Synthesis and evaluation of novel pyrazole-imidazole hybrids as antimicrobial candidates

Article abstract of DOI:10.1080/00397911.2021.1953532

The present study describes a facile and efficient synthesis of 1-(N-arylacetamide)-4-phenyl-2-pyrazolyl-imidazole 7a–7t by reacting 1-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole (3a)/3,5-dimethyl-1-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole (3b) with 2-bromo-N-

Full text of DOI:10.1080/00397911.2021.1953532

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
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Synthesis and evaluation of novel pyrazole-
imidazole hybrids as antimicrobial candidates
Suman Punia, Vikas Verma, Devinder Kumar, Ashwani Kumar, Laxmi Deswal
& Mahavir Parshad
To cite this article: Suman Punia, Vikas Verma, Devinder Kumar, Ashwani Kumar, Laxmi Deswal
& Mahavir Parshad (2021): Synthesis and evaluation of novel pyrazole-imidazole hybrids as
antimicrobial candidates, Synthetic Communications, DOI: 10.1080/00397911.2021.1953532
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SYNTHETIC COMMUNICATIONSV
https://doi.org/10.1080/00397911.2021.1953532
Synthesis and evaluation of novel pyrazole-imidazole
hybrids as antimicrobial candidates
Suman Puniaa, Vikas Vermaa, Devinder Kumara, Ashwani Kumarb, Laxmi Deswala,
and Mahavir Parshada
aDepartment of Chemistry, Guru Jambheshwar University of Science and Technology, Hisar, India;
bDepartment of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology,
Hisar, India
ABSTRACT
ARTICLE HISTORY
Received 8 March 2021
The present study describes a facile and efficient synthesis of 1-
(N-arylacetamide)-4-phenyl-2-pyrazolyl-imidazole 7a–7t by reacting
1-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole (3a)/3,5-dimethyl-1-(4-phe-
nyl-1H-imidazol-2-yl)-1H-pyrazole (3b) with 2-bromo-N-substituteda-
cetamide 6a–6j) in good yield. The structure of compounds 7a–7t
are confirmed by various spectral techniques. All the prepared com-
pounds are examined for in-vitro antimicrobial activities against three
bacterial strains; S. aureus, B. subtilis and E. coli and two fungal
strains; C. albicans and A. niger. The synthesized analogues show
moderate inhibitory activity against tested bacterial strains and the
compounds 7a, 7b, 7l, 7m and 7r exhibited good activity against C.
albicans. Most of the compounds obey the Lipinski rule of five. The
compound 7b is docked in the active site of C. albicans sterol-14a-
demethylase and DNA gyrase to find the binding conformation.
KEYWORDS
Amide; antimicrobial;
imidazole; molecular
modeling; pyrazole
GRAPHICAL ABSTRACT
Introduction
Imidazole and Pyrazole has received substantial attention due to their broad range of
pharmacological activities. Further, many drugs such as Butoconazole, Bifonazole,
CONTACT Vikas Verma
vikas_chem_pu@yahoo.com
Department of Chemistry, Guru Jambheshwar University of
Science and Technology, Hisar, India.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2021 Taylor & Francis Group, LLC

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S. PUNIA ET AL.
Figure 1. Design strategy for target compound. (A) Pyrazolylimidazole hybrid. (B) N-substituted-
phenyl-imidazole derivatives. (C) Imidazole-quinolin-2(1H)-one hybrid.
Ketoconazole, Metronidazole, containing imidazole framework as core unit are currently
used for the treatments of various types of bacterial and fungal infections. On other
hand, pyrazole provides a useful pharmacophore to some antibiotics like sulfaphenazo-
lum, etc. Moreover, some documented pyrazolylimidazole hybrids possess a number of
pharmacological activities such as, antidiabetic,[1] antifungal,[2] antibacterial,[3] anti-
cancer,[4] anti-tubercular,[5] etc.
Amide functionality adjoining to heteroaromatic ring especially imidazole, provide a
particular stereoelectronic effect to the molecule.[6] The medicinal chemistry database
reflected that 25% of the known drugs possess at least one amide unit such as captopril,
acetazolamide, bupivacaine and valsartan.[7] Moreover, the tendency of amide bond to
create hydrogen bond and dipole-dipole interactions with the enzyme and target recep-
tors increases the significance of amides in designing new drug molecules.[8] In general,
compounds bearing amide moiety have a broad range of pharmacological activities like
antimicrobial,[9] anti-inflammatory,[10] antimalarial[11] and anti-viral.[12]
It has been reported that 4-(4,5-diphenyl-1H-imidazol-2-yl)-3-(p-tolyl)-1H-pyra-
zole[13] ((A) ,Figure 1) a pyrazole-imidazole hybrid exhibited remarkable activity against
bacterial strains viz. S. aureus, methicillin resistant S. aureus, P. aeruginosa and E. coli.
Further, N-substitutedphenyl- imidazole derivatives[14] ((B), Figure 1) and amide teth-
ered imidazole-quinolin-2(1H)-one hybrid, 2-(1H-imidazol-1-yl)-N-(2-oxo-1,2,3,4-tetra-
hydroquinolin-4-yl)acetamide[14] ((C), Figure 1) has been reported to show promising
antimicrobial activity. Prompted by pharmacological importance of pyrazolylimidazole
and amide incorporated imidazole derivatives, the present study is designed to incorpor-
ate amide functionality on pyrazolylimidazole scaffold (Figure 1).

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3
Compound
R1
Compound
R1
H
H
H
H
H
H
H
H
H
H
Compound
R2
7a
7b
7c
7d
7e
7f
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
7k
7l
7a/7k
7b/7l
7c/7m
7d/7n
7e/7o
7f/7p
7g/7q
7h/7r
7i/7s
-4-CH3OC6H4
-4-CH3C6H4
-3-Cl-2-CH3C6H3
-4-ClC6H4
7m
7n
7o
7p
7q
7r
7s
-2-3-(Cl)2C6H3
-2-6-(Cl)2C6H3
-2-CH3COC6H4
-1-C10H7
7g
7h
7i
-2-NO2
7j
7t
7j/7t
-2-4-(NO2)2C6H3
Scheme 1. Synthesis of 1-(N-arylacetamide)-4-phenyl-2-pyrazolyl-imidazole compounds 7a–7t.
Thus, a new series of 1-(N-arylacetamide)-4-phenyl-2-pyrazolyl-imidazole compounds
7a–7t is synthesized and investigated as potential antimicrobial agents. The synthesized
1
compounds are characterized by using different spectral techniques, (FTIR, H NMR,
13C NMR and HRMS spectroscopy). Furthermore, the synthesized series is screened for
antimicrobial assay on three bacterial strains viz. B. subtilis, E. coli and S. aeureus and
two fungal strains viz. C. albicans and A. niger. The most active compound is docked in
the active site of E. coli DNA gyrase enzyme. DNA gyrase is one of two essential mem-
bers of the type II topoisomerase family in E. coli. It carries out ATP-dependent super-
coiling of chromosomal DNA, as well as potentially being involved in the decatenation
of newly synthesized chromosomal and plasmid DNA. It is an endorsed drug target for
the discovery of novel antibacterial candidates and the emergence of resistance for fluo-
roquinolones toward it necessitates the development of novel bacterial topoisomerase
inhibitors.[ref. https://doi.org/10.1016/j.bioorg.2020.104266].
Results and discussion
Chemistry
The protocol for the synthesis of 1-(N-arylacetamide)-4-phenyl-2-pyrazolyl-imidazole
compounds 7a–7t is depicted in Scheme 1. Initially, 1-(4-phenyl-1H-imidazol-2-yl)-1H-
pyrazole (3a) and 3,5-dimethyl-1-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole (3b) were
synthesized.[15] It involves the (3 þ 2) cyclo-condensation reaction of 1H-pyrazole-1-

4
S. PUNIA ET AL.
Scheme 2. Synthesis of pyrazolylimidazole 3a–3b.
Compound
4a/6a
R2
Compound
4f/6f
R2
4-CH3OC6H4
4-CH3C6H4
3-Cl-2-CH3C6H3
4-ClC6H4
-2-6-(Cl)2C6H3
-2-CH3COC6H4
-1-C10H7
4b/6b
4g/6g
4c/6c
4h/6h
4i/6i
4d/6d
-2-NO2
4e/6e
2-3-(Cl)2C6H3
4j/6j
-2-4-(NO2)2
Scheme 3. Synthesis of 2-bromo-N-(substituted) acetamide 6a–6j.
carboximidamide hydrochloride (1a) and 3,5-dimethyl-1H-pyrazole-1-carboximidamide
nitrate (1b) with 2-bromo-1-phenylethanone (2a) in presence of potassium carbonate
by using THF and water as a solvent in the ratio of 5:1 mL (Scheme 2).
Cyclization of carboxamidine 1a–1b moiety to imidazole ring 3a–3b was confirmed
by the appearance of a D2O exchangeable broad singlet at d 12.81–12.59 ppm (N–H of
1
imidazole) and another singlet at d 7.56–7.59 ppm (imidazole ring proton), in H NMR
spectrum of compound 3a and 3b.
Also, the compounds displayed 5 aromatic protons confirming the presence of phenyl
ring. Further, 2-bromo-N-(substituted)acetamide 6a–6j were synthesized by reacting the
corresponding substituted aniline derivatives and bromoacetylbromide in dichlorome-
thane using potassium carbonate as a base (Scheme 3).[16]
Afterwards, the novel hybrid 1-(N-arylacetamide)-4-phenyl-2-pyrazolyl-imidazole
compounds 7a–7t were synthesized in good yields by stirring the equimolar amount of
compound 3a/3b with 2-bromo-N-(substituted)acetamide in presence of potassium car-
ꢀ
bonate in dimethylformamide at 35–50 C (Scheme 1).

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5
Figure 2. 1H NMR and 13C NMR assignment of compound 7n.
An intense band at 1699–1657 cmꢁ1 and 1644–1604 cmꢁ1 corresponds to amide I
1
and II respectively. The H NMR spectrum of compound 7a–7t, exhibited disappear-
’
ance of singlet at d 12.81–12.59 ppm and displaying singlet in the span of 4.88–5.35(C1 )
ppm due to 2H confirmed N-alkylation of pyrazolylimidazole. In 13C NMR, a confirma-
tory sign for N-alkylation of imidazole derivatives was the appearance methylene carbon
’
’
around d 49.22–51.25(C1 ) and carbonyl carbon at d 150.28–172.18(C2 ) which was fur-
ther assured by DEPT spectrum of compound 7n. Furthermore, appearance of two dou-
blets in aromatic shift value in addition to 5 aromatic protons; (2H (d) around 7.77
1
(2H, t, H7/ H11) around 7.41(H10/H8) and 1H (t) around 7.26(H9) ppm in H NMR
spectrum of parent compounds; 3a/3b also support the fact of N-alkylation. In addition,
the D2O exchangeable singlet in the span of 12.57–9.83 ppm was assigned to N–H of
anilide group.
Structure of compounds 7a–7t was established from the 2 D [COSY, HSQC and
HMBC] spectroscopic data and 1H NMR and 13C NMR assignment of compound 7n
shown in Figure 2. The connectivity of all hydrogen atoms in 7n, through intervening
bond was established by COSY (Correlation Spectroscopy). In COSY peak at d 7.73
does not correlate with any other proton showing that it is the proton of imidazole ring
’’
(H5) and the signal at d 6.55 (t, J ¼ 2.4, 2.0 Hz, 1H, H4 ) correlate with 8.33 (d,
’’
’’
’’
J ¼ 2.4 Hz, 1H, H5 ) and 7.80 (bs, 1H, H3 ) justifying that it is due H4 of pyrazole ring.
Consequently, the protons at d 6.55, 8.33, and 7.80 are the protons of pyrazole ring.
’
’
Mutual correlation of signal at d 7.55 (d, J ¼ 8.8 Hz, 2H, H5 / H9 ) with signal at d 7.37
’
’
(d, J ¼ 8.8 Hz, 2H, H6 /H8 ) support our assignment of these signals to the p-chloroani-
lide. Further, the peak at d 7.41 (t, J ¼ 7.6, J ¼ 8.8 Hz, H10/H8) show correlation with d
7.77 (bs, H7/ H11), and 7.26 (t, J ¼ 7.2, J ¼ 7.6 Hz, H9) were assigned to phenyl ring in
the molecule.

6
S. PUNIA ET AL.
Table 1. In-vitro antibacterial activity of compounds 7a–7t (MIC in mmol/mL).
Compound
R1
R2
E. coli
S. aureus
B. subtilis
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
ꢁ4-CH3OC6H4
ꢁ4-CH3C6H4
ꢁ4-Cl-2-CH3C6H3
ꢁ4-ClC6H4
0.0622
0.0324
0.0595
0.0615
0.0595
0.0606
0.0604
0.0593
0.0600
0.0541
0.0669
0.0349
0.0637
0.0330
0.0606
0.0606
0.0649
0.0635
0.0643
0.0576
0.0047
0.0311
0.0324
0.0297
0.0307
0.0595
0.0151
0.0302
0.0296
0.0300
0.0220
0.0167
0.0349
0.0377
0.0661
0.0606
0.0303
0.0324
0.0317
0.0321
0.0288
0.0047
0.0311
0.0162
0.0297
0.0307
0.0297
0.0606
0.0302
0.0296
0.0300
0.0541
0.0334
0.0349
0.0637
0.0661
0.0303
0.0303
0.0324
0.0317
0.0321
0.0576
0.0047
ꢁ2-3-(Cl)2C6H3
ꢁ2-6-(Cl)2C6H3
ꢁ2-COCH3C6H4
ꢁ1-C10H7
ꢁ2-NO2C6H4
ꢁ2-4-(NO2)2C6H3
ꢁ4-CH3OC6H4
ꢁ4-CH3C6H4
ꢁ4-Cl-2-CH3C6H3
ꢁ4-ClC6H4
ꢁ2-3-(Cl)2C6H3
ꢁ2-6-(Cl)2C6H3
ꢁ2-COCH3C6H4
ꢁ1-C10H7
7q
7r
7s
7t
ꢁ2-NO2C6H4
ꢁ2-4-(NO2)2C6H3
–
H
–
Ciprofloxacin
13C NMR assignment of representative molecule indicated the presence of 16 signals.
In order to establish the assignment of each carbon HSQC (Heteronuclear Single-
Quantum Coherence Spectroscopy) and HMBC (Heteronuclear Multiple Bond
Correlation Spectroscopy) 2 D NMR experiments were carried out. HSQC helped the
assignment of carbon signals at d 142.32, 131.38, 129.25, 129.12, 127.19, 124.60, 121.08,
118.89, 107.74 and 50.29. These were assigned on 0t0he basis of key correlation found in
00
00
0
00
HSQC spectra as follow; d 8.33(H5 ) fi 131.38(C5 ), 7.80(H3 ) fi142.32(C3 ), 7.77(H5/
0
0
0
H11) fi 124.60(C5/C11), 7.73(H5) ! 118.89(C5), 7.55(H4 /H9 ) ! 121.08(C4 /C9 ),
7.41(H5 /H100) ! 129.25(C5 /C100), 7.26(H9) ! 127.19(C9), 6.55(H4 ) ! 107.74(C4 ),
0
0
0
0
0
0
5.18(H1 ) ! 50.29(C1 ) ppm.
DEPT 13513C NMR spectrum clearly shows the presence of 9 tertiary carbons and 1
secondary carbon thereby suggesting that remaining 6 signals were due to quaternary
carbons. HMBC experiment established the assignment of quaternary carbon signals
through two bond correlations at 165.82, 140.75, 137.17, 134.00, 129.82 and 127.50. The
00
above assignment was confirmed through the correlation of signals at d 8.33(H5 ) !
00
0
142.32(H2 ) and 107.74(C4 ); 7.73(H5) !137.17(C4), 140.75(C2) and 118.89(C5);
7.77(H5/H11) ! 129.25(H8/H10) and 127.50(C6); 7.41(H5 /H100) ! 124.60(C5/C11) and
0
0
0
0
0
127.19(C9); 7.55 ! 129.82(C4’) and 129.12(C6 /C8 ), 7.37(H6 /H8 ) ! 134.00(C7’),
0
121.08(C5’) and 5.18(H1 ) ! 118.89(C5), 140.75(C2) and 165.82(C2’) ppm.
Antimicrobial activity
Antibacterial activity
All the compounds of the series have shown considerable antibacterial activity ranging
from 0.0167 to 0.0669 mmol/mL as depicted in Table 1. Perusal of activity data in Table
1 indicates that compounds 7f (MIC, 0.0151 mmol/mL) has emerged as a most activeÙ
antibacterial agent against S. aureus followed by derivative 7k (MIC, 0.0167 mmol/mL).

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7
Table 2. In-vitro antifungal activity of compounds 7a–7t (MIC in mmol/mL).
Compounds
R1
R2
C. albicans
A. niger
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
ꢁ4-CH3OC6H4
ꢁ4-CH3C6H4
ꢁ4-Cl-2-CH3C6H3
ꢁ4-ClC6H4
0.0078
0.0084
0.0297
0.0307
0.0283
0.0142
0.0302
0.0146
0.0152
0.0541
0.0303
0.0079
0.0377
0.0082
0.0175
0.0175
0.0326
0.0080
0.0150
0.0878
0.0051
0.0156
0.0168
0.0594
0.0614
0.0567
0.0284
0.0604
0.0292
0.0304
0.1088
0.0606
0.0158
0.0636
0.0164
0.0350
0.0350
0.0652
0.0160
0.0300
0.1756
0.0102
ꢁ2-3-(Cl)2C6H3
ꢁ2-6-(Cl)2C6H3
ꢁ2-COCH3C6H4
ꢁ1-C10H7
ꢁ2-NO2C6H4
ꢁ2-4-(NO2)2C6H3
ꢁ4-CH3OC6H4
ꢁ4-CH3C6H4
ꢁ4-Cl-2-CH3C6H3
ꢁ4-ClC6H4
ꢁ2-3-(Cl)2C6H3
ꢁ2-6-(Cl)2C6H3
ꢁ2-COCH3C6H4
ꢁ1- C10H7
7q
7r
7s
7t
ꢁ2-NO2C6H4
ꢁ2-4-(NO2)2C6H3
–
H
–
Fluconazole
In case of B. subtilis, compound 7b (MIC, 0.0162 mmol/mL) exhibited highest activity
while 7c (MIC, 0.0297 mmol/mL), 7d (MIC, 0.0307 mmol/mL), 7e (MIC, 0.0297 mmol/
mL), 7g (MIC, 0.0302 mmol/mL), 7h (MIC, 0.0296 mmol/mL), 7i (MIC, 0.0300 mmol/
mL), 7o (MIC, 0.0303 mmol/mL) and 7p (MIC, 0.0303 mmol/mL) displayed significant
activity. Among the synthesized derivatives, compounds 7b, 7n and 7l showed signifi-
cant activity against E. coli with MIC value of 0.0324, 0.0330 and 0.0349 mmol/mL
respectively.
In amide incorporated imidazole-pyrazole hybrids having pyrazole bearing CH3
group, dichloro-derivatives have shown higher bactericidal efficacy against all the tested
bacterial strains than monochloro analogues. Moreover, compounds having 2-acetyl and
2-NO2 (7g, 7i, 7q and 7s) group at anilide ring has been found to show almost equal
potency against all the tested bacterial strains. From the results, it has been generalized
that compounds with methyl substituted pyrazole scaffold exhibited better activity in
comparison to compounds having unsubstituted pyrazole scaffold against the E. coli, S.
aureus and B. subtilis.
Antifungal activity
The synthesized hybrids 7a–7t were screened for antifungal activity against Candida
albicans and Aspergillus niger and the data is presented in Table 2. The synthesized
compounds showed good activity against the tested fungal strains, however they were
found more active against C. albicans than A. niger. Among the series, compounds 7a
(MIC, 0.0078 mmol/mL), 7b (MIC, 0.0084 mmol/mL), 7l (MIC, 0.0079 mmol/mL), 7n
(MIC, 0.0082 mmol/mL) and 7r (MIC, 0.0080 mmol/mL) exhibited good activity against
C. albicans. The derivatives 7a (MIC, 0.0156 mmol/mL) and 7l (MIC, 0.0158 mmol/mL)
showed highest and almost similar inhibitory efficacy toward the tested strain of fungus,
A. niger. Similarly, compounds 7f (MIC, 0.0142 mmol/mL) and 7h (MIC, 0.0146 mmol/
mL) were found almost equally potent against C. albican. The hybrids having two NO2

8
S. PUNIA ET AL.
Table 3. Molinspiration properties.
Compound
S þ logD
mi logP
MWt
HBDH
M_NO
TPSA
No. of violation
%ABS
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7I
7m
7n
7o
7p
7q
7r
3.596
3.994
2.909
3.691
4.245
3.166
4.273
3.053
2.543
3.166
2.974
4.029
2.909
3.074
3.655
2.553
3.681
2.452
3.753
2.543
4.079
4.294
4.153
4.079
4.293
3.319
4.297
3.972
3.784
4.226
3.645
4.358
4.153
3.645
3.864
2.886
3.864
3.546
4.151
3.784
385.472
419.917
461.44
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
6
12
6
6
7
6
7
9
9
6
6
12
6
6
7
6
7
64.74
64.74
156.38
64.74
64.74
73.97
64.74
81.81
110.56
110.56
64.74
64.74
156.38
64.74
64.74
73.97
64.74
81.81
64.74
110.56
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
86.6607
86.6607
55.0489
86.6607
86.6607
83.4803
86.6607
80.7755
67.5308
67.5308
86.6607
86.6607
55.0489
86.6607
86.6607
83.4803
86.6607
80.7755
86.6607
67.5308
405.89
440.335
401.471
440.335
413.482
388.388
416.442
357.417
407.478
489.44
377.835
412.28
373.417
412.28
385.428
393.451
388.388
7s
7t
6
9
groups were found to show least activity i.e., 7j (0.1088 mmol/mL), 7t (0.1756 mmol/mL)
against A. niger while compound 7t (0.0878 mmol /mL) against C. albicans.
The results depicted that compounds bearing electron donating groups (CH3, Cl,
OMe) at anilide ring leads to increase in activity against the tested fungal strains. In
general, it was observed that all synthesized imidazole hybrids possess more inhibitory
efficacy against C. albicans than A. niger. Moreover, N-naphthyl imidazole conjugates;
7h and 7r showed higher antifungal activity than most of substituted anilide analogues.
Further, in most of compounds, methyl group at pyrazole ring appeared to enhance the
activity against the fungal strain.
Drug likeness of synthesized imidazole derivatives 7a–7t
The pharmacokinetic parameters of amide linked pyrazolyimidazole hybrids were eval-
uated by using Lipinski’s rule of five (Table 3). These parameters act as filter for drug
likeness and indicate the physicochemical properties of synthesized compounds. It
includes absorption, distribution, metabolism and excretion from the human body.
ꢀ
The sum total of –OH and –NH groups (HBDH) showed that number of hydrogen
bond donor is less than 5.0 (1 for all). Also, the value of octanol-water partition coeffi-
cient (mi logP) of all synthesized derivatives is less than 5.00 (2.886–4.358) demonstrat-
ing their excellent permeability through the lipid bilayers of biological membrane and
high affinity. The molecular weight less than 500 for most of compounds showed easy
transportation, absorption and easy diffusion. In addition, the total number of hydrogen
bond acceptor (M_NO) are less than 10 for most of compound (except for 7c and 7 m).
The transport properties of biomolecules such as blood barrier penetration, intestinal
absorption etc. are predicted on the basis of total surface area values which should be
less than 160Å2 (64.74 Å2 to 156.38 Å2). The mathematical formula of %ABS is equal to
109-(0.345 x TPSA).[17] All the reported imidazole-amide conjugates show good absorp-
tion (%ABS ¼ 55.0489 to 86.6607).

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SYNTHETIC COMMUNICATIONSV
9
Figure 3. Compounds 7a and 7b interactions with active site residues of C. albicans sterol-14a-
demethylase.
It is evident from the above discussion that parameters of all of the tested compounds
were within the limits of Lipinski’s rule of five with violation number equal to zero for
most of the compounds except for 7c, 7m and have good drug likeliness property.
Docking simulation
Compared with other compounds, compound 7a and 7b had better antifungal activity in
in vitro results of the antifungal analysis. These compounds have been inserted in C. albi-
cans sterol-14 alpha-demethylase (PDB ID: 5TZ1) active site to determine binding integrity
and interactions. Figure 3 demonstrates the most favorable docked position of these com-
pounds in the active site. The pi electrons of phenyl ring of the compound 7a made pi-cat-
ion interactions with Lys90 and T-shaped pi-pi interactions with Tyr401. Phe233 residue
was stacked against the 1,3-imidazole ring while N-phenyl ring interacted with Met508
through pi-sulphur interactions. Other residues Ala61, Lue87, Leu88, Met92, Pro230 and
Val234 were engaged in other hydrophobic interactions with the different parts of the
compound 7a. In case compound 7b, phenyl ring made pi-pi stacked interactions with
Phe233 while imidazole ring was involved in T-shaped pi-pi interactions with Phe380 aro-
matic ring electrons. Leu87, Lys90, Tyr118, Pro230, Leu376 and Met 508 residues of the
enzyme created hydrophobic interactions with 7b. The binding affinity of the two com-
pounds was ꢁ10.8 and ꢁ10.7 kcal/mol respectively. This trend follows the experimental
observations and thus explaining the reason behind the activity difference. The protein
cartoon diagram including the mounted molecules is shown in Figure 4.
The compound 7b had better antibacterial activity in in vitro results of the antibacter-
ial analysis. This compound has been inserted in E. coli’s DNA gyrase (PDB ID: 1KZN)
active site to determine binding integrity and interactions. Figure 5 demonstrates the
docked position of this compound in the active site. The pi electrons of 1,2-imidazole
ꢀ
ring of the compound made pi-anion (3.84675 Å) interactions with Glu50. The peptide
ꢀ
bond between Asn46 and Ala47 (C … O … N, 5.09897 Å) formed an interaction with
ꢀ
the 1,3-imidazole ring while N-phenyl ring interacted with Ile90 (3.86114 A Å) through

10
S. PUNIA ET AL.
Figure 4. Compounds 7a (red) and 7b (yellow) in binding site of C. albicans sterol-14a-demethylase.
Figure 5. Compound 7b interactions with active site residues of E. coli DNA gyrase.
ꢀ
ꢀ
ꢀ
pi-sigma bond (3.86114 Å). Ala47 (5.07841 Å) and Ile78 (4.36772 Å) were involved in
hydrophobic interactions with the different rings. The protein cartoon diagram includ-
ing the mounted molecules and the co-crystallized CBN ligand is shown in Figure 6.
The docking study of ciprofloxacin showed that keto oxygen of quinolone ring made
hydrogen bonds with Arg136. Chlorophenyl ring interacited with Glu50 through pi-
anion interactions. Piprazine ring made carbon hydrogen bond with Asn46. Further,

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11
Figure 6. Compounds 7b (red) along with Clorobiocin (yellow) in binding site of E. coli DNA gyrase.
Figure 7. Fluconazole interactions with active site residues of C. albicans sterol-14a- demethylase.
Ile78, Pro79 and Ile90 anchored ciprofloxacin through hydrophobic interactions. It is
clear that our synthesized and docked molecule and ciprofloxacin share many amino
acid residues in their binding to the enzyme. The diagrammatic representation of bind-
ing site interactions of fluconazole and ciprofloxacin are given in Figures 7 and 8.
Conclusion
In conclusion, a new series of twenty pyrazolylimidazole hybrids 7a–7t with amide
functionality has been affordably synthesized by N-alkylation of 3a–3b with anilide
derivatives 6a–6j in good yields. The synthesized compounds exhibited good inhibitory
efficacy against C. albicans and A. niger and moderate activity against the bacterial

12
S. PUNIA ET AL.
Figure 8. Ciprofloxacin interactions with active site residues of E. coli DNA gyrase.
strains. The derivatives 7a (MIC, 0.0078 mmol/mL), 7b (MIC, 0.0084 mmol/mL), 7l
(MIC, 0.0079 mmol/mL), 7n (MIC, 0.0082 mmol/mL) and 7r (MIC, 0.0080 mmol/mL) dis-
played better inhibition of C. albicans. The derivative, 7b was found to show broad
spectrum of biological activity against the tested microbial strains. Inhibitory efficacy of
compound 7b was further supported using in silico docking analysis by calculating
binding energy of ligand in C. albicans sterol-14a-demethylase (PDB ID: 5TZ1) active
site (-10.7 kcal/mol). Further, antifungal activities of the prepared series were more pro-
lific than antibacterial activities. Moreover, ADME prediction of all compounds showed
good drug likeness properties.
Experimental
In the present study, chemicals were procured from Alfa-Aesar /Sigma Aldrich and
used as such without further purification. To detect the melting point of synthesized
compounds, open capillary method was used. Standard literature procedures were used
for drying the solvents. To perform thin layer chromatography (TLC), Pre-coated
Merck silica gel (SIL G/UV254, ALUGRAM) plates were used in ethyl acetate: hexane
mixture solvent system followed by observing spots under UV light (254 nm). The spec-
1
trum for H NMR (400 MHz) and 13C NMR (100 MHz) were taken in dimethylsulfox-
1
ide-d6 by Bruker Avance III. The chemical shifts of DMSO for H (d 2.50 ppm) and 13C
(d 39.50 ppm) were used as reference. Coupling constant values are given in Hertz.
1
Peaks splitting patterns in H NMR are reported as follow: s, singlet; bs, broad singlet;
d, doublet; bd, broad doublet; t, triplet; bt, broad triplet; m, multiplet. The IR spectra of

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SYNTHETIC COMMUNICATIONSV
13
synthesized compounds were recorded on Shimazdu IR Affinity-I using KBr powder as
a standard in the region of 4000–400 cmꢁ1. The high resolution mass spectra (HRMS)
were recorded by Mass spectrometer Esquire 3000 with ESI resource.
General method for synthesis of 1-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole (3a)
and 3,5-Dimethyl-1-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole (3b)
1H-pyrazole-1-carboximidamide hydrochloride (1a, 1 mmol) was dissolved in 10 mL of
the mixture of tetrahydrofuran and water (5:1) mL, and potassium carbonate
(2.5 mmol) was added into it in small portions. Then a solution of 2-bromo-1-phenyle-
thanone (2, 1.2 mmol) in tetrahydrofuran was constantly added drop wise via pressure
equalizing funnel over a period of 20 min while refluxing. The progress of reaction was
monitored by thin layer chromatography (TLC). After completion of reaction, crude
product was filtered and purified with the help of column chromatography (60–120
mesh silica, hexane/ethylacetate 90/10). Compound 3b was prepared by the same pro-
cedure from 3,5-dimethyl-1H-pyrazole-1-carboximidamide nitrate.
1-(4-Phenyl-1H-imidazol-2-yl)-1H-pyrazole 3a
1
ꢀ
White floppy solid; mp: 158–160 C; H NMR (400 MHz, DMSO): d ¼ 12.81 (s, 1H),
8.40 (bs, 1H), 7.82–7.83 (m, 2H), 7.80 (bs, 1H), 7.59 (s, 1H), 7.37 (t, J ¼ 7.6, Hz, 2H),
7.22 (t, J ¼ 7.6, 7.2 Hz, 1H), 6.58 (t, J ¼ 2.4, 2.0, 1H); 13C NMR (100 MHz, DMSO):
d ¼ 141.89, 141.77, 138.78, 134.56, 128.93, 128.42, 126.83, 124.70, 112.39, 108.25 ppm; IR
(KBr, max/cmꢁ1): 3445.30 (N-H str.); HRMS: m/z Cacld. [M þ H]þfor C12H10O4:
211.0978 founded 211.0975.
3,5-Dimethyl-1-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole 3b
1
ꢀ
Light Yellow solid; mp: 193–194 C; H NMR (400 MHz, DMSO): d ¼ 12.59 (s, 1H),
7.80 (d, J ¼ 7.6 Hz, 2H), 7.56 (bs, 1H), 7.36 (t, J ¼ 7.6 Hz, 2H), 7.21 (t, J ¼ 7.6, 7.2 Hz),
6.12 (s, 1H), 2.60 (s, 3H), 2.23 (s, 3H); 13C NMR (100 MHz, DMSO): d ¼ 149.41,
141.87, 140.86, 138.64, 134.83, 128.89, 126.66, 124.62, 111.78, 108.11, 13.71, 13.05 ppm;
IR (KBr, max/cmꢁ1): 3439.09 (N-H str.); HRMS: m/z Cacld. [M þ H]þfor C14H14N4:
239.1291 founded 239.1293.
General method for the preparation of 2-bromo-N-(substituted)acetamide (6a–6j)
Aromatic amine (4a–4j, 1 mmol) and 2-bromoacetyl bromide (5, 1.5 mmol) were stirred
ꢀ
with potassium carbonate in dichloromethane at 0–10 C for 3–4 h. After completion of
reaction, ice cold water was added to reaction mixture. Layer of dichloromethane was
separated and dried over anhydrous sodium sulfate. The excess of solvent was removed
under reduced pressure and the obtained compound was purified from chloroform.

14
S. PUNIA ET AL.
General method for the preparation of 1-(N-arylacetamide)-4-phenyl-2-pyrazolyl-
imidazole compounds (7a–7t)
The N-acetylamido imidazole derivatives, i.e., 7a–7t were synthesized by stirring the
corresponding pyrazolylimidazole 3 (1 mmol) with 2-bromo-N-(substituted)acetamide
(6, 1 mmol) and potassium carbonate (2.0 mmol), in the dimethylformamide at 35–50
ꢀC for 5–7 h. The workup of the reaction was done by pouring the reaction mixture
into cold water. The final product was filtered and dried over vacuum. The crude prod-
ucts were purified by crystallization in ethylacetate. All other derivatives were obtained
by the same procedure.
Antimicrobial activity
The antimicrobial activities of newly synthesized imidazole compounds were assessed in
vitro against bacterial and fungal strains as per the dilution method.[18] Ciprofloxacin
for antibacterial activity and Fluconazole for antifungal activity. Results were recorded
in terms of minimum inhibitory activity (MIC) expressed in mmol/mL in Tables 1
and 2.
Molinspiration calculations
The 2 D structures of the compounds were drawn online on the webpage of
Molinspiration (https://www.molinspiration.com/cgi-bin/properties). Then the properties
were calculated for each structure individually.
Molecular docking
The procedure for docking simulation was followed according to the protocols reported
by Kumar et al and the procedure[19] chosen in the present study. 3 D structure of the
molecules was made with Marvin Sketch 5.10 (Marvin sketch, 1998–2012)[20] which
were cleaned with gradient optimization. The X-ray crystallographic structure of E. coli
topoisomerase II DNA gyrase B was obtained from Brookhaven Protein Databank
(http://www.rcsb.org/pdb). Preparation of protein was accomplished with UCSF[21]
Chimera1.9. Incomplete side chains were completed with Dunbrack rotamer library[22]
and Gasteiger charges were assigned with Antechamber.[23] Structures of ligands and
proteins were transformed with the aid of AutoDock tools (AutoDock Tools, 2010).[24]
Results were pictured with the help of PyMol (PyMol, 2008)[25] and Discovery Studio
visualizer 3.5 (Discovery Studio Visualizer, 2005-2014).[26]
Acknowledgments
Authors are highly thankful to DST-PURSE (SR/PURSE Phase 2/40 (G)), New Delhi and Dr. APJ
Abdul Kalam CIL, GJUS&T, Hisar.

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