2,5-Disubstituted thiadiazoles as potent β-glucuronidase inhibitors; Synthesis, in vitro and in silico studies

Article abstract of DOI:10.1016/j.bioorg.2019.103126

Twenty-five thiadiazole derivatives 1–25 were synthesized from methyl 4-methoxybenzoate via hydrazide and thio-hydrazide intermediates, and evaluated for their potential against β-glucuronidase enzyme. Most of the compounds including 1 (IC₅₀ = 26.05 ± 0.60 μM), 2 (IC₅₀ = 42.53 ± 0.80 μM), 4 (IC₅₀ = 38.74 ± 0.70 μM), 5 (IC₅₀ = 9.30 ± 0.29 μM), 6 (IC₅₀ = 6.74 ± 0.26 μM), 7 (IC₅₀ = 18.40 ± 0.66 μM), and 15 (IC₅₀ = 18.10 ± 0.53 μM) exhibited superior activity potential than the standard D-saccharic acid-1,4-lactone (IC₅₀ = 48.4 ± 1.25 μM). Molecular docking studies were conducted to correlate the in vitro results and to identify possible mode of interaction with enzyme active site.

Full text of DOI:10.1016/j.bioorg.2019.103126

Accepted Manuscript
2,5-Disubstituted Thiadiazoles as Potent β-glucuronidase Inhibitors; Synthesis,
In Vitro and In Silico Studies
Muhammad Taha, Noor Barak Almandil, Umer Rashid, Muhammad Ali,
Mohamed Ibrahim, Mohammed Gollapalli, Ashik Mosaddik, Khalid
Mohammed Khan
PII:
S0045-2068(19)30345-1
https://doi.org/10.1016/j.bioorg.2019.103126
103126
DOI:
Article Number:
Reference:
YBIOO 103126
Bioorganic Chemistry
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Revised Date:
Accepted Date:
2 March 2019
5 July 2019
11 July 2019
Please cite this article as: M. Taha, N. Barak Almandil, U. Rashid, M. Ali, M. Ibrahim, M. Gollapalli, A. Mosaddik,
K. Mohammed Khan, 2,5-Disubstituted Thiadiazoles as Potent β-glucuronidase Inhibitors; Synthesis, In Vitro and
In Silico Studies, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103126
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2,5-Disubstituted Thiadiazoles as Potent β-glucuronidase Inhibitors; Synthesis, In Vitro and
In Silico Studies
Muhammad Taha,a Noor Barak Almandil,a Umer Rashid,b Muhammad Ali,c Mohamed
Ibrahim,a Mohammed Gollapalli,d Ashik Mosaddik,a Khalid Mohammed Khana,e
aDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box1982, Dammam 3144, Saudi Arabia
bDepartment of Chemistry, COMSATS Institute of Information Technology
cUoN Chair of Oman's Medicinal Plants and Marine Natural Products, University of Nizwa,
P.O. Box 33, Birkat Al Mauz, Nizwa 616, Oman
dCollege of Computer Science & Information Technology (CCSIT) Imam Abdulrahman Bin
Faisal University P.O. Box 1982, Dammam 31441, Saudi Arabia
eH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
Abstract: Twenty-five thiadiazole derivatives 1-25 were synthesized from methyl 4-
methoxybenzoate via hydrazide and thio-hydrazide intermediates, and evaluated for their potential
against β-glucuronidase enzyme. Most of the compounds including 1 (IC50 = 26.05 ± 0.60 μM), 2
(IC50 = 42.53 ± 0.80 μM), 4 (IC50 = 38.74 ± 0.70 μM), 5 (IC50 = 9.30 ± 0.29 μM), 6 (IC50 = 6.74 ±
0.26 μM), 7 (IC50 = 18.40 ± 0.66 μM), and 15 (IC50 = 18.10 ± 0.53 μM) exhibited superior activity
potential than the standard D-saccharic acid-1,4-lactone (IC50 = 48.4 ± 1.25 μM). Molecular
docking studies were conducted to correlate the in vitro results and to identify possible mode of
interaction with enzyme active site.
Keywords: Synthesis, thiadiazoles, docking study, β-glucuronidase inhibition, SAR.
Introduction
Thiadiazole nucleus is one of the most important and well-known heterocyclic nuclei that is
present in variety of medicinal agents as well as in natural products. Among four isomers of
thiadiazole, the isomer 1,3,4-thiadiazole constitutes an important class of heterocyclic compounds
due to its important biological activities. 1,3,4-Thiadiazole nucleus is present as a core structural
component in a range of medicinal compounds, agriculture, and other fields of technology [1].
Many 1,3,4-thiadiazoles have been patented in the agricultural field as herbicides and bactericides.
Further to this, a broad spectrum of biological potentials related to 1,3,4-thiadiazole nucleus has
E-mail: taha_hej@yahoo.com and mtaha@iau.edu.sa; Tel: 00966502057370
1

been reported that includes antimicrobial [2-15], antifungal [16-18], antidiabetic [19], anti-
inflammatory [20,21], antileishmanial [22], antituberculosis [23], anticancer [24], anti-HIV [25],
antioxidant/radio protective [26], carbonic anhydrase [27], and urease inhibitory activities [28].
β-Glucuronidase is a lysosomal enzyme, its specific task is to catalyze the cleavage of
glucuronosyl-O-bonds. It degrades glucuronic acid-containing glycosaminoglycan, such as
heparan sulfate, chondroitin sulfate and dermatan sulfate. Over-expression of this enzyme has
reported in various types of malignancies such as breast, lung, and gastrointestinal tract
carcinomas, and melanomas. Elevated level of β-glucuronidase in bronchial tumors has also been
observed. Elevated activity of this enzyme was also reported in borderline tuberculoid and
lepromatous patients [29].
Immense experience of our research group in the field of computer-aided drug design (CADD)
and synthesis of chemical entities as putative drugs for the treatment of various diseases prompted
us to further explore new inhibitors for β-glucuronidase as lead compounds. We have already
reported various classes of compounds including coumarin diones, thiadiazole containing aryl and
N-aryl rings, thiazole Schiff bases, and unsymmetrical heterocyclic thiourea as β-glucuronidase
inhibitors (Figure-1) [30-33]. Previously, we identified the 2,5-disubstituted thiadiazoles as new
class of β-glucuronidase inhibitors. In the current study, we adopted a chemical transformation
which leads to the 2,5-disubstitutes thiazole molecules, in which -NH part of N-aryl group is
lacking, in order to see its potential effect on the β-glucuronidase inhibitory activity. Furthermore,
we used different substitutions on ring B, while keeping the methoxy phenyl ring (Ring A) as
permanent feature (Figure-1). Thus the synthetic compounds having two aryl ring directly linked
with thiadiazole scaffold were synthesized and explored for their potential inhibitory activity
against β-glucuronidase enzyme. To the best of our knowledge these compounds have never been
tested against β-glucuronidase enzyme previously.
2

N
S
OH
OH
N
R
R
S
N
N
H
H
Thiourea Derivatives [32]
Thiazole Derivatives [33]
H
S
MeO
N
R
S
R
N
N
R
N
N
Previously Identified Thiadiazole
Newly Identified
-Glucuronidase Inhibitors
Derivatives as Potent -


Glucuronidase Inhibitors [31]
Figure-1: Previously and newly identified β-glucuronidase inhibitors
Results and Discussion
Chemistry
Methyl 4-methoxybenzoate was refluxed in methanolic hydrazine hydrate solution for 6 h to obtain
corresponding hydrazide. Resulting hydrazide upon treatment with Lawson's reagent in toluene
was refluxed to yield corresponding thio-analogue. Thiohydrazide was then treated with various
aryl aldehydes to form cyclized adducts 1-25 (Table-1) in the presence of POCl3 (Scheme-1).
O
N
N
O
a,b,c
H3CO
S
H3CO
R
i
1-25
Scheme-1: Synthesis of 2,5-disubstituted thiadiazoles;
Reagents and Conditions: a) NH2NH2.H2O, MeOH, Reflux, 6 h; b) Lawesson's Reagent, Toluene, Reflux; c)
Substituted aldehydes, POCl3, Pyridine
Table-1: β-Glucuronidase inhibitory potential of compounds 1-25
S. No.
1
R-
IC50 ± SEMa (μM)
S. No.
R-
IC50 ± SEMa (μM)
26.05 ± 0.60
14
cN.A.
OH
OH
2
42.53 ± 0.80
15
18.10 ± 0.53
HO
3

3
cN.A.
16
cN.A.
H3CO
OCH3
OH
4
5
6
38.74 ± 0.70
9.30 ± 0.29
6.74 ± 0.26
17
18
19
cN.A.
45.72 ± 0.98
cN.A.
Br
HO
OH
OH
N
OH
OH
OH
OCH3
O
7
8
9
18.40 ± 0.66
52.36 ± 1.29
48.95 ± 1.04
20
21
22
cN.A.
39.00 ± 0.88
cN.A.
OH
OH
Cl
Cl
OH
N
OCH3
OH
10
38.44 ± 0.76
23
cN.A.
OCH3
OCH3
11
12
cN.A.
24
25
cN.A.
cN.A.
N
NO2
OH
42.44 ± 0.88
H3CO
F
4

13
cN.A.
Standardb
48.4 ± 1.25
SEMa is the standard error of the mean; bD-saccharic acid-1,4-lactone is standard inhibitor for β-glucuronidase activity; cN.A. stands for “Not Active”
β-Glucuronidase inhibition
All the synthetic compounds 1-25 showed excellent β-glucuronidase inhibitory activity in the
range of IC50 = 6.74 ± 0.26 - 52.36 ± 1.29 μM compared to the standard D-saccharic acid-1,4-
lactone (IC50 = 48.4 ± 1.25 μM). Limited structure-activity relationship (SAR) was established by
analyzing the effect of varying features such as R group at ring B as shown in Figure-2.
N
N
Ring-A
H3CO
S
Ring-B
R
1-25
Figure-2: General representation of compounds 1-25 for SAR analysis
Amongst all synthesized derivatives, hydroxy substituted analogs were found to be excellent
inhibitors. It was observed that 2-hydroxy substituted derivative 15 (IC50 = 18.10 ± 0.53 μM) was
found to be more active than 4-hydroxy and 3-hydroxy substituted analogs 1 (IC50 = 26.05 ± 0.60
μM) and 2 (IC50 = 42.53 ± 0.80 μM), respectively. Which gives a perception that hydroxy at
position-2 and -4 interacted more efficiently with the active site of enzyme, while a sharp change
in activity was observed when the hydroxy group was at position-3. Amongst the di-hydroxy
substituted analogs, 2,4-dihydroxy substituted derivative 6 (IC50 = 6.74 ± 0.26 μM) was found to
be the most potent analog of the current library. Changing positions of the two hydroxy groups
brought changes in the inhibitory potential. Such as 2,3-dihydroxy substituted compound 5 (IC50
= 9.30 ± 0.29 μM) showed slightly decreased inhibitory activity as compared to the derivative 6.
Similarly, 2,5- and 3,4-dihydroxy analogs 4 (IC50 = 38.74 ± 0.70 μM) and 7 (IC50 = 18.40 ± 0.66
μM), respectively, showed superior activity than the standard the standard D-saccharic acid-1,4-
lactone (IC50 = 48.4 ± 1.25 μM) (Figure-3). Thus, it can be concluded that di hydroxy analogs were
found to have more potential in comparison with mono hydroxy compounds.
5

N
N
OH
S
H3CO
H3CO
H3CO
OH
OH
2
N
N
N
N
IC50 = 42.53 ± 0.80 µM
S
S
OH
OH
H3CO
H3CO
IC50 = 38.74 ± 0.70 µM
1
OH
N
N
4
IC50 = 26.05 ± 0.60 µM
S
OH
6
OH
N
N
N
N
IC50 = 6.74 ± 0.26 µM
OH
OH
S
S
H3CO
H3CO
OH
5
7
N
N
IC50 = 9.30 ± 0.29 µM
IC50 = 18.40 ± 0.66 µM
S
15
IC50 = 18.10 ± 0.53 µM
Figure-3: SAR of hydroxy substituted compounds
Compounds bearing solely methoxy groups such as 3, 19, and 23 were found to be completely
inactive. However, incorporation of hydroxy with methoxy substitution brought out inhibitory
activity. Such as compounds 9 (IC50 = 48.95 ± 1.04 μM), 10 (IC50 = 38.44 ± 0.76 μM), and 12
(IC50 = 42.44 ± 0.88 μM) bearing hydroxy along with methoxy at different positions showed
comparable inhibition potential to the standard D-saccharic acid-1,4-lactone (IC50 = 48.4 ± 1.25
μM). Among them, compound 10 with 2-hydroxy and 4-methoxy substituents was more active,
however, shifting of hydroxy group to position-3 and methoxy to position-5 in compounds 9 and
12, respectively, resulted in decreased inhibition (Figure-4).
6

OH
N
N
N
N
N
N
OCH3
OH
S
S
S
HO
H3CO
OCH3
OCH3
H3CO
H3CO
9
10
IC50 = 38.44 ± 0.76 µM
12
IC50 = 48.95 ± 1.04 µM
IC50 = 42.44 ± 0.88 µM
N
N
N
N
N
N
OCH3
OCH3
OCH3
S
S
S
OCH3
H3CO
H3CO
H3CO
3
19
Not Active
23
Not Active
Not Active
Figure-4: SAR of hydroxy and methoxy substituted compounds
Amongst the chloro substituted derivatives, 4-chloro substituted derivative 21 (IC50 = 39.00 ± 0.88
μM) showed superior activity than 3-chloro substituted analog 8 (IC50 = 52.36 ± 1.29 μM). Which
indicates that chloro at position-4 is playing a crucial role in the inhibitory potential. All methyl
substituted analogs 13, 14, and 16 were found to be completely inactive (Figure-5).
N
N
N
N
Cl
S
S
Cl
N
H3CO
H3CO
21
8
IC50 = 39.00 ± 0.88 µM
IC50 = 52.36 ± 1.29 µM
CH3
N
N
N
N
N
CH3
S
S
S
CH3
H3CO
H3CO
H3CO
13
14
Not Active
16
Not Active
Not Active
Figure-4: SAR of chloro and methyl substituted compounds
Among heterocyclic ring bearing analogs only compound 18 IC50 = 45.72 ± 0.98 μM) having 2-
pyridyl ring showed good activity. In general, it was deduced that hydroxy substituted derivatives
were found to be more potent than compounds bearing methoxy with hydroxy group. Furthermore,
chloro bearing derivatives also showed comparable inhibitory potential to the standard D-saccharic
acid-1,4-lactone (IC50 = 48.4 ± 1.25 μM).
7

Docking studies
Human β-glucuronidase acts as an exoglycosidase in lysosomes and is involved in stepwise
degradation of glucuronic acid-containing glycosaminoglycans (GAGs) including heparin sulfate,
dermitant sulfate, and chondroitin sulfate. The functional form of GUS is a tetramer of four
identical subunits of 75000 Da [34]. Computational analysis of designed inhibitors by docking
simulations was carried out to determine the binding orientation, affinity and the binding energy
of the tested inhibitors. These analyses were carried out by using Gold 5.4.1 [35].
Validation of GOLD accuracy
In order to check the reliability of the docking method, known physiological substrate p-
nitrophenyl β-glucuronide was docked into the active site of human β-glucuronidase (1BHG)
(http://www.rcsb.org/pdb). The docking results showed that the docked substrate was properly
oriented and showed interactions with catalytically important residues Glu451 and Glu540.
Binding affinities of synthesized compounds
Gold Score was selected as the criteria for the selection of compounds since it serves as fitness
function for the orientation and estimates of binding affinity. In general, thiadiazole derivatives
with R = aryldiols (compounds 4-7) exhibited highest binding affinities, i.e. lower binding energies
into the binding site of 1BHG. These compounds fitted properly into the groove of the binding site
(Figure-5).
Figure-5: Modelled mode of binding of a) Compound 5 (b) Compound 6 in 1BHG active site.
Compound 5, 6, and 7 exhibited GOLD fitness score -60.65, -61.97 and -58.94 respectively. These
lower binding energies are mainly attributed to hydrogen bond formation between the diols of the
8

docked molecules and the key amino acid residues. Figure-5 revealed that aryldiol ring of
compounds 5, 6, and 7 are involved in hydrogen bonding interactions and formed 3-4 hydrogen
bonds with Asp207, Glu451 and Glu541. Van der Waals interaction of thiadiazole ring also
enhances the fitting into the active site gorge. Compound 6 anchors itself in such a way that enables
a strong hydrogen bond Glu451 and Asp207 at a distance of 2.8 Å and 1.2 Å respectively) via its
oxygen of the hydroxyl group at 2 and 4-postion of ring B (Figure-6A). Similar interactions were
observed with all compounds containing aryldiol ring (Figure-6 B-C). The superimposed
structures of aryldiols are shown in Figure-6D.
Figure-6: 3D interactions of compounds 5, 6, and 7 and their superimposed structures in the
active site of 1BHG
Poor binding affinities are observed with inhibitors 8, 9, 10, and 12 that bear methoxy or chloro
substituents on the ring B. Binding modes of weakly active compounds (8 and 9) and their
superimposed structure with the most active compound 5 are shown in Figure 7A-C.
9

Figure-7: 3D interactions of weakly compounds 8 and 9 (A-B) and their superimposed
structures (C) in the active site of 1BHG
Conclusion
Thiadiazole derivatives 1-25 were synthesized and screened for their potential against β-
glucuronidase enzyme. A number of compounds such as 1, 2, 4, 5-7, 10, 12, 15, and 21 exhibited
potent β-glucuronidase inhibitory potential having IC50 values of 6.74 ± 0.26 to 42.53 ± 0.80 μM
compared to standard D-saccharic acid-1,4-lactone (IC50 = 48.4 ± 1.25 μM). Nevertheless,
compounds 8 (IC50 = 52.36 ± 1.29 μM) and 9 (IC50 = 48.95 ± 1.04 μM) displayed comparable
activity with the standard D-saccharic acid-1,4-lactone. Structure-activity relationship revealed
that hydroxy containing compounds exhibited superior inhibitory activity as compared to other
substituents. The molecular docking studies has identified many structural motifs actively
interacting within the enzyme’s active site thus taking part in the inhibition. This study has
identified some lead molecules those can be used in future research in order to get more potent β-
glucuronidase inhibitors.
10

Experimental
Materials and methods
Melting point was measured on Büchi M-560 melting point apparatus and are uncorrected. IR
spectra were recorded on a Spectrum One FT-IR spectrometer (Perkin Elmer), using KBr discs
and values were signified in cm-1. The 1H-NMR and 13C-NMR spectra were measured on a Bruker
500 Ultrashield Plus NMR (500 MHz) in DMSO-d6, using tetramethylsilane (TMS) as an internal
standard, and chemical shifts are expressed in ppm. ESI-MS were determined on Agilent 6330 Ion
Trap using positive/negative mode at Faculty of Pharmacy, UiTM Puncak Alam, Malaysia.
General procedure for the synthesis of thiadiazole derivatives 1-25
Methyl-4-methoxybenzoate (i) was refluxed with 5 mL of hydrazine hydrate solution in 15 mL of
methanol for 6 h. The excess hydrazine and methanol was then removed in vacuo to obtain crude
product which was then recrystallized from methanol to afford pure 4-methoxybenzohydrazide in
92% yield. Pure 4-methoxybenzohydrazide (20 mmol, 3.32 g) were refluxed with Lawesson's
reagent (20 mmol, 8.09 g) of in dry toluene for 8 h to get corresponding 4-
methoxybenzothiohydrazide. The crude product was washed with diethyl ether and crystallized
from methanol to get pure 4-methoxybenzothiohydrazide in 91% yield. In order to get thiadiazole
derivatives 1-25, a mixture of 4-methoxybenzothiohydrazide (0.5 mmol) and the corresponding
aromatic aldehyde (0.5 mmol) were taken together in a round-bottomed flask and was then added
drop wise POCl3 (5 mL) carefully. The reaction mixture was heated to reflux for 4-6 h, then cooled
to room temperature and poured onto crushed ice. NaHCO3 solution was added and the resulting
solid mass precipitated out was filtered, dried, and crystallized from methanol in good to excellent
yields.
4-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)phenol (1)
º
1
White needle shaped crystalline solid; M.p. = 256 C; Rf: 0.86 (ethyl acetate/hexanes, 3:7); H-
NMR (500 MHz, DMSO-d6):  9.80 (s, 1H, OH), 7.92 ( d, 2H, J = 8.0 Hz), 7.52 (d, 2H, J = 8.0Hz),
13
7.08 (d, 2H, J = 8.0 Hz), 6.82 (d, 2H, J = 8.5 Hz), 3.83 (s, 3H, OCH3); C-NMR (125 MHz,
DMSO-d6) δ 174.2, 174.2, 160.5, 158.4, 128.8, 128.8, 128.4, 128.4, 126.1, 125.7, 116.3, 116.3,
114.7, 114.7, 55.7, Anal Calcd for C15H12N2O2S, C, 63.36; H, 4.25; N, 9.85; Found: C, 63.34; H,
4.23; N, 9.84; HREI-MS: m/z Calcd for for C15H12N2O2S, [M]+ 284.0619; Found 284.0627.
11

3-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)phenol (2)
1
Light yellow granular crystalline solid; M.p. = 249 ºC; Rf: 0.76 (ethyl acetate/hexanes, 3:7); H-
NMR (500 MHz, DMSO-d6):  9.40 (s, 1H, OH), 7.90 (d, 2H, J= 8.5 Hz), 7.30 (t, 1H, J =8.5 Hz),
7.26 (s, 1H), 7.12 (d, 1H, J = 8.0 Hz), 7.04 (d, 2H, J = 8.5 Hz), 6.80 (d, 1H, J = 6.5 Hz, H-4),
3.86(s, 3H, OCH3); 13C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 160.5, 157.4, 134.8, 130.7,
128.4, 128.4, 125.7, 123.4, 115.8, 114.7, 114.7, 112.8, 55.7, Anal. Calcd for C15H12N2O2S, C,
63.36; H, 4.25; N, 9.85; Found: C, 63.34; H, 4.23; N, 9.84; HREI-MS: m/z Calcd for for
C15H12N2O2S, [M]+ 284.0619; Found 284.0611.
2-(3,4-Dimethoxyphenyl)-5-(4-methoxyphenyl)-1,3,4-thiadiazole (3)
Yellow needle like crystalline solid; M.p. = 220 ºC; Rf: 0.72 (ethyl acetate/hexanes, 3:7); 1H-NMR
(500 MHz, DMSO-d6):  7.94 (d, 2H, J = 8.0 Hz), 7.36 (s, 1H), 7.21 (d, 1H, J = 8.5 Hz), 7.07 (d,
2H, J = 8.5 Hz), 6.76 (d, 1H, J= Hz), 3.86 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.81 (s, 3H, OCH3);
13C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 160.5, 150.2, 149.7, 128.4, 128.4, 126.7, 125.7,
120.8, 114.7, 114.7, 112.2, 111.1, 56.1, 56.1, 55.7, Anal Calcd for C17H16N2O3S, C, 62.18; H, 4.91;
N, 8.53; Found C, 62.17; H, 4.90; N, 8.52; HREI-MS: m/z Calcd for for C17H16N2O3S, [M]+
328.0882; Found 328.0896.
2-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-1,4-diol (4)
Off white rod like solid; M.p. = 282 ºC; Rf: 0.76 (ethyl acetate/hexanes, 3:7); 1H-NMR (500 MHz,
DMSO-d6):  11.10 (s, 1H, OH), 9.15 (s, 1H, OH), 7.92 (d, 2H, J = 9.0 Hz), 7.06 (d, 2H, J = 9.0
Hz), 6.94 (dd, 1H, J = 8.0, J = 2.0 Hz), 6.83 (d, 1H, J = 2.0 Hz), 6.72 (d, 1H, J = 8.0 Hz), (s, 3H,
OCH3); 13C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 160.5, 150.0, 147.7, 128.4, 128.4, 125.7,
125.0, 117.7, 117.3, 114.7, 114.7, 114.2, 55.7; Anal. Calcd for C15H12N2O3S, C, 59.99; H, 4.03;
N, 9.33; Found C, 59.98; H, 4.01; N, 9.32; HREI-MS: m/z Calcd for for C15H12N2O3S, [M]+
300.0569; Found 300.0558.
12

3-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-1,2-diol (5)
º
1
Yellow massive crystalline solid; M.p. = 265 C; Rf: 0.84 (ethyl acetate/hexanes, 3:7); H-NMR
(500 MHz, DMSO-d6):  11.20 (s, 1H, OH), 9.50 (s, 1H, OH), 7.92 (d, 2H, J = 9.0 Hz), 7.12 (d,
2H, J = 9.0 Hz), 6.93 (dd, 1H, J = 6.5, J = 2.0 Hz), 6.88 (dd, 1H, J = 6.5, J = 2.0 Hz, H-6), 6.76 (t,
1H, J = 6.5 Hz, H-5), 3.85 (s, 3H, OCH3); 13C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 160.5,
143.8, 145.5, 125.2, 125.7, 114.7, 121.4, 128.4, 114.7, 117.2, 128.4, 123.1, 55.7; Anal Calcd for
C15H12N2O3S, C, 59.99; H, 4.03; N, 9.33; Found C, 59.97; H, 4.02; N, 9.31; HREI-MS: m/z Calcd
for for C15H12N2O3S, [M]+ 300.0569; Found 300.0576.
4-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (6)
Yellow square crystalline solid; M.p. = 270ºC; Rf: 0.82 (ethyl acetate/hexanes, 3:7); 1H-NMR (500
MHz, DMSO-d6):  11.30 (s, 1H, OH) 9.98 (s, 1H, OH), 7.94 (d, 2H, J = 8.0 Hz), 7.32 (d, 1H, J =
8.5 Hz), 7.06 (d, 2H, J = 8.0 Hz, H-3), 6.39 (dd, 1H, J= 8.5, 2.0 Hz), 6.30 (d, 1H, J = 2.0 Hz), 3.86
(s, 3H, OCH3); 13C-NMR (126 MHz, DMSO-d6) δ 174.2,174.2,160.5, 156.5, 159.8, 116.2, 125.7,
105.5, 114.7, 109.1, 130.2, 128.4, 114.7, 128.6, 55.9; Anal Calcd for C15H12N2O3S, C, 59.99; H,
4.03; N, 9.33; Found C, 59.98; H, 4.01; N, 9.32; HREI-MS: m/z Calcd for for C15H12N2O3S, [M]+
300.0569; Found 300.0582.
4-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)benzene-1,2-diol (7)
1
White, granular crystalline solid; M.p. = 240 ºC; Rf: 0.76 (ethyl acetate/hexanes, 3:7); H-NMR
(500 MHz, DMSO-d6):  9.30 (s, 2H, OH), 7.91 (d, 2H, J = 8.0 Hz), 7.40 (s, 1H), 7.08 (d, 2H, J =
13
8.0 Hz), 6.91 (d, 1H, J = 8.0 Hz), 6.76 (d, 1H, J = 8.0 Hz), 3.85 (s, 3H, OCH3); C-NMR (126
MHz, DMSO-d6) δ 174.2,174.2, 160.5, 145.8, 147.2, 127.4, 125.9, 114.2, 114.9, 116.1, 121.4,
128.4, 114.9, 128.4, 55.7; Anal Calcd for C15H12N2O3S, C, 59.99; H, 4.03; N, 9.33; Found C,
59.97; H, 4.01; N, 9.31; HREI-MS: m/z Calcd for for C15H12N2O3S, [M]+ 300.0569; Found
300.0580.
2-(3-Chlorophenyl)-5-(4-methoxyphenyl)-1,3,4-thiadiazole (8)
º
1
Yellow crystalline solid, M.p. = 221.6 C; Rf: 0.80 (ethyl acetate/hexanes, 3:7); H-NMR (500
MHz, DMSO-d6):  7.90 (d, 2H, J = 8.0 Hz), 7.40 (t, 1H, J = 7.5 Hz), 7.32-728 (m, 1H), 7.09 (d,
13

2H, J = 8.0 Hz), 6.80 (dd, 1H, J = 7.5, 2.0 Hz), 3.86 (s, 3H, OCH3); 13C-NMR (126 MHz, DMSO-
d6) δ 174.2, 174.2, 134.7, 160.5, 134.8, 125.7, 127.3, 114.7, 128.4, 129.1, 128.7, 114.7, 128.4,
129.4, 55.7; Anal Calcd for C15H11ClN2OS, C, 59.50; H, 3.66; N, 9.25; Found C, 59.49; H, 3.65;
N, 9.24; HREI-MS: m/z Calcd for for C15H11ClN2OS, [M]+ 302.0281; Found 302.0290.
2-Methoxy-5-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)phenol (9)
Light yellow crystalline solid; M.p. = 235 ºC; Rf: 0.72 (ethyl acetate/hexanes, 3:7); 1H-NMR (500
MHz, DMSO-d6):  9.55 (s, 1H, OH), 7.92 (d, 2H, J = 8.5 Hz, H-2), 7.30 (s, 1H), 7.07 (d, 1H, J =
8.0 Hz), 7.03 (d, 2H, J= 8.5 Hz), 6.80 (d, 1H, J= 8.0 Hz), 3.82 (s, 3H, OCH3), 3.88 (s, 3H, OCH3);
13C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 147.4, 160.5, 147.3, 127.1, 125.6, 113.8, 111.3,
114.9, 121.2, 128.6, 114.9, 128.3, 56.2, 55.7; Anal Calcd, C16H14N2O3S C, 61.13; H, 4.49; N, 8.91;
Found C, 61.12; H, 4.48; N, 8.90; HREI-MS: m/z Calcd for for C15H11ClN2OS, [M]+ 314.0725;
Found 314.0714.
5-Methoxy-2-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)phenol (10)
Yellow needle like solid, M.p. = 264ºC; Rf: 0.74 (ethyl acetate/hexanes, 3:7); 1H-NMR (500 MHz,
DMSO-d6):  9.70 (s, 1H, OH), 7.91 (d, 2H, J = 8.0 Hz), 7.30 (d, 1H, J = 8.5 Hz), 7.08 (d, 2H, J =
8.0 Hz, H-3), 6.42 (dd, 1H, J= 8.5, 2.0 Hz), 6.33 (d, 1H, J = 2.0 Hz), 3.88 (s, 3H, OCH3), 3.82 (s,
13
3H, OCH3); C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 162.1, 160.7, 156.1, 116.1, 125.7,
104.1, 107.3, 114.7, 129.8, 128.3, 114.9, 128.4, 55.6, 55.6; Anal Calcd, C16H14N2O3S C, 61.13; H,
4.49; N, 8.91; Found C, 61.11; H, 4.47; N, 8.91; HREI-MS: m/z Calcd for for C15H11ClN2OS,
[M]+ 314.0725; Found 314.0732.
2-(4-Methoxyphenyl)-5-(pyridin-4-yl)-1,3,4-thiadiazole (11)
1
Off white crystalline solid, M.p. = 216. ºC; Rf: 0.88 (ethyl acetate/hexanes, 3:7); H-NMR (500
MHz, DMSO-d6):  8.62 (d, 2H, J = 6.0 Hz), 7.90 (d, 2H, J = 8.5 Hz), 7.67 (d, 2H, J = 6.0 Hz),
13
7.11 (d, 2H, J = 8.5 Hz), 3.86 (s, 3H, OCH3); C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2,
160.4, 149.7, 149.7, 143.6, 125.7, 114.7, 121.2, 121.2, 128.4, 114.7, 128.4, 55.7; Anal Calcd for
C14H11N3OS, C, 62.43; H, 4.12; N, 15.60; Found C, 62.42; H, 4.11; N, 15.59; HREI-MS: m/z
Calcd for for C14H11N3OS, [M]+ 269.0623; Found 269.0614.
14

4-Methoxy-2-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)phenol (12)
Yellow granular crystalline solid, M.p. = 201.8 ºC; Rf: 0.76 (ethyl acetate/hexanes, 3:7); 1H-NMR
(500 MHz, DMSO-d6): 10.30 (s, 1H, OH), 7.91 (d, 2H, J = 8.5 Hz), 7.10 (d, 1H, J = 8.5 Hz), 7.07
(d, 2H, J = 8.5 Hz), 6.92 (dd, 1H, J = 8.5, 2.0 Hz), 6.85 (d, 1H, J = 2.0 Hz, H-6), 3.88 (s, 3H,
OCH3), 3.82(s, 3H, OCH3); 13C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 153.6, 160.5, 147.4,
124.6, 125.7, 115.6, 114.7, 117.3, 112.6, 128.3, 114.7, 128.3, 55.6, 55.6 ; Anal Calcd,
C16H14N2O3S C, 61.13; H, 4.49; N, 8.91; Found C, 61.12; H, 4.48; N, 8.90; HREI-MS: m/z Calcd
for for C15H11ClN2OS, [M]+ 314.0725; Found 314.0736.
2-(4-Methoxyphenyl)-5-o-tolyl-1,3,4-thiadiazole (13)
Yellow elongated crystalline solid, M.p. = 201 ºC; Rf: 0.90 (ethyl acetate/hexanes, 3:7); 1H-NMR
(500 MHz, DMSO-d6):  7.90 (d, 2H, J = 8.5 Hz), 7.58 (d, 1H, J = 8.0 Hz), 7.11 (d, 2H, J = 8.5
Hz), 7.30-7.26 (m, 2H), 3.86 (s, 3H, OCH3), 2.49 (s, 3H, CH3); 13C-NMR (126 MHz, DMSO-d6)
δ 174.2, 174.2, 160.5, 137.1, 125.7, 136.8, 114.7, 127.3, 128.4, 129.4, 114.7, 128.4, 126.1, 128.5,
55.7, 18.6; Anal Calcd C16H14N2OS, C, 68.06; H, 5.00; N, 9.92; Found C, 68.05; H, 5.01; N, 9.91;
HREI-MS: m/z Calcd for for C16H14N2OS, [M]+ 282.0827; Found 282.0838.
2-(4-Methoxyphenyl)-5-m-tolyl-1,3,4-thiadiazole (14)
1
Dark yellow granular solid, M.p. = 201 ºC; Rf: 0.86 (ethyl acetate/hexanes, 3:7); H-NMR (500
MHz, DMSO-d6):  7.92 (d, 2H, J = 8.5 Hz), 7.72 (dd, 1H, J = 8.0, 2.0 Hz), 7.64 (d, 1H, J = 2.0
Hz), 7.34 (t, 1H, J = 7.0 Hz), 7.17 (d, 1H, J = 20 Hz), 7.10 (d, 2H, J = 8.5 Hz), 3.84(s, 3H, OCH3),
13
2.46 (s, 3H, CH3); C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 160.5, 133.3, 125.7, 138.8,
114.7, 128.4, 127.8, 130.3, 114.7, 128.4, 129.1, 129.2, 55.7, 21.5; Anal Calcd C16H14N2OS, C,
68.06; H, 5.00; N, 9.92; Found C, 68.04; H, 5.02; N, 9.90; HREI-MS: m/z Calcd for for
C16H14N2OS, [M]+ 282.0827; Found 282.0832.
2-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)phenol (15)
1
Yellow amorphous solid, M.p. = 182.5 ºC; Rf: 0.75 (ethyl acetate/hexanes, 3:7); H-NMR (500
MHz, DMSO-d6):  11.20 (s, 1H, OH), 7.93 (d, 2H, J = 9.0 H), 7.53 (d, 1H, J = 7.5, H-3), 7.32 (t,
1H, J = 8.5 Hz), 7.06 (d, 2H, J = 9.0 Hz), 6.93-6.88 (m, 2H), 3.86 (s, 3H, OCH3); 13C-NMR (126
MHz, DMSO-d6) δ 174.2, 174.2, 160.5, 155.1, 130.0, 128.8, 128.4, 128.4, 125.7, 123.6, 121.7,
15

117.7, 114.7, 114.7, 55.7, Anal. Calcd for C15H12N2O2S, 63.36; H, 4.25; N, 9.85;; Found: C, 63.34;
H, 4.23; N, 9.84; HREI-MS: m/z Calcd for for C15H12N2O2S, [M]+ 284.0619; Found 284.0632.
2-(4-Methoxyphenyl)-5-p-tolyl-1,3,4-thiadiazole (16)
Light yellow granular crystalline solid, M.p. = 226. ºC; Rf: 0.84 (ethyl acetate/hexanes, 3:7); 1H-
NMR (500 MHz, DMSO-d6):  7.90 (d, 2H, J = 8.5 Hz), 7.55 (d, 2H, J = 8.0 Hz), 7.24 (d, 2H, J =
13
8.0 Hz), 7.10 (d, 2H, J = 8.5 Hz), 3.86 (s, 3H, OCH3), 2.40 (s, 3H, CH3), C-NMR (126 MHz,
DMSO-d6) δ 174.2, 174.2, 160.5, 131.1, 130.4, 129.4, 129.4, 128.4, 128.4, 127.3, 127.3, 114.7,
114.7, 55.7, 21.4; Anal Calcd, C16H14N2OS, C, 68.06; H, 5.00; N, 9.92; Found C, 68.04; H, 5.02;
N, 9.90; HREI-MS: m/z Calcd for for C16H14N2OS, [M]+ 282.0827; Found 282.0821.
2-Bromo-4-(5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)phenol (17)
Yellow crystalline solid, M.p. = 230 ºC; Rf: 0.75 (ethyl acetate/hexanes, 3:7); 1H-NMR (500 MHz,
DMSO-d6):  10.40 (s, 1H, OH), 7.80 (d, 2H, J = 8.5 Hz), 7.84 (s, 1H), 7.52 (d, 1H, J = 8.0 Hz),
13
7.08 (d, 2H, J = 9.0 Hz), 7.02 (d, 1H, J = 8.0 Hz), 3.84 (s, 3H, OCH3); C-NMR (126 MHz,
DMSO-d6) δ 174.2, 174.2, 160.5, 156.5, 134.4, 128.4, 128.4, 128.2, 127.8, 125.7, 118.5, 114.7,
114.7, 114.2, 55.7; Anal Calcd for C15H11BrN2O2S, C, 49.60; H, 3.05; N, 7.71 Found C, 49.58; H,
3.04; N, 7.70; HREI-MS: m/z Calcd for for C15H11BrN2O2S, [M]+ 361.9725; Found 361.9736.
2-(4-Methoxyphenyl)-5-(pyridin-2-yl)-1,3,4-thiadiazole (18)
1
White elongated crystalline solid, M.p. = 206 ºC; Rf: 0.81 (ethyl acetate/hexanes, 3:7); H-NMR
(500 MHz, DMSO-d6):  8.62 (d, 1H, J = 5.0 Hz), 7.96 (d, 1H, J = 8.0 Hz), 7.91 (d, 2H, J = 8.5
Hz), 7.84-7.80 (m, 1H, H-4), 7.55 (t, 1H, J = 8.0 Hz), 7.09 (d, 2H, J= 8.5 Hz), 3.87 (s, 3H, OCH3);
13C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 160.5, 157.3, 149.1, 137.1, 128.4, 128.4, 125.7,
124.1, 123.7, 114.7, 114.7, 114.2, 55.7; Anal Calcd C14H11N3OS, C, 62.43; H, 4.12; N, 15.60;
Found C, 62.42; H, 4.11; N, 15.59; HREI-MS: m/z Calcd for for C14H11N3OS, [M]+ 269.0623;
Found 269.0614.
16

2,5-Bis(4-methoxyphenyl)-1,3,4-thiadiazole (19)
Yellow amorphous solid, M.p. = 214 ºC; Rf: 0.65 (ethyl acetate/hexanes, 3:7); 1H-NMR (500 MHz,
DMSO-d6):  7.90 (d, 2H, J = 8.5 Hz), 7.66 (d, 2H, J = 8.0 Hz), 7.06 (d, 2H, J = 9.0 Hz), 7.03 (d,
2H, J = 8.0 Hz), 3.86 (s, 3H, OCH3), 3.82 (s, 3H, CH3); 13C-NMR (126 MHz, DMSO-d6) δ 174.2,
174.2, 160.5, 160.5, 128.4, 128.4, 128.4, 128.4, 125.7, 125.7, 114.7, 114.7, 114.7, 114.7, 55.7,
55.7; Anal Calcd C16H14N2O2S, C, 64.41; H, 4.73; N, 9.39; Found C, 64.40; H, 4.71; N, 9.37;
HREI-MS: m/z Calcd for for C16H14N2O2S, [M]+ 298.0776; Found 298.0762.
2-(Furan-2-yl)-5-(4-methoxyphenyl)-1,3,4-thiadiazole (20)
Off white needle like crystalline solid, M.p. = 206.8 ºC; Rf: 0.82 (ethyl acetate/hexanes, 3:7); 1H-
NMR (500 MHz, DMSO-d6):  7.91(d, 2H, J=9.0 Hz), 7.84 (s, 1H, H-3), 7.07 (d, 2H, J = 9.0 Hz),
6.91 (s, 1H), 6.64 (dd, 1H, J = 5.0, 2.0 Hz), 3.83 (s, 3H, OCH3); 13C-NMR (126 MHz, DMSO-d6)
δ 174.2, 174.2, 160.5, 146.1, 146.1, 128.4, 128.4, 125.7, 114.7, 114.7, 112.1, 112.1, 55.7,; Anal
Calcd C13H10N2O2S, C, 60.45; H, 3.90; N, 10.85; Found C, 60.45; H, 3.90; N, 10.85; HREI-MS:
m/z Calcd for for C13H10N2O2S, [M]+ 258.0463; Found 258.0450.
2-(4-Chlorophenyl)-5-(4-methoxyphenyl)-1,3,4-thiadiazole (21)
º
1
Light yellow solid, M.p. = 214 C; Rf: 0.68 (ethyl acetate/hexanes, 3:7); H-NMR (500 MHz,
DMSO-d6):  7.92 (d, 2H, J = 9.0 Hz), 7.76 (d, 2H, J = 8.5 Hz), 7.54 (d, 2H, J = 8.5 Hz), 7.07 (d,
2H, J = 9.0 Hz), 3.84 (s, 3H, OCH3); 13C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 160.5, 134.2,
131.5, 129.2, 129.2, 128.8. 128.8, 128.4, 128.4, 125.7, 114.7, 114.7, 55.7; Anal Calcd for
C15H11ClN2OS, C, 59.50; H, 3.66; N, 9.25; Found C, 59.48; H, 3.65; N, 9.23; HREI-MS: m/z
Calcd for for C15H11ClN2OS, [M]+ 302.0281; Found 302.0270.
2-(4-Methoxyphenyl)-5-(pyridin-3-yl)-1,3,4-thiadiazole (22)
Yellow granular solid, M.p. = 218 ºC; Rf: 0.76 (ethyl acetate/hexanes, 3:7); 1H-NMR (500 MHz,
DMSO-d6):  8.61 (d, 1H, J = 2.0 Hz), 8.16 (d, 1H, J = 8.0 Hz), 7.93 (d, 2H, J = 8.5 Hz), 7.51 (dd,
1H, J5,4 = 8.0, 5.0 Hz), 7.08 (d, 2H, J = 8.5 Hz), 3.88 (s, 3H, OCH3); 13C-NMR (126 MHz, DMSO-
d6) δ 174.2, 174.2, 160.5, 148.8, 147.8, 134.0, 133.3, 128.8. 128.8, 125.7, 124.0, 114.7, 114.7,
17

55.7; Anal Calcd for C14H11N3OS, C, 62.43; H, 4.12; N, 15.60; Found C, 62.42; H, 4.11; N, 15.58;
HREI-MS: m/z Calcd for for C14H11N3OS, [M]+ 269.0623; Found 269.0630.
2-(3-Methoxyphenyl)-5-(4-methoxyphenyl)-1,3,4-thiadiazole (23)
Yellow fine crystalline solid, M.p. = 218 ºC; Rf: 0.72 (ethyl acetate/hexanes, 3:7); 1H-NMR (500
MHz, DMSO-d6):  7.92 (d, 2H, J = 9.0 Hz), 7.38 (t, 1H, J = 7.5 Hz), 7.28-723 (m, 2H), 7.08 (d,
2H, J = 9.0 Hz), 6.81 (dd, 1H, J = 7.5, 2.0 Hz, H-6), 3.86 (s, 3H, OCH3), 3.82 (s, 3H, OCH3); 13C-
NMR (126 MHz, DMSO-d6) δ 174.2, 174.2, 160.5, 161.0, 134.4, 130.1, 128.8. 128.8, 125.7, 123.1,
114.7, 114.7, 114.2, 111.2, 55.7, 55.7; Anal Calcd for C16H14N2O2S, C, 64.41; H, 4.73; N, 9.39;
Found C, 64.40; H, 4.72; N, 9.37; HREI-MS: m/z Calcd for for C16H14N2O2S, [M]+ 298.0776;
Found 298.0784.
2-(4-Methoxyphenyl)-5-(4-nitrophenyl)-1,3,4-thiadiazole (24)
White square crystaline solid, M.p. = 240 ºC; Rf: 0.80 (ethyl acetate/hexanes, 3:7); 1H-NMR (500
MHz, DMSO-d6):  8.30 (d, 2H, J = 8.0 Hz), 8.02 (d, 2H, J = 8.0 Hz), 7.94 (d, 2H, J = 8.5 Hz ),
13
7.09 (d, 2H, J = 8.5 Hz), 3.85 (s, 3H, OCH3); C-NMR (126 MHz, DMSO-d6) δ 174.2, 174.2,
160.5, 147.8, 139.5, 128.8. 128.8, 128.4, 128.4, 125.7, 124.4, 124.4, 114.7, 114.7, 55.7; Anal Calcd
for C15H11N3O3S, C, 57.50; H, 3.54; N, 13.41; Found C, 57.49; H, 3.53; N, 13.40; HREI-MS: m/z
Calcd for for C15H11N3O3S [M]+ 313.0521; Found 313.0513.
2-(4-Fluorophenyl)-5-(4-methoxyphenyl)-1,3,4-thiadiazole (25)
Yellow amorphous solid, M.p. = 255 ºC; Rf: 0.86 (ethyl acetate/hexanes, 3:7); 1H-NMR (500 MHz,
DMSO-d6):  7.90 (d, 2H, J = 8.5 Hz), 7.82 (t, 2H, J = 7.0 Hz), 7.30 (t, 2H, J = 7.0 Hz), 7.09 (d,
2H, J = 8.5 Hz), 3.87 (s, 3H, OCH3); 13C-NMR (126 MHz, DMSO) δ 174.2, 174.2, 162.0, 160.5,
135.1, 128.8, 128.4, 127.5, 126.4, 125.7, 115.5, 115.5, 114.7, 114.7, 55.7; Anal Calcd for
C15H11FN2OS C, 62.92; H, 3.87; N, 9.78; Found C, 62.91; H, 3.85; N, 9.76; HREI-MS: m/z Calcd
for for C15H11FN2OS, [M]+ 286.0576; Found 286.0590.
Docking studies
Docking studies were carried out using GOLD 5.4.1. GOLD uses the Genetic algorithm (GA).
This method allows a partial flexibility of protein and full flexibility of ligand. To initialize the in-
silico studies, high resolution crystal structures of proteins were retrieved from the PDB (PDB ID
18

1BHG, 2.6 Å resolution). Hydrogen atoms were added to enzyme structure. The B-chain of protein
and hetero-atoms including cofactors were removed from the crystal structure [30]. To start the
docking simulations, a protein model was built by performing docking on choosing a known
substrate p-nitrophenyl β-glucuronide into the binding site. This model was then subjected to
energy minimization and further used for docking studies of the synthesized compounds. There
were four possible scoring functions: Gold score, Chemscore, Astex Statistical Potential (ASP)
and ChemPLP. GoldScore performs a force field-based scoring function and is made up of four
components: 1. Protein-ligand hydrogen bond energy (external H-bond); 2. Protein-ligand van der
Waals energy (external vdw); 3. Ligand internal van der Waals energy (internal vdw); 4. Ligand
intramolecular hydrogen bond energy (internal-H-bond). Binding site was defined as the residues
within 10 Å from the ligand. No water was present in any binding site. The default docking
protocol was applied (1.09 auto settings, 10 GA) and the best pose saved. Each experiment was
then repeated 10 times. Other docking parameters were set to the software’s default values. The
view of the docking results and analysis of their surface with graphical representations were done
using UCSF Chimera package [36].
Acknowledgments
Dr. Muhammad Taha wants to thank Department of Clinical Pharmacy, Institute for Research and
Medical Consultations (IRMC) for proving lab facilities. Dr. U. R. is thankful to The Cambridge
Crystallographic Data Centre (CCDC) for providing GOLD license. Dr. Khalid M. Khan is
thankful Pakistan Academy of Sciences for providing financial support Project No. (5-9/PAS/440).
References
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S.N. Swamy, Basappa, B.S. Priya, B. Prabhuswamy, B.H. Doreswamy, J. Shashidhara
Prasad, Kanchugarakoppal S. Rangappa, Eur. J. Med. Chem. 41 (2006) 531–538.
S. Hussain, J. Sharma, M. Amir, E-Jour. Chem. 5 (2008) 963–968.
A.A. Aly, R.E. Sayed, Chem. Pap. 60 (2006) 56-60.
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[10] A. Foroumadi, S. Emami, A. Hassanzadeh, M. Rajaee, K. Sokhanvar, M.H. Moshafi, A.
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[11] T. Onkol, D.S. Doruer, L. Uzun, S. Adak, S. Ozkan, M.F. Ahin, J. Enz. Inhib. Med. Chem.
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Graphical Abstract
2,5-Disubstituted Thiadiazoles as Potent -glucuronidase Inhibitors;

Synthesis,
and
Studies
In Silico
In Vitro
Muhammad Tahaa, Noor Barak Almandila, Umer Rashidb, Muhammad Alic, Mohamed Ibrahima, Mohammed Gollapallid,
Ashik Mosaddika, Khalid Mohammed Khana,e
aDepartment of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin
Faisal University, P.O. Box1982, Dammam 3144, Saudi Arabia
bDepartment of Chemistry, COMSATS Institute of Information Technology
cUoN Chair of Oman's Medicinal Plants and Marine Natural Products, University of Nizwa, P.O. Box 33, Birkat Al
Mauz, Nizwa 616, Oman
dCollege of Computer Science & Information Technology (CCSIT) Imam Abdulrahman Bin Faisal University P.O. Box
1982, Dammam 31441, Saudi Arabia
eH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of
Karachi, Karachi-75270, Pakistan
O
N
N
O
a,b,c
H3CO
S
H3CO
R
i
1-25
Scheme-1: Synthesis of 2,5-disubstituted thiadiazoles.
N
S
OH
N
R
R
S
N
N
OH
H
H
Thiourea Derivatives [32]
Thiazole Derivatives [33]
H
S
MeO
N
R
S
R
N
N
R
N
N
Previously Identified Thiadiazole
Derivatives as Potent -
Glucuronidase Inhibitors [31]
Newly Identified
-Glucuronidase Inhibitors
Figure-1: Previously and newly identified -glucuronidase inhibitors

Highlights:
1. Twenty-five thiadiazole derivatives 1-25 were synthesized.
2. The synthetic compounds were evaluated for their potential against β-glucuronidase
enzyme.
22

3. Most of the compounds e.g. 1, 2, 4, 5, 6, 7, and 15 exhibited superior activity potential
than the standard D-saccharic acid-1,4-lactone.
4. Molecular docking studies were conducted to correlate the in vitro results and to identify
possible mode of interaction with enzyme active site.
23