.5
>62.5
62.5
31.25
62.5
31.25
0.98
62.5
7.8
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
62.5
28.23±0.32
35.41±0.13
33.17±0.13
3.06±0.12
38.24±0.25
22.08±0.21
37.23±0.15
34.11±0.38
30.33±0.21
36.09±0.13
25.65±0.24
36.45±0.27
34.86±0.29
31.82±0.36
39.81±0.25
18.33±0.23
9.56±0.13
16.88±0.13
1.22±0.23
22.09±0.19
4.02±0.34
15.23±0.19
22.89±0.39
2.05±0.21
33.88±0.13
32.67±0.22
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
31.25
>62.5
62.5
62.5
62.5
>62.5
62.5
62.5
62.5
62.5
>62.5
62.5
62.5
62.5
62.5
62.5
62.5
0.98
62.5
1.95
62.5
62.5
1.95
>62.5
>62.5
>62.5
31.25
>62.5
>62.5
62.5
62.5
31.25
0.98
62.5
15.6
62.5
31.25
3.9
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
31.25
62.5
>62.5
31.25
>62.5
31.25
62.5
62.5
0.98
7.8
62.5
62.5
0.98
0.98
15.6
0.98
1.95
0.98
0.49
31.25
7.8
62.5
0.49
0.98
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
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>62.5
3.9
62.5
0.98
3.9
31.25
3.9
62.5
1.95
0.98
30.28±0.24
1.56±0.18
-
39
DDCP
1.95
0.98
Kanamycin B
a,b Values represent the average of three independent experiments run in triplicate.
2.3.2. E. coli FabH inhibitory activity of synthetic compounds
To generate data concerning the broad-spectrum potential of these novel
compounds in Table 1, FabH enzymes inhibition activities were determined by MTT
assay, and the IC50 values were summarized in Table 3. As shown, to some extent the
antibacterial potential is consistent with the enzyme inhibition of E. coli FabH, with a
few exceptions. In detail, compounds with low antibacterial activity showed poor
E.coli FabH enzyme inhibitory activity, with IC50 >25 µM; compounds possessing
potent activity against E. coli are generally endowed with significant potential against
the enzyme E. coli FabH, with IC50 of 1.22-22.89 µM.
The IC50 values of compounds 28-39 are generally lower than 25 µM, while IC50
of 13-27 is generally higher than 25 µM, which demonstrated that the introduction of
nicotinic acid groups enhances the inhibitory activity of E.coli FabH enzyme. From
the E.coli FabH enzyme inhibitory activity sequence 31 > 34 > 28 and 18 > 23 > 13, it
was concluded that the F substituent at R1 (halogen group) enhances the E.coli FabH
enzyme inhibitory activity. Besides, among compounds 28-39, H at R3 showed the
most potent E.coli FabH enzyme inhibitory activity. In addition, among compounds
13-27, electron donating group introduced at R2 has the lowest IC50 value and the
highest enzyme inhibitory activity. Further, during all the compounds, 31 exhibited
the most potent E.coli FabH enzyme inhibitory activity, with IC50 of 22.89 µM, which
was better than the positive control DDCP (IC50=1.56 µM). Then, it is concluded that
the antibacterial ability of the compounds is related to the inhibition of FabH enzyme,
for the FabH inhibition having a positive correlation with the antibacterial activity.
2.3.3. Antibacterial experiment on wild type and FabH mutant Xanthomonas
Campestris
To further explain that the antibacterial effect is achieved by inhibiting FabH,
wild type strain Xanthomonas oryzae pv. Oryza KACC10331 and mutant strain
Xanthomonas oryzae pv. Oryza KACC10331 ΔFabH were chosen for MIC
experiments.
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Table 4. MIC (µg/mL) of selected compounds against wild type and mutant
Xanthomonas Campestris strains.
Compounds
Xanthomonas oryzae pv.
Xanthomonas oryzae pv. Oryza
Oryza KACC10331
KACC10331 ∆FabH
15.63
0.49
>62.5
>62.5
>62.5
>62.5
>62.5
30
31
33
34
35
15.63
15.63
7.82
Fig. 4. 30, 31, 33, 34 and 35 were assessed against two types of Xanthomonas oryzae
pv. Oryza KACC10331 (wild type and mutant type). 5 µg/mL of each compound was
added onto two types of strains. (1) wild type strain without compound treating; (2),
(3), (4), (5), (7) 30, 33, 34, 35 and 31 acted on wild type strain, respectively; (6), (8)
mutant type of strain were treated with 35 and 31, respectively.
Five of the most potent analogues, 30, 31, 33, 34 and 35 were assessed against
the two strains (Table 4 and Fig. 4). As Table 4 depicted, they all showed significant
inhibition, especially 31 emerged as the most potent analogue with MIC of 0.49
µg/mL, however, all the selected compounds exhibited no inhibition against
FabH-deficient mutant strain. In addition, Fig. 4 exhibited the effect of 5 µg/mL of
each compound on two types of Xanthomonas oryzae pv. Oryza KACC10331 (wild
type and mutant type), and the results were in accordance with Table 4. Compound 31
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displayed the strongest inhibition against wild type bacterial, and no bacteria was seen
in the medium plate treated with 5 µg/mL of 31, then effect of 35 followed after 31.
Also, almost no inhibition was found in the acting on 31 and 35 against mutant type
bacterial. Consequently, this experiment firmly suggested the series of compounds
arrived at antibacterial activity via inhibiting FabH.
2.3.4. Cytotoxicity and hemolytic activities
For the sake of seeking for potent antibiotics, testing safety of these compounds
is significant. One of the main obstacles to the clinical use of compounds with
effective antibacterial activities is their degree of injury to mammalian cell that leads
to high hemolytic and cytotoxic activity dangerous to the host organism. Hemolytic
activity is conventionally used as a measure of cytotoxicity and model for mammalian
cells because red blood cells are, in general, extremely fragile. However, sometimes
compounds which show low hemolytic activities have the severe cytotoxicity against
mammalian cells. So the hemolysis and 293T cell (human kidney epithelial cell)
cytotoxicity assays were both tested. Overall, as shown in Table 5, these compounds
displayed low hemolytic activities. Besides, based on the data above, it showed that
the compounds with potent antibacterial inhibitory activity were low toxic.
Table 5. Human kidney epithelial cell cytotoxicity (CC50, µM) and hemolytic
activities (LC30, mg/mL) of tested compounds
293T
Hemolysis
293T
Hemolysis
Compounds
Compounds
(aCC50, µM)
203.53±0.22
175.76±0.12
195.47±0.14
175.75±0.31
215.78±0.28
179.62±0.11
188.59±0.18
147.61±0.31
178.43±0.43
178.89±0.15
168.37±0.25
bLC30 (mg/mL)
(aCC50, µM)
180.91±0.23
189.36±0.26
155.45±0.25
145.42±0.16
215.54±0.23
119.44±0.25
136.51±0.14
158.22±0.15
123.46±0.14
153.23±0.26
150.59±0.24
bLC30 (mg/mL)
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
13
14
15
16
17
18
19
20
21
22
23
27
28
29
30
31
32
33
34
35
36
37
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179.34±0.31
154.46±0.22
168.62±0.44
>10
>10
>10
134.56±0.35
153.64±0.16
201.23±0.12
>10
>10
>10
24
25
26
38
39
DDCP
a,b Values are the average of three independent experiments run in triplicate.
2.4. ADMET model
For in silico study, the ADMET (Absorption, Distribution, Metabolism,
Excretion, Toxicity) simulation [28-30] for 13-39 was initially depicted in Fig. 5 The
parameters of AlogP (the partition coefficient of drug in octanol/aqueous solution
calculated by ACD/PhysChem Suite Software) and PSA_2D (the fast calculated polar
surface area from the 2D structure) were used to predict the Absorption (human
intestinal absorption) and BBB (blood-brain barrier penetration) including 95% and
99% confidence ellipses as referenced [30]. All the compounds suggested good
potential in druggability, which are potential for oral administration with low side
effects.
Fig. 5. ADMET properties predicted for compounds 13-29. Compounds located inside
the innermost oval are better for this parameter. Only 32, 35 and 38 were outside the
innermost oval. The percentages in the figure legend meant the 95% and 99%
confidence ellipses.
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2.5. Molecular docking
Docking is an effective and reliable approach to simulate the probable binding
mode of ligands and proteins. To visualize the possible binding model of interactions
between a protein (enzyme) and small molecules (ligands) were the molecular
docking techniques used [31]. Molecular modelling in this study was conducted by
using CDOCKER protocol in Discovery Studio 3.5 (Discovery Studio 3.5, Accelrys,
Inc. San Diego, CA). All twenty-seven compounds were docked into the active site of
the receptor FabH (PDB code: 1HNJ), and effective docking scores of all the
compounds have been recorded. As depicted in Fig. 6, the CDOCKER Interaction
Energy (interaction energy between the ligand and the receptor) was coincident with
the E.coli FabH enzyme inhibitory activity trend for all compounds, and compound
31 showed the lowest energy value (-54.46 kcal/mol), which represented the most
potent binding affinity. This result hinted the consistency of the SAR and molecular
docking. Furthermore, the interacting model between protein receptor 1HNJ and the
most potent compound 31 was investigated. 2D and 3D maps of them were depicted
in Fig. 7, showing both the detailed surrounding situation and the laconic binding site.
Seen in Fig. 7, 31 formed three hydrogen bonds with ARG36 (O…H-N: 3.37 Å),
ARG36 (O…H-N: 3.04 Å) and ASN210 (O…H-N: 3.22 Å), indicating benzimidazole
amide was favorable for the activity increase. Amide-ꢀ interaction between pyrazole
and GLY209 (distance: 4.91 Å), ꢀ –ꢀ interaction between benzene ring neighboring
to pyrazole and PHE213 (distance: 5.11 Å) and ꢀ –cation interaction between the
same benzene ring and ARG249 (distance: 4.12 Å) maintained the basic binding
pattern of this series. Besides, the high affinity of compound 31 with 1HNJ may also
be caused by some weak interactions, such as Van der Waals’ force.
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Fig. 6. The histogram about CDOCKER_INTERACTION_ENERGY (-kcal/mol) of
compounds (13 - 39 and DDCP) for FabH, in the binding mode, compound 31 had a
best estimated binding free energy of -54.46 kcal/mol.
Fig. 7. Docking models of representative compound 31. (A) 2D molecular docking
modeling of compound 31 and surrounding residues of E. coli FabH (PDB code:
1HNJ). (B) 3D molecular docking modeling of compound 31 with E. coli FabH.
2.6 Lipinski's rule-of-five
In addition, this class of compounds were evaluated compliance with Lipinski's
rule-of-five (Molecular weight (MW)≤500D，Number of hydrogen bond donors
(HBD)≤5, Number of hydrogen bond acceptors (HBA)≤10, -2≤calculated lipophilicity
(logP)≤5, Number of rotatable bonds (NRB)≤10) for the preliminary screening. The
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indicator results were depicted in Table 6. According to this analysis, one can conclude
that this series of compounds showed excellent pharmacokinetic properties, and could
be used to improve novel attractive compounds.
Table 6 Calculated percentiles of main descriptors for these compounds.
Descriptor
Data
MW
NRB
logP
HBA
1-5
HBD
0-6
454.53-542.95 6-10
4.2-7.8
3. Conclusion
Overall, we have designed and synthesized twenty-seven new FabH inhibitors.
They exhibited excellent activity against both Gram-positive and Gram-negative
bacteria, as well as FabH inhibitory activity. The cytotoxicity test employing human
kidney epithelial cell 293T and hemolysis test indicated high safety. Of all these
compounds, compound 31 showed the most potent inhibition activity against four
bacteria strains (with MIC of 0.98, 0.49, 0.98, 0.98 µg/mL, respectively against E.
coli, P. aeruginosa, B. subtilis and S. aureus) and FabH (with IC50 of 1.22 µM). FabH
mutant Xanthomonas Campestris experiment validated that compounds binding site
outcomes FabH. Additionally, in silico ADMET evaluation also pointed out that these
compounds are potential for oral administration with low side effects. Furthermore,
the probable binding mode proposed by the docking simulation may be a good
explanation of the impressive performance of 31, in which 31 binds well with FabH
via three hydrogen bonds, an amide-ꢀ interaction, a ꢀ –ꢀ interaction and a ꢀ –cation
interaction.
4. Experimental section
4.1. Materials and measurements
All chemicals and reagents used in current study were analytical grade. Thin
layer chromatography (TLC), proton nuclear magnetic resnance (1H NMR) and
elemental microanalyses (CHN) were usually used. Analytical thin-layer
chromatography (TLC) was performed on the glass-backed silica gel sheets (silica gel
60 Å GF254). All compounds were detected using UV light (254 nm or 365 nm).
Separation of the compounds by column chromatography was carried out with silica
17

ACCEPTED MANUSCRIPT
gel 60 (200 – 300 mesh ASTM, E. Merck). The quantity of silica gel used was 50-100
times the weight charged on the column. Melting points were determined on a XT4
1
MP apparatus (Taike Corp., Beijing, China). H NMR spectra were measured on a
Bruker DPX 400 spectrometer at 25 ℃ and referenced to Me4Si. Chemical shifts are
reported in ppm (δ) using the residual solvent line as internal standard. Splitting
patterns are designed as s, singlet; d, doublet; t, triplet; m, multiplet. ESI-MS spectra
were recorded on a Mariner System 5304 Mass spectrometer. Elemental analyses
were performed on a CHN-O-Rapid instrument and were within ± 0.4% of the
theoretical values.
4.2. General procedure for preparation of 1-phenyl-2-(1-(p-tolyl)ethylidene)
hydrazine (4) [32, 33]
To prepared compound 4, p-methylacetophenone (compound 1, 6.71 g, 50 mmol)
was reacted with phenylhydrazine hydrochloride (8.68 g, 60 mmol) at the presence of
sodium acetate (10.21 g, 75 mmol) in water (120 mL). After stirring at room
temperature for 3.5 h, a large amount of yellow solid emerged and was filtered to give
compound
4
(yields
95
%).
The
corresponding
substituted
1-phenyl-2-(1-phenylethylidene) hydrazines followed this way to prepare pure
compounds 5 and 6 with yields of 91% and 96%.
4.3.
General
procedure
for
preparation
of
1-phenyl-3-(p-tolyl)-1H-pyrazole-4-carbaldehyde (7) [32, 33]
Compound 4 (8.97 g, 40 mmol) was treated with Vilsmeier-Haack reagent
(DMF-POCl3, 30 mL DMF and 10 mL POCl3). After stirring at 55 °C for 6 h, the
mixture was poured into ice-cold water, with a saturated solution of sodium hydroxide
being added to neutralize the mixture. Then a large amount of solid emerged, which
was filtered, washed with water, and dried to give compound 7 (yields 91 %). The
corresponding
substituted
1,3-diphenyl-1H-pyrazole-4-carbaldehydes
were
synthesized following this way to receive pure compounds 8 and 9 with a yield of
88% and 93%.
4.4.
General
procedure
for
preparation
of
2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (10) [34]
O-phenylenediamine (3.24 g, 30 mmol) was coupled with compound 7 (10.51 g,
30 mmol) in DMF (70 mL) at the presence of sodium pyrosulfite (11.41 g, 60 mmol).
The heterogeneous mixture was stirred at 110 ℃ for 4 h, then cooled and poured onto
18

ACCEPTED MANUSCRIPT
a lot of ice. A large amount of solid would emerge, and was filtered to give
2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazole (10),
powder, yielding 83%. The corresponding
a
yellow
substituted
2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazoles were synthesized following
this way, and we got pure compounds 11 and 12 with a yield of 82% and 85%.
4.5.
General
synthesis
method
of
(2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)(phenyl)methanone
(13)
To obtain compound 13, the intermediate product 10 (0.35 g, 1mmol) coupled
with benzoic acid (0.18 g, 1.5 mmol) in 30 mL dichloromethane at room temperature
for 4.5 h, with the help of triethylamine (2 - 3 drops), EDC·HCl (0.25 g, 1.3mmol)
and DMAP (0.16 g, 1.3mmol). The reaction mixture was then extracted with
dichloromethane (15 mL × 3) and distilled water (15 mL). After the solvent was
evaporated under reduced pressure, products were purified by column
chromatography on silica gel (200 - 300 mesh) using ethyl acetate / petroleum ether
(v/v = 1:6) as eluent to yield pure compound 13 (yields 68 %). The corresponding
substituted
(2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)(phenyl)methanone
compounds,
(2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)(pyridin-3-yl)methanon
e
compounds
and
(2-(1,3-diphenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)(pyridin-4-yl)methanon
e compounds were prepared following this way, with yields of 56 - 82% for pure
compounds 14 – 39.
4.5.1. phenyl(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)
methanone (13)
1
White powder, yield: 87%. M. p: 146 - 147 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.27 (s, 3H); 7.04 (d, J = 8, 2H); 7.25 - 7.31 (m, 4H); 7.36 - 7.40 (m, 4H); 7.47 - 7.50
(m, 3H); 7.52 - 7.55 (m, 2H); 7.79 (d, J = 5.2, 2H); 7.86 (d, J = 5.2, 1H); 8.84 (s, 1H).
ESI-MS: 454.53 (C30H22N4O, [M+H]+).
4.5.2. (2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)
(o-tolyl)methanone (14)
19

ACCEPTED MANUSCRIPT
1
White powder, yield: 69%. M. p: 165 - 167 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.34 (s, 3H); 2.48 (s, 3H); 7.05 (s, 2H); 7.21 - 7.25 (m, 3H); 7.28 (s, 2H); 7.37 - 7.42
(m, 3H); 7.52 - 7.57 (m, 4H); 7.68 (s, 2H); 7.80 (s, 1H); 8.44 (s, 1H). ESI-MS: 468.56
(C31H24N4O, [M+H]+).
4.5.3. (2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)
(m-tolyl)methanone (15)
1
Yellow powder, yield: 87%. M. p: 183 - 184 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.34 (s, 3H); 2.37 (s, 3H); 6.66 - 6.68 (m, 1H); 6.76 (d, J = 5.2, 1H); 7.07 - 7.09 (m,
1H); 7.21 (d, J = 4.8, 1H); 7.26 (d, J = 5.2, 2H); 7.29 - 7.31 (m, 1H); 7.38 (d, J = 3.2,
2H); 7.49 - 7.51 (m, 2H); 7.70 (d, J = 5.6, 2H); 7.76 (d, J = 12, 2H); 7.82 (d, J = 5.2,
2H); 8.51 (s, 1H). ESI-MS: 468.56 (C31H24N4O, M+H)+).
4.5.4. (2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)
(p-tolyl)methanone (16)
1
White powder, yield: 60%. M. p: 159 - 161 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.24 (d, J = 2.8, 6H); 7.11 (d, J = 5.2, 4H); 7.28 - 7.31 (m, 4H); 7.34 - 7.53 (m, 4H);
7.54 - 7.55 (m, 2H); 7.80 - 7.85 (m, 3H); 8.85 (s, 1H). ESI-MS: 468.56 (C31H24N4O,
[M+H]+). Anal.
4.5.5. (4-chlorophenyl)(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]
imidazol-1-yl)methanone (17)
1
Yellow powder, yield: 65%. M. p: 181 - 182 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.21 (s, 3H); 7.08 (d, J = 5.6, 2H); 7.12 - 7.14 (m, 2H); 7.32 (s, 3H); 7.36 - 7.39 (m,
2H); 7.41 - 7.46 (m, 2H); 7.51 - 7.66 (m, 3H); 7.75 (d, J = 5.2, 2H); 7.83 (d, J = 5.6,
1H); 8.74 (s, 1H). ESI-MS: 488.98 (C30H21ClN4O, [M+H]+).
4.5.6. (2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-
yl)(phenyl)methanone (18)
1
White powder, yield: 66%. M. p: 172 - 174 ℃, H NMR (DMSO-d6, 400 MHz) δ:
7.18 - 7.21 (m, 2H); 7.29 - 7.31 (m, 2H); 7.37 - 7.39 (m, 2H); 7.42 - 7.55 (m, 8H);
7.80 (d, J = 5.2, 2H); 7.86 (d, J = 5.2, 2H); 8.87 (s, 1H). ESI-MS: 458.50
(C29H19FN4O, [M+H]+). Anal.
4.5.7. (2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-
yl)(o-tolyl)methanone (19)
1
Yellow powder, yield: 66%. M. p: 156 - 157 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.02 (s, 3H); 7.09 - 7.12 (m, 2H); 7.22 - 7.28 (m, 3H); 7.37 - 7.40 (m, 2H); 7.43 - 7.47
20

ACCEPTED MANUSCRIPT
(m, 2H); 7.54 - 7.59 (m, 4H); 7.65 (d, J = 5.2, 1H); 7.77 (d, J = 5.2, 2H); 7.88 (d, J =
4.8, 1H); 8.81 (s, 1H). ESI-MS: 472.52 (C30H21FN4O, M+H)+).
4.5.8. (2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-
yl)(m-tolyl)methanone (20)
1
Yellow powder, yield: 67%. M. p: 214 - 215 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.09 (s, 3H); 7.16 - 7.24 (m, 5H); 7.28 (d, J = 4.8, 1H); 7.37 - 7.40 (m, 2H); 7.43 -
7.44 (m, 1H); 7.47 - 7.50 (m, 2H); 7.53 - 7.55 (m, 3H); 7.78 (d, J = 5.2, 2H); 7.86 (d,
J = 5.2, 1H); 8.85 (s, 1H). ESI-MS: 472.52 (C30H21FN4O, M+H)+).
4.5.9. (2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-
yl)(p-tolyl)methanone (21)
1
White powder, yield: 70%. M. p: 187 - 189 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.25 (s, 3H); 7.13 - 7.19 (m, 4H); 7.34 - 7.43 (m, 6H); 7.47 - 7.50 (m, 2H); 7.53 - 7.56
(m, 2H); 7.81 - 7.86 (m, 3H); 8.87 (s, 1H). ESI-MS: 472.52 (C30H21FN4O, M+H)+).
4.5.10. (4-chlorophenyl)(2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-
benzo[d]imidazol-1-yl)methanone (22)
1
Yellow powder, yield: 67%. M. p: 177 - 178 ℃, H NMR (DMSO-d6, 400 MHz) δ:
7.17 - 7.20 (m, 2H); 7.36 - 7.41 (m, 4H); 7.46 - 7.50 (m, 5H); 7.52 - 7.57 (m, 3H);
7.80 - 7.82 (m, 3H); 7.86 (d, J = 5.2, 1H). ESI-MS: 492.94 (C29H18ClFN4O, M+H)+).
4.5.11. phenyl(2-(1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-
benzo[d]imidazol-1-yl)methanone (23)
1
Yellow powder, yield: 66%. M. p: 192 - 194 ℃, H NMR (DMSO-d6, 400 MHz) δ:
7.29 - 7.32 (m, 2H); 7.36 - 7.45 (m, 4H); 7.49 - 7.52 (m, 3H); 7.54 - 7.57 (m, 2H);
7.71 - 7.75 (m, 4H); 7.82 - 7.87 (m, 3H); 8.94 (s, 1H). ESI-MS: 508.50 (C30H19F3N4O,
[M+H]+).
4.5.12. (2-(1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-benzo[d]
imidazol-1-yl)(o-tolyl)methanone (24)
1
Yellow powder, yield: 56%. M. p: 171 - 172 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.04 (s, 3H); 6.92 - 6.95 (m, 1H); 7.11 - 7.15 (m, 2H); 7.26 - 7.29 (m, 1H); 7.40 - 7.49
(m, 3H); 7.56 - 7.61 (m, 3H); 7.78 - 7.81 (m, 6H); 7.88 (d, J = 5.2, 1H); 8.88 (s, 1H).
ESI-MS: 522.53 (C31H21F3N4O, M+H)+).
4.5.13. (2-(1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-benzo[d]
imidazol-1-yl)(m-tolyl)methanone (25)
21

ACCEPTED MANUSCRIPT
1
White powder, yield: 66%. M. p: 199 - 201 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.08 (s, 3H); 7.16 - 7.19 (m, 1H); 7.25 - 7.30 (m, 3H); 7.38 - 7.46 (m, 3H); 7.51 - 7.58
(m, 3H); 7.74 (d, J = 1.6, 4H); 7.82 - 7.83 (m, 2H); 7.87 (d, J = 5.2, 1H); 8.94 (s, 1H).
ESI-MS: 522.53 (C31H21F3N4O, M+H)+).
4.5.14. (2-(1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-benzo
[d]imidazol-1-yl)(p-tolyl)methanone (26)
1
Yellow powder, yield: 76%. M. p: 188 - 189 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.25 (s, 3H); 7.14 (d, J = 5.2, 2H); 7.30 - 7.36 (m, 2H); 7.39 - 7.43 (m, 4H); 7.55 -
7.58 (m, 2H); 7.69 (d, J = 1.6, 4H); 7.86 (d, J = 5.6, 3H); 8.96 (s, 1H). ESI-MS:
522.53 (C31H21F3N4O, M+H)+).
4.5.15. (4-chlorophenyl)(2-(1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-
yl)-1H-benzo[d]imidazol-1-yl)methanone (27)
1
White powder, yield: 68%. M. p: 211 - 212 ℃, H NMR (DMSO-d6, 400 MHz) δ:
7.38 - 7.46 (m, 6H); 7.51 - 7.53 (m, 2H); 7.56 - 7.58 (m, 2H); 7.68 - 7.72 (m, 4H);
7.84 - 7.87 (m, 3H); 8.96 (d, J = 7.6, 1H). ESI-MS: 542.95 (C30H18ClF3N4O, M+H)+).
4.5.16. (2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)
(pyridin-3-yl)methanone (28)
1
Yellow powder, yield: 60%. M. p: 191 - 192 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.28 (s, 3H); 7.15 (d, J = 5.2, 2H); 7.24 - 7.27 (m, 3H); 7.37 - 7.39 (m, 1H); 7.41 -
7.48 (m, 2H); 7.53 - 7.55 (m, 2H); 7.70 - 7.75 (m, 2H); 7.78 (d, J = 5.2, 2H); 7.88 (d,
J = 5.2, 1H); 8.49 (s, 1H); 8.57 (d, J = 3.2, 1H); 8.87 (s, 1H). ESI-MS: 455.52
(C29H21N5O, [M+H]+).
4.5.17. (5-methylpyridin-3-yl)(2-(1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-
pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)methanone (29)
1
White powder, yield: 61%. M. p: 173 - 174 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.29 (s, 3H); 7.15 - 7.16 (m, 2H); 7.22 - 7.24 (m, 2H); 7.37 - 7.40 (m, 1H); 7.43 - 7.47
(m, 2H); 7.48 - 7.50 (m, 1H); 7.53 - 7.56 (m, 2H); 7.75 - 7.79 (m, 3H); 7.87 - 7.88 (m,
13
1H); 8.26 (d, J = 1.2, 1H); 8.39 (d, J = 0.8, 1H); 8.87 (s, 1H). C NMR (DMSO-d6,
100 MHz) δ: 17.72, 21.28, 112.35, 114.69, 119.10, 120.26, 125.34, 125.59, 127.49,
127.62, 128.51, 128.54, 129.03, 129.31, 129.72, 130.12, 130.22, 132.08, 132.47,
134.23, 137.80, 138.53, 139.25, 143.08, 146.58, 147.73, 150.71, 153.71, 167.21.
ESI-MS: 523.52 (C30H20F3N5O, [M+H]+).
4.5.18. (6-methylpyridin-3-yl)(2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo
22

ACCEPTED MANUSCRIPT
[d]imidazol-1-yl)methanone (30)
1
Yellow powder, yield: 71%. M. p: 189 - 191 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.25 (s, 3H); 2.36 (s, 3H); 7.11 - 7.14 (m, 3H); 7.22 (d, J = 5.2, 2H); 7.37 - 7.41 (m,
2H); 7.44 - 7.46 (m, 1H); 7.53 - 7.56 (m, 2H); 7.60 - 7.62 (m, 2H); 7.79 (d, J = 5.2,
13
2H); 7.86 (d, J = 5.2, 1H); 8.38 (d, J = 1.6, 1H); 8.85 (s, 1H). C NMR (DMSO-d6,
100 MHz) δ: 31.21, 33.43, 112.31, 114.27, 119.32, 120.41, 122.52, 125.21, 125.37,
127.42, 127.71, 129.13, 129.37, 129.72, 132.06, 134.28, 137.90, 138.12, 139.39,
143.41, 146.85, 150.22, 151.15. ESI-MS: 469.55 (C30H23N5O, [M+H]+).
4.5.19. (2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-
yl)(pyridin-3-yl)methanone (31)
1
Yellow powder, yield: 72%. M. p: 169 - 170 ℃, H NMR (DMSO-d6, 400 MHz) δ:
7.20 - 7.22 (m, 2H); 7.28 - 7.30 (m, 1H); 7.38 - 7.40 (m, 1H); 7.43 - 7.49 (m, 4H);
7.53 - 7.56 (m, 2H); 7.72 (d, J = 5.2, 1H); 7.78 (d, J = 5.6, 2H); 7.82 (d, J = 5.2, 1H);
7.88 (d, J = 5.2, 1H); 8.56 - 8.58 (m, 1H); 8.58 - 8.59 (m, 1H); 8.88 (s, 1H). ESI-MS:
459.48 (C28H18FN5O, [M+H]+).
4.5.20. (2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-
yl)(5-methylpyridin-3-yl)methanone (32)
1
White powder, yield: 70%. M. p: 157 - 158 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.05 (s, 3H); 7.20 - 7.23 (m, 2H); 7.38 - 7.41 (m, 1H); 7.43 - 7.49 (m, 4H); 7.54 - 7.57
(m, 3H); 7.76 - 7.79 (m, 3H); 7.88 (d, J = 5.2, 1H); 8.33 - 8.40 (m, 2H); 8.89 (s, 1H).
ESI-MS: 473.51 (C29H20FN5O, [M+H]+).
4.5.21. (2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-
yl)(6-methylpyridin-3-yl)methanone (33)
1
Yellow powder, yield: 74%. M. p: 149 - 150 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.37 (s, 3H); 7.15 - 7.20 (m, 3H); 7.38 - 7.43 (m, 2H); 7.44 - 7.47 (m, 3H); 7.54 - 7.56
(m, 2H); 7.62 (d, J = 5.2, 1H); 7.68 - 7.70 (m, 1H); 7.79 (d, J = 5.2, 2H); 7.87 (d, J =
5.2, 1H); 8.45 (s, 1H); 8.87 (s, 1H). ESI-MS: 473.51 (C29H20FN5O, [M+H]+).
4.5.22. (trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-
yl)(pyridin-3-yl)methanone (34)
1
White powder, yield: 56%. M. p: 182 - 184 ℃, H NMR (DMSO-d6, 400 MHz) δ:
7.31 (s, 1H); 7.41 -7.48 (m, 3H); 7.55 (d, J = 6.8; 2H); 7.67 - 7.88 (m, 9H); 8.61 (d, J
= 8, 2H); 8.95 (s, 1H). ESI-MS: 509.49 (C29H18F3N5O, [M+H]+).
4.5.23. (5-methylpyridin-3-yl)(2-(1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-
23

ACCEPTED MANUSCRIPT
pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)methanone (35)
1
Yellow powder, yield: 65%. M. p: 193 - 195 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.05 (s, 3H); 7.42 - 7.48 (m, 3H); 7.55 - 7.59 (m, 2H); 7.64 (s, 1H); 7.74 (s, 5H)；7.81
(d, J = 8, 2H); 7.88 - 7.90 (m, 1H); 8.38 - 8.42 (m, 2H); 8.95 (s, 1H). ESI-MS:
523.52 (C30H20F3N5O, [M+H]+).
4.5.24. (6-methylpyridin-3-yl)(2-(1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-
pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)methanone (36)
1
Yellow powder, yield: 75%. M. p: 188 - 190 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.38 (s, 3H); 7.17 (d, J = 8, 1H); 7.40 - 7.46 (m, 3H); 7.54 - 7.59 (m, 3H); 7.69 (s, 4H);
7.72 - 7.75 (m, 1H); 7.82 - 7.88 (m, 3H); 8.51 (s, 1H); 8.96 (s, 1H). ESI-MS: 523.52
(C30H20F3N5O, [M+H]+).
4.5.25. (2-(3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)-1H-benzo[d]imidazole
-1-yl)(pyridin-4-yl)methanone (37)
1
White powder, yield: 76%. M. p: 221 - 222 ℃, H NMR (DMSO-d6, 400 MHz) δ:
7.21 - 7.31(m, 4H); 7.42 - 7.57 (m, 5H); 7.68 (s, 2H); 7.86 - 7.92 (m, 4H); 8.44 (s,
1H); 8.87 (s, 2H). ESI-MS: 459.48 (C28H18FN5O, [M+H]+).
4.5.26. (2-(1-phenyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)-1H-benzo[d]
imidazol-1-yl)(pyridin-4-yl)methanone (38)
1
Yellow powder, yield: 65%. M. p: 149 - 150 ℃, H NMR (DMSO-d6, 400 MHz) δ:
7.20 - 7.23 (m, 3H); 7.43 - 7.45 (m, 1H); 7.53 (d, J = 4.8, 1H); 7.60 - 7.65 (m, 4H);
7.83 (d, J = 5.6, 3H); 7.98 - 8.00 (m, 2H); 8.19 (d, J = 5.6, 2H); 9.16 (s, 1H); 12.62 (s,
1H). ESI-MS: 509.49 (C29H18F3N5O, [M+H]+).
4.5.27. (2-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)
(pyridin-4-yl)methanone (39)
1
Yellow powder, yield: 71%. M. p: 186 - 187 ℃, H NMR (DMSO-d6, 400 MHz) δ:
2.35 (s, 3H); 7.29 - 7.31 (m, 2H); 7.37 - 7.39 (m, 3H); 7.42 - 7.55 (m, 9H); 7.80 (d, J
= 5.2, 2H); 7.86 (d, J = 5.2, 1H); 8.87 (s, 1H). ESI-MS: 455.52 (C29H21N5O, [M+H]+).
4.6. Minimum inhibitory concentration (MIC) assay using TTC double dilution
method
The MTT is defined as the lowest concentration of the substance that prevents
visible bacterial growth. Two Gram-negative bacterial strains: E. coli ATCC 25922
and P. aeruginosa ATCC 27853 and two Gram-positive bacterial strains: B. subtilis
ATCC 530 and S. aureus ATCC 25923 were employed in the antibacterial activities
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test, using the method recommended by National Committee for Clinical Laboratory
Standards (NCCLS). By TTC double dilution method, the in vitro activities of the
compounds were tested in nutrient broth (NB) for bacteria. Seeded broth (broth
containing microbial spores) was prepared in NB from 24 h old bacterial cultures on
nutrient agar (Hi-media) at 37 C. The bacterial suspension was adjusted with sterile
saline to a concentration of 1×104 to 1×105 CFU/mL. The tested compounds and
reference drugs were prepared by two- fold serial dilution to obtain the required
concentrations of 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 µg/mL. The tubes
were incubated in BOD incubators at 37 ℃ for bacteria. The MICs were recorded by
visual observations after 24 h (for bacteria) of incubation. Antibiotic DDCP and
kanamycin B were used as standards for bacterial. The observed MICs are presented
in Table 3.
4.7. FabH inhibition activity
The potency of FabH inhibition (IC50) was determined using [3H]- or
[14C]-radiolabeled substrates (IC50 values correspond to the concentration at which
half of the enzyme activity is inhibited by the compound). This was accomplished at
fixed concentrations of acetyl-CoA in a coupled FabD/FabH assay system that
generates the substrate malonyl-ACP in situ. Subsequently, the FabH acetylation
reaction was performed to exclude the possible contribution of undesired FabD
inhibitory activity. Compounds were included in cocrystallization trials with
Entercoccus faecalis and Haemophilus in- fluenzae FabH proteins and the derived
protein-inhibitor cocrystal structures were used to further optimize the inhibitors. The
FabH inhibition activity results are presented in Table 3.
4.8. Cytotoxicity test and Hemolysis test
The cytotoxic activity in vitro was measured against mammalian cells, human
macrophage using the MTT assay. The cell was grown in DMEM medium
supplemented with 10% PBS and 1× antimycotic and antibacterial solution (sigma
USA) at 37 °C, in humidified atmosphere having 5% CO2. 100 µL of the confluent
fibroblast stock suspension was dispensed in 96-well tissue culture plate. The original
medium from the wells was replaced with 100 µL serum free DMEM when the cells
reached 90% confluence after 5 h incubation in a CO2 incubator. Various
concentrations of the compounds were added to the growing cells and incubated for
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24 h. The absorbance was measured at a wavelength of 570 nm (OD570 nm) on an
ELISA microplate reader. Three replicate wells were used for each concentration and
each assay was measured three times, after which the average of CC50 was calculated.
The cytotoxicity of each compound was expressed as the concentration of compound
that reduced cell viability to 50% (CC50).
Hemolytic activity was assayed using fresh capillary human blood. Erythrocytes
were collected by centrifuging the blood three times in chilled phosphate buffered
saline (PBS at 4 °C) at 1000 × g for 10 min. The final pellet was resuspended in PBS
to give a 2% w/v solution. Using a microtitre plate, 100 mL of the erythrocyte
solution was added to dextran, PLL, stearyl-PLL or stearyl-PLL t LDL
(1-1000/mg/mL) in a volume of 100 mL. Samples were then incubated for 3 h and the
microtitre plate was centrifuged then at 1000 × g for 10 min and the supernatants (100
mL) transferred into a new microtitre plate. Hemoglobin release was determined
spectrophotometrically using a microtitre plate reader (absorbance at 550 nm). Results
were expressed as the amount of released hemoglobin induced by the compounds as a
percentage of the total.
4.9. Protocol of ADMET study
The three-dimensional structures of the aforementioned compounds were
constructed using Chem. 3D ultra 15.0 software [Chemical Structure Drawing
Standard; Cambridge Soft corporation, USA (2016)]. Then they were minimized a
CHARMm based force field the same as in docking study. The ADMET study was
conducted using the Calculate Molecular Properties in Small Molecules module of the
Discovery Studio (version 3.5). The ADMET properties map and data were provided
by the ADMET Descriptors tool.
4.8 Experimental protocol for docking study
Molecular docking was carried out using the Discovery Studio (version 3.5) as
implemented through the graphical user interface DS-CDOCKER protocol. The
three-dimensional structures of the aforementioned compounds were constructed
using Chem. 3D ultra 15.0 software [Chemical Structure Drawing Standard;
Cambridge Soft corporation, USA (2016)], then they were energetically minimized by
using MMFF94 with 5000 iterations and minimum RMS gradient of 0.10. The crystal
structures of protein complex were retrieved from the RCSB Protein Data Bank
(http:// www.rcsb.org/pdb/home/home.do). All bound water and ligands were
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eliminated from the protein. The molecular docking in this study was performed by
inserting compound 31 into the binding pocket of E. coli FabH (PDB code: 1HNJ)
based on the binding mode. Types of interactions of the docked protein with
ligand-based pharmacophore model were analyzed after the end of molecular
docking.
Acknowledgments
This work was supported by National Key R&D Program of China
(SQ2017YFNC060022-05, 2017YFC0506005) and China Postdoctoral Science
Foundation funded project (No. 2018M641927).
References
1.
2.
3.
4.
Otvos JL, Wade JD, Lin F et al. Designer antibacterial peptides kill
fluoroquinolone-resistant clinical isolates. Journal of Medicinal Chemistry
2005; 48: 5349-5359.
Li ZL, Li QS, Zhang HJ et al. Design, synthesis and biological evaluation of
urea derivatives from -hydroxybenzylamines and phenylisocyanate as
potential FabH inhibitors. Bioorg Med Chem 2011; 19: 4413-4420.
Cui Y, Dang YY, Zhang S, Ji R. Syntheses and antibacterial activity of a
series of 3-(pyridine-3-yl)-2-oxazolidinone. European Journal of Medicinal
Chemistry 2005; 40: 209-214.
Lee JY, Jeong KW, Shin S et al. Antimicrobial natural products as
beta-ketoacyl-acyl carrier protein synthase III inhibitors. Bioorganic &
Medicinal Chemistry 2009; 17: 5408-5413.
5.
6.
HJ Z, ZL L, HL Z. Advances in the research of β-ketoacyl-ACP synthase III
(FabH) inhibitors. Current Medicinal Chemistry 2009; 19: 1225-1237.
Clough RC, Matthis AL, Barnum SR, Jaworski JG. Purification and
characterization of 3-ketoacyl-acyl carrier protein synthase III from spinach. A
condensing enzyme utilizing acetyl-coenzyme A to initiate fatty acid synthesis.
Journal of Biological Chemistry 1992; 267: 20992-20998.
27

ACCEPTED MANUSCRIPT
7.
Khandekar SS, Daines RA, Lonsdale JT. Bacterial beta-ketoacyl-acyl carrier
protein synthases as targets for antibacterial agents. Current Protein & Peptide
Science 2003; 4: 21-29.
8.
9.
Heath RJ, Rock CO. The Claisen Condensation in Biology. Natural Product
Reports 2002; 19: 581-596.
Heath RJ, Rock CO. Inhibition of beta-ketoacyl-acyl carrier protein synthase
III (FabH) by acyl-acyl carrier protein in Escherichia coli. Journal of
Biological Chemistry 1996; 271: 10996-11000.
10.
Shi L, Fang RQ, Zhu ZW et al. Design and synthesis of potent inhibitors of β
-ketoacyl-acyl carrier protein synthase III (FabH) as potential antibacterial
agents. European Journal of Medicinal Chemistry 2010; 45: 4358-4364.
Jun W, Stephen M S, Katherine Y et al. Platensimycin is a selective FabF
inhibitor with potent antibiotic properties. Nature 2006; 441: 358-361.
Young K, Jayasuriya H, Ondeyka JG et al. Discovery of FabH/FabF inhibitors
from natural products. Antimicrob Agents Chemother 2006; 50: 519-526.
Zhang C, Ondeyka J, Herath K et al. Platensimycin and platencin congeners
from Streptomyces platensis. Journal of Natural Products 2011; 74: 329-340.
Allahverdiyev AM, Bagirova M, Abamor ES et al. The use of platensimycin
and platencin to fight antibiotic resistance. Infection & Drug Resistance 2013;
6: 99-114.
11.
12.
13.
14.
15.
16.
Evan M, Demain AL. Platensimycin and platencin: promising antibiotics for
future application in human medicine. Journal of Antibiotics 2011; 64:
705-710.
Musayev F, Sachdeva S, Scarsdale JN et al. Crystal structure of a substrate
complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein
synthase III (FabH) with lauroyl-coenzyme A. Journal of Molecular Biology
2005; 346: 1313-1321.
17.
18.
Jones PB, Parrish NM, Houston TA et al. A new class of antituberculosis
agents. Journal of Medicinal Chemistry 2000; 43: 3304-3314.
Mckinney DC, Eyermann CJ, Gu RF et al. Antibacterial FabH Inhibitors with
Mode of Action Validated in Haemophilus influenzae by in Vitro Resistance
Mutation Mapping. Acs Infectious Diseases 2016; 2: 456-464.
28

ACCEPTED MANUSCRIPT
19.
20.
Li P. Drug design and the mechanism of ꢁ-ketoacyl-acyl carrier protein
synthase III (FabH) inhibitors; 2017.
Choudhari PB, Subramani PP, Khare SV et al. Investigation on quantitative
structure activity relationships of benzoylamino benzoic acid derivatives as
β-ketoacyl-acyl carrier protein synthase III (FabH) Inhibitors. Marmara
Pharmaceutical Journal 2017; 21: 631-643.
21.
Ranjith PK, Rajeesh P, Haridas KR et al. Design and synthesis of positional
isomers of 5 and 6-bromo-1-[(phenyl)sulfonyl]-2-[(4-nitrophenoxy)methyl]-
1H -benzimidazoles as possible antimicrobial and antitubercular agents.
Bioorganic & Medicinal Chemistry Letters 2013; 23: 5228-5234.
Zhou B, Li B, Yi W et al. Synthesis, antioxidant, and antimicrobial evaluation
of some 2-arylbenzimidazole derivatives. Bioorganic & Medicinal Chemistry
Letters 2013; 23: 3759-3763.
22.
23.
24.
Elie CR, David G, Schmitzer AR. Strong antibacterial properties of anion
transporters: a result of depolarization and weakening of the bacterial
membrane. Journal of Medicinal Chemistry 2015; 58: 2358-2366.
Braccio MD, Grossi G, Signorello MG et al. Synthesis, invitro antiplatelet
activity
and
molecular
modelling
studies
of
10-substituted
2-(1-piperazinyl)pyrimido[1,2- a ]benzimidazol-4(10 H )-ones. European
Journal of Medicinal Chemistry 2013; 62: 564-578.
25.
Xin-Mei P, Gui-Xin C, Cheng-He Z. Recent developments in azole
compounds as antibacterial and antifungal agents. Current Topics in
Medicinal Chemistry 2013; 13: 1963-2010.
26.
27.
Zhou C, Kegui YU, Meng J et al. Dibenzimidazole and onium compound
thereof, preparation method and medical use thereof. CN Patent, 2009.
Lan R, Liu Q, Fan P et al. Structure-activity relationships of pyrazole
derivatives as cannabinoid receptor antagonists. Journal of Medicinal
Chemistry 1999; 42: 769-776.
28.
29.
Norinder U, Bergström CAS. Prediction of ADMET Properties.
Chemmedchem 2006; 1: 920-937.
Gleeson MP. Generation of a set of simple, interpretable ADMET rules of
thumb. Journal of Medicinal Chemistry 2008; 51: 817-834.
29