Structure based design, synthesis, and biological evaluation of imidazole derivatives targeting dihydropteroate synthase enzyme

Article abstract of DOI:10.1016/j.bmcl.2021.127819

In this study, we have designed and synthesized 2-((5-acetyl-1-(phenyl)-4-methyl-1H-imidazol-2-yl)thio)-N-(4-((benzyl)oxy)phenyl) acetamide derivatives. Antimicrobial activities of all the imidazole derivatives have been examined against Gram-positive and Gram-negative bacteria and results showed that the conjugates have appreciable antibacterial activity. Besides, several analogous were evaluated for their in vitro antiresistant bacterial strains such as Extended-spectrum beta-lactamases (ESBL), Vancomycin-resistant Enterococcus (VRE), and Methicillin-resistant Staphylococcus aureus (MRSA). The SAR revealed that the 12l compound resulted in potency against all bacterial strains as well as ESBL, VRE, and MRSA strains. Lipinski's rule of five, and ADME studies were preformed for all the synthesized compounds with Staphylococcus aureus dihydropteroate synthase (saDHPS) protein (PDB ID: 6CLV) and were found standard drug-likeness properties of conjugates. Moreover, the binding mode of the ligands with the protein study has been examined by molecular docking and results are quite promising. Besides, all the analogous were tested for their in vitro antituberculosis, antimalarial, and antioxidant activity.

Full text of DOI:10.1016/j.bmcl.2021.127819

Bioorg. Med. Chem. Lett. 36 (2021) 127819
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure based design, synthesis, and biological evaluation of imidazole
derivatives targeting dihydropteroate synthase enzyme
Drashti G. Darajia, Dhanji P. Rajanib, Smita D. Rajanib, Edwin A. Pithawalac,
,
*
Sivaraman Jayanthid, Hitesh D. Patela
a Department of Chemistry, School of Sciences, Gujarat University, Navarangpura, Gujarat, India
b Microcare Laboratory and TRC, Surat, India
c Microbiology and Biotechnology, Khyati Institute of Science, Palodia, Ahmedabad, Gujarat, India
d School of Bio Sciences and Technology, Vellore Institute of Technology, Vellore, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
In this study, we have designed and synthesized 2-((5-acetyl-1-(phenyl)-4-methyl-1H-imidazol-2-yl)thio)-N-(4-
((benzyl)oxy)phenyl) acetamide derivatives. Antimicrobial activities of all the imidazole derivatives have been
examined against Gram-positive and Gram-negative bacteria and results showed that the conjugates have
appreciable antibacterial activity. Besides, several analogous were evaluated for their in vitro antiresistant bac-
terial strains such as Extended-spectrum beta-lactamases (ESBL), Vancomycin-resistant Enterococcus (VRE), and
Methicillin-resistant Staphylococcus aureus (MRSA). The SAR revealed that the 12l compound resulted in potency
against all bacterial strains as well as ESBL, VRE, and MRSA strains. Lipinski’s rule of five, and ADME studies
were preformed for all the synthesized compounds with Staphylococcus aureus dihydropteroate synthase
(saDHPS) protein (PDB ID: 6CLV) and were found standard drug-likeness properties of conjugates. Moreover, the
binding mode of the ligands with the protein study has been examined by molecular docking and results are quite
promising. Besides, all the analogous were tested for their in vitro antituberculosis, antimalarial, and antioxidant
activity.
Imidazole
Antiresistant bacterial strains
saDHPS
Molecular docking
ADME
Infectious microbial disease remains a pressing crisis at worldwide,
due to inaccurate diagnosis and increasing use or abuse of antibacterial
agents as well as lack of development of new classes of antibacterial
drugs.1,2 Therefore, problems of multidrug-resistant (MDR) microor-
ganisms have become an alarming point in many countries around the
world.3–5 In drug-resistant microbes, vancomycin-resistant, MRSA, and
azole-resistant Candida species are familiar examples. Mostly, infections
treatment caused to complicate by these microbes especially in the case
of immune-compromised patients.6 Resistance can arise either through
the appearance of new strains that fall outside of compounds or the
attainment of specific mechanisms (target mutation, efflux, or drug
modifying enzymes). Among all the organisms, Staphylococcus aureus
displays extensive resistance profiles, specifically the MRSA.7,8
specific risk factors such as urine, lungs, bloodstream, and surgical sites,
while CA-MRSA happens in healthy individuals that do not have
affecting factors which primarily affects skin and skin structure in-
fections.10,11 MRSA resist all members of the β-lactam class of antibiotic,
sulfamethoxazole-trimethoprim (Bactrim)12 thereby disarming all pre-
vious mainstay treatment against Staphylococcus aureus.13 From the last
several years, VRE has emerged as a persistent nosocomial pathogen
with exaggeration of resistant genes from other bacteria even.14
Furthermore, recent studies suggest that targeting DHPS may be effec-
tive substitutes for the treatment of MRSA bacterial infections.15
Dihydropteroate synthase (DHPS) is recognized to be a validated
drug target to obstruct folate production in bacterial cells. For the
mechanism, DHPS specific enzyme catalyzes condensation between 7,8-
dihydropterine pyrophosphate (DHPP) and p-aminobenzoic acid
(PABA) to form tetrahydrofolate.16–20 Consequently, sulfonamide21,22
was competed with the main target of the natural substrate PABA to stop
the folate synthesis.23–27 Clinically speaking, the presence of PABA or
sulfa drugs binding site close to the flexible protein loops that are
Worldwide, MRSA has become one of the major health threats for the
last two decades. It has been projected that more than half percent of
Staphylococcus infections are due to MRSA.9 MRSA infections are clas-
sified into two major types, (i) hospital-acquired (HA) MRSA and (ii)
community-acquired (CA) MRSA. HA-MRSA occurs in patients due to
* Corresponding author.
E-mail address: drhiteshpatel1@gmail.com (H.D. Patel).
https://doi.org/10.1016/j.bmcl.2021.127819
Received 26 November 2020; Received in revised form 8 January 2021; Accepted 19 January 2021
Available online 26 January 2021
0960-894X/© 2021 Elsevier Ltd. All rights reserved.

D.G. Daraji et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127819
Scheme 1. Synthesis route of 2-((5-acetyl-1-(phenyl)-4-methyl-1H-imidazol-2-yl)thio)-N-(4-((benzyl)oxy)phenyl) acetamide derivatives.
agreeable and tolerant to mutations were the main drive toward this
bacterial resistance,28 but the predominant mechanism is mutation of
the folP gene29 that encodes DHPS.30 Traditionally, the sulfonamides
have been used for Gram-positive and Gram-negative bacterial in-
fections,21 combination with dihydropteroate reductase (DHFR) in-
hibitors such as trimethoprim which catalyzes an ensuing step in folate
synthesis.31,32
urgent need to develop new approaches that can overcome the problems
of microbial resistance.36 So, discovery and developing a new class of
antimicrobial agents are essential to fight against the increasing danger
of drug-resistant microbes.37,38 To discover new drugs, there are two
main strategies; either to search for novel lead compounds or to modify
the structure of a known drug.23
Using a structure-based approach, we have developed a novel lead
series of analogous that displays micromolar inhibition of MRSA, ESBL,
and VRE strains isolated from clinical samples. These compounds are
characterized by various analytical methods like 1HNMR, mass, and IR
spectroscopy. All conjugates were further tested against in vitro anti-
microbial, antimalarial, antituberculosis, and antioxidant activities. For
computational evaluation of all the analogous, analyses with Lipinski’s
rule of five (LRF), ADME and molecular docking study were exhibited.
We implemented a modest and efficient synthesis strategy to succeed
the title compounds (12a-12o) as depicted in Scheme 1 (Table 1). Initial
Since 1940s, sulfonamides were the first successful antimicrobial
agents and have been continuously used to treat a wide variety of bac-
terial infections such as Toxoplasma gondii encephalitis, Pneumocystis jir-
ovecii pneumonia, Staphylococcus aureus,33 and Shigellosis, and other
disease like protozoal infections, malaria, and tuberculosis.34 notwith-
standing, its widespread application but found limited use of sulfa drugs
against several infections attributable to rigorous immunological re-
actions and toxicity that causes fever, nausea, skin rashes, headache,
35
breathing trouble, vomiting, loss of appetite, etc…
For that reason,
2

D.G. Daraji et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127819
–
stretching vibrations of –NH and –C O, respectively noticeable the
Table 1
–
Synthesized
2-((5-acetyl-1-(phenyl)-4-methyl-1H-imidazol-2-yl)thio)-N-(4-
formation of amide via condensation of amine and chloroacetyl chlo-
ride. The IR spectrum of aromatic rings displayed peaks at 1546.10 and
((benzyl)oxy)phenyl) acetamide derivatives.
1474.60 cmꢀ 1 attributable to C C stretching. Moreover, the appear-
–
–
Compound
Code
R/R1
R2
Yield
(%)a
78
Rection time
M. P.
(oC)b
162.4
–
(h)
5.5
ance of a characteristic peak corresponding to C N stretching in the
region 1080–1360 cmꢀ 1; in the IR spectra of imidazole provided evi-
12a
4-F
4-
OCH3
4-F
4-Cl
4-Br
4-Cl
H
dence for the formation of imidazole ring. IR peak was observed at
12b
12c
12d
12e
12f
4-F
72
76
68
79
80
73
71
67
5.0
6.0
6.5
4.5
4.0
4.0
4.5
5.5
181.7
199.7
193.0
177.4
146.2
181.7
200.7
196.1
1162.51 cmꢀ 1 for C O of compound contain ether group and showed at
–
4-F
1664 cmꢀ 1 for C O of ketone group. Peak 1096.73 cmꢀ 1 observed due
–
–
4-OCH3
4-Cl
4-Cl
H
–
to the presence of C S group. The IR spectrum of substituted ring dis-
played peaks at 1012.27 and 760.71 cmꢀ 1 attributable to C–F and C–Cl
stretching band, respectively. The 1H NMR spectrum of compound 12c
peaks at; 8.924 ppm (s) showed the presence of –NH of CONH group.
Compound displayed two singlet peaks in the region of 4.5–5.5 ppm due
to the presence of two CH2 functional groups. Molecular weight of 12c is
523.11 g/mol (ChemDraw Ultra); which was confirmed by mass spec-
troscopy. It showed a molecular ion peak at m/z for 523.4 [M]+, 524.4
[M + 1]+, 525.4 [M + 2]+.
12g
12h
12i
H
H
4-F
4-
OCH3
12j
4-F
70
65
62
69
4.0
5.0
5.0
4.5
197.6
177.6
162.5
177.7
12k
12l
4-Br
All the synthesized compounds were tested for their antimicrobial
activity and the results presented in Table 2. Interestingly, some of the
imidazole derivatives displayed better antibacterial activity compared
to standards. It is worth mentioning that all molecules exhibited sig-
nificant activity against Pseudomonas aeruginosa and good to moderate
activity against Escherichia coli and Staphylococcus aureus strains. Com-
pounds 12a and 12l (12.5 µg/mL) showed more inhibition in Escherichia
coli as compared to standard drug ciprofloxacin and 12c (50 µg/mL)
being equipotent to reference antibiotic chloramphenicol. Analogous
12f, 12h, 12j, 12k, and 12m displayed equipotent to ampicillin against
Escherichia coli strain. Whereas Streptococcus pyogenes strain, analogous
12a, 12c, 12l, and 12f (12.5 µg/mL) were found to be more active than
ciprofloxacin and 12d, and 12g (50 µg/mL) equipotent to chloram-
phenicol. Among all the compounds, derivatives 12h, and 12j were not
found active and 12b, 12g, and 12l (62.5 µg/mL, 25 µg/mL, and 25 µg/
mL, respectively) disclosed significant activity against Staphylococcus
aureus strain. Analogous 12b bearing 4-F substitution on both aryl rings
showed less inhibition as compared to non-substituted 12g. Compounds
12f (62.5 µg/mL) and 12l (50 µg/mL) showed good activity against
Streptococcus pyogenes.
4-
OCH3
12m
4-F
12n
12o
4-Cl Ph
4-Cl Ph
4-Br
4-Cl
55
62
5.0
4.5
199.5
231.9
a: isolated yield.
b: melting point.
compounds
1-[1-(phenyl)-2-mercapto-4-methyl-1H-imidazol-5-yl]-
ethanone (11a-e) were obtained from substituted aniline, 3-chloro-2,4-
pentanedione, and potassium thiocyanate with good yield.39 Treatment
of p-nitro phenol (1) with benzyl chlorides (2a-d) in the presence of
potassium carbonate yielded 1-(benzyloxy)-4-nitrobenzene (3a-d). Re-
action between p-nitro phenol (1) and benzoyl chloride (7a-e) were
reacted using various catalysts such as potassium carbonate, triethyl
amine (TEA) and different solvents like DMF, acetone, THF provided
intermediate (8a-e). However, triethyl amine (TEA) as a catalyst and
THF as a solvent gave the good yield of compounds (8a-e). Reduction of
compounds (3a-d) or (8a-e) were performed using iron powder and
hydrochloric acid (HCl) at 60 ◦C. Consequently, we had to use
SnCl2⋅2H2O as a reducing agent for the synthesis of (8a-e) compounds at
room temperature due to the presence of ester group in compounds. All
the synthesized analogous were confirmed by the ninhydrin spray re-
agent on TLC. Finally, the following compounds (4a-d) or (9a-e) were
reacted with chloro acetyl chloride (5) in a catalytic amount of triethyl
amine (TEA) in THF to afford the N-(4-(benzyloxy)phenyl)-2-chlor-
oacetamide (6a-d) or 4-(2-chloroacetamido)phenyl benzoate (10a-e) in
good to excellent yield. The nucleophilic compound 1-[1-(phenyl)-2-
mercapto-4-methyl-1H-imidazol-5-yl]-ethanone (11a-e); sulfur group
reacted with compounds 6a-d or 10a-e with potassium carbonate as a
base catalyst to generate desire products (12a-o) in good yield. All the
title analogous were well purified by the crystallization method using
ethanol solvent.
Derivatives of compound 12a, and 12l showed better results than
other compounds, probably due to the presence of 4-OCH3 substitution
on aryl imidazole moiety. Similarly, compounds 12c and 12f, bearing 4-
Cl aryl chain showed good antibacterial activity. Series of compounds,
bearing benzyl chain displayed significant activity against antibacterial
activity compared to benzoyl contained analogous. In the bacterial ac-
tivity, 12l compound exposed the best active in all the bacterial strains.
Interestingly, compound 12l bearing 4-OCH3-aryl imidazole moiety,
revealed a prominent inhibition pattern ranging from potent to
considerable activity against the entire set of tested microorganisms. In
this series, compound 12j, bearing 4-F aryl imidazole moiety and furan
substituted chain, showed the least antibacterial activity against all
tested strains. Furthermore, analogous showed less active against anti-
fungal strains. Compounds 12i (250 µg/mL) derivatives bearing 4-
OCH3-aryl imidazole scaffold with furan side chain was the most active
against Candida albicans compared to griseofulvin. Analogous 12a, 12b,
12d, 12g, 12j, and 12m were exhibited equipotent to griseofulvin
against Candida albicans strain. Both side 4-Cl substituted 12o derivative
was displayed less active against all antifungal strains. SAR study clearly
showed that compounds bearing 4-OCH3-aryl imidazole moiety, i.e.
12a, 12l, and 12i displayed higher antimicrobial activity, probably due
to the presence of donating substituent. 4-F-aryl imidazole moiety is
more hydrophobic than other halo substituted groups. Comparison of
Synthesized
2-((5-acetyl-1-(phenyl)-4-methyl-1H-imidazol-2-yl)
thio)-N-(4-((benzyl)oxy)phenyl) acetamide compound was character-
ized by IR, 1H NMR, and mass spectroscopy after purification by crys-
tallization using ethanol solvent. The appearance of characteristic
absorption bands at 3294.21 and 1613.34 cmꢀ 1 attributed to the
3

D.G. Daraji et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127819
Table 2
Biological activity.
Compound Code
Antibacterial activity
MICa µg/mL
Antifungal activity
MFCb µg/mL
Antituberculosis activity
Antimalarial Activity
ECd
PAe
SAf
SPg
CAh
ANi
ACj
MICa µg/mL
ICc50 µg/mL
12a
12.5
125
50
25
100
62.5
100
250
100
250
25
100
125
100
125
100
62.5
125
250
125
250
250
50
500
500
1000
500
1000
>1000
500
1000
250
500
1000
1000
500
1000
>1000
–
500
1000
1000
>1000
>1000
500
1000
>1000
>1000
500
500
500
500
500
>1000
–
500
500
1000
1000
>1000
500
1000
>1000
>1000
500
500
500
250
500
>1000
–
50
100
62.5
250
25
12.5
500
62.5
62.5
250
25
25
100
12.5
125
–
1.40
0.82
0.73
1.25
0.56
1.45
0.93
0.45
0.78
0.36
1.18
0.45
1.14
1.04
0.46
–
12b
100
25
12c
12d
125
250
100
125
100
125
100
100
12.5
100
125
250
0.05
100
50
50
12e
125
25
12f
12g
50
12h
125
100
250
250
12.5
62.5
100
100
1
500
250
500
100
25
12i
12j
12k
12l
12m
125
250
125
0.25
250
50
125
100
100
0.5
100
50
12n
12o
Gentamicink
Ampicillink
Chloramphenicolk
Ciprofloxacink
Norfloxacink
Nystatink
Griseofulvink
Isoniazidk
Chloroquinek
Quininek
100
50
–
–
–
–
–
–
–
–
–
–
25
25
50
50
–
–
–
–
–
10
10
10
10
–
–
–
–
–
–
–
–
–
100
500
–
100
100
–
100
100
–
–
–
–
–
–
–
–
–
–
–
–
–
0.20
–
–
–
–
–
–
–
–
–
–
–
–
0.020
0.268
–
–
–
–
–
a: Minimum Inhibition Concentration.
b: Minimum Fungicidal Concentration.
c:Half maximal inhibitory concentration.
d: Escherichia coli (MTCC 443).
e: Pseudomonas aeruginosa (MTCC 1688).
f: Staphylococcus aureus (MTCC 96).
g: Streptococcus pyogenes (MTCC 442).
h: Candida albicans (MTCC 227).
i: Aspergillus niger (MTCC 282).
j: Aspergillus clavatus (MTCC 1323).
k: Standard drug.
Fig. 1. Graphical representations of antibacterial and antifungal activity, respectively.
4

D.G. Daraji et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127819
Fig. 2. Graphical representations of antibacterial and antifungal activity, respectively.
compound 12a with the MIC value 25 µg/mL and 12c, 12l with the MIC
value 62.5 µg/mL were found best active against ESBL strain. The
analogous 12b, 12d, 12g, 12h, 12j, 12k, 12m, and 12n exhibited
moderate antimicrobial activity against ESBL and VRE tested strains
with MIC values 100, 125, and 250 µg/mL. Subsequently, 12c derivative
(62.5 µg/mL) was demonstrated excellent activity and compounds 12a,
12f, and 12l (100 µg/mL) were good active against VRE strain. Besides,
MRSA tested strain was found less active as compared to other two
resisted bacterial strains. Compounds 12b, 12f, 12g, 12k, and 12l were
displayed good active for anti-MRSA. From all the tested compounds,
12l bearing 4-OCH3 aryl imidazole derivative found the best activity for
the all resistant bacterial strains where 12o, both side 4-Cl substitutions
were brought into being the lowest activity. In general, 4th position of
phenyl imidazole fragment increased the activity with the functional
group such as 4-OCH3 > 4-H > 4-Cl > 4-F > 4-Br. The resistant bacterial
strains SAR of all synthesized compounds were mentioned in Fig 3.
All the tested molecules were not showing better activity than the
standard drug to inhibit H37Rv strains in Table 2. All the synthesized
compounds were evaluated for their in vitro antimalarial activity against
plasmodium falciparum. All analogous were found to moderate activity
against plasmodium falciparum. Among all the compounds, analogous
12h, 12j, and 12l were exhibited good activity as compared with
reference drug Quinine (Table 2).
Table 3
Biological activity for resistant bacterial strains.
Compound Code
Resisted bacterial strain
Minimal Inhibition Concentration (µg/mL)
ESBLa
VREb
MRSAc
12a
12b
12c
12d
12e
12f
25
100
125
62.5
250
500
100
500
250
250
500
250
100
125
500
500
125
100
125
500
125
100
100
1000
500
1000
100
100
250
500
500
250
62.5
250
500
125
250
125
500
125
250
62.5
125
250
1000
12g
12h
12i
12j
12k
12l
12m
12n
12o
a = Extended spectrum beta-lactamases.
b = Vancomycin-resistant enterococci.
c = Methicillin-resistant Staphylococcus aureus.
All synthesized titled compounds were more explored for their in
vitro antioxidant property by 1,1-diphenylpicrylhydrazyl (DPPH), nitric
oxide (NO), and hydrogen peroxide (H2O2) radical scavenging assay
which was summarized in Table 4. Compounds 12h, and 12o showed
inhibitory radical scavenging activity in all three methods due to the
presence of mild electron-withdrawing groups. Though the DPPH
radical scavenging abilities of all the imidazole derivatives were
significantly lower than those of ascorbic acid (91.73 µg/ml), it was
antibacterial and antifungal activity of compounds with reference drugs
has been shown in Figs. 1 & 2.
In the primary study, all the synthesized compounds were found
good active against Escherichia coli and Staphylococcus aureus strains
then we had gone for further study of resisted bacterial strain against
ESBL, VRE, and MRSA. All the bacterial strains were listed in Table 3.
MIC values of all the targeted compounds were determined by the
above-mentioned method at a micro-care laboratory, Surat. Firstly,
Fig. 3. The resistant bacterial strains SAR of all synthesized compounds.
5

D.G. Daraji et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127819
Table 4
Antioxidant activity of synthesized compounds.
No*
DPPH method
NO method
H2O2 method
Concentration (µg/ml)
Concentration (µg/ml)
Concentration (µg/ml)
0
25
50
75
100
0
25
50
75
100
0
25
50
75
100
12a
12b
12c
12d
12e
12f
26.4
22.0
31.0
26.1
31.4
28.4
26.4
27.9
28.1
27.5
33.3
28.1
34.7
29.4
26.9
40.2
31.6
34.8
42.9
34.8
41.2
52.1
36.4
46.8
32.5
34.6
43.2
46.8
44.3
45.2
36.9
75.1
54.2
56.4
58.6
41.3
51.7
68.2
48.6
56.2
47.1
48.2
57.1
56.1
51.4
52.1
49.3
80.6
67.2
71.6
67.0
59.7
58.6
76
84.3
89.4
91.0
73.2
85.7
84.7
84.3
84.7
85.2
82.1
88.7
81.6
89.6
83.1
84.5
91.7
34.0
36.4
40.5
40.5
41.6
35.8
37.9
35.7
25.8
34.2
28.7
29.8
40.3
26.5
31.0
40.2
45.8
46.8
50.8
51.7
47.3
49.5
48.3
45.7
37.7
45.2
49.3
42.3
50.6
38.0
41.8
75.1
63.4
60.4
58.3
65.4
64.3
61.4
64.2
60.8
59.3
67.4
65.3
69.1
59.3
62.4
58.3
80.6
81.4
70.3
84.3
75.3
80.6
76.9
80.3
72.4
78.9
79.6
75.8
76.8
79.5
82.3
81.9
89.0
90.5
89.6
89.5
84.3
88.8
88.4
87.4
85.9
84.5
81.7
86.7
81.7
89.1
90.8
89.4
91.7
32.4
29.5
34.0
31.4
26.3
36.1
31.6
28
54
68.1
53.4
68.4
60.7
59.4
59.4
58.0
63.7
69.9
64.8
68.7
61.0
59.3
54.2
66.6
80.6
79.8
63.1
74.5
71.0
75.2
67.3
69.4
84.0
81.0
76.5
79.0
76.4
67.6
68.9
80
84.3
71.2
88.1
84.3
89.7
79.6
81.9
91.8
90.0
85.1
86.5
82.3
79.6
86.1
93.6
91.7
46.1
51.0
47.0
47.8
42.6
43.8
47.4
59.3
55.9
51.7
39.4
48.6
38.7
50.8
75.1
12g
12h
12i
60.0
62.3
66.2
66.7
64.8
68.1
65.4
65.2
64.9
89.0
39.2
43.1
32.8
27.3
39.0
28.0
38.0
40.2
12j
12k
12l
12m
12n
12o
Control
89.0
No*= Sample Code.
Std* = Ascorbic acid.
Fig. 4. Antioxidant activity of synthesized compounds.
evident that they show reducing ability. Among tested sample 12c
showed the most prominent scavenging capacity with 100 dilution
value. Also, the activity is dose-dependent to scavenge the radical. From
all tested compounds 12b, and 12m exhibited good antioxidant activity,
with 100 dilution values in the range of 89.44 and 89.64 µg/ml, while
100 dilution value of ascorbic acid was 91.73 µg/ml, respectively.
In NO radical scavenging method, compounds 12a, 12b, 12c, 12m,
12o, and 12n displayed high activity as compared with the standard
ascorbic acid. Compounds 12h, and 12o exhibited more potent activity
against hydrogen peroxide as compared to standard reference. On the
other hand, the compounds 12c, and 12e were less active. Similarly to
previous DPPH assay compound 12b, 12c, and 12m showed to be the
6

D.G. Daraji et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127819
antibacterial agents. The results summarized in Table 5 revealed that
compounds possessed the drug-like characteristics, as standard anti-
bacterial agents.
Table 5
In silico Lipinski’s rule of five properties of all compounds.
Compound code
MW
donorHB
accptHB
QPlogPo/
In silico, pharmacokinetics ADME parameters of all synthesized
compounds with saDHPS protein (PDB ID: 6CLV) are predicted using
Schrodinger software maestro 11.0 and summarized in Table 6. All the
titled compounds have good percent of oral absorptions. Compound
12g, 12h, and 12j have 100% oral absorption, which is greater than
reference drugs sulfametoxydiazine (69.708%) and Sulfasalazine
(58.943%). All the compounds and standard drugs were found good
results against the blood barrier potential, polar surface area, non-active
transport QPPCaco, and QPlogKhsa in the permissible range. The
aqueous solubility property was displayed moderate values of the
analogous. Compound 12i and reference drugs were showed good value
in the permissible range. All ADME parameters including absorption,
distribution, metabolism, and excretion were found to be favorable in
the acceptable range for all the derivatives and in some cases, even
better results than reference drugs were observed.
(130.0–725.0
gm/mol)
450.404
507.554
524.008
532.588
540.463
506.018
471.573
471.573
505.544
493.508
554.414
521.605
509.569
571.545
554.447
280.301
398.392
(<5)
(<10)
w (<5)
12a
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
9.7
2.191
6.017
6.499
4.147
6.923
6.43
12b
6.75
6.75
10.5
6.75
6.75
6.75
6.75
9.75
9
12c
12d
12e
12f
12g
6.043
6.043
4.048
4.396
4.779
4.757
4.874
5.831
5.697
6.023
2.467
12h
12i
12j
12k
9
12l
9.25
8.5
12m
12n
8.5
12o
8.5
sulfametoxydiazine
Sulfasalazine
9
8.25
To study the binding of the designed ligands to the receptor binding
site of saDHPS protein (PDB ID: 6CLV), a significant computational
method, viz. molecular docking, was performed using Schrodinger
maestro 11. All molecules 12a-12o were docked within the active site of
the receptor to evaluate the scoring functions and measure their mode of
interactions. During the analysis of the results, compound 12a showed
the highest interaction energy, i.e. binding energy of 12a was ꢀ 51.288
kcal/mol, whereas compound 12h displayed the lowest interaction en-
ergy, binding energy of 12h was ꢀ 38.000 kcal/mol. The rest of the
compounds showed moderate binding free energy. Here, synthesized
derivatives displayed the lowest binding free energy compared to the
reference drug. During in silico studies, Dock Score (D.S.), again sup-
ported a better interaction of designed ligands with the protein DHPS.
The designed imidazole derivatives showed significant to excellent dock
score value ranging from ꢀ 8.18 to ꢀ 3.50, whereas sulfametoxydiazine,
and Sulfasalazine exhibited dock score ꢀ 5.29, and ꢀ 4.843, respectively.
Molecular docking results were explained on the basis of hydrogen
bonding and non-covalent interactions, which stabilized the ligand–-
protein complex. Molecular docking results of all compounds with
saDHPS protein were shown in Table 7. Docking results showed that
compounds were accommodated well in the binding pocket of saDHPS.
Standard marketed drugs Sulfasalazine formed four H-bonds with amino
acids LYN203, ARG239, ARG219, and ARG204 and sulfametoxydiazine
formed two H-bonds with amino acids ASN103, LYN203. A close in-
spection of docking results revealed that CONH group of all designed
ligands formed 2 to 4-H bonds with amino acids. Compound 12l formed
H-bonds with amino acid ARG204, and GLY171; compound 12i with
MW: Molecular weight, donorHB: number of H bond donors,
accptHB: number of H bond acceptors, QPlogPo/w = log of the octanol–water
partition coefficient.
more potent as a scavenger of NO method. Meanwhile, 12l exhibited the
weakest scavenging capacities in all the radical scavenging activity.
Apart from this, the results also directed that radical scavenging activity
in all the three methods increases with increase in concentration. Three
methods of antioxidant activity were portrayed in graphically form in
Fig 4.
A good number of N-based heterocyclic imidazole derivatives have
been designed as probable antibacterial agents using in silico structure-
based approach. The physicochemical data of all compounds and stan-
dard antibacterial agents were calculated using Schrodinger software.
To find out the drug-like characteristics, molecules were evaluated using
Lipinski’s rule of five, which specifies that a probable drug molecule
should have <5 log P, <500 Dalton molecular weight, <10 hydrogen
bond acceptors and <5 hydrogen bond donor. As the rule of five
compliance ensures the bioavailability, the molecules in the designed
library were assumed to have better intestinal permeability. The pres-
ence of atoms allowed these molecules to function as H-bond acceptors
as well as H-bond donors. The lipophilicity of log P of compounds was
indicated that compounds should have no problem to passage through
cell membrane. Therefore, results revealed that none of the designed
ligand violated the rule of five and may be developed as potent drug-like
Table 6
ADME results of ligands with saDHPS receptor.
Compound code
PHOA (>80% high, < 25%
QPlogBB (ꢀ 3.0 to
1.2)
QPPCaco (<25 poor, >500
great)
QPlogKhsa (ꢀ 1.5 to
1.5)
PSA (70–200
Å)
QPlogS (ꢀ 6.5 to
0.5)
poor)
12a
90.965
89.86
94.301
95.01
96.773
94.125
100
ꢀ 0.988
ꢀ 0.846
ꢀ 0.708
ꢀ 0.717
ꢀ 0.691
ꢀ 0.557
ꢀ 0.758
ꢀ 0.942
ꢀ 1.592
ꢀ 1.397
ꢀ 1.261
ꢀ 1.418
ꢀ 1.308
ꢀ 0.988
ꢀ 1.112
ꢀ 1.374
ꢀ 2.659
1105.567
988.084
1.009
94.323
87.366
84.679
91.171
83.9
ꢀ 8.06
12b
0.99
ꢀ 8.045
ꢀ 8.681
ꢀ 8.101
ꢀ 9.403
ꢀ 7.442
ꢀ 7.883
ꢀ 7.707
ꢀ 6.248
ꢀ 6.87
12c
1217.029
1477.303
1214.874
1695.736
1875.59
1.124
12d
1.055
12e
1.253
12f
0.947
73.97
12g
0.989
80.343
85.165
132.157
123.277
121.11
121.655
115.073
110.324
112.624
106.593
152.039
12h
100
1024.378
338.73
0.948
12i
82.971
100
0.371
12j
392.507
0.462
12k
89.643
88.615
88.359
83.043
81.693
69.708
58.943
461.085
0.55
ꢀ 7.383
ꢀ 7.064
ꢀ 7.334
ꢀ 8.241
ꢀ 8.334
ꢀ 2.401
ꢀ 4.679
12l
410.418
0.596
12m
363.722
0.643
12n
507.723
0.866
12o
439.592
0.879
sulfametoxydiazine
Sulfasalazine
209.539
ꢀ 0.696
ꢀ 0.29
9.567
7

D.G. Daraji et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127819
Table 7
Docking results: molecular docking interaction of ligands with saDHPS receptor.
Compound code
Docking score
ꢀ 7.69
Gevdw
No. of H-B/Amino acid in H-B (distance Å)
No. of π-B/Amino acid in π-B
12a
12b
12c
12d
12e
12f
ꢀ 51.288
ꢀ 42.625
ꢀ 46.741
ꢀ 48.993
ꢀ 46.511
ꢀ 46.332
ꢀ 45.667
ꢀ 38.000
ꢀ 44.905
2/ARG204 (2.23), GLY171 (2.26)
4/ARG176, PHE172, ARG52, ARG204
5/ARG52, PHE172, PHE172, ARG204, ARG176
5/ARG52, PHE172, PHE172, ARG176, ARG204
1/ARG52
ꢀ 6.67
2/ARG204 (2.22), GLY171 (2.18)
ꢀ 6.54
3/ARG176 (1.86), ARG204 (2.20), GLY171 (2.37)
4/ARG204 (2.18), GLY171 (2.41), ASH84 (1.99), GLY54 (2.07)
3/ARG176 (2.06), ARG204 (2.18), GLY171 (2.39)
3/ARG176 (1.96), ARG204 (2.34), GLY171 (2.28)
3/ARG176 (1.98), ARG204 (2.32), GLY171 (2.28)
2/ARG176 (2.01), ARG204 (2.20)
ꢀ 7.43
ꢀ 6.52
4/ARG52, PHE172, ARG176, ARG204
3/ARG52, PHE172, PHE172
ꢀ 5.34
12g
12h
12i
ꢀ 3.50
3/ARG52, PHE172, PHE172
ꢀ 5.27
5/ARG204, ARG176, ARG52, ARG52, PHE172
6/ARG176, ARG204, ARG52, PHE172, ARG52,
PHE172
ꢀ 8.18
4/ARG176 (1.91), ARG204 (2.21), GLY171 (2.18), ARG239 (2.39)
12j
ꢀ 5.89
ꢀ 6.22
ꢀ 6.57
ꢀ 6.94
ꢀ 7.55
ꢀ 7.51
ꢀ 5.29
ꢀ 4.843
ꢀ 47.837
ꢀ 44.67
4/ARG176 (2.02), ARG204 (2.30), GLY171 (2.34), ARG239 (2.53)
2/GLY171 (2.22), ARG204 (2.27)
5/ARG176, ARG204, ARG52, PHE172, PHE172
4/PHE172, ARG52, ARG176, ARG204
4/ARG52, PHE172, ARG52, ARG204
5/ARG204, ARG176, ARG52, PHE172, PHE172
3/PHE172, PHE172, ARG52
12k
12l
ꢀ 49.217
ꢀ 44.471
ꢀ 49.926
ꢀ 47.158
ꢀ 35.694
ꢀ 30.136
2/ARG204 (2.41), GLY171 (2.12)
12m
2/ARG204 (2.25), ARG239 (2.59)
12n
3/GLY54 (1.99), ARG204 (2.18), GLY171 (2.60)
3/ARG176 (1.86), ARG204 (2.24), GLY171 (2.29)
2/ASN103 (2.04), LYN203 (2.12)
12o
4/ARG204, ARG176, ARG52, PHE176
3/ARG52, PHE172, PHE172
sulfametoxydiazine
Sulfasalazine
4/LYN203 (2.20), ARG239 (2.31), ARG219 (2.16), ARG204 (2.12)
1/ARG204
Fig. 5. a and b are 2D binding pose and c and d are 3D binding pose of compounds 12i and sulfasalazine, respectively (PDB ID: 6CLV).
ARG176, ARG204, GLY171, and ARG239.
six
and PHE172. Analogous 12l displayed 4
ARG52, PHE172, and ARG52. 12b, 12c, and 12m showed the same
interactions with ARG52, PHE172, PHE172, ARG204, and ARG176.
Compounds, additionally, showed two interactions with ARG176,
and ARG204, which was not observed in reference drugs. Molecular
π
-
π
interactions with ARG176, ARG204, ARG52, PHE172, ARG52,
Most notably, docking results revealed that all compounds interacted
π-π
interactions with ARG204,
with the active site of saDHPS enzyme and formed 2 to 6
π
-
π
interactions
interaction with
ARG204 amino acid while sulfametoxydiazine interacted with ARG52,
PHE172, and PHE172 to form interactions. Compound 12i showed
π-π
with amino acid residues. Sulfasalazine formed one π-π
π-π
π-π
8

D.G. Daraji et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127819
Fig. 6. (A) RMSD (B) Protein RMSF (C) Ligand RMSF and (D) Protein-Ligand Contacts of protein 6CLV-compound 12 l complex during the MD simulations of 100 ns.
docking interactions of some selected imidazole derivatives and the
reference drugs with the active site of saDHPS binding pocket are shown
in Fig 5.
For scrutinization of intermolecular H-bonding patterns in the
saDHPS-compound 12l complex was not displayed in the system. The
protein–ligand contacts during the simulation, a few H-bonds were
found during contacts; hence, ionic bonds were not shown in the com-
plex. Compound 12l was exhibited H-bond, hydrophobic, and water
bridge interaction in the stable region of ARG-52, and ARG-204. H-bond
found to be formed majorly with ARG-52, GLY-54, ALA-173, ARG-204,
and hydrophobic interaction was dominated by SER-16, PRO-53, PHE-
172, ARG-204 throughout the dynamic simulation. The residues of
ASP-84, GLN-105, GLU-179, and ARG-239 were not found any interac-
tion due to fluctuation (Fig 6D). A mean RMSD of 2.5 Å of compound
indicated good conformational modification; high polar solvent area
(PSA) (105–135 Å), solvent accessible surface area (SASA) (150–600),
and molecular surface area (MolSA) (420–460 Å) of the compound
during simulation time which is further supported its stabilization dur-
ing 100 ns molecular dynamic simulation (Fig. 7).
MD simulation offered to probe the behavioral dynamics of bio-
macromolecules including protein–ligand complex from nanometer to
micrometer timescales. In this study, the MD simulation of the
ligand–protein complex was considered to explore in detail of in-
teractions of ligand 12l with saDHPS enzyme (PDB ID: 6CLV) individ-
ually at a 100 ns. The Root Mean Square Deviation (RMSD) of the
enzyme backbone with rapidly increased up to 3.0 Å during the initial
15 ns (ns) then a relatively constant value of 3.0–3.5 Å for the rest of the
trajectory. Moreover, the Ligand RMSD of all system initially increased
up to 4.5 Å during 10 ns then stable up to 60 ns after that increased value
with fluctuation which ends at 6.0 Å (Fig. 6A). On this RMSF plot, peaks
indicated area of the protein that fluctuates the most during the simu-
lation. Typically, the plot was observed that the tails (N- and C-terminal)
fluctuated more than any other part of the protein. Secondary structure
elements like alpha helices and beta strands were usually more rigid
than the unstructured part of the protein, and thus fluctuate less than the
loop regions (Fig. 6B). Ligand RMSF showed the ligand’s fluctuations
broken down by atom, corresponding to the 2D structure in the top
panel. In the bottom panel, the ‘Fit Ligand on Protein’ line displayed the
ligand fluctuations, concerning the protein (Fig. 6C).
In summary, all the synthesized compounds with imidazole scaffold
were prepared and evaluated for their in vitro antimicrobial activity
including drug-resistant strains were assessed in this work. We have
synthesized novel compounds for saDHPS enzyme target, including
MRSA, ESBL, and VRE. Among all the compounds, compound 12l (MIC
= 62.5, 100, 100 µg/mL) exhibited broad-spectrum antibacterial activ-
ity against all ESBL, VRE, and MRSA strains, respectively. The SAR
9

D.G. Daraji et al.
Bioorganic & Medicinal Chemistry Letters 36 (2021) 127819
Fig. 7. Ligand properties of the protein–ligand complex during the MD simulations of 100 ns.
revealed that the substituent OCH3 at 4th position resulted in potency
against resistant bacterial strains. In addition, compound 12a (MIC = 25
µg/mL) as efficacious against ESBL strain. Meanwhile, in silico study
confirmed that all the compounds possessed good docking scores be-
tween ꢀ 8.18 to ꢀ 3.50, and found drug-likeness properties. Further-
more, all the compounds were tested for their in vitro antituberculosis,
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.127819.
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