Design, synthesis and molecular docking studies of novel pyrazole benzimidazole derivatives as potent antibacterial agents

Article abstract of DOI:10.14233/ajchem.2019.22137

A novel series of pyrazole benzimidazole derivatives were synthesized and the structure of the final targets 4a-h were confirmed by IR, Mass, ¹³C NMR and ¹H NMR spectral analysis. The new pyrazole core with imidazole and benzimidazol

Full text of DOI:10.14233/ajchem.2019.22137

Asian Journal of Chemistry; Vol. 31, No. 12 (2019), 2733-2739
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2019.22137
Design, Synthesis and Molecular Docking Studies of Novel Pyrazole
Benzimidazole Derivatives as Potent Antibacterial Agents
3
2
1
SRINIVASA RAO DASARI1,2,*, SUBBAIAH TONDEPU , LAKSHMANA RAO VADALI and NARESHVARMA SEELAM
1Department of Chemistry, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur-522502, India
2Mylan Laboratories Ltd, Hyderabad-500049, India
3Department of Chemical Engineering, Vignan’s Foundation for Science, Technology and Research, Vadlamudi, Guntur-522213, India
*Corresponding author: E-mail: dasari43.srinivas@gmail.com
Received: 16 April 2019;
Accepted: 16 June 2019;
Published online: 16 November 2019;
AJC-19612
A novel series of pyrazole benzimidazole derivatives were synthesized and the structure of the final targets 4a-h were confirmed by IR,
Mass, 13C NMR and 1H NMR spectral analysis. The new pyrazole core with imidazole and benzimidazoles derivatives were evaluated for
in vitro antibacterial, antifungal activity against six bacterial strains significantly. In dispersion, 4c, 4f and 4g had the highest antibacterial
activities on these microorganisms Bacillus subtilis B29, Escherichia coli E266, with zone of inhibition 21, 19 and 19 mm, respectively.
Compounds 4a, 4c, 4h shows good antifungal activity against A. niger, Fusarium oxysporum fungal strains. Further, molecular docking
for protein ligands interactions was performed using the crystal structure of C(30) carotenoid dehydrosqualene synthase from Staphylococcus
aureus complexed with bisphosphonate BPH-700. Among the final compounds 4e, 4g and 4h show highest binding energy ∆G = -7.89,
-7.48 and -7.08 Kcal/mol, respectively and amino acid interactions Lys273, Asp27.
Keywords: Imidazole, Pyrazole, Antibacterial activity, Antifungal activity, Molecular modeling, 2ZCS.
as anti-inflammatory and antipyretic. Even though the pyra-
zoles are very much familiar for 80 years, their chemistry explo-
ration is on slow pace. Pyrazole compounds have the potential
biological activities or have shown good pharmacological
effects such as, anti-inflammatory [3], antiviral [4], antimicro-
bial [5], anticonvulsant [6], antitumor [7], fungicidal activities
[8] and antihistaminic [9]. The pyrazole moiety has important
role in some drugs structure, for example, some aryl pyrazole
derivatives have anti-HIV-1 activity [10-12] and some pyrazole-
3-carboxamide moiety has anti-CB1 cannabinoid ability [13].
Moreover, they are used as anti-inflammotary, antitumor,
antipsychotic, antimicrobial and analgesic agents [14]. It is a
fact that pyrazole derivatives are an important class of comp-
ounds for medicines, biological systems, agrochemicals and
many fields of industrial products.
INTRODUCTION
One of the most complex branches of organic chemistry
is the chemistry of heterocyclic composites. It is equally compul-
sive for its “theoretical implications”, “diversified synthetic
procedures” and for the “physiological and industrial impor-
tance” of heterocyclic compounds [1]. Heterocyclic comp-
ounds in particular, will be widely studied for cause of their
intrinsic interest as well as many medicines, natural products,
dyestuffs and drugs belong to their group [2]. Heterocycles
are the cyclic organic compounds with at least one atom other
than the carbon atom in their ring system. More than one third
of the organic compounds are likely to be heterocycles. Majority
of alkaloids, vitamins, synthetic medicines, antibiotics and
substances like nucleic acids which are more closely associated
with the human life evolution belongs to heterocyclic compounds
[1,2].
Benzimidazole derivatives are a blend of many varieties
of pharmacodynamic and pharmacokinetic characteristics.
Benzimidazoles belong to the most bioactive heterocyclic
group that demonstrates a wide range of biological activities
exclusively as a vitamin B12 constituent [15]. The pharmacolo-
Pyrazoles exhibit the typical aromatic system properties,
which are in fact rather prominent in their structures having
insecticide, herbicide properties and pharmaceutical interest
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2734 Dasari et al.
Asian J. Chem.
gical properties of benzimidazoles containing mebendazole,
albendazole and thiabendazole moieties have been well docum-
ented and extensively used as anthelmintic drugs [16]. More-
over, benzimidazole have natural abundance of nucleotide
structural entity and thus it can interacts more easily with bio-
polymers of the living system. This nature leads to its enormous
biological aspects like antihelminthic [17], antifungal [18],
antiallergic [19], antimicrobial [20,21], antiviral [22] and
antineoplastic activities [23]. The inclusion of benzimidazole
nucleus became the vital synthetic strategy for drug discovery,
biological studies and many applications of crop protection
chemistry as the proteinase is being associated to a majority
of disease states like inflammation, thrombosis, tumor growth,
bronchoconstiction and invasion [24].
fungal activity against two microorganisms. For molecular
docking, it was found that the target compounds has potential
to inhibit nitrate reductase protein (PDB code, 2ZCS) which
showed kinetic energies and good affinity by using auto-dock
4.2 software. By comparing the free energies equivalent to
binding of title compounds with target protein, it has been
revealed that most of the compounds interact with receptors.
Almost all molecules are exhibiting lower free energy values,
indicating more thermodynamically favoured interaction.
Therefore, these lead molecules are synthesizing, exhibit better
activity to improve further efficacy and specificity as a potent
antibacterial and antifungal agents.
EXPERIMENTAL
This work deals with the synthesis and characterization
of novel N-alkyl imidazole pyrazole derivatives 4a-h starting
with 2-butyl-4-chloro-1H-imidazole-5-carbaldehyde (1). Ini-
tially n-alkyl-2-butyl-4-chloro-1H-imidazole-5-carbaldehydes
(2a-d) reacts with acetophenone/2-acetyl benzimdazole in
20 % sodium hydroxide solution to give 3a-h. The resulted
compounds cyclized with hydrazine hydrate in methanol to
give the desired products 4a-h showed in Scheme-I. The synthe-
However, pyrazole core with benzimidazoles, imidazole
are rarely found in nature and that makes the pyrazole synthesis
crucial. In this view, we report the synthesis of novel N-alkyl
imidazole pyrazole derivatives (4a-h) incorporated with 2-butyl-
4-chloro-1H-imidazole-5-carbaldehyde and acetophenone/2-
acetyl benzimidazole which obsessed a broad range of biological
activity cheering to antibacterial activity against Gram-positive,
Gram-negative organisms.Additionally, we also report the anti-
Cl
Cl
R1
Cl
N
O
N
N
(i)
(ii)
H3C
O
+
CHO
CHO
H3C
N
H3C
N
H3C
R1
N
R
H
R
1
3a-h
2a-d
Cl
H
N
R = CH3, C2H5, C3H7, C6H5CH2
N
(iii)
N
H3C
N
N
R
R1= Ph,
R1
N
H
4a-h
Cl
H
Cl
Cl
H
N
H
N
N
N
N
N
N
N
N
H3C
H3C
H3C
N
N
N
N
N
N
HN
HN
HN
4a
Cl
4b
Cl
4c
H
N
Cl
N
H
N
H
N
N
N
N
H3C
N
N
N
H3C
H3C
N
N
N
Ph
HN
4f
4e
4d
Cl
Cl
H
N
H
N
N
N
N
N
H3C
H3C
N
N
Ph
4g
4h
Scheme-I: Synthesis of novel pyrazole benzimidazole derivatives 4a-h; Reagent and conditions: (i) PEG, alkylating agent, 50 °C, 15 min, 85
% (ii) acetophenone/2-acetyl benzimdazole, 20 % aq. NaOH, room temperature, 7-9 h, 72-86 % (iii) NH2NH2, acetic acid,
methanol, 74-89 %

Vol. 31, No. 12 (2019)
Design, Synthesis and Molecular Docking Studies of Novel Pyrazole Benzimidazole Derivatives 2735
1
sized compounds structures were confirmed by FT-IR, H
NMR 13C NMR, elemental and mass spectral analysis.
General procedure for the preparation of E-imidazole-
propene-1-one intermediates (3a-h): A solution of 2-butyl-
4-chloro-1H-imidazole-5-carbaldehyde (1) (5 g, 26 mmol), is
added with corresponding 2-acetyl benzimdazole (4.3 g, 26
mmol), acetophenone (3.3 g, 27 mmol) in 20 % sodium hydro-
xide solution (10 mL) and methanol (5 mL) at ambient tempe-
rature and stirred for about 7-9 h. When the reaction comp-
leted as shown by TLC analysis by using EtOAc:hexane as
mobile phase, the reaction mass was poured into ice water
(50 mL) to get the product precipitated out as a solid. It was
then filtered and dried to obtain the corresponding imidazole-
propene-1-one intermediates 3a-h with good yield.
(E)-1-(1H-Benzo[d]imidazol-2-yl)-3-(2-butyl-4-chloro-
1-methyl-1H-imidazol-5-yl)prop-2-en-1-one (3a): Yield 84
%; Off-white solid, m.p. 178-179 °C; Elemental analysis calcd.
(found) % of C18H19N4OCl: C, 63.06 (63.90); H, 5.59 (6.23);
Cl, 10.34 (10.01); N, 16.34 (15.44). IR (KBr, νmax, cm-1): 824
(CCl str.), 1605, 1554 (Ar-C=C str.), 1760 (CO str.), 2846,
2926 (CH str.), 3052 (=CH str.), 3420 (NH str.); 1H NMR (400
MHz, DMSO/TMS): δppm 1.08 (t, 3H, J = 6.2 Hz, CH3), 1.28-
1.36 (sex, 2H, CH2), 1.67-1.68 (quin, 2H, CH2), 2.68-2.69 (t,
2H, J = 6.5 Hz, CH2), 3.8 (s, 3H, NCH3), 7.37-7.39 (m, 2H,
vinyl proton & ArH), 7.66-7.74 (m, 3H, ArH), 7.84-7.88 (d,
1H, J = 7.0 Hz, vinyl proton).
(E)-1-(1H-Benzo[d]imidazol-2-yl)-3-(2-butyl-4-chloro-
1-ethyl-1H-imidazol-5-yl)prop-2-en-1-one (3b): Yield 85 %;
Off-white solid, m.p. 184-187 °C; Elemental analysis calcd.
(found) % of C19H21N4OCl: C, 63.95 (64.75); H, 5.93 (6.83);
Cl, 9.94 (9.54); N, 15.70 (14.82). IR (KBr, νmax, cm-1): 832
(CCl str.), 1604, 1524 (Ar-C=C str.), 1760 (CO str.), 2915, 2962
(CH str.), 3065 (=CH str.), 3305 (NH str.); 1H NMR (400 MHz,
DMSO/TMS): δppm 1.15 (t, 3H, J = 6.5 Hz, CH3), 1.29-1.35
(sex, 2H, CH2), 1.62-1.68 (quin, 2H, CH2), 2.06 (t, 3H, J = 7.0
Hz, NCH2CH3), 2.64-2.67 (t, 2H, J = 6.5 Hz, CH2), 4.14 (q,
2H, J = 7.5 Hz, NCH2),7.34-7.36 (m, 2H, vinyl proton &ArH),
7.61-7.69 (m, 3H, ArH), 7.89-7.93 (d, 1H, J = 7.0 Hz, vinyl
proton).
(E)-1-(1H-Benzo[d]imidazol-2-yl)-3-(2-butyl-4-chloro-
1-propyl-1H-imidazol-5-yl)prop-2-en-1-one (3c): Yield 83
%; Off-white solid, m.p. 184-187 °C; Elemental analysis calcd.
(found) % of C20H23N4OCl: C, 64.77 (65.69); H, 6.25 (6.95);
Cl, 9.56 (9.06); N, 15.11 (14.05). IR (KBr, νmax, cm-1): 845 (CCl
str.), 1621, 1546 (Ar-C=C str.), 1780 (CO str.), 2893, 2962
(CH str.), 3062 (=CH str.), 3325 (NH str.); 1H NMR (400 MHz,
DMSO/TMS): δppm 0.88 (t, 3H, J = 7.0 Hz, CH3), 1.26-1.34
(sex, 2H, CH2), 1.57-1.63 (quin, 2H, CH2), 2.58-2.69 (t, 2H, J
= 7.0 Hz, CH2), 2.9 (t, 3H, J = 6.5 Hz, CH3), 7.47-7.52 (m,
2H, vinyl proton & ArH), 7.72-7.76 (m, 3H, ArH), 7.94-7.98
(d, 1H, J = 6.8 Hz, vinyl proton), LC-MS: m/z: 407 (M+1)+.
(E)-1-(1H-Benzo[d]imidazol-2-yl)-3-(1-benzyl-2-butyl-
4-chloro-1H-imidazol-5-yl)prop-2-en-1-one (3d): Yield 85
%; Pale yellow solid, m.p. 189-195 °C; Elemental analysis
calcd. (found) % of C20H23N4OCl: C, 68.81 (68.75); H, 5.53
(5.83); Cl, 8.46 (8.26); N, 13.37 (13.19). IR (KBr, νmax, cm-1):
841 (CCl str.), 1622, 1578 (Ar-C=C str.), 1795 (CO str.), 2890,
2955 (CH str.), 3054 (=CH str.), 3250 (NH str.); 1H NMR (400
MHz, DMSO/TMS): δppm 0.89 (t, 3H, J = 6.8 Hz, CH3), 1.30-
1.36 (sex, 2H, CH2), 1.62-1.70 (quin, 2H, CH2), 2.65-2.69 (t,
2H, CH2), 7.4-7.5 (d, 1H, vinyl proton), 7.59-7.67 (m, 4H,ArH),
8.0 (m, 2H, vinyl proton & ArH), 12.84 (s, 1H, NH). LC-MS:
m/z: 289 (M+1)+.
(E)-3-(2-Butyl-4-chloro-1-methyl-1H-imidazol-5-yl)-1-
phenylprop-2-en-1-one (3e):Yield 85 %; Off-white solid, m.p.
189-195 °C; Elemental analysis calcd. (found) % of C17H19N2OCl:
C 67.43 (67.25); H, 6.32 (6.91); Cl, 11.71 (11.67); N, 9.25
(9.02). IR (KBr, νmax, cm-1): 825 (CCl str.), 1608, 1554 (Ar-
C=C str.), 1785 (CO str.), 2885, 2951 (CH str.), 3051 (=CH
str.); 1H NMR (400 MHz, DMSO/TMS): δppm 1.22 (t, 3H, J =
7.0 Hz, CH3), 1.29-1.34 (sex, 2H, CH2),1.64-1.72 (quin, 2H,
CH2), 2.64-2.69 (t, 2H, CH2), 7.34-7.35 (d, 1H, vinyl proton),
7.62-7.69 (m, 4H, ArH), 8.20-8.25 (m, 2H, vinyl proton &
ArH); LC-MS: m/z: 289 (M+1)+.
General procedure for the preparation of imidazole
containing pyrazole compounds 4a-h: A solution of 3a (5 g,
17 mmol) in methanol (30 mL) was added with hydrazine
hydrate (1 g, 20 mmol), acetic acid (0.2 mL) and the mixture
was heated to reflux temperature, stirred for about 3 h till the
completion of the starting materials. After the reaction got
completed, it was cooled to room temperature and added into
ice cold water (50 mL). The precipitated solid was filtered
and washed twice with chilled water (2 × 20 mL) and dried to
get 2-(5-(2-butyl-4-chloro-1-methyl-1H-imidazol-5-yl)-4,5-
dihydro-1H-pyrazol-3-yl)-1H-benzo[d]imidazole (4a). The
remaining targets were prepared same as above procedure with
different quantities for desired products.
2-(5-(2-Butyl-4-chloro-1-methyl-1H-imidazol-5-yl)-
4,5-dihydro-1H-pyrazol-3-yl)-1H-benzo[d]imidazole (4a):
Yield (4.1 g, 79 %); Off-white solid, m.p. 202-203 °C; Elem-
ental analysis calcd. (found) % of C18H21N6Cl: C, 60.58 (61.53);
H, 5.93 (6.25); Cl, 9.93 (9.56); N, 23.55 (22.66). IR (KBr, νmax
,
cm-1): 857 (CCl str.), 1392 (C-N str.), 1452 (C=N str.), 1608,
1505 (Ar-C=C str.), 1739 (CO str.), 2926, 2952 (CH str.), 3092
(=CH str.), 3260 (NH str.); 1H NMR (400 MHz, CDCl3/TMS):
δ
ppm 1.13 (t, 3H, J = 6.5 Hz, CH3), 1.28-1.31 (sex, 2H, CH2),
1.41-1.58 (quin, 2H, CH2), 2.66 (t, 2H, J = 6.5 Hz, CH2), 3.58
(d, 2H, J = 7.0 Hz, pyrazol proton), 3.78 (s, 3H, NCH3), 5.17 (t,
1H, J = 7.0 Hz, pyrzole proton), 6.16 (s, 1H, benzimdazole-
NH), 7.26-7.53 (m, 4H, ArH), 9.33 (s, 1H, pyrazole-NH); 13C
NMR (100 MHz, DMSO-d6) δppm 160.00, 138.27, 136.88,
131.15, 127.77, 125.76, 124.98, 121.67, 120.19, 115.00, 54.83,
53.63, 48.56, 35.27, 30.71, 23.07. LC-MS: m/z: 356 (M+1)+.
2-(5-(2-Butyl-4-chloro-1-ethyl-1H-imidazol-5-yl)-4,5-
dihydro-1H-pyrazol-3-yl)-1H-benzo[d]imidazole (4b):
Yield (85 %); Off-white solid, m.p. 197-198 °C; Elemental
analysis calcd. (found) % of C19H23N6Cl: C, 61.53 (61.53); H,
6.25 (6.25); Cl, 9.56 (9.56); N, 22.66 (22.66). IR (KBr, νmax
,
cm-1): 836 (CCl str.), 1376 (C-N str.), 1458 (C=N str.), 1517,
1610 (Ar-C=C str.), 1658 (CO str.), 2932, 2957 (CH str.), 3071
(=CH str.), 3277 (NH str.); 1H NMR (400 MHz, DMSO/TMS):
δ
ppm 1.02 (t, 3H, J = 6.5 Hz, CH3), 1.23-1.28 (sex, 2H, CH2),
1.42-1.60 (quint, 2H, CH2), 2.60 (t, 2H, J = 6.5 Hz, CH2), 3.23
(t, 3H, J = 6.5 Hz, NCH2CH3), 3.56 (d, 2H, J = 7.0 Hz, pyrazol
proton), 4.23 (t, 2H, J = 6.5 Hz, NCH2), 5.21 (t, 1H, J = 6.5
Hz, pyrzole proton), 6.28 (s, 1H, benzimdazole-NH), 7.29-7.55

2736 Dasari et al.
Asian J. Chem.
(m, 4H,ArH), 9.38 (s, 1H, pyrazole-NH); 13C NMR (100 MHz,
DMSO-d6) δppm 155.16, 147.79, 138.19, 134.27, 132.31,
127.81, 124.71, 122.94, 121.80, 116.94, 55.72, 45.30, 44.24,
42.85, 34.91, 29.34, 24.83, 21.90. LC-MS: m/z: 370 (M+1)+.
2-(5-(2-Butyl-4-chloro-1-propyl-1H-imidazol-5-yl)-
4,5-dihydro-1H-pyrazol-3-yl)-1H-benzo[d]imidazole (4c):
Yield (3.8 g, 74 %); Off-white solid, m.p. 194-195 °C; Elemental
analysis calcd. (found) % of C19H23N6Cl: C, 62.41 (62.35); H,
Cl, 10.72 (10.22); N, 16.93 (16.53). IR (KBr, νmax, cm-1): 836
(CCl str.), 1368 (C-N str.), 1452 (C=N str.), 1612, 1536 (Ar-
C=C str.), 2922, 2965 (CH str.), 3072 (=CH str.), 3310 (NH
str.); 1H NMR (400 MHz, CDCl3/TMS): δppm 0.96 (t, 3H, J =
6.5 Hz, CH3), 1.29-1.31 (sex, 2H, CH2), 1.52-1.59 (m, 4H, CH2),
2.67 (t, 2H, J = 6.5 Hz, CH2), 2.87 (t, 3H, J = 6.5 Hz, CH3),
3.73 (d, 2H, J = 7.0 Hz, pyrazole proton), 3.79 (t, 1H, J = 6.5
Hz, pyrazol proton), 4.19 (q, 2H, J = 7.0 Hz, NCH2), 7.53-7.66
(m, 3H, Ar-H), 8.02 (d, 2H, J = 7.0 Hz, Ar-H), 9.46 (s, 1H,
pyrazole-NH); 13C NMR (100 MHz, DMSO-d6) δppm 155.63,
138.38, 136.87, 131.00, 127.80, 125.77, 121.66, 120.16, 54.86,
48.56, 42.01, 30.24, 28.33, 20.71, 14.04. LC-MS: m/z: 384
(M++1).
5-(2-Butyl-4-chloro-1-propyl-1H-imidazol-5-yl)-3-
phenyl-4,5-dihydro-1H-pyrazole (4g): Yield (3.8 g, 74 %);
Off-white solid, m.p. 142-143 °C; Elemental analysis calcd.
(found) % of C19H25N4Cl: C, 66.17 (65.17); H, 7.31 (7.72);
Cl, 10.28 (10.46); N, 16.25 (16.18). IR (KBr, νmax, cm-1): 848
(CCl str.), 1345 (C-N str.), 1473 (C=N str.), 1584 (Ar-C=C
str.), 2960 (CH str.), 3061 (=CH str.), 3312 (NH str.); 1H NMR
(400 MHz, CDCl3/TMS): δppm 1.05 (t, 6H, J = 6.5 Hz, CH3),
1.25 (sex, 2H, CH2), 1.89 (m, 4H, CH2), 2.54 (t, 2H, J = 6.5
Hz, CH2), 3.62 (d, 2H, J = 7.0 Hz, pyrazole proton), 3.88 (t,
1H, J = 6.5 Hz, pyrazol proton), 4.19 (t, 2H, J =6.5 Hz, NCH2),
7.28-7.37 (m, 3H, Ar-H), 7.92-7.99 (d, 2H, J = 7.0 Hz, Ar-H),
9.62 (s, 1H, pyrazole-NH); 13C NMR (100 MHz, CDCl3): δ154.12,
138.28, 133.15, 131.20, 130.81, 128.60, 127.15, 126.48, 52.6,
36.12, 33.89, 28.3, 25.37, 21.2, 16.39. LC-MS: m/z: 345 (M++1).
5-(1-Benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-3-
phenyl-4,5-dihydro-1H-pyrazole (4h): Yield (4.7 g, 89 %);
Off-white solid, m.p. 147-148 °C; Elemental analysis calcd.
(found) % of C19H25N4Cl: C, 70.31 (71.15); H, 6.41 (6.28);
Cl, 9.02 (9.19); N, 14.26 (14.18). IR (KBr, νmax, cm-1): 860
(CCl str.), 1398 (C-N str.), 1462 (C=N str.), 1605 (Ar-C=C
str.), 2860, 2945 (CH str.), 3058 (=CH str.), 3321 (NH str.); 1H
NMR (400 MHz, CDCl3/TMS): δppm 1.12 (t, 3H, J = 6.5 Hz,
CH3), 1.38-1.42 (sex, 2H, CH2), 1.56-1.63 (quin, 2H, CH2),
2.65 (t, 2H, J = 6.5 Hz, CH2), 3.52 (t, 1H, J = 6.5 Hz, pyrazole
proton), 3.86 (d, 2H, J = 6.5 Hz, pyrazol proton), 4.92 (q, 2H,
J = 7.0 Hz, NCH2), 7.42-7.58 (m, 4H, ArH), 7.91-7.99 (d, 2H,
J = 6.5 Hz,ArH), 8.18-8.29 (m, 4H,ArH), 9.15 (s, 1H, pyrazole-
NH); 13C NMR (100 MHz, DMSO-d6) δppm 161.02, 147.61,
138.22, 133.80, 132.71, 131.15, 129.34, 128.46, 127.71,
126.37, 124.91, 121.94, 120.43, 116.43, 48.42, 36.45, 32.16,
29.37, 24.82, 20.91, 18.62. LC-MS: m/z 393 (M+1)+.
6.55 (6.50); Cl, 9.21 (9.17); N, 21.83 (21.80). IR (KBr, νmax
,
cm-1): 853 (CCl str.), 1378 (C-N str.), 1449 (C=N str.), 1601
(Ar-C=C str.), 1786 (CO str.), 2931, 2955 (CH str.), 3076 (=CH
str.), 3290 (NH str.); 1H NMR (400 MHz, CDCl3/TMS): δppm
0.91 (t, 3H, J = 6.5 Hz, CH3), 1.21-1.29 (sex, 2H, CH2), 1.39-
1.56 (m, 4H, CH2), 2.68 (t, 2H, J = 6.5 Hz, CH2), 2.78 (t, 3H,
J = 6.5 Hz, CH3), 3.36 (d, 1H, J = 7.0 Hz, pyrazole proton),
3.59 (d, 2H, J = 7.0 Hz, pyrazol proton), 5.19 (t, 1H, pyrzole
proton), 6.17 (s, 1H, benzimdazole-NH), 7.32-7.54 (m, 4H,Ar-
H), 9.42 (s, 1H, pyrazole-NH); 13C NMR (100 MHz, DMSO-d6)
δ
ppm 155.22, 138.40, 135.49, 132.07, 128.28, 126.72, 125.21,
121.40, 118.14, 58.73, 52.67, 46.18, 42.84, 36.24, 30.18,
29.31, 28.27, 22.46. LC-MS: m/z: 384 (M++1).
2-(5-(1-Benzyl-2-butyl-4-chloro-1H-imidazol-5-yl)-4,5-
dihydro-1H-pyrazol-3-yl)-1H-benzo[d]imidazole (4d): Yield
(4.7 g, 89 %); Pale yellow solid, m.p. 174-175 °C; Elemental
analysis calcd. (found) % of C24H25N6Cl: C, 66.58 (66.42); H,
5.82 (5.71); Cl, 8.19 (8.20); N, 19.41 (19.24). IR (KBr, νmax
,
cm-1): 836 (CCl str.), 1392 (C-N str.), 1456 (C=N str.), 1612
(Ar-C=C str.), 2856, 2942 (CH str.), 3052 (=CH str.), 3320
(NH str.); 1H NMR (400 MHz, CDCl3/TMS): δppm 1.05 (t, 3H,
J = 6.5 Hz, CH3), 1.32-1.37 (sex, 2H, CH2), 1.46-1.57 (quin,
2H, CH2), 2.72-2.79 (t, 2H, J = 6.5 Hz, CH2), 3.52 (t, 1H, J =
6.5 Hz, pyrazole proton), 3.66 (d, 2H, J = 6.5 Hz, pyrazol
proton), 4.98 (q, 2H, J = 7.0 Hz, NCH2), 7.42-7.61 (m, 5H,
ArH), 8.12-8.31 (m, 4H,ArH), 8.91 (s, 1H, pyrazole-NH), 11.26
(s, 1H, imdazole proton); 13C NMR (100 MHz, DMSO-d6)
δppm 160.18, 137.44, 135.84, 132.77, 130.86, 129.79, 126.83,
124.22, 122.78, 120.82, 119.34, 115.99, 109.37, 53.64, 48.15,
34.28, 32.74, 29.38, 25.37, 22.31. LC-MS: m/z 432 (M+1)+.
5-(2-Butyl-4-chloro-1-methyl-1H-imidazol-5-yl)-3-
phenyl-4,5-dihydro-1H-pyrazole (4e): Yield (3.9 g, 74 %);
Off-white solid, m.p. 192-193 °C; Elemental analysis calcd.
(found) % of C17H21N4Cl: C, 64.45 (64.24); H, 6.68 (6.72);
Cl, 11.19 (11.42); N, 17.68 (17.91). IR (KBr, νmax, cm-1): 848
(CCl str.), 1368 (C-N str.), 1460 (C=N str.), 1612, 1564 (Ar-
C=C str.), 2945 (CH str.), 3050 (=CH str.), 3325 (NH str.); 1H
NMR (400 MHz, CDCl3/TMS): δppm 1.09 (t, 3H, J = 6.5 Hz,
CH3), 1.28-1.34 (sex, 2H, CH2), 1.62-1.69 (quin, 2H, CH2),
2.84 (t, 2H, J = 6.5 Hz, CH2), 3.43 (t, 1H, J = 6.5 Hz, pyrazole
proton), 3.62 (d, 2H, J = 7.0 Hz, pyrazol proton), 3.79 (s, 3H,
NCH3), 7.32-7.49 (m, 3H, Ar-H), 7.93-8.02 (m, 2H, Ar-H),
9.52 (s, 1H, pyrazole-NH); 13C NMR (100 MHz, DMSO-d6)
Biological evaluation: By using streptomycin (100 µg/
mL) as a reference antibacterial agent, all the final compounds
were tested for in vitro antibacterial activity against three Gram-
positive strains and three Gram-negative bacteria by adopting
cup plate agar diffusion method [25]. The sterilized mediums
[1 mg/mL] (autoclaved at 110 °C for 45 min) was inoculated
with suspensions of the microorganisms (105 cfu mL-1) as
harmonized to McFarland barium sulphate solution and filled
into petri-dishes to get a depth of 3-4 mm. For about 18 to 24
h, the mediums were solidified, inverted and incubated at 30-
37 °C till the adequate growth was noticed. After incubation,
each plate was scrutinized and measured for complete zones
of inhibition. The zone of inhibition was measured using
δ
ppm 154.16, 138.40, 135.49, 132.07, 128.28, 126.72, 125.21,
121.70, 119.14, 59.78, 53.64, 48.15, 42.81, 32.52, 29.38,
25.37, 20.39. LC-MS: m/z: 316 (M+1)+.
5-(2-Butyl-4-chloro-1-ethyl-1H-imidazol-5-yl)-3-
phenyl-4,5-dihydro-1H-pyrazole (4f): Yield (3.8 g, 74 %);
Off-white solid, m.p. 164-165 °C; Elemental analysis calcd.
(found) % of C17H21N4Cl: C, 65.34 (65.99); H, 7.01 (7.62);

Vol. 31, No. 12 (2019)
Design, Synthesis and Molecular Docking Studies of Novel Pyrazole Benzimidazole Derivatives 2737
sliding calipers to the nearest whole millimeter (mm). All the
target compounds were screened for their antifungal activity
against two pathogenic fungi, Fusarium oxysporum and Asper-
gillus niger by the poison plate technique [26].
From the above results, it can be understood that the
compound 4c was found to exhibit excellent activity against
Bacillus subtilis B29 and Staphylococcus aureus S276 and
also shows good activity against P. aeruginosa ATCC 15442,
Escherichia coli E266. A capable activity against all the
bacterial strains has been exhibited by Imidazole-pyrazoles
containing electron donor properties. An increased zone of
inhibition 21, 20 and 16 mm has been exhibited by compounds
4c, 4f and 4g, respectively when compared to the standard
drug streptomycin (18 mm) against Bacillus subtilis B29 strain.
Compounds 4f (19 mm) and 4g (19 mm) showed a good activity
against the Gram-negative bacteria, Escherichia coli E266.
The antifungal screening results of all the compounds were
compared with standard antifungal drug, the fluconazole and
observed that all the compounds have showed moderate activity.
The activity of compounds 4a, 4c against Fusarium oxysporum
was slightly higher with zone of inhibition 13, 12 mm, respec-
tively. Target compounds 4h, 4a shows very good antifungal
activity against A. niger strain with a zone of inhibition 15 mm.
Docking: Docking studies of all the synthesized comp-
ounds into the binding site of the proteins was performed to
determine the binding accord of the complex. It is a significant
part of the structure based drug design process “crystal struc-
ture of the C(30) carotenoid dehydrosqualene synthase from
Staphylococcus aureus complexed with bisphosphonate BPH-
700” (2ZCS). Molecular docking studies was most extensively
used method for the calculation of protein-ligand interactions
and all the synthesized compounds are summarized in Table-
2. In this study, some of the new ligands (6) have identified as
potent antibacterial inhibitors from binding free energy assess-
ment to assign the best binding conformation byAutoDock4.2
version and it is an efficient method to predict the potential
Docking studies: The binding modes of synthesized
compounds with a protein have been explained by molecular
docking.All the final targets were docked individually by using
Autodock 4.2 software [27,28]. After receiving the modeled
three dimensional structure of nitrate reductase protein to
Autodock 4.2, it was structurally optimized by adding hydrogen
to protein designated with kollaman charges. The model was
then saved in PDBQT format after adding hydrogen. Later
the ligands were prepared by adjusting the torsion angles and
saved them in PDBQT format. Potential binding sites for all
the proteins was selected by using PDBSUM[29].Around binding
site of protein, a grid was generated to identify the xyz coordi-
nates. Lamarckian GeneticAlgorithm (LGA) was selected for
docking, freezing and default parameters used in autodock4.2.
The structural intercommunications between PDB with two
inhibitors were docked separately. The binding modes of
protein and their binding energies of ligands were commonly
estimated by performing Docking studies. X,Y,Z coordinates
of PDB were selected by using SPDBV.
RESULTS AND DISCUSSION
Antibacterial activity: By considering streptomycin as
a standard drug, the in vitro antibacterial studies of newly
synthesized compounds were assessed against two Gram-
positive strains and two Gram-negative bacteria (Table-1). The
antibacterial activity of the final compounds varied from mode-
rate to excellent over the bacterial strains used. But some of
the target compounds did not show activity.
TABLE-1
ANTIBACTERIAL, ANTIFUNGAL ACTIVITY OF SYNTHESIZED COMPOUNDS 4a-h
Zone of inhibition in mm at 25 µg/mL
Gram-positive bacteria
Gram-negative bacteria
Fungal strains
Compound No.
Bacillus subtilis
Staphylococcus
P. aeruginosa
Escherichia coli
Fusarium
Aspergillus
niger
B29
14
12
21
13
09
20
16
–
aureus S276
ATCC 15442
E266
oxysporum
4a
4b
4c
4d
4e
4f
4g
16
13
20
–
08
17
18
15
15
–
12
15
18
12
11
18
14
17
18
–
13
10
15
08
–
19
19
14
20
–
13
–
15
12
14
08
13
09
10
15
–
12
10
06
10
07
12
–
4h
Streptomycin
Flucanazole
18
–
15
18
TABLE-2
MOLECULAR DOCKING INTERACTIONS OF SOME OF THE DERIVATIVES WITH THEIR ANTIBACTERIAL PROTEIN
Dissociation constant
Binding energy ∆G
Compd. No.
PDB
Interacting amino acids Grid X-Y-Z coordinates
(KI) (µM)
(Kcal/mol)
4a
4c
4e
4f
4g
4h
2ZCS
2ZCS
2ZCS
2ZCS
2ZCS
2ZCS
2ZCS
Asp27, Lys273
Asp49, Arg265
Lys273, Asp27
Asp27, Lys273
Lys273
56.026, 5.999, 60.238
56.026, 5.999, 60.238
56.026, 5.999, 60.238
56.026, 5.999, 60.238
56.026, 5.999, 60.238
56.026, 5.999, 60.238
56.026, 5.999, 60.238
-6.34
-6.24
-7.08
-5.77
-7.89
-7.48
-5.85
22.68
26.47
6.47
58.77
1.66
Lys273
Ser21
3.27
51.74
Streptomycin

2738 Dasari et al.
Asian J. Chem.
4a
4c
4e
4f
4g
4h
Fig. 1. Docking conformations complexed with bisphosphonate BPH-700 (2ZCS)
ligand interactions. Experimental activities and predicted
values by Lamarckian Genetic Algorithm and the derivatives
selected for molecular docking have some collective structural
features.
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