Facile synthesis, biological evaluation and molecular docking studies of novel substituted azole derivatives

Article abstract of DOI:10.1016/j.molstruc.2017.03.013

In this study, we synthesized the series of novel azole derivatives and evaluated for enzyme inhibition assays, corresponding kinetic analysis and molecular modeling. Among the investigated bioassays, the oxadiazole derivatives 4a-k were found potent α-glucosidase inhibitors while the Schiff base derivatives 7a-k exhibited considerable potential toward urease inhibition. The inhibition kinetics for the most active compounds were analyzed by the Lineweaver–Burk plots to investigate the possible binding modes of the synthesized compounds toward the tested proteins. Moreover, the detailed docking studies were performed on the synthesized library of 4a-k and 7a-k to study the molecular interaction and binding mode in the active site of the modeled yeast α-glucosidase and Jack Bean Urease, respectively. It could be inferred from docking results that theoretical studies are in close agreement to that of the experimental results. The structure of one of the compound 7k was characterized by the single crystal X-ray diffraction analysis in order to find out the predominant conformation of the molecules.

Full text of DOI:10.1016/j.molstruc.2017.03.013

Accepted Manuscript
Facile synthesis, biological evaluation and molecular docking studies of novel
substituted azole derivatives
Muhammad Rafiq, Muhammad Saleem, Farukh Jabeen, Muhammad Hanif, Sung-
Yum Seo, Sung Kwon Kang, Ki Hwan Lee
PII:
S0022-2860(17)30274-0
10.1016/j.molstruc.2017.03.013
MOLSTR 23509
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 24 December 2016
Revised Date: 1 March 2017
Accepted Date: 2 March 2017
Please cite this article as: M. Rafiq, M. Saleem, F. Jabeen, M. Hanif, S.-Y. Seo, S.K. Kang, K.H.
Lee, Facile synthesis, biological evaluation and molecular docking studies of novel substituted azole
derivatives, Journal of Molecular Structure (2017), doi: 10.1016/j.molstruc.2017.03.013.
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ACCEPTED MANUSCRIPT
Graphical abstract
Facile synthesis, biological evaluation and molecular docking studies of novel
substituted azole derivatives
Muhammad Rafiq, Muhammad Saleem, Muhammad Hanif, Farukh Jabeen, Sung-Yum Seo, Sung Kwon Kang, Ki
Hwan Lee
In this study, we synthesized the series of oxadiazole derivatives 4a-k as well as triazole Schiff base derivatives
7a-k. The synthesized compounds were evaluated for bioactivities, kinetic analysis and molecular modeling.

ACCEPTED MANUSCRIPT
Facile synthesis, biological evaluation and molecular docking studies of novel
substituted azole derivatives
A running title: Synthesis, biological evaluation and molecular docking studies
Muhammad Rafiqa,b, Muhammad Saleemc,d, Farukh Jabeene,f, Muhammad Hanifg, Sung-Yum Seoa, Sung Kwon
Kangh, Ki Hwan Leec,*
aDepartment of Biology, Kongju National University, Gongju, Chungnam 32588, Republic of Korea
bDepartment of Biochemistry and Biotechnology (Baghdad-ul-Jadeed Campus), The Islamia University of
Bahawalpur 63100, Pakistan
cDepartment of Chemistry, Kongju National University, Gongju, Chungnam 32588, Republic of Korea
dDepartment of Chemistry, University of Sargodha, Sub-campus Mianwali, Pakistan
eFlorida Center of Heterocyclic Compounds, University of Florida, Gainesville, Fl, 32601, USA
fCenter for Computationally Assisted Science and Technology, North Dakota State University, Fargo, ND, 58102,
USA
gDepartment of Chemistry, GC University Faisalabad (sub campus layyah), Pakistan
hDepartment of Chemistry Chungnam National University Daejeon 34134, Republic of Korea
*Corresponding author. Fax: +82-41-856-8613; E-mail address: khlee@kongju.ac.kr
Abstract
In this study, we synthesized the series of novel azole derivatives and evaluated for enzyme inhibition assays,
corresponding kinetic analysis and molecular modeling. Among the investigated bioassays, the oxadiazole
derivatives 4a-k were found potent α-glucosidase inhibitors while the Schiff base derivatives 7a-k exhibited
considerable potential toward urease inhibition. The inhibition kinetics for the most active compounds were
analyzed by the Lineweaver–Burk plots to investigate the possible binding modes of the synthesized compounds
toward the tested proteins. Moreover, the detailed docking studies were performed on the synthesized library of 4a-k
and 7a-k to study the molecular interaction and binding mode in the active site of the modeled yeast α-glucosidase
and Jack Bean Urease, respectively. It could be inferred from docking results that theoretical studies are in close
agreement to that of the experimental results. The structure of one of the compound 7k was characterized by the
single crystal X-ray diffraction analysis in order to find out the predominant conformation of the molecules.
Keywords: triazole Schiff bases; biological evaluation; kinetic analysis; molecular modeling; single crystal XRD.
Introduction
Development of new therapeutic agents utilizing natural or non-toxic precursor is one of the fundamental goals in
medicinal chemistry for the design and development of novel drugs. The five-membered heteroaromatic rings are
important building blocks of a wide range of biologically active molecules and have become a substantial
construction motif in drug discovery [1,2]. The 1,3,4-oxadiazoles are influential heterocyclic systems in the field of
synthetic, medicinal and materials chemistry due to their remarkable biological and pharmacological properties such
as anti-bacterial [3,4], antimicrobials [5], antifungal [6], anti-oxidant [7], anticancer [8], anticonvulsants [9],
antiproliferative [10], anti-inflammatory [11] and insecticidal activities [12], as well as conspicuous enzyme
inhibitions against varieties of enzymes including superoxide dismutase [13], gluconeogenesis [14], telomerase [15],
α-glucosidase [16], α-amylase [17], histone deacetylase [18] and thymidine phosphorylase [19]. The substituted
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oxadiazole derivative also have attracted attention in photochemistry for the development of optical sensor, staining
agent and storage material due to their consequential light harvesting potential [20,21].
Guo et al. [22] patented the heterocyclic skeleton containing oxadiazole nucleus a (Fig. 1) as effective for type 2
diabetes Mellitus, and of conditions that are often associated with this disease, including hyperglycemia, insulin
resistance, obesity, lipid disorders, and hypertension. Shingalapur et al. [23], reported the excellent anti-diabetic
potential for the 1,3,4-oxadiazoles derivatives b (Fig. 1). Recently, Nazreen et al. [24] and Kashtoh et al. [25] found
1,3,4-oxadiazole containing heterocycles c, d and e (Fig. 1) exhibiting significant blood glucose lowering effect and
α-glucosidase inhibition efficacy comparable with that of reference standard. Keeping in view the recent developed
structure activity relationship of 1,3,4-oxadiazole compounds, we have synthesized a series of eleven oxadiazole
derivatives 4a-k and evaluated their α-glucosidase inhibition activity by spectrophotometric methods along with the
kinetic analysis by Lineweaver–Burk plots.
Similarly, the 1,2,4-triazole containing compounds have been shown to exhibit a variety of interesting biological
actions, including anti-cancer [25,26], anti-inflammatory [27], anti-diabetic [28], anti-malarial [29], anti-
proliferative [30], antimitotic, anti-vascular [31], anticonvulsant [32], anti-fungal and enzyme inhibition potential
such as urease [33] and α-fucosidase [34]. The alterations in chemical structure of substituted 1,2,4-triazoles
strongly change their activities and affect their interaction with cells and tissues, thereby leading to different
biological effects [35]. Schiff bases have been widely used in several fields of chemical production and scientific
research as chelating agents, stabilizers, biologically active agents, analysis and catalytic reagents [36]. Schiff bases
derived from various heterocycles were reported to possess diverse biological activities [37]. Alongside, the Schiff
base containing 1,2,4-triazole nucleus have attracted great and growing interest in chemistry and biology for many
years due to their facile synthesis and broad range of biological activities [38-41], including antitumor [40],
antiviral [42], anti-bacterial [43] and corrosive inhibitor [44, 45].
Amtul et al. [46], reported a series of twenty five 1,2,4-triazole-3-thiones derivatives f (Fig. 2) exhibiting urease
inhibition potential. Ibrar et al. [47] summarizes a library of triazole scaffold g-m (Fig. 2) as potent urease inhibitors.
Recently, Mentese et al. [48] reported microwave assisted synthesis of relatively complex triazole derivatives n (Fig.
2) as urease inhibitors. Prompted by recent findings, the present study was undertaken to develop novel Schiff base
derivatives containing 1,2,4-triazole nucleus and employed them against urease inhibition assay by
spectrophotometric methods using thiourea as reference inhibitor alongside kinetic investigation to get mechanistic
insight. The commendable urease inhibition potential was observed by the designed Schiff bases of 1,2,4-triazole in
comparison to the previously reported isolated triazole based molecules o (Fig. 2) [49] or those Schiff base
derivatives where there was no direct connection of imine bond with that of triazole skeleton e. g., p (Fig. 2) [50].
Moreover, the predominant conformational structures adopted by the compounds 7a-k were checked out by growing
the fine crystal of one of the compounds among the series with the subsequent characterization with the single
crystal X-ray diffraction analysis.
Fig. 1 and Fig. 2
Experimental
Substrate and reagents
Phenyl, aryl and aralkyl carboxylic acids were purchased from Alfa Aesar and Aldrich. Ethanol, methanol,
chloroform, water (Samchun Chemicals, Korea), H2SO4, HCl (Jin Chemical & Pharmaceutical Co. Ltd., Korea)
were used in these experiments. Urease from Jack bean (EC 3.5.1.5), α-glucosidase from Saccharomyees Cerevisiae,
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(EC 3.2.1.2.0), thiourea, EDTA, sodium nitroprusside and active chloride, were purchased from Sigma Aldrich. All
the reagents used for bioassays were of analytical grade.
Instrumentations
The reaction progress was monitored by thin layer chromatographic (TLC) analysis and the Rf values were
determined by employing pre–coated silica gel aluminum plates, Kieselgel 60 F254 from Merck (Germany). TLC
was visualized under a UV lamp (VL–4 LC, France). The melting points were determined on a Fisher Scientific
(USA) melting point apparatus and are uncorrected. The FT-IR spectra were recorded in KBr pellets on a Shimadzu
FTIR–8400S spectrometer (Kyoto, Japan). Proton and carbon nuclear magnetic resonance (1H NMR & 13C NMR)
spectra were recorded on a Bruker Avance 400 MHz spectrometer. The chemical shifts are reported as
δ values in
ppm downfield from the internal tetramethylsilane of the indicated organic solution. Peak multiplicities are
expressed as follows: s, singlet; d, doublet and m, multiplet. Single crystal data was obtained by using Bruker
SMART APEX II X–ray Diffractometer CCD equipped with Mo X–ray tube and graphite monochromator at the
ambient temperature. The schematic representation of synthetic route adapted to obtained target compounds 4a-k
and 7a-k are shown in Scheme 1.
Scheme 1
Synthesis of aralkanoic acid hydrazides 3
Aralkanoic acid chlorides 2 were synthesized by the reaction of aralkanoic acids 1 (1 mmol) in the presence of
1,2–dichloroethane (12 mL) solvent and phosphorous oxychloride (0.4 mL) as chlorinating agent under reflux for 3
h. Then, the resulting solution was cooled to room temperature, and the solvent was removed under reduced pressure
to afford aralkanoic acid chlorides 2, which was directly used in the next step without further purification.
Aralkanoic acid chlorides 2 was dissolved in acetonitrile (80 mL), added drop wise to a solution containing
hydrazine hydrate (1 mmol), TEA (0.5 mL) and acetonitrile (20 mL) and allowed to reflux for 3 h with monitoring
by TLC. After consumption of the starting material, the reaction mixture was cooled to room temperature.
Evaporation of the solvent under reduced pressure yielded crude substituted aromatic acid hydrazides 3 as a white
solid on cooling, which was purified by column chromatography and crystallized on methanol.
Synthesis of 2,5-disubstituted-1,3,4-oxadiazoles 4a-k
An equimolar mixture of aralkanoic acid hydrazides 3 and substituted aromatic acids were mixed in phosphorous
oxychloride and heated under reflux at 107 °C for 3 h. The reaction progress was monitored by thin layer
chromatography. After consumption of the starting material; the reaction mixture was cooled to room temperature
and poured into crushed ice which becomes precipitated. These precipitates was filtered, washed with cold water
and diethyl ether, filtered and crystallized on methanol to get 2,5-disubstituted-1,3,4-oxadiazoles 4a-k. The product
was purified with column chromatography.
2,5-Bis(4-methoxybenzyl)-1,3,4-oxadiazole (4a)
Off white solid; yield: 71 %; Rf: 0.64 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3026 (sp2 CH), 2945 (sp3 CH),
1579 (C=N), 1551, 1498 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
δ 7.19-7.00 (m, 4H, aromatic
proton), 6.98-6.89 (m, 2H, aromatic proton), 6.88-6.68 (m, 2H, aromatic proton), 3.66 (s, 2H, aliphatic CH2), 3.64
(s, 3H, methoxy proton), 3.60 (s, 2H, aliphatic CH2); 13C NMR (100 MHz, DMSO-d6)
δ
158.4, 155.1, 149.3, 147.0,
130.0, 127.6, 125.8, 120.5, 114.1, 120.0, 56.1, 55.9, 31.5.
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2-(4-Fluorophenethyl)-5-(4-methoxybenzyl)-1,3,4-oxadiazole (4b)
Brown solid; yield: 77 %; Rf: 0.62 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3041 (sp2 CH), 2947 (sp3 CH),
1579 (C=N), 1568, 1478 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
δ 7.35-7.31 (m, 1H, aromatic
proton), 7.25-7.23 (m, 1H, aromatic proton), 7.03-7.01 (m, 1H, aromatic proton), 6.97-6.91 (m, 1H, aromatic
proton), 6.88-6.81 (m, 2H, aromatic proton), 6.78-6.73 (m, 2H, aromatic proton), 3.69 (t, 2H, aliphatic CH2), 3.66 (s,
3H, OCH3), 2.34 (t, 2H, aliphatic CH2); 13C NMR (100 MHz, DMSO-d6)
δ 158.4, 154.9, 146.7, 139.2, 133.2, 130.1,
129.6, 128.4, 127.4, 124.9, 114.1, 55.4, 31.6, 21.1.
2-(3-Fluorophenethyl)-5-(4-methoxybenzyl)-1,3,4-oxadiazole (4c)
Off white solid; yield: 76 %; Rf: 0.64 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3044 (sp2 CH), 2947 (sp3 CH),
1581 (C=N), 1565, 1514, 1497 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
7.50-7.44 (m, 1H, aromatic
δ
proton), 7.31-7.27 (m, 1H, aromatic proton), 7.25-7.18 (m, 1H, aromatic proton), 7.12-7.10 (m, 1H, aromatic
proton), 6.90-6.85 (m, 2H, aromatic proton), 6.77-6.75 (m, 2H, aromatic proton), 3.78 (s, 2H, aliphatic CH2), 3.73 (t,
2H, J = 6.4 Hz, aliphatic CH2), 3.72 (s, 3H, aliphatic OCH3), 1.25 (t, 2H, J = 7.2 Hz, aliphatic CH2); 13C NMR (100
MHz, DMSO-d6)
δ 161.1, 158.4, 155.6, 154.6, 153.8, 146.4, 131.3, 130.8, 129.0, 128.0, 124.1, 116.0, 55.4, 31.6,
29.2, 14.9.
2-(2-Fluorophenethyl)-5-(4-methoxybenzyl)-1,3,4-oxadiazole (4d)
Brown solid; yield: 77 %; Rf: 0.60 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3055 (sp2 CH), 2936 (sp3 CH),
1588 (C=N), 1556, 1533, 1501 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
δ 7.29-7.23 (m, 1H, aromatic
proton), 7.14-7.00 (m, 4H, aromatic proton), 6.99-6.98 (m, 2H, aromatic proton), 6.82-6.80 (m, 2H, aromatic
proton), 3.99 (s, 2H, aliphatic CH2), 3.73 (t, 2H, J = 6.4 Hz, aliphatic CH2), 3.68 (s, 3H, aliphatic OCH3), 1.24 (t,
2H, J = 7.2 Hz, aliphatic CH2); 13C NMR (100 MHz, DMSO-d6)
δ 158.2, 154.1, 146.7, 139.4, 133.2, 130.9, 129.6,
128.4, 127.4, 124.9, 114.1, 55.4, 31.6, 29.2, 14.9.
2-(4-Methoxybenzyl)-5-(2-methoxyphenethyl)-1,3,4-oxadiazole (4e)
yellow solid; yield: 76 %; Rf: 0.65 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3045 (sp2 CH), 2951 (sp3 CH),
1584 (C=N), 1556, 1511, 1494 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
δ 7.48-7.22 (m, 4H, aromatic
proton), 7.12-7.10 (m, 2H, aromatic proton), 6.89-6.74 (m, 2H, aromatic proton), 3.86 (s, 2H, aliphatic, CH2), 3.72
(t, 2H, J = 6.0 Hz, aliphatic CH2), 3.63 (s, 3H, aliphatic, OCH3), 3.34 (s, 3H, aliphatic, OCH3), 1.15 (t, 2H, J = 7.2
Hz, aliphatic CH2); 13C NMR (100 MHz, DMSO-d6)
δ 158.7, 153.2, 151.4, 146.7, 139.4, 133.2, 130.9, 129.4, 128.4,
127.4, 124.9, 114.1, 112.0, 56.1, 55.9, 31.4, 29.4, 14.1.
2-(4-Methoxybenzyl)-5-(4-methoxyphenethyl)-1,3,4-oxadiazole (4f)
Yellowish solid; yield: 74 %; Rf: 0.61 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3033 (sp2 CH), 2951 (sp3 CH),
1584 (C=N), 1563, 1510, 1489 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
δ 7.36-7.23 (m, 2H, aromatic
proton), 7.14-7.10 (m, 2H, aromatic proton), 7.09-7.05 (m, 4H, aromatic proton), 3.87 (s, 2H, aliphatic CH2), 3.72 (t,
2H, J = 6.8 Hz, aliphatic CH2), 3.63 (s, 3H, aliphatic, OCH3), 3.13 (s, 3H, aliphatic, OCH3), 1.09 (t, 2H, J = 6.4 Hz,
aliphatic CH2); 13C NMR (100 MHz, DMSO-d6)
δ 159.4, 154.8, 145.3, 139.3, 133.0, 131.6, 129.6, 128.3, 124.8,
122.5, 115.6, 55.4, 25.9, 21.1.
2-(2-Fluorobenzyl)-5-(2-methoxyphenethyl)-1,3,4-oxadiazole (4g)
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Off white solid; yield: 74 %; Rf: 0.60 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3037 (sp2 CH), 2945 (sp3 CH),
1584 (C=N), 1571, 1522, 1501 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
δ 7.50-7.44 (m, 1H, aromatic
proton), 7.31-7.18 (m, 2H, aromatic proton), 7.12-7.10 (m, 3H, aromatic proton), 6.90-6.75 (m, 2H, aromatic
proton), 3.78 (s, 2H, aliphatic CH2), 3.72 (t, 2H, aliphatic, J = 6.4 Hz, CH2), 3.68 (s, 3H, aliphatic, OCH3), 1.25 (t,
2H, J = 7.2 Hz, CH2); 13C NMR (100 MHz, DMSO-d6)
δ 158.5, 154.9, 146.7, 139.2, 133.2, 130.1, 129.6, 128.4,
127.4, 124.9, 114.1, 55.4, 31.6, 29.2, 14.9.
2-(2-Fluorophenethyl)-5-(2-methoxyphenethyl)-1,3,4-oxadiazole (4h)
Off white solid; yield: 65 %; Rf: 0.54 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3041 (sp2 CH), 2942 (sp3 CH),
1584 (C=N), 1548, 1511, 1489 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
δ 7.58-7.41 (m, 1H, aromatic
proton), 7.32-7.23 (m, 2H, aromatic proton), 7.19-7.11 (m, 2H, aromatic proton), 6.90-6.74 (m, 3H, aromatic
proton), 3.78 (s, 2H, aliphatic, CH2), 3.72 (t, 2H, J = 6.4 Hz, aliphatic, CH2), 3.65 (s, 3H, aliphatic, OCH3), 1.25 (t,
2H, J = 8.0 Hz, aliphatic, CH2); 13C NMR (100 MHz, DMSO-d6)
δ 158.4, 155.1, 149.3, 147.0, 139.2, 133.2, 130.1,
129.6, 128.4, 127.4, 124.9, 114.1, 112.0, 55.4, 31.6, 29.2, 14.9.
2,5-Bis(2-methoxyphenethyl)-1,3,4-oxadiazole (4i)
Off white solid; yield: 63 %; Rf: 0.56 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3038 (sp2 CH), 2937 (sp3 CH),
1587 (C=N), 1554, 1522, 1499 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
7.56-7.39 (aromatic, 4H,
δ
m), 7.25-6.94 (aromatic, 4H, m), 3.56 (s, 6H, OCH3), 2.90 (t, 4H, J = 6.0 Hz, CH2), 2.88 (t, 4H, J = 6.0 Hz, CH2);
13C NMR (100 MHz, DMSO-d6)
δ 166.5, 161.9, 159.4, 159.0, 137.9, 135.1, 133.4, 127.5, 120.1, 119.4, 116.5, 56.0,
35.4, 33.7.
2-(2-Methoxyphenethyl)-5-phenyl-1,3,4-oxadiazole (4j)
Brown solid; yield: 76 %; Rf: 0.61 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3051 (sp2 CH), 2939 (sp3 CH),
1585 (C=N), 1561, 1511, 1499 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
δ 7.36-7.35 (m, 1H, aromatic
proton), 7.29-7.23 (m, 2H, aromatic proton), 7.14-7.12 (m, 1H, aromatic proton), 7.10-7.05 (m, 4H, aromatic
proton), 3.72 (t, 2H, J = 6.4 Hz, aliphatic, CH2), 3.68 (s, 3H, aliphatic, OCH3), 1.25 (t, 2H, J = 7.2 Hz, aliphatic,
CH2); 13C NMR (100 MHz, DMSO-d6)
δ 158.4, 154.8, 145.3, 139.3, 133.0, 131.6, 131.5, 129.6, 129.5, 129.4, 128.3,
124.8, 124.7, 124.6, 122.5, 122.3, 55.4, 25.8, 21.6.
2-(2-Methoxybenzyl)-5-(2-methoxyphenethyl)-1,3,4-oxadiazole (4k)
Brown solid; yield: 69 %; Rf: 0.60 (n-hexane : ethyl acetate, 8:2); FT–IR (υ /cm-1): 3041 (sp2 CH), 2948 (sp3 CH),
1589 (C=N), 1560, 1510, 1484 (C=C of phenyl ring); 1H NMR (400 M Hz, DMSO-d6)
δ 7.29-7.23 (m, 2H, aromatic
proton), 7.14-7.11 (m, 2H, aromatic proton), 7.10-7.00 (m, 2H, aromatic proton), 6.99-6.80 (m, 2H, aromatic
proton), 3.78 (s, 2H, aliphatic, CH2), 3.73 (t, 2H, J = 6.0 Hz, aliphatic, CH2), 3.72 (s, 3H, aliphatic, OCH3), 3.71 (s,
3H, aliphatic, OCH3), 1.24 (t, 2H, J = 7.2 Hz, aliphatic, CH2); 13C NMR (100 MHz, DMSO-d6)
δ 158.7, 154.8,
151.4, 146.7, 133.3, 131.2, 129.7, 129.0, 114.1, 112.0, 56.1, 55.9, 25.9, 21.1.
Synthesis of substituted 1,2,4-triazole derivatives 6
Potassium hydroxide (0.125 mol, 1eq) was dissolved in dry methanol (50 mL). To the solution, substituted acid
hydrazides 3 (0.125 mol, 1eq) was added and cooled the solution in ice. To this, carbon disulfide (0.125 mol, 1eq)
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was added with constant stirring for 1–2 h. The solid product of potassium salt of hydrazinecarbodithioates 5
formed, was filtered, washed with chilled diethyl ether and dried. It was directly used for next step without further
purification. The potassium salt of hydrazinecarbodithioates 5 was taken in deionized water (50 mL) and hydrazine
hydrate (0.250 mol) was added, followed by reflux overnight. The reaction mixture turned to yellowish green with
evolution of hydrogen sulfide and finally it became homogeneous. It was then poured in crushed ice and neutralized
with hydrochloric acid to afford white precipitates of substituted triazole derivatives 6 which was filtered, washed
with cold water and crystallized on methanol.
Synthesis of Schiff base derivatives 7a-k
The corresponding substituted triazole derivatives 6 (0.125 mol, 1eq) was dissolved in methanol (20 mL) and
different substituted aldehydes (0.125 mol, 1eq) was separately dissolved in methanol (20 mL) and both solutions
were mixed together prior to exposure with microwave for 10 min with constant monitoring the reaction progress by
TLC after regular interval of two min. On complete consumption of starting materials, the extra solvent was
evaporated on reduced pressure and resulting powder was crystallized on methanol. The purification of product was
done by column chromatography with n-hexane: ethyl acetate solvent system and crystallized on methanol.
(E)-3-(2-fluorophenyl)-4-[(thiophen-2-ylmethylene)amino]-1H-1,2,4-triazole-5(4H)-thione (7a)
Light pink; yield: 83 %; Rf: 0.54 (chloroform : methanol, 9:1); 1H NMR (400 MHz, DMSO–d6) δ 13.88 (s, 1H, NH),
10.16 (s, 1H, imine proton), 7.96-7.95 (m, 1H, aromatic proton), 7.79-7.77 (m, 1H, aromatic proton), 7.38-7.29 (m,
2H, aromatic proton), 7.27-7.26 (m, 1H, aromatic proton), 7.25-7.13 (m, 1H, aromatic proton); 13C NMR (100 MHz,
DMSO–d6) δ 162.1, 159.6, 157.7, 149.5, 136.8, 136.2, 133.4, 131.9, 129.7, 128.9, 124.9, 122.3, 115.8.
(E)-3-(2-bromophenyl)-4-[(thiophen-2-ylmethylene)amino]-1H-1,2,4-triazole-5(4H)-thione (7b)
Light gray; yield: 84 %; Rf: 0.54 (chloroform : methanol, 9:1); 1H NMR (400 MHz, DMSO–d6) δ 13.78 (s, 1H, NH),
10.18 (s, 1H, imine proton), 7.95-7.72 (m, 1H, aromatic proton), 7.57-7.49 (m, 1H, aromatic proton), 7.47-7.27 (m,
2H, aromatic proton), 7.27-7.20 (m, 2H, aromatic proton); 13C NMR (100 MHz, DMSO–d6) δ 162.7, 162.0, 157.4,
151.4, 150.2, 136.9, 136.1, 135.3, 134.9, 133.3, 131.8, 129.0, 120.5.
(E)-3-(2-methoxyphenyl)-4-[(thiophen-2-ylmethylene)amino]-1H-1,2,4-triazole-5(4H)-thione (7c)
Light pink; yield: 86 %; Rf: 0.53 (chloroform : methanol, 9:1); 1H NMR (400 MHz, DMSO–d6) δ 13.71 (s, 1H, NH),
10.16 (s, 1H, imine proton), 7.97-7.80 (m, 1H, aromatic proton), 7.79-7.64 (m, 1H, aromatic proton), 7.28-7.18 (m,
3H, aromatic proton), 6.96-6.84 (m, 2H, aromatic proton), 3.73 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO–d6) δ
161.9, 158.6, 157.3, 151.0, 136.9, 136.1, 133.3, 130.5, 129.0, 128.7, 127.2, 114.3, 55.5.
(E)-4-[{(1H-pyrrol-2-yl)methylene}amino]-3-(2-fluorophenyl)-1H-1,2,4-triazole-5(4H)-thione (7d)
Off white; yield: 86 %; Rf: 0.53 (chloroform : methanol, 9:1); 1H NMR (400 MHz, DMSO–d6) δ 13.78 (s, 1H, NH),
9.57 (s, 1H, imine proton), 7.57-7.50 (m, 2H, aromatic proton), 7.49-7.45 (m, 3H, aromatic proton), 7.29-7.17 (m,
1H, aromatic proton), 6.82-6.80 (m, 1H, aromatic proton), 6.24-6.22 (m, 1H, aromatic proton), 5.55 (s, 1H, NH); 13
C
NMR (100 MHz, DMSO–d6) δ 161.8, 156.4, 150.0, 135.1, 132.0, 131.8, 131.5, 125.5, 120.4, 120.2, 109.9.
(E)-4-[{(1H-pyrrol-2-yl)methylene}amino]-3-(2-methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione (7e)
Ivory; yield: 83 %; Rf: 0.52 (chloroform : methanol, 9:1); 1H NMR (400 MHz, DMSO–d6) δ 13.76 (s, 1H, NH), 9.57
(s, 1H, imine proton), 7.43-7.30 (m, 2H, aromatic proton), 7.27-7.11 (m, 3H, aromatic proton), 6.86-3.81 (m, 1H,
6

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aromatic proton), 6.29-6.22 (m, 1H, aromatic proton), 5.59 (s, 1H, NH), 3.81 (s, 3H, OCH3); 13C NMR (100 MHz,
DMSO–d6) δ 162.0, 159.6, 156.4, 149.4, 132.0, 129.7, 125.5, 124.9, 122.5, 120.2, 115.8, 109.9, 55.5.
(E)-3-(3-fluorophenyl)-4-[(2-phenylpropylidene)amino]-1H-1,2,4-triazole-5(4H)-thione (7f)
1
Brown; yield: 81 %; Rf: 0.56 (chloroform : methanol, 9:1); H NMR (400 MHz, DMSO–d6) δ 13.56 (s, 1H, NH),
10.14 (s, 1H, imine proton), 7.86-7.50 (m, 3H, aromatic proton), 7.46-7.11 (m, 5H, aromatic proton), 2.99-2.90 (m,
1H, aliphatic proton), 1.94 (d, 3H, J = 6.4, aliphatic proton); 13C NMR (100 MHz, DMSO–d6) δ 166.6, 151.4, 135.3,
131.8, 131.6, 125.5, 120.4, 114.4, 30.09, 18.4.
(E)-3-(3-bromophenyl)-4-[(2-phenylpropylidene)amino]-1H-1,2,4-triazole-5(4H)-thione (7g)
1
Antique white; yield: 82 %; Rf: 0.56 (chloroform : methanol, 9:1); H NMR (400 MHz, DMSO–d6) δ 13.66 (s, 1H,
NH), 10.18 (s, 1H, imine proton), 7.96-7.95 (m, 1H, aromatic proton), 7.78-7.72 (m, 1H, aromatic proton), 7.57-7.44
(m, 3H, aromatic proton), 7.32-7.20 (m, 4H, aromatic proton), 2.85-2.72 (m, 1H, aliphatic proton), 1.24 (d, 3H,
aliphatic proton); 13C NMR (100 MHz, DMSO–d6) δ 162.0, 157.4, 151.4, 150.2, 136.9, 136.1, 135.3, 134.9, 133.3,
131.7, 129.0, 120.5, 30.5, 19.4.
(E)-3-(3-methoxyphenyl)-4-[(2-phenylethylidene)amino]-1H-1,2,4-triazole-5(4H)-thione (7h)
1
Violet; yield: 81 %; Rf: 0.52 (chloroform : methanol, 9:1); H NMR (400 MHz, DMSO–d6) δ 13.73 (s, 1H, NH),
10.16 (s, 1H, imine proton), 7.97-7.95 (m, 1H, aromatic proton), 7.81-7.64 (m, 1H, aromatic proton), 7.28-7.18 (m,
5H, aromatic proton), 6.90-6.84 (m, 2H, aromatic proton), 4.02 (s, 2H, aliphatic), 3.96 (s, 3H, OCH3); 13C NMR
(100 MHz, DMSO–d6) δ 161.9, 158.6, 157.3, 152.1, 138.9, 136.9, 136.1, 134.2, 133.3, 131.4, 130.5, 129.0, 127.7,
114.3, 55.5, 30.3.
(Z)-4-[(2-fluorobenzylidene)amino]-3-(4-fluorobenzyl)-1H-1,2,4-triazole-5(4H)-thione (7i)
1
Antique white; yield: 83 %; Rf: 0.54 (chloroform : methanol, 9:1); H NMR (400 MHz, DMSO–d6) δ 13.89 (s, 1H,
NH), 10.04 (s, 1H, imine proton), 7.98-7.86 (m, 2H, aromatic proton), 7.39-7.32 (m, 2H, aromatic proton), 7.31-7.27
(m, 2H, aromatic proton), 7.22-7.11 (m, 2H, aromatic proton), 4.10 (s, 2H, aliphatic CH2); 13C NMR (100 MHz,
DMSO–d6) δ 163.3, 163.0, 162.4, 162.0, 159.6, 149.8, 131.9, 131.6, 129.8, 129.2, 124.9, 116.9, 115.8, 30.1.
(Z)-4-[(2-fluorobenzylidene)amino]-3-(4-methoxyphenyl)-1H-1,2,4-triazole-5(4H)-thione (7j)
1
Thistle; yield: 81 %; Rf: 0.51 (chloroform : methanol, 9:1); H NMR (400 MHz, DMSO–d6) δ 13.53 (s, 1H, NH),
10.03 (s, 1H, imine proton), 7.99-7.91 (m, 2H, aromatic proton), 7.51-7.34 (m, 2H, aromatic proton), 7.22-7.13 (m,
2H, aromatic proton), 6.89-6.80 (m, 2H, aromatic proton), 3.71 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO–d6) δ
162.2, 160.8, 158.6, 152.1, 151.1, 131.6, 131.1, 130.4, 129.3, 127.7, 127.3, 116.9, 114.4, 55.5.
(E)-3-(4-methoxybenzyl)-4-[(thiophen-2-ylmethylene)amino]-1H-1,2,4-triazole-5(4H)-thione (7k)
1
Yellow; yield: 89 %; Rf: 0.52 (chloroform : methanol, 9:1); H NMR (400 MHz, DMSO–d6) δ 13.88 (s, 1H, NH),
10.16 (s, 1H, imine proton), 7.96-7.94 (m, 1H, aromatic proton), 7.79-7.77 (m, 1H, aromatic proton), 7.38-7.29 (m,
2H, aromatic proton), 7.27-7.26 (m, 1H, aromatic proton), 7.26-7.13 (m, 2H, aromatic proton), 4.18 (s, 3H, OCH3),
3.40 (s, 2H, aliphatic, CH2); 13C NMR (100 MHz, DMSO–d6) δ 162.1, 159.6, 157.7, 149.5, 136.8, 136.2, 133.4,
131.9, 129.7, 128.9, 124.9, 122.3, 115.8, 55.5.
Single crystal X-ray diffraction analysis
The crystallographic data obtained for compound 7k are shown in Table 1
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Table 1
Structure solution and refinements
Data collection: APEX II (Bruker, 2005); cell refinement: SAINT (Bruker, 2005); data reduction: SAINT (Bruker,
2005); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure:
SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); computing molecular graphics
and material was performed by Ortep-3 for Windows (Farrugia, 2012)and WinGX publication routines (Farrugia,
2012). The CIF file for crystal is given in supporting information. The crystallographic data have been deposited
with the Cambridge Crystallographic Data Centre as supplementary publication No. CCDC-1033154.
Pharmacology
Protocol for in vitro bioassay
α-Glucosidase inhibition assay protocol
The enzyme inhibition activity for α-glucosidase was determined by the reported method with slight modification
[51]. The assay solution consisted of 50 µL of (0.1 M) sodium phosphate buffer pH 7.0, 25 µL of (0.5 mM)
substrate (p-nitrophenyl-α-D-glucopyranoside), 20 µL of increasing concentration of test compounds and 25 µL of
enzyme solution. This reaction mixture was then incubated for 30 min at 37 °C. Then, the reaction was stopped by
the addition of 100 µL of (0.2 M) sodium carbonate solution followed by the measurement of absorbance at 405 nm.
For non-enzymatic reaction, the assays were carried out with a blank containing all components except enzyme. The
assay measurements were carried out by using a micro plate reader (OPTI Max, Tunable). The reaction rates of the
tested compounds were compared with the reference drug and the percent inhibition was calculated by using the
formula 100−(Abstestwell/Abscontrol)×100 [52]. Acarbose was used as a reference inhibitor. Each concentration was
analyzed in three independent experiments run in triplicate. The IC50 values determined by the Data analysis and
graphing software, Origin 8.6, 64-bit.
Kinetic analysis for the α-glucosidase inhibition assay
A series of experiments were performed to determine the inhibition kinetics by following the reported method [51-
53]. Briefly, for each of three different concentrations of inhibitor compounds 4b (0 mM, 1.6 mM and 3.2 mM) and
4c (0 mM, 0.096 mM and 1.58 mM), respectively, the substrate p-nitrophenol-α-D-glucopyranoside (p-NPG)
concentration was varied in the order of 1.25 mM, 2.5 mM and 5 mM, respectively. Pre-incubation and
measurement time was the same as discussed in α-Glucosidase inhibition assay protocol. Maximal initial velocity
was determined from initial linear portion of absorbance up to five minutes after addition of α-Glucosidase at a 15s
interval. The inhibition type on the enzyme was assayed by Lineweaver–Burk plots of inverse of velocities (1/V)
versus inverse of substrate concentration1/[S] mM-1, and the inhibition constant Ki was determined by secondary
plot of Km versus the concentration of inhibitor 4b. In case of compound 4c the enzyme inhibitor (EI) dissociation
constant Ki was determined by secondary plot of slope versus 4c concentrations while enzyme substrate inhibitor
(ESI) dissociation constant Kiʹ was determined by intercept versus 4c concentrations.
Molecular Docking studies
Preparation of receptor
Molecular Operating Environment (MOE 2014) by Chemical Computing Group (CCG) was used for current studies
[54]. The protein preparation step involved 3D protonation, energy minimization and active site identification. The
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crystal structure of Jack Bean Urease was obtained from protein data bank (PDB code:3AL4) [55]. The co-
crystallized bound compounds and water molecules were stripped off from the crystal structure. Protein was energy
minimized and 3-D protonated by using the structure preparation module of MOE. Since, the protein was devoid of
co-crystallized ligand therefore active site was identified by using site finder module of the MOE. The chain with
the both nickel ions was selected to find the active site. The pocket was found to be deep cleft lined with the two
nickel ions and both hydrophobic and hydrophilic amino acid residues.
Preparation of Ligands
The ligand files for molecular docking studies were prepared in MOE-2014 and were followed by energy
optimization at standard MMFF94 force field level, with a 0.0001 kcal/mol energy gradient convergence criterion
[55, 56]. The optimized geometries were saved in molecular data base (mdb) file for further studies.
Molecular Docking studies against α-glucosidase
The optimized ligands were docked with the modeled yeast α-glucosidase protein, which was carried out in another
studies [57], using the MOE-Dock suite. The Placement, Rescoring 1 and Rescoring 2 were set as Triangle Matcher,
London dG and affinity dG, respectively. Algorithm with two rescoring functions London dG and GBVI/WSA dG
were utilized to generate 30 poses of each compound. As a result of docking run mdb output files were generated
enclosing all docking results with scoring and multiple conformations of ligands. The resulting conformations and
their energies were recorded to a molecular database file. The top ranked conformation (best scored pose) for each
compound was chosen for further studies of interaction evolution and validation. The 2D ligand-protein interactions
were visualized using the MOE ligand interactions program. Since, the 3D structure of yeast α-glucosidase has not
yet been determined by any experimental methods, we took the 3D coordinates of the modeled yeast α-glucosidase
which was used as our target protein [57].
Urease inhibition assay protocol
The urease activity was determined by measuring amount of ammonia produced by indophenols method [58, 59].
Briefly, the assay mixture, containing 10 µL of enzyme (5 U/mL) and 10 ꢀL of test samples in 40 ꢀL buffer (100
mM urea, 0.01 M K2HPO4, 1 mM EDTA and 0.01 M LiCl2, pH 8.2), were incubated for 30 min at 37 ºC in 96-well
plate. The 40 ꢀL each of phenol reagents (1 %, w/v phenol and 0.005 %, w/v sodium nitroprusside) and 40 ꢀL of
alkali reagent (0.5 %, w/v NaOH and 0.1 % active chloride NaOCl) were added to each well. The absorbance at 625
nm was measured after 30 min, using a microplate reader (OPTI Max, Tunable). All reactions were performed in
triplicate and thiourea was used as the standard inhibitor of urease.
Kinetic analysis for the Urease inhibition assay
Lineweaver-Burk plots of 1/absorbance versus 1/Urea were used to determine the type of inhibition. Urease
inhibition was measured by varying the concentration of urea in the presence of different concentrations (0, 13.7,
27.5 and 55 µM) of the tested compound (7b). The EI dissociation constant (Ki) was determined by secondary replot
of slope versus inhibitors concentrations while ESI dissociation constant (Kiʹ) was determined as the intersection on
the X-axis of the secondary replot of 1/V (y-intercept of Lineweaver-Burk plot) versus inhibitor concentrations.
Briefly the urea concentration was changed from 3.12 to 100 mM for urease kinetics studies and remaining
procedure was same as describes in urease inhibition assay protocol. Urease activity was determined by measuring
ammonia production using the indophenol method as reported previously [60]. The results (change in absorbance
per min) were processed by using SoftMaxPro software.
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Molecular Docking studies against Urease
The optimized ligands were docked with the Jack Bean Urease (PDB code:3AL4) protein using the MOE-Dock
program [54]. A total of 30 independent docking runs were performed using MOE docking simulation program. All
the docked conformations were analyzed and the best scored pose for each compound was opted for further studies
of interaction evaluation. The 2D ligand-protein interactions were visualized using the MOE ligand interactions
program. Default MOE docking parameters i.e., Triangle Matcher, Algorithm with two rescoring functions London
dG and GBVI/WSA dG were utilized to generate 30 poses of each compound. As a result of docking run mdb output
files were generated enclosing all docking results with scoring and multiple conformations of ligands.
Results and discussion
Synthesis of target compounds 4a-k and 7a-k
The conversion of substituted carboxylic acids 1 to the substituted acid chlorides 2 were indicated in the FT-IR
spectral data by the disappearance of broad signal in the range of 3400-2500 cm-1 due to acid hydroxyl group. The
formation of substituted acid hydrazides 3 were indicated in the IR spectra by the appearance of new signal at 3346,
3296 and 3156 due to primary and secondary amino group of acid hydrazides. Meanwhile there was slight shift in
the carbonyl stretching vibration from 1733-1634 cm-1 indicating the successful conversion of substituted acid
1
chlorides 2 into substituted aromatic acid hydrazides 3. In the H NMR spectrum, the appearance of broad singlet
due to primary and secondary amino group at 9.23 ppm as well as 4.25 ppm, respectively, further confirms the
successful synthesis of 3. The coupling of substituted aromatic acid hydrazides 3 with several substituted carboxylic
acids were carried out by refluxing with phosphorus oxychloride at 107 °C which afford corresponding 2,5-
disubstituted 1,3,4-oxadiazole derivatives 4a-k. The disappearance of signal at 3346, 3296 and 3156 due to primary
and secondary amino group of acid hydrazides gives indication for the formation of substituted oxadiazole
derivatives. The further confirmation was done by NMR spectral analysis.
1
Meanwhile, the formation of substituted triazole derivatives 6 were characterized by the FT-IR, H NMR and 13C
NMR spectral data. In the FT-IR spectra, a relatively broad peak in the range of 3280, 3169 cm-1 with shoulder for
NH2 stretching vibration and a new signal at 1321 cm-1 for C=S stretching vibration indicate the transformation of 3
1
into 6. In the H NMR, a characteristic highly downfield signal at 13.57 ppm due to triazole ring secondary amino
proton resolution confirms the formation of 6. The condensations of 6 with different substituted aldehydes were
confirms by NMR (1H NMR & 13C NMR) spectral analysis. The slight down field shift in the NH signal from 13.57
ppm to 13.88 ppm and complete disappearance of NH2 signal at 5.60 ppm confirms the Schiff base formation. In the
meantime, the appearance of down field singlet in the range of 10.03-10.18 ppm due to imine proton and emergence
of additional signals in both carbon and proton NMR spectra further confirmed the successful conversion of 6 into 7.
Thione-thiol tautomerism in the compounds 7a-k
The target compounds 7a-k have labile hydrogen atom in the 1,2,4-triazole nucleus and there is possibility for this
hydrogen atom to stay whether on the ring nitrogen atom or it may stay on the sulfur atom connected to the 5-
membered ring structure. Due to this replaceable nature of hydrogen atom, theoretically, the two tautomeric
conformational structures of the resulting molecules are possible as shown in the Scheme 2. The preliminary
investigations about the predominant conformational structure of the synthesized analogues were done by recording
the FT-IR spectra. There were clear indications about the thione tautomer of the compounds 7a-k in the FT-IR
spectral analysis as there was no signal appearance in the range of 2500-2400 cm−1 due to terminal thiol strectching
viabration. The absence of –SH signal in the FT-IR spectra support the thione conformatinal structure of the
synthesized molecules. For better understanding about the predominant isomer of 1,2,4-triazole's Schiff bases, one
of the compound 7k was characterized by the single crystal X-ray diffraction analysis and from the single crystal
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data of the molecule 7k, it can be clearly found that molecule predominantly exist in thione tautomeric conformation
as shown in Fig. 3.
Scheme 2, Fig. 3
The packing diagram of 7k showed the formation of molecular chains parallel to the short x axis via classical
inversion-symmetric H bonding systems. The exclusive presence of the NH–C=S tautomer is confirmed by the
relevant molecular dimensions and by the direct localization and free refinement of the NH hydrogen. In the crystal
structure, intermolecular N—H---S hydrogen bonds (Fig. 4) link the molecule to infinite chain, which seems to be
effective in the stabilization of the crystals. The bond length [Å] and bond angle [°] for hydrogen bond of 7k are
shown in Table 2.
Fig. 4 and Table 2
Molecular geometry
The crystal suitable for X-ray diffraction analysis was obtained by the slow evaporation of methanolic solution of 7k
at room temperature and the single crystal data was obtained by using Bruker SMART APEX II X–ray
diffractometer equipped with Mo X–ray tube, graphite monochromator and CCD detector at 100(1) K. The H atom
of the NH group was refined freely and methyl groups were refined as an idealized rigid groups allowed to rotate but
not tip; other H atoms were included using a riding model. The final wR2 was 0.1038 and 0.1111 for all reflections
with conventional R1 values of 0.0410 and 0.0488. The experimental structural parameters including bond length
and bond angles of the molecule are tabulated in the Table 3, 4 and in accordance, the atom numbering scheme are
given in Fig. 3. Briefly, in the target compound 7k, the N(1)-C(5) and N(1)-C(2) bond length was 1.378(2) and
1.3811(19) Å, respectively, which lies in between double and single bond suggesting the delocalization of electronic
cloud inside the triazole ring. Meanwhile, the C(2)-S(13) bond length was 1.6795(18) Å which represent the C=S
character as the bond length for C-SH has been reported as 1.80 Å [61]. These facts give solid evidence about the
involvement of molecule into thione-thiol tautomerism but the predominant conformation of compound 7k is the
thione conformation as shown by the molecular structure of 7k (Fig. 3). The rest of bond length for triazole skeleton
was N(4)-C(5); 1.299(2), C(2)-N(3); 1.338(2), N(3)-N(4); 1.377(2) and N(3)-H(3); 0.85(2) Å. Furthermore, the
N(1)-N(6) exhibited single bond character as the bond length values reside at 1.4037(18) while the bond length
values for the N(6)-C(7) was observed at 1.281(2) clearly showing the double bond character and this is the linking
site via imine bond formation between triazole skeleton and the side coupled group. In the same manner, the bond
length for the C(8)-S(9) and S(9)-C(10) was 1.7242(17) and 1.703(2), respectively, completely match with the
reported thiophene C-S bond length. While the bond length for the rest of thiophene skeleton i. e., C(8)-C(12);
1.370(2), C(10)-C(11); 1.345(3), C(11)-C(12); 1.415(3) exhibited slight deviation from the standard bond length for
the single and double bonds which is due to the delocalization of the electronic cloud inside the thiophene moiety.
Moreover, the bond length for the rest of molecules was within the normal range as shown in the Table 3.
Table 3
The bond angles in the molecule 7k were within the normal range. Briefly, the bond angles for methoxy substituent
including O(21)-C(22)-H(22A), O(21)-C(22)-H(22B), H(22A)-C(22)-H(22B), O(21)-C(22)-H(22C), H(22A)-C(22)-
H(22C) and H(22B)-C(22)-H(22C) fall at 109.5 degree showing the tetrahedral geometry. All the bond angles for
the benzene ring was within the normal range while the bond angles for triazole ring exhibited slight deviation due
to presence of strain in the five member ring structure. The C(5)-N(1)-C(2) and N(3)-C(2)-N(1) bond angle
exhibited more distortion with the values close to the tetrahedral geometry in former case i.e., 108.62(12) while
more shortness in later case i. e., 102.57(13). The lower values for the internal bond angles of triazole nucleus was
observed for the C(2)-N(3)-N(4); 114.00(14) and C(5)-N(4)-N(3); 104.23(13) as compare to the external bond
11

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angles involving the triazole nucleus i. e., C(5)-N(1)-N(6); 122.02(12), C(2)-N(1)-N(6); 128.33(13), N(3)-C(2)-
S(13); 128.50(12), N(1)-C(2)-S(13); 128.92(11), C(2)-N(3)-H(3); 126.2(14), N(4)-N(3)-H(3); 119.7(14), N(4)-C(5)-
C(14); 125.40(15) and N(1)-C(5)-C(14); 124.04(14). The bond angles for the imine junction was slightly shortened
from the trigonal geometry as the experimental values for C(7)-N(6)-N(1) was 114.93(12). Meanwhile, very short
bond angles was observed for the C(10)-S(9)-C(8); 91.57(9) due to presence of ring strain in the thiophene skeleton.
Similarly, the shortening of the internal bond angles was observed for the thiophene nucleus i. e., C(11)-C(10)-S(9);
112.46(14), C(8)-C(12)-C(11); 112.42(15) and C(11)-C(10)-S(9); 112.46(14) while the external bond angles was
close to the trigonal bond angles with the observed values of C(11)-C(10)-H(10); 123.8, S(9)-C(10)-H(10); 123.8,
C(10)-C(11)-H(11); 123.7, C(12)-C(11)-H(11); 123.7, C(8)-C(12)-H(12); 123.8, C(11)-C(12)-H(12); 123.8 and
C(7)-C(8)-S(9); 122.53(12). Furthermore, the bond angles for the rest of molecules were within the normal range as
shown in the Table 4.
Table 4
Bioevaluation
α-Glucosidase inhibition activity of 2,5-disubstituted 1,3,4-oxadiazole derivatives 4a-k
Diabetes mellitus is one of the most serious and chronic diseases associated with an increase in both obesity and
ageing in the general population. The management of blood glucose levels after a meal is a critical strategy in the
control of diabetes and its complications. One of the therapeutic approaches for decreasing postprandial
hyperglycemia is to retard the absorption of glucose in blood return to its normal limits by inhibiting carbohydrate
hydrolyzing enzymes (i.e., alpha‐glucosidase) in the digestive organs [62]. Inhibitor drugs with α-glucosidase
inhibitory activities could be considered potential candidates to manage the early stages of hyperglycemia risk
linked to type 2 diabetes. Based on these facts, we have synthesized a new series of 2,5-disubstituted 1,3,4-
oxadiazole derivatives 4a-k and screened for their α-glucosidase inhibition activity. Acarbose with IC50 value of
0.382 ± 0.023 mM was used as a reference inhibitor. The compounds 4b and 4c were the most potent inhibitor of α-
glucosidase with IC50 values of 0.641 ± 0.12 and 0.873 ± 0.18 mM, respectively. The compound 4b has 4-OCH3
group as substituent R while strong electron withdrawing 4-F group as substituent X with n values of 1 and n' = 2
and the compound 4c exhibited electron donating 4-OCH3 group as substituent R and electron withdrawing 3-F
group as substituent X with the same number of carbon atoms in between oxadiazole ring and side coupled aryl
group. Meanwhile, the compound 4d with variation in the substituent X as 2-F group possess slight lower α-
glucosidase inhibition activity i. e., IC50 values of 1.021 ± 0.13 mM but still the third most active compound among
the series. All of the other compounds exhibited moderate to good α-glucosidase inhibition activities with IC50
values ranging from 1.161 ± 0.09 to 1.44 ± 0.05 mM. The least active compound among the series was 4k with IC50
values of 1.44 ± 0.05 mM and it possess 2-OCH3 group as substituent R and X with the n = 2 and n' = 1. In general,
we have synthesized a new class of compounds with excellent anti-diabetic potential compare to that of reference
drug. From these findings, it was concluded that the compounds substituted with electron withdrawing fluoro group
were found to be more potent α-glucosidase inhibitor while those with electron donating group exhibited moderate
inhibition potential against tested bioassay. The results of α-glucosidase inhibition assay for the 2,5-disubstituted
1,3,4-oxadiazole derivatives 4a-k are tabulated in Table 5.
Table 5
Mechanism underlying inhibitory effect of compounds 4b and 4c on α-glucosidase
Since 4b and 4c were the most potent inhibitors in hand, we therefore further study mechanism underlying this
inhibitory effect. The kinetic studies of the enzyme by the Lineweaver–Burk plot of 1/V versus 1/[S] in the presence
of different concentrations of 4b and 4c gave a series of straight lines as shown in Fig. 5 and Fig. 6, respectively.
The results of Fig. 5a showed that the compound 4b was a competitive inhibitor since increasing its concentration
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result the same Vmax but different Km values. The equilibrium constant Ki for inhibitor binding with free enzyme was
obtained from a plot of the inhibitor concentration [4b] versus the apparent Michaelis–Menten constant (Km) which
is a linear as shown in the inset (Fig. 5b). The obtained Ki of 4b was 1.5 × 10−4 M. The results of kinetic constants
and the inhibition constants are summarized in Table 6.
Fig. 5 and Table 6
The results of Fig. 6a showed that by increasing the concentration of substrate yielded straight lines, all of which
intersected within the second quadrant. The analysis showed that Vmax decreased with increasing Km in the presence
of increasing concentrations of 4c. This behavior of inhibitor compound 4c indicated that it inhibits α-glucosidase by
two different pathways; forming enzyme substrate inhibitor (ESI) complex in noncompetitive manner and enzyme
inhibitor (EI) complex in competitive manner. The plot of slope against concentration of 4c showed EI dissociation
constant values Ki = 2.5 × 10−4 and ESI dissociation constant values of Kiʹ= 4.0 × 10−3 (Fig. 6b and c). The lower
dissociation constant (Ki) (Table 7) pointed out stronger binding between enzyme and 4c which suggested preferred
competitive over noncompetitive manners.
Fig. 6 and Table 7
Moreover, the concentration dependent enzyme inhibition potential was observed for the compounds 4b and 4c
displaying the comparable α-glucosidase inhibition with that of reference acarbose as shown in Fig. 7. However, the
complete suppression of activity was not observed at any concentration of the inhibitor drug.
Fig. 7
Molecular docking studies
The ligand-protein binding modes and interactions are vital to understand the catalytic activity as the preferred
orientation of ligands in 3D space into the protein binding site determines the activity of that ligand. Therefore, all
the 30 conformations of ligands were docked inside the pocket despite the different level of binding energy and
interactions with key residues. The docked poses were prioritized on the bases of binding free energy and ligand
interaction. MOE ranked the docked conformations on the bases of binding energy (London dG) and binding free
energy in the “S” field. Detailed docking results are tabulated in Table T1 (Supporting information). These results
are recorded on the bases of 2D interactions of the most preferred docked poses. All the preferred docked poses
stacked beautifully inside the cavity.
Fig. 8
The most proffered docked poses of all the ligand showed that none of the ligand expressed any hydrogen bonding
and Pi-H bonding with the side chain of the amino acids or back bone residues. Pi-Pi stacking is also established by
only ligand 4e and 4g with HSD239. However 4e and 4g anchored over binding site by lesser number of the non-
bonded interactions as compared to the ligands which showed better activity. All the ligands bind in active site
through fewer non-bonded interaction with that of polar residues and hydrophobic residues. The finding obtained in
docking computations is in close agreement with the experimental studies. Fig. 9 depicts the interaction of the most
active ligands (4b, 4c and 4d) with amino acid residues of active site in 2 dimensional (2D) space while 2-D
interaction of all the docking poses of most preferred conformation of the series (4a-k) along with the control drug
used in this studies are shown in Fig. S1.
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All the inhibitors showed binding free energy in S filed (-6.9187 to -7.6047 Kcal/mol) and binding energy, London
dG in Escore-1 field (-9.5093 to -9.7277 Kcal/mol) in narrow range. In current studies the theoretical results
synchronized well the experimental results. The compounds 4b, 4c and 4d inhibited the catalytic activities of the α-
glucosidase by fewer non-bonded interactions.
Fig. 9
Taking into account the peculiarities of the active-site residues in the α-glucosidase catalysis, it can be concluded
that some of the compounds of 4a-k series bind in active site through non-bonded interactions with Phe 157, Phe
177, Phe 300 Arg 312, Arg439 and Asp349.
Urease inhibition activity of the triazole Schiff base derivatives 7a-k
All the synthesized compounds 7a-k were evaluated for their urease inhibition activities by employing thiourea as
reference inhibitor with the IC50 value of 20.9 ± 0.92 µM. All the IC50 values were recorded in triplicate and this
data is presented as + SEM (standard error of the mean, n=3). The compounds 7g and 7b with the IC50 value of
8.02 ± 0.59 and 17.02 ± 4.63 µM, respectively, were most potent with the higher inhibition in comparison to the IC50
value of the reference inhibitor thiourea as shown in Table 8. The compound 7b was further employed for the
kinetic analysis in order to trace the mechanism underlying during the assay measurement. The Lineweaver-Burk
plot represent the mixed type inhibition by the compound 7b with the EI and ESI dissociation constant values of 3
and 42 ꢀM, respectively. The compounds 7g and 7b possessed the 2-ethylphenyl and 2-thiophenyl group as
substituent Y while 3-bromo and 2-bromo group as substituent R, respectively. There was no extra carbon between
the triazole skeleton and the side coupled aryl group. The compound 7h with the IC50 value of 20.29 ± 2.76 µM
exhibiting comparable urease inhibition potential with that of reference thiourea. The compound 7h has 3-OCH3
group as substituent R while benzyl group as substituent Y representing that the replacement of bromo group at
position 3 of side coupled aryl ring caused the reduction of urease inhibition activity of the resulting analogue.
Meanwhile, the compounds 7j, 7e, 7c and 7a with the IC50 values of 25.45 ± 3.09, 35.71 ± 6.28, 39.30 ± 5.03 and
42.59 ± 4.35 µM, respectively, showed moderate urease inhibition potential. In all of these compounds, there is no
bromo substitution over side coupled aryl rings. The activities of rest of compounds were in the range of 77.34 ±
7.92 to 186.8 ± 16.95 µM. The compound 7d with the IC50 value of 186.8 ± 16.95 µM was the least active
compound among the investigated series. This compound possessed the fluoro group at position 2 of the side
coupled aryl ring and 2-pyrroyl substitution as the group Y connected to the central nucleus via Schiff base spacer.
The overall structure activity relationship of the synthesized compounds in comparison to their inhibition potential
reveals that the presence of bromo substituent at position 3 and 2 of the aryl ring as well as 2-ethylphenyl group
coupled via imine formation with the central triazole nucleus impart potent inhibitory characters to the compounds.
The methoxy substitution to the side coupled aryl group induces the moderate inhibition while the least inhibition
efficacy was observed for the analogues possessing strong electron withdrawing fluoro substitution on side coupled
aryl ring. On the other hand, the 2-ethylphenyl group as substituent Y coupled to the central triazole skeleton via
imine linkage was found more effective in imparting inhibitory characters toward urease while 2-pyrroyl group
reduced the activities of the compounds.
Table 8
Mechanism underlying inhibitory effect of compound 7b
The kinetic investigation for the urease inhibition analysis was accomplished by the Lineweaver–Burk plot of (1/V)
versus 1/[S] in the presence of different concentrations of inhibitor (7b) which gave a series of straight lines as
shown in Fig. 10. Furthermore, the EI dissociation constant (Ki) and ESI dissociation constant (Ki´) were determined
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to gain insightful pathway by secondary replots. By increasing the concentration of substrate (urea) gave a family of
straight lines, all of which intersected within the second quadrant. The analysis showed that Vmax (reaction velocity)
decreased with increasing Km (Michaelise-Menten constant) in the presence of increasing concentrations of
compound (7b). This behavior of compound (7b) indicated that it is a mixed type inhibitor with respect to the
substrate urea exhibiting the Ki and Ki´ values of 3 and 42 µM, respectively, as shown in Fig. 10 (b, c) [51].
Fig. 10
Molecular docking studies
Molecular docking of 7a-k series into the crystal structure of jack bean urease was performed by using the MOE
program. The most favorable docking conformations of all the compounds were stacked well inside the active site
cavity. The active site was architectured with both the hydrophobic and hydrophilic amino acids. The hydrophobic
part was constructed by Leu523, Ala 636, Ala440, Met 637 and Met 588 while hydrophilic portion contained
Arg439, His 492, Asp494, His519, His593, Glu493and Arg609 amino acids. It has been observed that two Ni ions
played a significant role by bridging the amino acids residues inside the active site. All the 30 conformations of
ligands were docked inside the pocket despite the different level of binding energy and interactions with amino acids
residues inside the active site .The docked poses were prioritized on the bases of binding free energy and ligand
interaction. MOE ranked the docked conformations on the bases of binding energy (London dG) and binding free
energy in the “S” field. Detailed docking results are tabulated in Table T2 (Supporting information). These results
are recorded on the bases of 2D interactions of the most preferred docked poses. All the preferred docked poses
stacked beautifully inside the cavity as shown in Fig. 11.
Fig. 11
An insight into the binding mode analysis of the most preferred docked conformations revealed that the ligands 7b,
7g and 7h were tightly anchored over the active-site cavity through hydrogen bonding. A very significant hydrogen
bonding interactions with the Met637, Arg439 and Met588, has been noticed. Ni ions played a significant role in
orientation of the residues inside the close vicinity of active site through chelation as shown in Fig. 12. The Fig. 12
depicted the 3-D interaction of the most significant inhibitors 7b, 7g and 7h. The binding pattern adopted by these
ligands made them engage the active site residues, using typical hydrogen bonds to anchor tightly with the side
chain and backbone of the enzyme. The phenyl ring found to be involved in the arene-H interaction in case of the
most active ligands. Only ligands 7c showed arene-arene stacking.
Fig. 12
All the inhibitors showed binding free energy in S filed (-5.0312 to -6.0760 Kcal/mol) and binding energy, London
dG in Escore-1 field (-8.5129 to -9.7104 Kcal/mol) in narrow range. The theoretical results synchronized well the
experimental results in current studies. The compounds which didn’t show any significant interaction were found
inactive in experimental screening such as 7i and 7d. The compounds 7b, 7g and 7h presented remarkable
interactions with amino acid residues in active site these were found to exhibit far better activity than that of
standard drug in experimental screening. This fact could be attributed to the multiple strong binding interactions by
these ligands with amino acid residues of the jack bean urease as compared to the standard drug thiourea. The
docking mode of the most potent compounds, 7b, 7g and 7h inhibited the catalytic activities of the urease by
anchoring firmly through strong hydrogen bonding, Pi-H interactions and tremendous polar interactions in active
site. Fig. 13 depicts the interaction of the most active ligands (7b, 7g and 7h) with key residues of active site in 2
dimensional (2D) space while 2-D interaction of all the docking poses of most preferred conformation of the series
(7a to 7k) along with the control drug used in this studies are shown in Fig. S2 (Supporting information).
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Fig. 13
Taking into account the distinctiveness of the active-site residues in the urease catalysis, it can be concluded that
some of the compounds of 7a-k made strong contacts with the side chains of urease residues, which was reflected in
their enhanced affinity to the Arg439, Ala 636, Ala440, Met 637, His592, Asp494, Arg439, Met588 and Met637
residues [63,64]. Besides being directly involved in the architecture of the active site, the nickel ions had a vital role
in positioning other key residues in the active site appropriately for the catalysis through various interactions such as
hydrogen bonding and chelation.
Conclusion
In summary, we have synthesized the series of substituted oxadiazole derivatives 4a-k as well as triazole Schiff base
1
derivatives 7a-k. The structures of these compounds were characterized by FT-IR, H NMR, 13C NMR and single
crystal X-ray diffraction analysis. The X-ray single crystal structure of compound 7k showed that molecule
predominantly exist in thione conformation. The synthesized compounds were evaluated for bioactivities and kinetic
analysis. The oxadiazole derivatives 4a-k were found as potent α–glucosidase inhibitors while the Schiff base
derivatives 7a-k exhibited considerable potential toward urease inhibition. The compounds 4b and 4c were the most
potent inhibitor of α-glucosidase with the IC50 values of 0.641 ± 0.12 and 0.873 ± 0.18 mM, respectively, exhibiting
more inhibition potency in comparison to the IC50 values of reference acarbose (IC50 value of 0.382 ± 0.023 mM).
Meanwhile, the compound 4d was screened as 3rd most active compound among the tested series with the IC50
values of 1.021 ± 0.13 mM. The inhibition kinetics analyzed by Lineweaver–Burk plots against α–glucosidase
revealed that the compound 4b act as competitive while 4c as mixed inhibitors with the inhibition constant values of
1.5 × 10−4 and 4.0 × 10−3 M, respectively. In case of Schiff base series 7a-k, the compounds 7g and 7b with the IC50
values of 8.02 ± 0.59 and 17.02 ± 4.63 µM, respectively, showed excellent inhibition potential toward the urease
even higher inhibition than the reference thiourea (IC50 = 20.9 ± 0.92 µM). For mechanistic insight, the compound
7b was further employed for the kinetic analysis via Lineweaver-Burk plot which represents the mixed type
inhibition by the compound 7b alongside the EI and ESI dissociation constant values of 3 and 42 ꢀM, respectively.
The 3rd most active urease inhibitor among the tested series was 7h with the IC50 value of 20.29 ± 2.76 µM
exhibiting comparable urease inhibition with that of reference thiourea. Meanwhile, the compounds 7j, 7e, 7c and 7a
with the IC50 values of 25.45 ± 3.09, 35.71 ± 6.28, 39.30 ± 5.03 and 42.59 ± 4.35 µM, respectively, showed
moderate urease inhibition activity. The commendable urease inhibition was observed by the designed Schiff bases
of 1,2,4-triazole in comparison to previously reported isolated triazole based molecules or Schiff base derivatives
where there was no direct connection of imine bond with that of triazole skeleton. Moreover, according to the
systematic investigation on the binding mode of the α-glucosidase and urease inhibition by the compounds 4a-k and
7a-k, respectively, it could be deduced that the compounds 4b, 4c, 7b, 7g and 7h were competitive inhibitors
targeting amino acids in the active site of the corresponding protein. The docking results and experimental screening
results were found in close agreement.
Conflict of interest
All authors declare that they have no conflicts of interest.
Acknowledgements
This work was supported by a grant from Mid-Career Researcher Program (NRF-2016R1A2B4016552) through the
National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning(MSIP).
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We are extremely grateful for the cooperation of Dr. Charles Dennis Hall and Prof Alan Roy Katritzky (late)
(University of Florida) for the MOE software license. Department of Energy, United State of America through Grant
No. DE-SC0001717 is gratefully acknowledged.
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Schemes, Figures and Tables along with captions
Scheme 1
Scheme 1 Synthesis of target compounds 4a-k and 7a-k: Reagents and conditions: (i) POCl3, ClCH2CH2Cl, reflux
3 h; (ii) hydrazine hydrate, TEA, MeCN, reflux 3 h; (iii) substituted aromatic acid, POCl3, reflux, 3 h; (iv) CS2,
KOH, methanol, stirring, 0 °C, 1 h; (v) Hydrazine hydrate (80 %), water, reflux, 10-12 h; (vi) different substituted
triazole derivatives, substituted aldehydes, methanol, microwave pulse (10 min).
Scheme 2
Scheme 2 Thione-thiol tautomerism in the compounds 7a-k
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Fig. 1
Fig. 1 Molecular structure of known anti-diabetic compounds
Fig. 2
Fig. 2 Molecular structure of the reported 1,2,4-triazole based urease inhibitors.
Fig. 3
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Fig. 3 Single crystal X–ray structure of 7k with the atom-numbering scheme
Fig. 4
Fig. 4 The packing diagram for 7k in the regular crystal lattice; the dash line represent the intermolecular hydrogen
bonding within the compound 7k.
Fig. 5
Fig. 5 Lineweaver–Burk plots for 4b against α-glucosidase inhibition assay; (a) concentration of 4b was 0 mM, 1.6
mM and 3.2 mM; substrate concentration was 1.25 mM, 2.5 mM and 5 mM, respectively; (b) Inset graph represents
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the plot of Km versus the concentration of 4b in order to determine the inhibition constant (Ki). The line was drawn
using linear least-squares fitting method.
Fig. 6
Fig. 6 Lineweaver–Burk plots against α-glucosidase inhibition in the presence of compound 4c; the concentrations
of 4c was 0 mM, 0.096 mM and 1.58 mM, respectively while substrate concentration was 1.25 mM, 2.5 mM and
5 mM, respectively; the insets represent the plot of the slope and the vertical intercepts (1/Vmax app) versus 4c
concentrations in order to determine inhibition constants which was drawn by using linear least squares fitting
method (inset graph b and c).
Fig. 7
Fig. 7 Concentration-dependent α-Glucosidase inhibition by the compounds 4b, 4c and reference Acarbose
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Fig. 8
Fig. 8: Superimposition of the ligands inside the active site of homology modeled α-glucosidase; (a) Homology
model of the yeast α-glucosidase (blue, yellow and red color) showing the ligand cluster (cyan color; stick mode)
into the binding site. The green color indicates the amino acid residues (labeled in red) surrounding the binding site;
(b) Close view of the active site, pocket is marked with surfaces (line mode); c) Binding site cavity with the ligand
cluster (amino acid residues are shown in elemental color, solid surfaces).
Fig. 9
Fig. 9: Docked poses of the most ligands inside the active site in 2-D space; a) 4b; b) 4c and c) 4d. (Legend about
color and rest of the pattern is given in Fig. S1 in supporting information).
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Fig. 10
Fig. 10. The double reciprocal Lineweaver–Burk plots for the inhibition of urease in the presence of compound (7b)
(a); the concentrations of 7b were 0, 13.7, 27.5 and 55 µM, respectively using the urea as substrate. The urea
concentrations were 3.12, 6.25, 12.5, 25, 50 and 100 mM, respectively. The insets represent the plot of the slope
(inset, b); and the vertical intercepts (inset, c), versus inhibitor (7b) concentrations in order to find out the inhibition
constants. The lines were drawn using linear least squares fit.
Fig. 11
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Fig. 11. Superimposition of the ligands inside the active site; (a) Overlaying of all the minimum energy docked
poses inside the pocket. Ligands are shown in cyan color in stick mode while key residues are shown in green color
in stick mode. Backbone receptor is shown in cartoon and ribbon form. (b) Close view of the active site, pocket is
marked with surfaces (line mode).
Fig. 12
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Fig. 12. 3D Interaction of ligand 2a (a-b) and 7a (e-f) 8a (g-h). Ligands are shown in cyan color (stick n ball mode),
key residues of the active site are shown in green and element color (stick mode) nickel ions are shown as cyan dots.
Hydrogen bonding and other interactions are shown as solid green color lines, marked with the distance. Interaction
of amino acids with each other is shown in cyan color dotted lines; a) Interaction of S atom with atoms Arg 439; b)
Pi-H contact of phenyl ring with Ala440, (shown in red colored dotted line) 7a (c-f); c) Interaction of Br atom with
Met588 and Met637; d) Hydrogen bonding of ligand atom with Cme592 (shown in blue color); e) Hydrogen
bonding of ligand atom with Arg439; f) Contact of Hydrogen with S atom of Met 637 8h (g-i); g) Interaction of S
atom with side chain of Arg 609, Met 493 and Glu439; h) Hydrogen bonding of ligand atoms with Met637; i)
Hydrogen bonding of ligand atoms with Arg439
Fig. 13
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Fig. 13: Docked poses of the most ligands inside the active site in 2-D space; a) 7b, b) 7g and c) 7h. Interactions are
shown in green dotted lines (Legend about color and rest of the pattern is given in Fig. S2 in Supporting
information).
Tables along with captions
Table 1
Table 1 Crystallographic data for compound 7k
Description
Empirical formula
Experimental values Description
C15H14N4OS2 Cell formula unit, z
Experimental values
4
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