Biology-oriented drug synthesis (BIODS): In vitro β-glucuronidase inhibitory and in silico studies on 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.11.031

Current study is based on the biology-oriented drug synthesis (BIODS) of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate derivatives 1–26, by treating metronidazole with different aryl and hetero-aryl carboxylic acids in the presence of 1,1'-carbonyl diimidazole (CDI) as a coupling agent. Structures of all synthetic derivatives were confirmed with the help of various spectroscopic techniques such as EI-MS,¹H -NMR and¹³C NMR. CHN elemental analyses were also found in agreement with the calculated values. Synthetic derivatives were evaluated to check their β-glucuronidase inhibitory activity which revealed that except few derivatives, all demonstrated good inhibition in the range of IC₅₀= 1.20 ± 0.01–60.30 ± 1.40 μM as compared to the standard D-saccharic acid 1,4-lactone (IC₅₀= 48.38 ± 1.05 μM). Compounds 1, 3, 4, 6, 9–19, and 21–24 were found to be potent analogs and showed superior activity than standard. Limited structure-activity relationship is suggested that the molecules having electron withdrawing groups like NO₂, F, Cl, and Br, were displayed better activity than the compounds with electron donating groups such as Me, OMe and BuO. To verify these interpretations, in silico study was also performed, a good correlation was observed between bioactivities and docking studies.

Full text of DOI:10.1016/j.ejmech.2016.11.031

Accepted Manuscript
Biology-oriented drug synthesis (BIODS): In vitro β-glucuronidase inhibitory and in
silico studies on 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate derivatives
Uzma Salar, Khalid Mohammed Khan, Muhammad Taha, Nor Hadiani Ismail,
Basharat Ali, Qurat-ul-Ain, Shahnaz Perveen, Mehreen Ghufran, Abdul Wadood
PII:
S0223-5234(16)30969-2
10.1016/j.ejmech.2016.11.031
EJMECH 9064
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 September 2016
Revised Date: 25 October 2016
Accepted Date: 13 November 2016
Please cite this article as: U. Salar, K.M. Khan, M. Taha, N.H. Ismail, B. Ali, Qurat-ul-Ain, S. Perveen,
M. Ghufran, A. Wadood, Biology-oriented drug synthesis (BIODS): In vitro β-glucuronidase inhibitory
and in silico studies on 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate derivatives, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.031.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Biology-Oriented Drug Synthesis (BIODS): In Vitro β-Glucuronidase Inhibitory and In
Silico Studies on 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate Derivatives
a
Uzma Salar,a Khalid Mohammed Khan, Muhammad Taha,b,c Nor Hadiani Ismail,b Basharat
Ali,a Qurat-ul-Ain,a Shahnaz Perveen,d Mehreen Ghufrane, Abdul Wadoode
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
bAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),
Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E., Malaysia
cFaculty of Applied Science, Universiti Teknologi MARA, Shah Alam 40450, Selangor D. E.,
Malaysia
dPCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
eDepartment of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University, Mardan, Pakistan
Abstract: Current study is based on the biology-oriented drug synthesis (BIODS) of 2-(2-
methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate derivatives 1-26, by treating
metronidazole with different aryl and hetero-aryl carboxylic acids in the presence of 1,1ꞌ-
carbonyl diimidazole (CDI) as a coupling agent. Structures of all synthetic derivatives were
1
13
confirmed with the help of various spectroscopic techniques such as EI-MS, H -NMR and C-
NMR. CHN elemental analyses were also found in agreement with the calculated values.
Synthetic derivatives were evaluated to check their β-glucuronidase inhibitory activity which
revealed that except few derivatives, all demonstrated good inhibition in the range of IC50 = 1.20
± 0.01-60.30 ± 1.40 ꢀM as compared to the standard
1.05 ꢀM). Compounds 1, 3, 4, 6, 9-19, and 21-24 were found to be potent analogs and showed
superior activity than standard Limited structure-activity relationship is suggested that the
D-saccharic acid 1,4-lactone (IC50 = 48.38 ±
.
molecules having electron withdrawing groups like NO2, F, Cl, and Br, were displayed better
activity than the compounds with electron donating groups such as Me, OMe and BuO. To verify
these interpretations, in silico study was also performed, a good correlation was observed
between bioactivities and docking studies.
Keywords: Synthesis; in vitro; β-glucuronidase; in silico; structure-activity relationship.
*Corresponding Author: khalid.khan@iccs.edu, hassaan2@super.net.pk, Tel.: 0092-21-34824910; Fax: 0092-21-
34819018
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Introduction
Nitroimidazole derived compounds have a diverse spectrum of biological activities [1]. Amongst
them, 5-nitroimidazole is the privileged scaffold of many marketed drugs such as secnidazole,
ketoconazole, miconazole, and metronidazole (Flagyl®) which are being clinically used to treat
the bacterial infections since many years (Figure-1). Toxicology and metabolism of
nitroimidazoles have been well characterized and documented [2-4]. Metronidazole is one of the
most important nitroimidazole derivatives which has been commonly used as antimicrobial
prescription [4-10].
Cl
O
Cl
Cl
O
O
N
N
O
N
Cl
N
O
N
Cl
N
Cl
Miconazole
Ketoconazole
N
N
NO2
N
H3C
NO2
OH
H3C
N
OH
Me
Metronidazole
Secnidazole
Figure-1: Marketed Analogs of Imidazole
β-Glucuronidase enzyme (EC 3.2.1.31) is commonly found in anaerobic bacteroides, Clostridia,
Escherichia, and Peptostreptococcus. It is also found in human body fluids, serum, blood cells,
gastric juice, bile, urine, spleen, and in organs like kidney, liver, lung, and muscles [11-13]. This
enzyme catalyzes the breaking of β-glucuronosyl-O-bonds [14]. Insufficiency of this enzyme
causes Sly syndrome in humans which is related to the increase level of glycosaminoglycans in
cells [15,16]. Similarly, over-expression is associated with many disorders such as urinary tract
infections [17-20], active pyelonephritis and acute renal necrosis [21]. Certain pathological
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conditions including epilepsy, cancer, acquired immune deficiency syndrome (AIDS), renal
diseases, neoplasm of breast, larynx, bladder, and testes, and inflammation in joints and hepatic
[22-24], also leads to over-expression of β-glucuronidase. Hence, it is the topmost task to inhibit
this enzyme for curing several pathologies.
Based on our previously introduced terminology biology-oriented drug synthesis (BIODS) [25]
which aims to design and synthesis of libraries of compounds on the skeleton of authentic drugs
(marketed drugs) with more and diversified biological activities.
We have already identified many heterocycles as potential class of β-glucuronidase inhibitors
[26-33]. Since, we have reported the β-glucuronidase inhibitory activity of benzimidazoles [32]
and recently published the ester derivatives of dihydropyrimidone as potent inhibitors of β-
glucuronidase enzyme [26], therefore, we decided to screen metronidazole esters for their β-
glucuronidase inhibitory activity due to their appreciable structural similarity with benzimidazole
as well as the esters of dihydropyrimidones (Figure-2). Herein, this manuscript describes the
BIODS of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate derivatives 1-26 and
evaluation of their β-glucuronidase inhibition studies. To the best of our information, these
compounds have never been reported for β-glucuronidase inhibitory activity.
N
O
R
Cl
Me
O2N
O
CH3
N
O
NH
N
O
Cl
Me
N
H
O
N
H
R
1-26
1-37
IC50 = 6.33 ± 0.40 µM
IC50 = 1.20 ± 0.01-60.30 ± 1.40
µ
M
IC50 = 9.38-78.16 µM
N
N
N
N
NO2
NO2
NO2
N
NO2
N
N
N
Me
Me
Me
Me
O
O
O
O
O
O
O
O
Br
Cl
Cl
F
Cl
Cl
F
F
Cl
16
15
11
12
IC50 = 6.30 ± 0.1 µM
IC50 = 1.20 ± 0.01
µM
IC50 = 3.60 ± 0.10 µM
IC50 = 6.20 ± 0.10 µM
Standard = -Saccharic acid 1,4-lactone (IC50 = 48.38 ± 1.05 µM)
D
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Figure-2: Rationale of the Current Study
Results and Discussion
Chemistry
All derivatives 1-26 were synthesized by reacting metronidazole with different aryl and hetero-
aryl carboxylic acid in the presence of 1,1ꞌ-carbonyl diimidazole (CDI) as a coupling agent.
Reactions were performed in tetrahydrofuran (THF) at room temperature. CDI was readily
reacted with the carboxylic acid derivative to form acid imidazole intermediate which was
immediately coupled with metronidazole to afford products 1-26. All synthetic molecules were
1
13
structurally characterized by spectroscopic techniques such as, EI-MS, H-NMR and C-NMR.
CHN analysis was also performed and found satisfactory.
Scheme-1: Syntheses of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl carboxylate derivatives
1-26
In Vitro β-Glucuronidase Inhibitory Activity
Synthetic compounds 1-26 were screened for their in vitro β-glucuronidase inhibitory activity. It
is worth mentioning that except compounds 2, 8, 25, and 26, all compounds were demonstrated
good inhibitory activity in the range of IC50 = 1.20 ± 0.01-60.30 ± 1.40 ꢀM as compared to the
standard D-saccharic acid 1,4-lactone (IC50 = 48.38 ± 1.05 ꢀM) (Table-1). Compounds 1, 3, 4, 6,
9-19 and 21-24 were exhibited superior β-glucuronidase inhibitory activity than standard as
given in Table-1.
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Table-1: In vitro β-glucuronidase inhibitory activity and docking scores of synthetic derivatives
1-26
N
H3C
NO2
O
N
O
R
1-26
IC50 ± SEMa [
µM]
R
Comp. No.
1
Docking Score (S)
Me
44.16 ± 0.90
NAb
-7.1356
-6.7176
-7.5236
2
3
35.50 ± 0.80
31.10 ± 0.75
60.16 ± 1.40
-7.6320
-7.7753
4
5
24.20 ± 0.50
60.30 ± 1.40
NAb
-8.0149
-7.7583
-6.5987
6
7
8
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16.30 ± 0.20
19.30 ± 0.30
-7.9033
-7.9216
9
10
3.60 ± 0.10
-9.3028
11
6.20 ± 0.10
8.10 ± 0.10
-8.6108
-8.4793
12
13
12.40 ± 0.20
6.30 ± 0.1
-7.9357
-9.0313
14
15
1.20 ± 0.01
27.60 ± 0.70
8.90 ± 0.20
-9.7108
-7.6861
-8.2156
16
17
18
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7.30 ± 0.10
48.50 ± 1.10
42.61 ± 0.90
-8.4376
-7.5368
-7.5297
19
20
21
2.10 ± 0.10
32.60 ± 0.80
8.40 ± 0.10
-9.6250
-7.5639
-8.6232
22
23
24
NAb
NAb
-7.3328
-7.2938
25
26
Standardc = D-saccharic acid 1,4-lactone
48.38 ± 1.05
aIC50 values (mean ± standard error of mean); bNA (Not active); cStandard inhibitor for β-glucuronidase.
Structure-Activity Relationship (SAR)
Limited structure-activity relationship (SAR) is suggested that apparently all structural features
of compounds 1-26 as shown in Figure-3 are taking part in the inhibitory activity, however, the
variation in the activity of different compound was due to the nature and position of substituents
at ring “R”.
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Figure-3: Structure of compound 16 with numbering
As shown in table-1, compound 20 (IC50 = 48.50 ± 1.10 ꢀM) with ortho bromo substitution at
ring “R”, showed inhibitory activity comparable to standard D-saccharic acid 1,4-lactone (IC50 =
48.38 ± 1.05 ꢀM). Switching of bromo residue from ortho to para as in compound 21 (IC50 =
42.61 ± 0.9 ꢀM), a slight increased activity was observed. It proves our hypothesis that the
position of a substituent also influence the activity. Structure of compound 15 (IC50 = 6.30 ± 0.1
ꢀM) is closely resembles to compound 20 (IC50 = 48.50 ± 1.10 ꢀM), only the addition of fluoro
group at para position of 15, displayed an eight-fold better inhibitory activity (Figure-4).
Figure-4: Comparison of structure-activity relationship of compound 21 with 20 and 15.
Amongst the chloro substituted derivatives, compounds 11 (IC50 = 3.60 ± 0.10 ꢀM) with dichloro
substituents ortho to each other, was found to be the third most potent and fifteen-fold more
active compound than the standard D-saccharic acid 1,4-lactone. Compound 12 (IC50 = 6.20 ±
0.10 ꢀM) is also a dichloro substituted derivative, but both chloro substitutions are para to each
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other, showed almost half inhibitory activity than compound 11 (Figure-5). It verifies that the
position of substituents highly effects the inhibitory potential. Mono chloro substituted
compounds such as 9 (IC50 = 16.30 ± 0.20 ꢀM) and 10 (IC50 = 19.30 ± 0.30 ꢀM) showed lesser
inhibitory activity than dichloro substitution 11 and 12 as shown in Figure-5.
Figure-5: Comparison of structure-activity relationship of compound 11 with 12, 9, and 10.
It is worth mentioning that compound 16 (IC50 = 1.20 ± 0.01 ꢀM), the most potent compound of
this series and distinctly similar in structure to compound 10 (IC50 = 19.30 ± 0.30 ꢀM), but the
presence of two fluoro substituents were greatly enhanced the activity of compound 16. Amongst
the mono-fluorinated compounds, compound 13 (IC50 = 8.10 ± 0.10 ꢀM) which has fluoro at
meta position was more active than its para counterpart 14 (IC50 = 12.40 ± 0.20 ꢀM) (Figure-6).
Figure-6: Comparison of structure-activity relationship of compound 16 with 15, 14, and 13.
More critically, it was monitored that halogens were played vital roles in the inhibitory potential,
especially, fluoro groups were enhanced the inhibitory activity.
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Compounds containing strong electron withdrawing nitro group such as 17-19 were found to be
potent inhibitors too. Amongst them, compound 19 (IC50 = 7.30 ± 0.10 ꢀM) having 3-methyl-4-
nitro substitutions at “R” was found to be one of the most potent compounds, about seven fold
more active than standard. Absence of methyl group as in analog 18 (IC50 = 8.90 ± 0.20 ꢀM),
displayed slightly lowered activity. Similarly, switching of nitro group from para to meta as in
compound 17 (IC50 = 27.60 ± 0.70 ꢀM) sharply declined the activity. It showed that nitro group
at para position played a vital role in enhancing the inhibitory activity (Figure-7).
Figure-7: Comparison of structure-activity relationship of compound 19 with 18, and 17.
Compounds 22-24 having heterocyclic ring as “R” were displayed potent inhibition than
standard. Out of them, compound 22 (IC50 = 2.10 ± 0.10 ꢀM) having indole ring was found to be
the potent derivative and demonstrated twenty-four times more activity than standard.
Compound 24 (IC50 = 8.40 ± 0.10 ꢀM) having benzothiazole ring as “R”, was exhibited six times
better potential than standard. It was observed that both compounds 22 and 24 have bicyclic
aromatic rings as “R”, which might involve in strong π-π interactions with the active site of
enzyme, however, the lesser activity of compound 23 (IC50 = 32.60 ± 0.80 ꢀM) with pyridine
ring due to comparatively weak π-π interactions with active site as compared to compounds 22
and 24 (Figure-8).
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Figure-8: Comparison of structure-activity relationship of compound 22 with 24, and 23.
Compounds 1, 3, and 6 having electron donating methyl substituents were also demonstrated
better activity, but lesser than the compounds having electron withdrawing groups. Out of them,
compound 1 (IC50 = 44.16 ± 0.90 ꢀM) having methyl at ortho position showed good inhibition.
Switching of methyl group from ortho to meta as in compound 2, leads to complete loss of
activity, however, from ortho to para as in compound 3 (IC50 = 35.50 ± 0.80 ꢀM), brings further
better inhibitory activity. Compound 6 (IC50 = 24.20 ± 0.50 ꢀM) having dimethyl substituents at
meta positions, showed much better activity than mono substituted compounds (Figure-9).
N
N
N
N
NO2
O
NO2
O
NO2
O
NO2
N
N
N
N
Me
Me
Me
Me
O
O
O
O
O
Me
Me
Me
Me
Me
1
2
3
6
IC50 = 44.16 ± 0.90 µM
Not Active
IC50 = 35.50 ± 0.80 µM
IC50 = 24.20 ± 0.50 µM
Figure-9: Comparison of structure-activity relationship of compound 1 with 2, 3, and 6.
Methoxy containing derivatives 4, 5, and 7 demonstrated good to moderate inhibition. Such as
compound 4 (IC50 = 31.10 ± 0.75 ꢀM) was found to be good inhibitor as compared to standard.
Its activity can compare with compound 5 (IC50 = 60.16 ± 1.40 ꢀM) which only lacks the olefin
moiety, a sharp decline in the activity was observed. Decreased activity of compound 5 was
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might be due to the lesser π-π interactions. Compound 7 (IC50 = 60.30 ± 1.40 ꢀM) having
dimethoxy groups at meta positions, also demonstrated moderate inhibition, however, para-
butoxy derivative 8 was found to be completely inactive (Figure-10).
N
N
NO2
O
N
N
NO2
O
NO2
O
NO2
O
N
Me
N
N
N
Me
Me
Me
O
O
O
O
OMe
MeO
OMe
OBu
OMe
4
5
7
8
IC50 = 31.10 ± 0.75 µM
IC50 = 60.16 ± 1.40 µM
IC50 = 60.30 ± 1.40 µM
Not Active
Figure-10: Comparison of structure-activity relationship of compound 4 with 5, 7, and 8.
In a nut shell, careful assessment of the influence of groups on activity is revealed that
compounds having electron withdrawing groups were found to be more potent than the
compounds having electron donating groups except few (Table-1). However, in order to get in-
depth insights regarding the SAR as well as molecular interactions of compounds with the active
sides of β-glucuronidase enzyme, in silico studies were also performed on all compounds and
discussed in the next section.
In Silico Studies
p-Nitro phenyl β-D-glucuronide, the known substrate molecule [34], was docked into the active
site of the enzyme using MOE-Dock program prior to docking of the synthetic molecules.
Similar to our previous results [27,33], the modeled three dimensional substrate-bound structure
of human β-D-glucuronidase was displayed that the glycoside bond of p-nitrophenyl β-D-
glucuronide was properly oriented towards the catalytic residues such as Glu540, Glu451, and
Tyr504 (Figure-11). The substrate molecule was demonstrated appropriate binding with the
active site residues; Asp207, Glu451, Glu540, His385, Tyr508, Tyr504 and Arg600 [30]. This
3D protein structure was used in the assessment of the binding mode of the synthetic derivatives.
The docking of the compounds was carried out via MOE with the parameters i.e., Placement:
Triangle Matcher, Rescoring: London dG. For each ligand, ten conformations were generated
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and on the basis of docking score (S) the top-ranked conformation of each compound was used
for further analysis. The docking score is the binding free energy calculated by the GBVI/WSA
scoring function and the lower score indicates more favorable poses. The unit for all scoring
functions is kcal/mol [35].
The docking study predicted that the compounds with electron withdrawing substituents i.e. F,
Cl, Br, NO2 showed good in silico inhibition. It was observed that the number of these
substituents and their respective positions may affect the orientation and binding pattern of the
compound in the active site of the enzyme. For example, compound 22 with 1H-indole group
showed the best interactions mode (Figure-12) among these synthetic compounds. Asp207,
His385, and Asn450 were interacted through a polar bond with the imidazole moiety. Arg600
was showed arene-cation contacts with 2-methyl-1H-indole residue.
Figure-11: Molecular interactions of substrate with human β-glucuronidase.
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Figure-12: Molecular interactions of compound 22.
Similarly, compound 11 with dichlorobenzene (meta-para dichloro) showed three interactions
with the active site residues Tyr508 and Arg600 as shown in Figure-13. Changing the positions
of chlorine groups in compound 12 i.e. ortho and opposite meta, resulted in mild interaction
mode which was in accordance to its biological activity. The docking study was predicted almost
the same behavior for the different halogen groups as experienced in biological evaluations.
However, some contrast behavior was observed for the most active compound 16 (IC50 = 1.20 ±
0.01 µM) in the docking study. This compound was unpredictably showed two hydrogen
bonding with Thr599 and Arg600 (Figure-14). The contact i.e. hydrogen bonding with Arg600 is
shown by compounds 14, 15 and 19. Whereas, compound 24 showed arene-cation interaction
with Arg600.
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Figure-13: Molecular interactions of compound 11.
Figure-14: Molecular interactions of compound 16.
Compound 13 with fluorine at meta position of ring “R” showed good mode of interactions with
Asn484 and Arg600. Asn484 was formed hydrogen bonding with the N of imidazole group and
basic Arg600 made hydrogen bonding with the carbonyl oxygen (Figure-15). Nitro substituted
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compound 18 was also showed a single arene-arene contact with Tyr508 through nitrobenzene
substituent. In case of non-active/poor-active compounds and the compounds with methyl (Me)
or methoxy (MeO) substituents, no valuable binding network with the enzyme was observed.
Figure-15: Molecular interaction of compound 13.
Overall a good correlation was observed between the docking study and biological evaluation of
active compounds. The correlation graph and the correlation coefficient value are given in
Figure-16.
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Figure-16: A correlation graph for docking predicted activity and IC50 values.
Conclusion
β-Glucuronidase inhibitory activity of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl aryl
carboxylate derivatives 1-26 was examined which revealed that almost all compounds were
demonstrated potent inhibition except few. Limited structure-activity relationship and in silico
study rationalized that compounds having electron withdrawing groups such as NO2, F, Cl, and
Br were more prone to inhibit the β-glucuronidase enzyme as compared to the compounds
having electron donating groups e.g. Me, OMe and BuO. This study identified a set of lead
compounds for further research in order to get good β-glucuronidase inhibitors.
Experimental
Material and Methods
Reagents were purchased from Sigma-Aldrich, USA. All reagents and solvents were of
analytical grade and used as received. Thin layer chromatography was performed on pre-coated
silica gel, GF-254 (Merck, Germany). Spots were visualized under ultraviolet light at 254 and
366 nm or iodine vapors. Mass spectra were recorded under electron impact (EI) on MAT 312
1
and MAT 113D mass spectrometers. The H, 13CNMR were recorded on Bruker AM
spectrometers, operating at 300, 400 and 500 MHz. The chemical shift are presented in ppm (δ),
relative to tetramethylsilane (TMS) as an internal standard and the coupling constant (J) are in
Hz. Elemental analysis was performed on a Carlo Erba Strumentazione-Mod-1106, Italy.
General Experimental Procedure for the Synthesis of Compounds 1-26
Carboxylic acids (1 mmol) and 1,1ꞌ-carbonyl diimidazole (1 mmol) were taken in THF (15 mL)
in a round-bottommed flask (100 mL) and stirred for 30 minutes in order to activate the
carboxylic acids. Then metronidazole (1 mmol) was added into the reaction mixture with
constant stirring for 24 h. Reaction progress was monitored by TLC (6:4 = EtOAc:Hexane).
Reaction mixture was poured onto crushed ice (100 mL), precipitates appeared immediately
which were filtered and dried in air. The precipitates were crystallized from ethanol. Products
1
were characterized by spectroscopic techniques such as EIMS, H-NMR and 13C-NMR. CHN
analysis was also performed.
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β-Glucuronidase Inhibitory Assay
Bioassay protocol was used according to literature report [36]. β-Glucuronidase enzyme (E.C.
3.2.1.31), from bovine liver (G-0251), and p-nitrophenyl-β-D-glucuronide (N-1627) were
obtained from Sigma-Aldrich USA.
β-Glucuronidase activity was determined spectrophotometrically by quantifying the absorbance
at 405 nm of p-nitrophenol formed during the reaction. The total reaction volume was 250 µL.
DMSO (100%) was used to dissolve the compound (5 µL) which become 2% in the final assay
(250 µL) and the same conditions were used for standard (D-saccharic acid 1,4-lactone). The
reaction mixture, contained 0.1 M acetate buffer (185 µL), test compound solution (5 µL), and
°
enzyme solution (10 µL), was incubated at 37 C for 30 minutes. The plates were read on a
multiplate reader (SpectraMax plus 384, USA) at 405 nm after the addition of 50 µL of 0.4 mM
p-nitrophenyl-β-D-glucuronide. All assays were run in triplicate.
Molecular Docking Study
The molecular docking program is widely used to predict the binding interaction of the
compounds in the binding pocket of the enzyme. The crystal structure of the human β-
glucuronidase was obtained from the protein databank (PDB ID: 1BHG) [37] having 80%
sequence similarity with bovine β-D-glucuronidase [30] as the 3D structure of bovine β-D-
glucuronidase is not reported yet. From the original protein data bank file, the B-chain of protein
and hetero-atoms including cofactors were removed. Using MOE (Molecular Operating
Environment) software (www.chemcomp.com) the retrieved protein molecule was 3D
protonated and the energy minimization was carried out using the default parameters of MOE
[the Amber99 force field and gradient: 0.05]. The structures of the synthetic compounds 1-26
were built in MOE and were energy minimized.
Spectral Analysis of Compounds (1-26)
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-methylbenzoate (1)
Solid; Yield: 73%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.04 (s, 1H, H-4), 7.67 (d, J6′,5′ = 7.6 Hz,
1H, H-6′), 7.49 (t, J5′(4′,6′) = 7.2 Hz, 1H, H-5′), 7.31 (m, 2H, H-3′, H-4′), 4.72 (t, J2′′,1′′ = 5.2 Hz,
2H, CH2-2ʹʹ), 4.60 (t, J1′′,2′′ = 5.2 Hz, 2H, CH2-1ʹʹ), 2.41 (s, 3H, CH3), 2.40 (s, 3H, CH3); 13C
18

ACCEPTED MANUSCRIPT
NMR (75 MHz, DMSO-d6):
δ 167.6, 151.6, 139.5, 138.3, 132.7, 132.3, 131.1, 129.9, 129.7,
125.5, 62.4, 40.6, 21.6, 12.8; EI-MS: m/z (rel. abund. %), 288 (M+, 18), 272 (25), 260 (7), 243
(15), 199 (4), 177 (19), 163 (33), 149 (15), 138 (21), 119 (100); Anal. Calcd for C14H15N3O4: C =
58.13, H = 5.23, N = 14.53; Found: C = 58.16, H = 5.25, N = 14.51.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-methylbenzoate (2)
Solid; Yield: 78%; 1H-NMR: (300 MHz, DMSO-d6): δ 8.04 (s, 1H, H-4), 7.63 (s, 1H, H-2′), 7.63
(d, J6′,5′ = 8.1 Hz, 1H, H-6′), 7.47 (d, J4′,5′ = 7.2 Hz, 1H, H-4′), 7.41 (t, J5′(4′,6′) = 7.2 Hz, 1H, H-5′),
4.74 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.62 (t, J1′′,2′′ = 5.1 Hz, 2H, CH2-1ʹʹ), 2.44 (s, 3H, CH3), 2.34
(s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
δ 165.9, 151.5, 138.6, 138.4, 133.4, 132.2, 130.4,
130.0, 128.6, 126.8, 62.4, 40.7, 21.4, 12.7; EI-MS: m/z (rel. abund. %), 289 (M+, 5), 243 (23),
163 (35), 136 (5), 119 (100), 91 (69); Anal. Calcd for C14H15N3O4: C = 58.13, H = 5.23, N =
14.53; Found: C = 58.16, H = 5.25, N = 14.51.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3,5-dimethylbenzoate (3)
1
Solid; Yield: 79%; H-NMR: (400 MHz, DMSO-d6): δ 8.03 (s, 1H, H-4), 7.43 (s, 2H, H-2′, H-
6′), 7.28 (s, 1H, H-4′), 4.73 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.60 (t, J1′′,2′′ = 4.8 Hz, 2H, CH2-1ʹʹ),
13
2.44 (s, 3H, CH3), 2.30 (s, 6H, 2CH3); C NMR (75 MHz, DMSO-d6):
δ 165.8, 151.6, 138.5,
138.3, 138.3, 135.4, 132.1, 129.7, 127.0, 127.0, 62.4, 40.7, 21.5, 21.5, 12.8; EI-MS: m/z (rel.
abund. %), 303 (M+, 8), 257 (17), 191 (3), 176 (21), 149 (3), 133 (100); Anal. Calcd for
C15H17N3O4: C = 59.40, H = 5.65, N = 13.85; Found: C = 59.43, H = 5.62, N = 13.88.
(E)-2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(4-methoxyphenyl)acrylate (4)
1
Solid; Yield: 78%; H-NMR: (400 MHz, DMSO-d6): δ 8.03 (s, 1H, H-4), 7.66 (d, J2′,3′/6′,5′ = 8.8
Hz, 2H, H-2′, H-6′), 7.54 (d, J1′′′,2′′′ = 16.0 Hz, 1H, H-1′′′), 6.98 (d, J3′,2′/5′,6′ = 8.8 Hz, 2H, H-3′, H-
5′), 6.42 (d, J2′′′,1′′′ = 16.0 Hz, 1H, H-2′′′), 4.64 (t, J2′′,1′′ = 5.2 Hz, 2H, CH2-2ʹʹ), 4.49 (t, J1′′,2′′ = 5.2
Hz, 2H, CH2-1ʹʹ), 3.79 (s, 3H, OCH3), 2.47 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
δ
166.4, 159.7, 151.6, 145.2, 138.5, 132.3, 130.3, 130.3, 127.6, 116.3, 114.0, 114.0, 62.5, 55.7,
40.6, 12.7; EI-MS: m/z (rel. abund. %), 331 (M+, 32), 285 (48), 244 (15), 216 (4), 205 (21), 161
(100); Anal. Calcd for C16H17N3O5: C = 58.00, H = 5.17, N = 12.68; Found: C = 58.02, H = 5.15,
N = 12.65.
19

ACCEPTED MANUSCRIPT
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-methoxybenzoate (5)
1
Solid; Yield: 82%; H-NMR: (400 MHz, DMSO-d6): δ 8.02 (s, 1H, H-4), 7.79 (d, J2′,3′/6′,5′ = 8.4
Hz, 2H, H-2′, H-6′), 7.03 (d, J3′,2′/5′,6′ = 7.2 Hz, 2H, H-3′, H-5′), 4.71 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-
13
2ʹʹ), 4.60 (t, J1′′,2′′ = 4.8 Hz, 2H, CH2-1ʹʹ), 3.82 (s, 3H, OCH3), 2.44 (s, 3H, CH3); C NMR (75
MHz, DMSO-d6):
δ 165.8, 164.6, 151.5, 138.5, 132.3, 130.8, 130.8, 122.3, 114.4, 114.4, 62.5,
55.7, 40.7, 12.6; EI-MS: m/z (rel. abund. %), 305 (M+, 25), 259 (100), 218 (8), 179 (68), 152
(32), 135 (100); Anal. Calcd for C14H15N3O5: C = 55.08, H = 4.95, N = 13.76; Found: C = 55.05,
H = 4.97, N = 13.79.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-methylbenzoate (6)
1
Solid; Yield: 76%; H-NMR: (400 MHz, DMSO-d6): δ 8.03 (s, 1H, H-4), 7.73 (d, J2′,3′/6′,5′ = 8.0
Hz, 2H, H-2′, H-6′), 7.32 (d, J3′,2′/5′,6′ = 7.6 Hz, 2H, H-3′, H-5′), 4.73 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-
13
2ʹʹ), 4.62 (t, J1′′,2′′ = 4.8 Hz, 2H, CH2-1ʹʹ), 2.44 (s, 3H, CH3), 2.36 (s, 3H, CH3); C NMR (75
MHz, DMSO-d6):
δ 165.9, 151.6, 142.9, 138.6, 132.3, 129.7, 129.7, 128.6, 128.6, 127.2, 62.4,
40.6, 21.4, 12.8; EI-MS: m/z (rel. abund. %), 289 (M+, 8), 243 (79), 217 (11), 173 (32), 163 (39),
119 (100), 91 (41); Anal. Calcd for C14H15N3O4: C = 58.13, H = 5.23, N = 14.53; Found: C =
58.16, H = 5.25, N = 14.51.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3,5-dimethoxybenzoate (7)
1
Solid; Yield: 80%; H-NMR: (400 MHz, DMSO-d6): δ 8.04 (s, 1H, H-4), 6.92 (d, J2′,4′/6′,4′ = 2.4
Hz, 2H, H-2′, H-6′), 6.76 (s, 1H, H-4′), 4.75 (t, J2′′,1′′ = 5.2 Hz, 2H, CH2-2ʹʹ), 4.61 (t, J1′′,2′′ = 5.2
13
Hz, 2H, CH2-1ʹʹ), 3.77 (s, 6H, 2OCH3), 2.49 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6):
δ
165.8, 161.4, 161.4, 151.8, 138.5, 132.3, 108.8, 106.4, 106.4, 104.5, 62.5, 55.6, 55.6, 40.7, 12.7;
EI-MS: m/z (rel. abund. %), 335 (M+, 42), 318 (45), 305 (4), 289 (8), 209 (30), 165 (100), 151
(20), 137 (48); Anal. Calcd for C15H17N3O6: C = 53.73, H = 5.11, N = 12.53; Found: C = 53.76,
H = 5.14, N = 12.55.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-butoxybenzoate (8)
1
Solid; Yield: 77%; H-NMR: (400 MHz, DMSO-d6): δ 8.03 (s, 1H, H-4), 7.78 (d, J2′,3′/6′,5′ = 8.8
Hz, 2H, H-2′, H-6′), 7.02 (d, J3′,2′/5′,6′ = 8.8 Hz, 2H, H-3′, H-5′), 4.70 (t, J2′′,1′′ = 5.2 Hz, 2H, CH2-
2ʹʹ), 4.60 (t, J1′′,2′′ = 6.4 Hz, 2H, CH2-1ʹʹ), 4.05 (t, 2H, CH2), 2.44 (s, 3H, CH3), 1.71 (m, 2H, CH2),
20

ACCEPTED MANUSCRIPT
13
1.45 (m, 2H, CH2), 0.94 (t, 3H, CH3); C NMR (75 MHz, DMSO-d6):
δ 165.8, 163.6, 151.8,
138.3, 132.3, 130.6, 130.6, 121.8, 114.4, 114.4, 68.3, 62.5, 40.7, 31.9, 19.2, 12.7, 14.0; EI-MS:
m/z (rel. abund. %), 347 (M+, 6), 301 (91), 245 (23), 221 (10), 177 (100), 165 (33), 138 (20), 121
(100); Anal. Calcd for C17H21N3O5: C = 58.78, H = 6.09, N = 12.10; Found: C = 58.76, H = 6.07,
N = 12.13.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-chlorobenzoate (9)
Solid; Yield: 82%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.03 (s, 1H, H-4), 7.79 (s, 1H, H-2′), 7.79
(d, J4′,5′ = 6.8 Hz, 1H, H-4′), 7.75 (d, J6′,5′ = 8.0 Hz, 1H, H-6′), 7.58 (t, J5′(4′,6′) = 8.0 Hz, 1H, H-5′),
4.75 (t, J2′′,1′′ = 5.2 Hz, 2H, CH2-2ʹʹ), 4.64 (t, J1′′,2′′ = 5.2 Hz, 2H, CH2-1ʹʹ), 2.49 (s, 3H, CH3); 13C
NMR (75 MHz, DMSO-d6):
δ 165.7, 151.8, 138.3, 134.6, 134.3, 133.2, 132.0, 130.2, 129.7,
128.1, 62.5, 40.6, 12.8; EI-MS: m/z (rel. abund. %), 309 (M+, 4), 311 (M+2, 2), 263 (19), 183
(31), 139 (100), 111 (19); Anal. Calcd for C13H12ClN3O4: C = 50.42, H = 3.91, N = 13.57;
Found: C = 50.45, H = 3.93, N = 13.54.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-chlorobenzoate (10)
Solid; Yield: 78%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.04 (s, 1H, H-4), 7.68 (d, J6′,5′ = 7.6 Hz,
1H, H-6′), 7.58 (m, 1H, H-4′, H-5′), 7.47 (m, 1H, H-3′), 4.73 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.63
(t, J1′′,2′′ = 4.8 Hz, 2H, CH2-1ʹʹ), 2.42 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
δ 165.7,
151.8, 138.3, 134.3, 133.4, 133.2, 132.1, 131.2, 129.8, 126.6, 62.4, 40.7, 12.9; EI-MS: m/z (rel.
abund. %), 309 (M+, 1), 311 (M+2), 274 (8), 263 (9), 183 (28), 153 (3), 139 (100), 111 (20);
Anal. Calcd for C13H12ClN3O4: C = 50.42, H = 3.91, N = 13.57; Found: C = 50.43, H = 3.91, N =
13.52.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3,4-dichlorobenzoate (11)
Solid; Yield: 86%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.03 (s, 1H, H-4), 7.96 (d, J2′,6′ = 1.6 Hz,
1H, H-2′), 7.82 (m, 2H, H-5′, H-6′), 4.74 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.65 (t, J1′′,2′′ = 4.8 Hz,
13
2H, CH2-1ʹʹ), 2.47 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6):
δ 165.8, 151.6, 138.5, 137.8,
132.4, 132.1, 130.0, 129.8, 129.5, 129.3, 62.4, 40.6, 12.8; EI-MS: m/z (rel. abund. %), 343 (M+,
5), 345 (M+2, 3), 217 (38), 190 (2), 173 (100), 145 (18); Anal. Calcd for C13H11Cl2N3O4: C =
45.37, H = 3.22, N = 12.21; Found: C = 45.34, H = 3.25, N = 12.23.
21

ACCEPTED MANUSCRIPT
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 2,5-dichlorobenzoate (12)
Solid; Yield: 88%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.04 (s, 1H, H-4), 7.72 (d, J6′,4′ = 2.4 Hz,
1H, H-6′), 7.68 (dd, J4′,6′ = 2.4, J4′,3′ = 8.4 Hz, 1H, H-4′), 7.61 (d, J3′,4′ = 8.4 Hz, 1H, H-3′), 4.73 (t,
13
J2′′,1′′ = 5.2 Hz, 2H, CH2-2ʹʹ), 4.62 (t, J1′′,2′′ = 5.2 Hz, 2H, CH2-1ʹʹ), 2.43 (s, 3H, CH3); C NMR
(75 MHz, DMSO-d6):
δ 165.7, 151.6, 138.5, 133.2, 132.4, 132.0, 131.5, 130.4, 129.8, 128.6,
62.4, 40.7, 12.7; EI-MS: m/z (rel. abund. %), 343 (M+, 55), 345 (M+2, 37), 308 (81), 297 (73),
217 (100), 173 (78); Anal. Calcd for C13H11Cl2N3O4: C = 45.37, H = 3.22, N = 12.21; Found: C
= 45.35, H = 3.20, N = 12.23.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-fluorobenzoate (13)
Solid; Yield: 79%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.03 (s, 1H, H-4), 7.68 (d, J6′,5′ = 7.6 Hz,
1H, H-6′), 7.59 (m, 3H, H-2′, H-4′, H-5′), 4.74 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.65 (t, J1′′,2′′ = 4.8
Hz, 2H, CH2-1ʹʹ), 2.45 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
δ 165.8, 162.7, 151.8,
138.5, 132.3, 131.8, 130.3, 125.6, 119.7, 114.6, 62.4, 40.7, 12.8; EI-MS: m/z (rel. abund. %), 293
(M+, 11), 247 (48), 167 (72), 154 (4), 123 (100), 95 (47); Anal. Calcd for C13H12FN3O4: C =
53.24, H = 4.12, N = 14.33; Found: C = 53.26, H = 4.14, N = 14.30.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-fluorobenzoate (14)
1
Solid; Yield: 82%; H-NMR: (400 MHz, DMSO-d6): δ 8.03 (s, 1H, H-4), 7.92 (m, 2H, H-2′, H-
6′), 7.38 (m, 2H, H-3′, H-5′), 4.72 (t, J2′′,1′′ = 4.5 Hz, 2H, CH2-2ʹʹ), 4.64 (t, J1′′,2′′ = 5.4 Hz, 2H,
CH2-1ʹʹ), 2.44 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
δ 167.3, 165.8, 151.6, 138.5, 132.3,
131.4, 131.4, 125.6, 115.3, 115.3, 62.4, 40.6, 12.7; EI-MS: m/z (rel. abund. %), 293 (M+, 16),
247 (64), 167 (58), 123 (100), 95 (43); Anal. Calcd for C13H12FN3O4: C = 53.24, H = 4.12, N =
14.33; Found: C = 53.21, H = 4.15, N = 14.30.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-bromo-4-fluorobenzoate (15)
1
Solid; Yield: 90%; H-NMR: (400 MHz, DMSO-d6): δ 8.03 (s, 1H, H-4), 7.76 (m, 2H, H-5′, H-
6′), 7.42 (m, 1H, H-3′), 4.72 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.62 (t, J1′′,2′′ = 4.8 Hz, 2H, CH2-1ʹʹ),
13
2.42 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6):
δ 169.5, 165.8, 151.6, 138.3, 133.6, 132.3,
127.4, 123.6, 120.4, 114.5, 62.5, 40.7, 12.8; EI-MS: m/z (rel. abund. %), 371 (M+, 15), 373
22

ACCEPTED MANUSCRIPT
(M+2, 14), 325 (16), 292 (16), 245 (29), 203 (100), 173 (14); Anal. Calcd for C13H11BrFN3O4: C
= 41.96, H = 2.98, N = 11.29; Found: C = 41.94, H = 2.96, N = 11.27.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-chloro-4,5-difluorobenzoate (16)
1
Solid; Yield: 78%; H-NMR: (400 MHz, DMSO-d6): δ 8.04 (s, 1H, H-4), 7.90 (m, 1H, H-6′),
7.82 (m, 1H, H-3′), 4.72 (t, J2′′,1′′ = 5.2 Hz, 2H, CH2-2ʹʹ), 4.62 (t, J1′′,2′′ = 5.2 Hz, 2H, CH2-1ʹʹ),
13
2.42 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6):
δ 165.7, 155.3, 151.6, 147.4, 138.5, 132.3,
131.6, 128.7, 119.4, 117.8, 62.5, 40.7, 12.8; EI-MS: m/z (rel. abund. %), 345 (M+, 5), 347 (M+2,
2), 310 (9), 299 (19), 219 (45), 175 (100), 147 (20); Anal. Calcd for C13H10ClF2N3O4: C = 45.17,
H = 2.92, N = 12.16; Found: C = 45.15, H = 2.95, N = 12.14.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-nitrobenzoate (17)
Solid; Yield: 88%; 1H-NMR: (300 MHz, DMSO-d6): δ 8.52 (s, 1H, H-2′), 8.50 (d, J4′,5′ = 8.0 Hz,
1H, H-4′), 8.24 (d, J6′,5′ = 8.0 Hz, 1H, H-6′), 8.04 (s, 1H, H-4), 7.85 (t, J5′(4′,6′) = 8.0 Hz, 1H, H-5′),
4.77 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.71 (t, J1′′,2′′ = 5.2 Hz, 2H, CH2-1ʹʹ), 2.48 (s, 3H, CH3); 13C
NMR (75 MHz, DMSO-d6):
δ 165.8, 151.6, 147.9, 138.5, 136.1, 132.3, 129.4, 129.4, 128.3,
123.4, 62.4, 40.7, 12.8; EI-MS: m/z (rel. abund. %), 320 (M+, 6), 274 (50), 194 (83), 150 (100),
123 (5), 104 (17); Anal. Calcd for C13H12N4O6: C = 48.75, H = 3.78, N = 17.49; Found: C =
48.73, H = 3.75, N = 17.47.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-nitrobenzoate (18)
Solid; Yield: 85%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.35 (d, J2′,3′/6′,5′ = 9.0 Hz, 2H, H-2′, H-6′),
8.08 (d, J3′,2′/5′,6′ = 9.0 Hz, 2H, H-3′, H-5′), 8.04 (s, 1H, H-4), 4.74 (t, J2′′,1′′ = 4.2 Hz, 2H, CH2-2ʹʹ),
4.70 (t, J1′′,2′′ = 4.2 Hz, 2H, CH2-1ʹʹ), 2.46 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
δ 165.8,
152.3, 151.6, 138.3, 136.3, 132.1, 130.7, 130.7, 123.9, 123.9, 62.5, 40.7, 12.7; EI-MS: m/z (rel.
abund. %), 320 (M+, 28), 274 (48), 194 (68), 164 (7), 150 (100), 104 (58); Anal. Calcd for
C13H12N4O6: C = 48.75, H = 3.78, N = 17.49; Found: C = 48.73, H = 3.80, N = 17.47.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-methyl-4-nitrobenzoate (19)
Solid; Yield: 82%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.07 (d, J5′,6′ = 8.4 Hz, 1H, H-5′), 8.04 (s,
1H, H-4), 7.91 (s, 1H, H-2′), 7.85 (d, J6′,5′ = 8.0 Hz, 1H, H-6′), 4.74 (t, J2′′,1′′ = 4.4 Hz, 2H, CH2-
23

ACCEPTED MANUSCRIPT
13
2ʹʹ), 4.67 (t, J1′′,2′′ = 4.4 Hz, 2H, CH2-1ʹʹ), 2.52 (s, 3H, CH3), 2.46 (s, 3H, CH3); C NMR (75
MHz, DMSO-d6): 165.7, 153.5, 151.6, 138.3, 136.2, 132.3, 132.1, 131.0, 127.7, 123.6, 62.4,
δ
40.7, 18.4, 12.7; EI-MS: m/z (rel. abund. %), 334 (M+, 12), 288 (41), 208 (68), 164 (100), 135
(7), 118 (17); Anal. Calcd for C14H14N4O6: C = 50.30, H = 4.22, N = 16.76; Found: C = 50.32, H
= 4.20, N = 16.79.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-bromobenzoate (20)
1
Solid; Yield: 72; H-NMR: (400 MHz, DMSO-d6): δ 8.04 (s, 1H, H-4), 7.75 (m, 1H, H-4′), 7.64
(m, 1H, H-5′), 7.49 (m, 2H, H-3′, H-6′), 4.72 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.63 (t, J1′′,2′′ = 4.8
Hz, 2H, CH2-1ʹʹ), 2.42 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
δ 165.8, 151.6, 138.5,
132.7, 132.3, 132.0, 131.8, 127.5, 126.4, 121.2, 62.5, 40.7, 12.7; EI-MS: m/z (rel. abund. %), 353
(M+, 4), 355 (M+2, 5), 307 (13), 274 (37), 227 (35), 183 (17); Anal. Calcd for C13H12BrN3O4: C
= 44.09, H = 3.42, N = 11.86; Found: C = 44.06, H = 3.45, N = 11.83.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-bromobenzoate (21)
Solid; Yield: 79%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.02 (s, 1H, H-4), 7.74 (s, 4H, H-2′, H-3′,
H-5′, H-6′), 4.73 (t, J2′′,1′′ = 5.1 Hz, 2H, CH2-2ʹʹ), 4.65 (t, J1′′,2′′ = 5.1 Hz, 2H, CH2-1ʹʹ), 2.44 (s, 3H,
13
CH3); C NMR (75 MHz, DMSO-d6):
δ 165.7, 151.8, 138.5, 132.3, 132.2, 132.2, 131.4, 131.4,
129.0, 127.3, 62.4, 40.7, 12.8; EI-MS: m/z (rel. abund. %), 353 (M+, 4), 355 (M+2, 4), 307 (21),
229 (20), 183 (100), 155 (13); Anal. Calcd for C13H12BrN3O4: C = 44.09, H = 3.42, N = 11.86;
Found: C = 44.07, H = 3.45, N = 11.82.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 1H-indole-2-carboxylate (22)
Solid; Yield: 80%; 1H-NMR: (400 MHz, DMSO-d6): δ 11.89 (s, 1H, NH), 8.04 (s, 1H, H-4), 7.66
(d, J7′,6′ = 8.0 Hz, 1H, H-7′), 7.43 (d, J4′,5′ = 8.4 Hz,1H, H-4′), 7.27 (t, J5′(4′,6′) = 7.6 Hz, 1H, H-5′),
7.09 (t, J6′(5′,7′) = 7.6 Hz, 1H, H-6′), 7.03 (s, 1H, H-3′), 4.71 (t, J2′′,1′′ = 4.4 Hz, 2H, CH2-2ʹʹ), 4.68
(t, J1′′,2′′ = 4.4 Hz, 2H, CH2-1ʹʹ), 2.47 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
δ 158.3,
151.6, 138.5, 132.3, 131.5, 130.2, 125.2, 121.8, 120.6, 119.7, 112.4, 111.0, 62.4, 40.7, 12.8; EI-
MS: m/z (rel. abund. %), 314 (M+, 67), 297 (5), 286 (28), 268 (8), 187 (53), 154 (26), 144 (100),
115 (44); Anal. Calcd for C15H14N4O4: C = 57.32, H = 4.49, N = 17.83, Found: C = 57.30, H =
4.46, N = 17.85.
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2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl nicotinate (23)
Solid; Yield: 83%; 1H-NMR: (300 MHz, DMSO-d6): δ 8.95 (d, J2′,4′ = 1.6 Hz, 1H, H-2′), 8.81 (d,
J4′,5′ = 3.6 Hz, 1H, H-4′), 8.17 (d, J6′,5′ = 8.0 Hz, 1H, H-6′), 8.04 (s, 1H, H-4), 7.58 (m, 1H, H-5′),
4.75 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.67 (t, J1′′,2′′ = 4.8 Hz, 2H, CH2-1ʹʹ), 2.46 (s, 3H, CH3); 13C
NMR (75 MHz, DMSO-d6):
δ 165.8, 151.6, 151.3, 150.5, 138.6, 136.3, 132.1, 126.2, 122.0,
62.5, 40.7, 12.7; EI-MS: m/z (rel. abund. %), 276 (M+, 4), 230 (17), 150 (49), 137 (4), 124 (8),
106 (100), 85 (20); Anal. Calcd for C12H12N4O4: C = 52.17, H = 4.38, N = 20.28; Found: C =
52.14, H = 4.36, N = 20.25.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl benzo[d]thiazole-6-carboxylate (24)
Solid; Yield: 76%; 1H-NMR: (400 MHz, DMSO-d6): δ 9.60 (s, 1H, H-2′), 8.73 (s, 1H, H-7′), 8.19
(d, J4′,5′ = 8.0 Hz, 1H, H-4′), 8.04 (s, 1H, H-4), 7.98 (d, J5′,4′ = 8.4 Hz,1H, H-5′), 4.75 (t, J2′′,1′′
4.8 Hz, 2H, CH2-2ʹʹ), 4.70 (t, J1′′,2′′ = 4.8 Hz, 2H, CH2-1ʹʹ), 2.49 (s, 3H, CH3); 13C NMR (75 MHz,
DMSO-d6): 165.7, 155.9, 154.7, 151.8, 138.5, 133.7, 132.0, 126.7, 126.2, 123.5, 121.8, 62.4,
=
δ
40.9, 12.8; EI-MS: m/z (rel. abund. %), 332 (M+, 28), 286 (74), 206 (63), 179 (10), 162 (100),
143 (2), 134 (93); Anal. Calcd for C14H12N4O4S: C = 50.60, H = 3.64, N = 16.86; Found: C =
50.63, H = 3.61, N = 16.89.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 1-naphthoate (25)
Solid; Yield: 82%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.57 (d, J2′,3′ = 8.4 Hz, 1H, H-2′), 8.20 (d,
J8′,7′ = 8.4 Hz,1H, H-8′), 8.05 (s, 1H, H-4), 8.03 (d, J4′,3′ = 8.8 Hz, 1H, H-4′), 8.00 (d, J5′,6′ = 7.2
Hz, 1H, H-5′), 7.61 (m, 3H, H-3′, H-6′, H-7′), 4.78 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.73 (t, J1′′,2′′
=
13
4.4 Hz, 2H, CH2-1ʹʹ), 2.41 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6):
δ 167.8, 151.8, 138.5,
134.4, 133.6, 133.4, 132.2, 129.8, 129.1, 128.3, 127.7, 126.9, 126.7, 126.4, 62.4, 40.7, 12.8; EI-
MS: m/z (rel. abund. %), 325 (M+, 53), 308 (3), 279 (26), 199 (17), 172 (10), 155 (100), 139 (5),
127 (58); Anal. Calcd for C17H15N3O4: C = 62.76, H = 4.65, N = 12.92; Found: C = 62.74, H =
4.67, N = 12.95.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-naphthoate (26)
Solid; Yield: 78%; 1H-NMR: (400 MHz, DMSO-d6): δ 8.49 (s, 1H, H-1′), 8.09 (d, J4′,3′ = 8.4 Hz,
1H, H-4′), 8.05 (s, 1H, H-4), 8.03 (d, J8′,7′ = 8.4 Hz,1H, H-8′), 8.01 (d, J5′,6′ = 8.4 Hz, 1H, H-5′),
25

ACCEPTED MANUSCRIPT
7.86 (dd, J3′,1′ = 1.6, J3′,2′ = 8.4, 1H, H-3′), 7.70 (t, J6′(5′,7′) = 8.0 Hz, 1H, H-6′), 7.64 (t, J7′(6′,8′)
=
7.6 Hz, 1H, H-7′), 4.79 (t, J2′′,1′′ = 4.8 Hz, 2H, CH2-2ʹʹ), 4.70 (t, J1′′,2′′ = 4.8 Hz, 2H, CH2-1ʹʹ), 2.49
(s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6):
165.8, 151.6, 138.5, 135.6, 132.7, 132.3, 130.6,
δ
129.4, 128.7, 128.4, 128.2, 128.2, 127.2, 125.8, 62.4, 40.7, 12.8; EI-MS: m/z (rel. abund. %), 325
(M+, 31), 279 (30), 199 (21), 172 (12), 155 (100), 127 (63); Anal. Calcd for C17H15N3O4: C =
62.76, H = 4.65, N = 12.92; Found: C = 62.79, H = 4.67, N = 12.95.
Acknowledgement: This work was supported by the Higher Education Commission (HEC)
Pakistan (Project No. 20-2073), under the National Research Program for Universities.
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