Synthesis of chromen-4-one-oxadiazole substituted analogs as potent β-glucuronidase inhibitors

Article abstract of DOI:10.3390/molecules24081528

Chromen-4-one substituted oxadiazole analogs 1–19 have been synthesized, characterized and evaluated for β-glucuronidase inhibition. All analogs exhibited a variable degree of β-glucuronidase inhibitory activity with IC₅₀ values ranging in betw

Full text of DOI:10.3390/molecules24081528

molecules
Article
Synthesis of Chromen-4-One-Oxadiazole Substituted
Analogs as Potent β-Glucuronidase Inhibitors
Muhammad Taha 1,*, Fazal Rahim 2, Muhammad Ali 3, Muhammad Naseem Khan 4,
Mohammed A. Alqahtani 5, Yasser A. Bamarouf 5, Mohammed Gollapalli 5, Rai Khalid Farooq 6,
Syed Adnan Ali Shah 7,8 , Qamar Uddin Ahmed 9 and Zainul Amiruddin Zakaria 10,11,
*
1
Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam
Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
Department of Chemistry, Hazara University, Mansehra 21300, Khyber Pakhtunkhwa, Pakistan;
fazalstar@gmail.com
Natural and Medical Sciences Research Center, University of Nizwa, P.O. Box 33, Birkat Al Mauz, Nizwa 616,
Sultanate of Oman; m_alisaad@yahoo.com
2
3
4
5
Department of Chemistry, COMSATS Institute of Information Technology, University Road,
Abbottabad 22060, KPK, Pakistan; naseemkhan.tareen@gmail.com
Department of Computer Information Systems, College of Computer Science & Information Technology,
Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia;
maqhtani@iau.edu.sa (M.A.A.); yabamarouf@iau.edu.sa (Y.A.B.); magollapalli@iau.edu.sa (M.G.)
Department of Neuroscience Research, Institute for Research and Medical Consultations (IRMC), Imam
Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 3144, Saudi Arabia; rkfarooq@iau.edu.sa
Faculty of Pharmacy, Universiti Teknologi MARA Puncak Alam Campus, 42300 Bandar Puncak Alam,
Selangor D.E., Malaysia; benzene301@yahoo.com
Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA Puncak
Alam Campus, 42300 Bandar Puncak Alam, Selangor D.E., Malaysia
Department of Pharmaceutical Chemistry, Kulliyyah of Pharmacy, International Islamic University Malaysia,
25200 Kuantan, Pahang DM, Malaysia; quahmed@iium.edu.my
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,
43400 Serdang, Selangor, Malaysia
Halal Institute Research Institute, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Correspondence: mtaha@iau.edu.sa (M.T.); zaz@upm.edu.my (Z.A.Z.); Tel.: +00966502057370 (M.T.);
+603-8947-2111 (Z.A.Z.); Fax: +603-8943-6178 (Z.A.Z.)
6
7
8
9
10
11
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 December 2018; Accepted: 2 February 2019; Published: 18 April 2019
Abstract: Chromen-4-one substituted oxadiazole analogs
and evaluated for -glucuronidase inhibition. All analogs exhibited a variable degree of
-glucuronidase inhibitory activity with IC50 values ranging in between 0.8
1–19 have been synthesized, characterized
β
β
±
0.1–42.3
±
0.8
µ
M when
compared with the standard d-saccharic acid 1,4 lactone (IC50 = 48.1
relationship has been established for all compounds. Molecular docking studies were performed to
predict the binding interaction of the compounds with the active site of enzyme.
±
1.2
µM). Structure activity
Keywords: chromen-4-one; oxadiazole; synthesis; β-glucuronidase inhibition; molecular docking;
SAR
1. Introduction
β
-Glucuronidase (E.C.3.2.1.31) is one of the most extensively studied enzymes in the metabolic
hydrolysis of conjugated compounds, and eliminates large number of toxic compounds from
body as glucuronides [ ]. It acts as a glycoside hydrolase enzyme which induces to breaking of
glucuronosyl-O-bonds [
1
2
]. In human body
β
-glucuronidase is present in many body fluids and
Molecules 2019, 24, 1528; doi:10.3390/molecules24081528
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1528
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organs [
renal disorders [
bladder [ ] and breast cancers [
3
]. Increased activity of
β
-glucuronidase has resulted in various health issues that include
4], urinary tract infections [
5
], epilepsy [
6
], renal transplant rejection [7], neoplasm of
8
9
]. Bacterial
β
-glucuronidase and β
-glucosidase in the human colon
are involved in the metabolism and activation of xenobiotics derived from dietary compounds [10].
The enzymes are identified to be mediators of colon cancer (CRC). Many carcinogenic dietary
compounds are metabolized in the liver and then conjugated to glucuronic acid before being excreted
with bile into the small intestine. In the colon, bacterial β-glucuronidase hydrolyzes the conjugates,
there by releasing the parent compounds or their activated hepatic metabolites [11]. Epidemiological
studies have shown that populations at high risk for colorectal cancer have high levels of fecal
β
-glucuronidase activity and CRC patients have significantly higher levels of the enzyme than healthy
controls [12].
Chromone (1,4-benzopyrone) is a derivative of benzopyran with a substituted keto group on the
pyran ring. Chromones (4H-chromen-4-ones) constitute a well-known class of naturally occurring
oxygen-containing heterocycles. These compounds exhibit important biological properties such as
anticancer [13], cytotoxic [14], antioxidant [15], anti-inflammatory [16], antifungal activities [17].
1,3,4-Oxadiazole containing compounds have remarkable biological and pharmacological
properties. Recently, differently substituted 1,3,4-oxadiazoles have been reported for their biological
activities such as antibacterial [18], antifungal [19], insecticidal [20], antiviral [21], inflammatory [22],
antitubercular [23], antitumor [24] and analgesic activity [25].
Our research group has been working on synthesis of variety of heterocycles and explored a huge
biological potential of these compounds [26
protocol that has been developed for the synthesis of a series of chromone substituted oxadiazole
pharmacophores in another effort towards developing more novel agents against -glucuronidase
–32]. Present work belongs to a simple and reliable synthetic
β
enzyme. This is beyond any doubt that combination of two or more biologically active compounds
grants newer molecules with complementary pharmacophoric potentials or with different mechanisms
of actions. The unity of pharmacophoric approach, as a result, could possibly address the active site of
different targets more effectively and could offer the possibility to overcome the issue of drug resistance
and unwanted effects [33–36]. The outcomes of this merger have already been the subject of attention
in finding new antimicrobials [37–41].
2. Result and Discussions
2.1. Chemistry
The synthesis of the novel series of chromene-based oxadiazole β-glucuronidase inhibitors 1–19
commenced with the reaction of 2-hydroxyacetophenone and p-formylbenzoic acid in ethanolic
potassium hydroxide at room temperature (Scheme 1).
COOH
O
O
I2-DMSO,
reflux
COOH
O
O
+
Ethanolic-KOH,
room temperature
OH
OHC
OH
COOH
(I)
(II)
R
MeOH, H2SO4,
reflux
O
O
NH2
N
COOMe
N
N
Hydrazine hydrate,
MeOH, reflux [30]
H
O
O
O
RCHO
O
O
POCl3/NCH3)3 [31].
O
(IV)
(III)
(1-19)
Scheme 1. Synthesis of chromene based oxadiazole derivatives 1–19.

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The adduct
I
underwent oxidative cyclization to form 4-substituted chromenone analog II. This
acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding
hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions
formed the desired oxadiazoles
various spectroscopic methods.
1–19 Figure 1. All synthesized compounds were characterized using
Figure 1. Structure of the basic skeleton of the chromen-4-one based oxadiazole derivatives 1–19.
2.2. β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives
1–19 were synthesized and evaluated for
β
-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential,
ranging between 0.8
lactone (IC50 value = 48.1
on the basis of substitution pattern on the phenyl ring.
±
0.1
µ
M to 42.3
±
0.8 µM when compared with the standard d-saccharic acid 1,4
±
1.2 M) (Table 1). The structure activity relationship has been established
µ
Table 1. Chromen-4-one based oxadiazole derivatives 1–19.
S. No
1
R
IC50 ± SEM a
S. No
R
IC50 ±SEM a
26.1 ± 0.5
2.6 ± 0.1
11
2
5.1 ± 0.2
12
17.2 ± 0.4
NO2
3
4
5
6
7
2.1 ± 0.1
1.1 ± 0.05
3.8 ± 0.2
0.8 ± 0.1
9.4 ± 0.3
13
14
15
16
17
32.20 ± 0.6
39.5 ± 0.7
20.1 ± 0.5
14.9 ± 0.4
34.7 ± 0.6
Cl
8
9
13.0 ± 0.3
8.6 ± 0.3
18
19
24.5 ± 0.5
42.3 ± 0.8
10
21.3 ± 0.4
d-saccharic acid 1,4 lactone c
48.1 ± 1.2
a
standard error mean; c standard drug.

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The compound
6
(IC50 = 0.8
±
0.1
µ
M), an ortho-fluoro analog was found to be the most potent
among the series. The greater potential shown by this analog might be due to the electron withdrawing
fluoro group. If we compare it with other fluoro analogs like compound (IC50 = 1.1 0.05 M) a
para-fluoro analog and compound (IC50 = 3.8 0.2 M), a meta-fluoro analog, the analog was found
superior than the analogs and . The little bit difference in their potential is appears to be due to the
difference in the position of fluoro groups on phenyl ring Figure 2.
4
±
µ
5
±
µ
6
4
5
Figure 2. ortho-Fluoro and para-fluoro analogs (most potent ones).
The compound 3, an ortho-chloro analog (IC50= 2.1
most potent analog among the series if we compare it with other chloro analogs like compound
(IC50 = 2.6 0.1 M) a para-chloro analog and compound (IC50 = 5.1 0.2 M) a meta-flouro analog.
The greater potential is again seemingly due to position of substituent(s) on phenyl ring.
Similarly, compound , an ortho methyl analog (IC50= 8.6 0.3 M) showed outstanding inhibitory
potential. If we compare it with other methyl analogs like analog (IC50 = 9.4 0.3 M) a para-methyl
analog and compound 8 (IC50 = 13.0 ± 0.3 µM) a meta-methyl analog, compound 9 is superior.
Similarly compound 12 (IC50 = 17.2 0.4 M), an ortho-nitro analog showed greater potential if
we compare it with other nitro analogs like compound 11 (IC50 = 26.1 0.5 M) a meta-nitro analog
and compound 10 (IC50 = 21.3 0.4 M) a para-nitro analog. The same pattern was observed in other
analogs with different substituents.
±
0.1 µM) was found to be the second
1
±
µ
2
±
µ
9
±
µ
7
±
µ
±
µ
±
µ
±
µ
A closer look upon the structures of compounds and their corresponding IC50 values revealed
that the presence of different substitutions i.e., fluoro. chloro, methyl, nitro, methoxy and pyridinyl on
the aromatic side chain is of great importance. It was also observed that either an EWG or EDG on the
phenyl part showed great potential but the slight difference in potential was mainly affected by the
position of the substituent.
Further to above, it is evident from the results that the presence of halogens or alkyl groups on
benzene ring influenced the inhibitory potentials significantly. Similarly, nitro substitutions reduced the
potential of inhibition. Apart from direct interactions of these attachments, it can be easily argued that
these substitutions significantly affected the electronic distributions and intensities at the active sites.
In many cases, oxadiazole part also played a significant role in forming hydrogen bond interactions
with the active site of enzyme. Most important of these positions are Asp 207, Glu 451 and Glu 540.
To better understand the binding interactions of the most active analogs, molecular docking analysis
were performed.
2.3. Molecular Docking Studies
Experimental results were further validated by carrying out molecular docking studies and more
information was obtained concerning binding modes of synthesized compounds within the active
site of
β-Glucuronidase. The X-ray structure of β-glucuronidase was retrieved from the Protein Data
Bank with PDB ID: 1BHG. All synthesized compounds were docked into the active site of protein and

Molecules 2019, 24, 1528
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conformations selected for each docked compound were of least energy and were pictured to further
understand the interactions between ligands and receptor.
Binding mode of compound
6
, the most active member (IC50 = 0.80
±
0.10 µM) among the series
was analyzed. Three conventional hydrogen bond interactions were observed between the fluorine
atom at ortho position and side chain amino acids including Tyr 508 and Lys 606 with a internuclear
distance of 2.63 Å, 2.66 Å and 1.83 Å respectively. Meanwhile, C-H and N-H groups of Asn 484 were
involved in forming carbon hydrogen bond with ether oxygen of chromene-4-one ring (2.88 Å) and
π
-donor hydrogen bond with the central arene ring of compound
Asp 207, Glu 451 and Glu 540 were not involved in hydrogen bonding due to absence of hydrogen
bond donor groups on compound instead, these residues were involved in -anion interactions as
shown in Figure 3a. Furthermore, the ligand /receptor complex was further stabilized by T-shaped
and π-π Stacked interactions between indole ring of Trp 587 and phenol ring of Tyr 508 respectively.
Compound , the second most active member with IC50 = 1.10 0.05 M was evaluated and a
6 (3.14 Å). Significant residues like
6
π
π-π
4
±
µ
conventional hydrogen bond between fluorine atom at para position and N-H group of Tyr 487 with a
internuclear distance of 2.22 Å and carbon hydrogen bond between nitrogen atom of pyrazole ring of
compound 4 and C-H (at imidazole ring) of His 509 (2.58 Å) were detected. Meanwhile, the catalytic
residue Glu 451, as in case of compound
the chromene-4-one ring of compound
6
was also involved in forming two -Anion interactions with
π
4. Side chain residues involved in π-π T-Shaped interactions
with compound 4 were Tyr 504 and Tyr 508 as shown in Figure 3b.
Analysis of interactions between third most active compound
site of -Glucuronidase revealed that it was not involved in conventional hydrogen bonding. It was
found that the ether oxygen atom of chromene-4-one ring and central arene ring of compound were
involved in forming carbon hydrogen bond (2.77 Å) and -Donor hydrogen bond (3.26 Å) with the C-H
3
(IC50 = 2.10
±
0.10 µM) and active
β
3
π
and N-H groups of Asn 484 respectively. The chlorine atom presents at ortho position formed so-called
halogen bond of distance 2.94 Å and bond angle ~153◦ with the carboxyl oxygen of Glu 451. Other
interactions shown by compound 3 were almost the same as those shown by compound 6 (Figure 3c).
The least active compound 19 (IC50 = 42.30
interactions as those made by active compounds including conventional hydrogen bonding and
halogen bonding. It was involved in forming carbon hydrogen and -Donor hydrogen bond with
Asn 484 as formed by compound but with one longer bond distance i.e., 2.89 Å and 3.2 Å. Other
interactions resulted between compound 19 and Asp 207, Glu 451, Tyr 508, Glu 540, Trp 587 of the side
chain were -Anion (3.87 Å), -Anion (4.11 Å), Stacked (4.27 Å), -Anion (3.51) and T-shaped
(5.19 Å and 5.29 Å) respectively (Figure 3d).
±
0.80 µM) among the series failed to form many
π
3
π
π
π
-π
π
π-π
Figure 3. Cont.

Molecules 2019, 24, 1528
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Figure 3. Ligand–protein interactions of compound (a) 6, (b) 4, (c) 3 and (d) 19 with the active site of
β
-glucuronidase (1BHG) created by using Discovery Studio 17.2. The left side displays 3D maps of the
hydrogen bonds of the compounds in the binding site. The right side shows the 2D interaction patterns.
Dashed lines show the interactions among the ligand and the amino acids of the protein. Distances are
in Angstrom.
3. Conclusions
Chromen-4-one based oxadiazole derivatives
-glucuronidase inhibitory potential. All analogs displayed potent
ranging between 0.8 0.1 to 42.3 0.8 M as a compare to standard d-saccharic acid 1,4 lactone
(IC50 = 48.1 1.2 M). The fluoro derivatives were found to be the more potent among the series.
A molecular docking study indicated that the top ranked conformations of almost all compounds
1
–
19 have been synthesized and evaluated for
-glucuronidase inhibitory potential
β
β
±
±
µ
±
µ

Molecules 2019, 24, 1528
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were well accommodated inside the active site of
β
-glucuronidase enzyme and they were involved in
various types of interactions with the active site residues of the β-glucuronidase enzyme.
4. Experimental
4.1. General Information
The intermediates 4-(4-oxo-4H-chromen-2-yl)benzoic acid (II), methyl 4-(4-oxo-4H-chromen-2-
yl)benzoate (III) and 4-(4-oxo-4H-chromen-2-yl)benzohydrazide (IV) were synthesized as reported in
our previous paper [42].
4.2. General Procedure for Synthesis of Flavone-Based Oxadiazoles
Compound IV (0.5 mmol) and a substituted arylcarboxylic acid (0.5 mmol) were mixed in a 50 mL
round bottom flask then POCl3 (5 mL) was added dropwise [33]. The mixture was refluxed for 4–5 h
while the reaction progress was monitored using TLC until completion, when the reaction mixture
cooled to room temperature and poured onto crushed ice. NaHCO3 solution was added and the
resulting solid mass that precipitated out was filtered, dried, and recrystallized from methanol in good
to excellent yields.
4.3. 2-(4-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (1)
Solid; Yield 75%; m.p. 288–289 ◦C; 1H-NMR (MeOD-d4, 500 MHz):
(d, J = 8.5 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H), 7.75 (dd, J = 8.0, 2.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.20
(s, 1H), 7.17 (td, J = 8.5 Hz, 1.0 Hz, 1H), 6.94–6.91 (m, 2H); 13C-NMR (125 MHz, DMSO-d6):
177.4,
δ 8.05 (d, J = 8.5 Hz, 2H), 7.96
δ
164.5, 164.4, 163.6, 156.2, 135.2, 132.6, 132.2, 132.2, 131.6, 130.1, 128.5, 128.5, 127.8, 127.8, 127.1, 127.1,
126.0, 125.1, 124.0, 123.2, 116.0, 104.6; HR-EI-MS: m/z Calcd for C23H13ClN2O3, [M]+ 414.0770; found
414.0752.
4.4. 2-(4-(5-(3-Chlorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (2)
Solid; Yield 79%; m.p. 282–283 ◦C; 1H-NMR (DMSO-d6, 500 MHz):
7.72–7.70 (m, 2H), 7.47 (d, J = 4.5 Hz, 2H), 7.31 (s, 1H), 7.16 (t, J = 8.5 Hz, 1H), 6.94 (d, J = 8.5 Hz, 1H),
6.85 (t, J = 8.0 Hz, 1H); 13C-NMR (125 MHz, DMSO-d6):
177.3, 166.2, 164.4, 156.1, 137.8, 135.1, 134.1,
δ 8.04 (s, 4H), 7.83 (s, 1H),
δ
130.1, 129.9, 129.8, 127.6, 127.6, 127.2, 127.2, 127.0, 126.1, 125.5, 125.5, 125.2, 123.6, 124.2, 116.3, 104.6;
HR-EI-MS: m/z Calcd for C23H13ClN2O3, [M]+ 414.0771; found 414.0751.
4.5. 2-(4-(5-(2-Chlorobenzyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (3)
◦
1
Solid; Yield 81%; m.p. 276–277 C; H-NMR (DMSO-d6, 500 MHz):
500 MHz): 8.24 (dd, J = 8.0, 2.0 Hz, 1H), 8.02 (d, J = 8.5 Hz, 2H), 7.96 (d, J = 8.5 Hz, 2H), 7.70 (dd,
J = 8.0, 2.0 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.43–7.40 (m, 2H), 7.22–7.18 (m, 2H), 6.94–6.91 (m, 2H);
13C-NMR (125 MHz, DMSO-d6):
177.4, 166.3, 164.1, 156.0, 138.0, 135.3, 134.5, 130.8, 130.6, 129.1, 127.5,
δ
1H-NMR (MeOD-d4,
δ
δ
127.5, 127.1, 127.1, 127.0, 126.9, 126.0, 125.2, 124.2, 123.7,123.2, 116.2, 104.7; HR-EI-MS: m/z Calcd for
C23H13ClN2O3, [M]+ 414.0772; found 414.0754.
4.6. 2-(4-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (4)
Solid; Yield 82%; m.p. 299–300 ◦C; 1H-NMR (DMSO-d6, 500 MHz):
(m, 3H), 7.33–7.30 (m, 3H), 7.18 (t, J = 7.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.88 (t, J = 7.0 Hz, 1H);
13C-NMR (125 MHz, DMSO-d6):
177.8, 164.6, 164.6, 163.8, 163.0, 156.1, 135.0, 130.5, 129.3, 129.3, 127.6,
δ 8.00–7.96 (m, 4H), 7.78–7.75
δ
127.6, 126.8, 126.8, 125.6, 125.5, 124.1, 123.6, 121.5, 116.3, 115.8, 115.8, 104.7; HR-EI-MS: m/z Calcd for
C23H13FN2O3, [M]+ 384.0910; found 384.0894.

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4.7. 2-(4-(5-(3-Fluorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (5)
Solid; Yield 80%; m.p. 294–295 ◦C; 1H-NMR (DMSO-d6, 500 MHz):
(d, J = 7.5 Hz, 2H), 7.74–7.71 (m, 2H), 7.60 (d, J = 7.0 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 7.20–7.17 (m,
3H), 6.96–6.982 (m, 2H); 13C-NMR (125 MHz, DMSO-d6):
177.6, 164.4, 164.4, 163.5, 162.1, 135.1, 130.4,
δ 8.02 (d, J = 7.5 Hz, 2H), 87.96
δ
128.0, 127.6, 127.5, 127.5, 127.3, 127.3, 127.1, 127.1, 125.9, 124.1, 123.6, 123.0, 116.0, 115.8, 115.3, 104.3;
HR-EI-MS: m/z Calcd for C23H13FN2O3, [M]+ 384.0911; found 384.0892.
4.8. 2-(4-(5-(2-Fluorophenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (6)
Solid; Yield 78%; m.p. 291–292 ◦C; 1H-NMR (MeOD-d4, 500 MHz):
(d, J = 8.0 Hz, 2H), 7.97 (d, J = 8.0 Hz, 2H), 7.72 (dd, J = 7.0, 20 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.25 (t,
J = 8.0 Hz, 1H), 7.22–7.18 (m, 3H), 6.97–6.95 (m, 2H); 13C-NMR (125 MHz, DMSO-d6):
177.5, 164.4,
δ 8.20 (t, J = 7.0 Hz, 1H), 8.03
δ
164.4, 163.5, 158.2, 156.1, 135.0, 130.1, 129.0, 127.2, 127.2, 126.8, 126.8, 125.6, 125.2, 125.0, 124.9, 123.8,
123.4, 123.3, 116.0, 114.5, 104.4; HR-EI-MS: m/z Calcd for C23H13FN2O3, [M]+ 384.0912; found 384.0895.
4.9. 2-(4-(5-(p-Tolyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (7)
Solid; Yield 83%; m.p. 270–271 ◦C, 1H-NMR (MeOD-d4, 500 MHz):
(d, J = 8.0 Hz, 2H), 7.74–7.71 (m, 3H), 7.25 (d, J = 8.0 Hz, 2H), 7.23–7.20 (m, 2H), 6.97–6.95 (m, 2H), 2.43
(s, 3H); 13C-NMR (125 MHz, DMSO-d6):
177.4, 164.6, 164.6, 163.5, 156.0, 142.1, 135.1, 131.5, 130.2,
δ 8.03 (d, J = 8.0 Hz, 2H), 7.96
δ
127.3, 127.3, 127.2, 127.2, 126.8, 126.8, 126.2, 126.2, 125.6, 125.2, 123.7, 123.5, 116.0, 104.3, 21.2; HR-EI-MS:
m/z Calcd for C24H16N2O3, [M]+ 380.1161; found 380.1145.
4.10. 2-(4-(5-(m-Tolyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (8)
Solid; Yield 85%; m.p. 264–265 ◦C, 1H-NMR (DMSO-d6, 500 MHz):
δ 8.03 (m, 4H), 7.72(s, 1H),
7.56 (s, 1H), 7.51 (d, J = 7.0 Hz, 1H), 7.42 (s, 1H), 7.32 (t, J = 7.0 Hz, 1H), 7.24 (d, J = 7.0 Hz, 1H), 7.16
(t, J = 8.5 Hz, 1H), 6.95(d, J = 7.5 Hz, 1H), 6.89 (t, J = 7.5 Hz, 1H), 2.39 (s, 3H); 13C-NMR (125 MHz,
DMSO-d6):
δ 177.5, 164.5, 164.5, 163.6, 156.2, 139.0, 135.2, 130.3, 129.1, 129.0, 128.8, 127.3, 127.3, 126.7,
126.7, 126.0, 125.9, 125.2, 124.5, 123.9, 123.2, 116.0, 104.3, 21.5; HR-EI-MS: m/z Calcd for C24H16N2O3,
[M]+ 380.1160; found 380.1143.
4.11. 2-(4-(5-(o-Tolyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (9)
Solid; Yield 80%; m.p. 260–261 ◦C; 1H-NMR (DMSO-d6, 500 MHz):
(m, 4H), 7.85 (d, J = 7.0 Hz, 1H), 7.16 (t, J = 7.0 Hz, 1H), 6.94 (s, 1H), 6.88 (t, J = 7.0 Hz, 1H), 2.44 (s, 3H);
13C-NMR (125 MHz, DMSO-d6):
177.3, 164.3, 164.3, 163.5, 156.1, 137.1, 136.8, 135.1, 130.2, 129.4, 128.5,
δ 8.02–7.97 (m, 5H), 7.31–7.28
δ
127.3, 127.3, 127.2, 126.7, 126.7, 126.1, 125.8, 125.2, 123.6, 123.2, 116.0, 104.4, 18.5; HR-EI-MS: m/z Calcd
for C24H16N2O3, [M]+ 380.1161; found 380.1141.
4.12. 2-(4-(5-(4-Nitrophenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (10)
Solid; Yield 85%; m.p. 304–305 ◦C; 1H-NMR (DMSO-d6, 500 MHz):
(m, 6H), 7.72 (d, J = 7.5 Hz, 1H), 7.34 (s, 1H), 7.17 (t, J = 7.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.89
(t, J = 7.0 Hz, 1H); 13C-NMR (125 MHz, DMSO-d6):
177.4, 164.4, 164.4, 163.4, 156.0, 147.8, 135.1,
δ 8.29–8.25 (m, 2H), 8.03–7.99
δ
132.0, 131.0, 131.0, 130.2, 128.6, 128.6, 127.2, 127.2, 127.0, 127.0, 125.6, 125.2, 123.7, 123.3, 116.0, 104.5;
HR-EI-MS: m/z Calcd for C23H13N3O5, [M]+ 411.0855; found 411.0839.
4.13. 2-(4-(5-(3-Nitrophenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (11)
Solid; Yield 82%; m.p. 296–297 ◦C; 1H-NMR (DMSO-d6, 500 MHz):
J = 7.0 Hz, 1H), 8.14 (d, J = 7.0 Hz, 1H), 8.03–7.98 (m, 3H), 7.74–7.71 (m, 2H), 7.35 (s, 1H), 7.15 (t, J = 7.5
Hz, 1H), 6.94 (d, J = 7.0 Hz, 1H), 6.88 (t, J = 7.0 Hz, 1H); 13C-NMR (125 MHz, DMSO-d6):
177.5, 164.4,
δ 8.56–8.52 (m, 2H), 8.27(d,
δ
164.4, 163.4, 156.0, 148.3, 135.1, 133.8, 130.2, 130.1, 127.4, 127.4, 127.0, 126.8, 126.8, 125.8, 125.2, 123.8,
123.8, 123.2, 122.6, 116.0, 104.4; HR-EI-MS: m/z Calcd for C23H13N3O5, [M]+ 411.0855; found 411.0837.

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4.14. 2-(4-(5-(2-Nitrophenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (12)
Solid; Yield 84%; m.p. 287–288 ◦C; 1H-NMR (MeOD-d4, 500 MHz):
(m, 3H), 7.32 (s, 1H), 7.15 (t, J = 7.0 Hz, 1H), 6.94 (d, J = 7.0 Hz, 1H), 6.86 (t, J = 7.0 Hz, 1H);
13C-NMR (125 MHz, DMSO-d6):
177.6, 164.5, 164.5, 163.5, 156.1, 146.8, 135.2, 135.1, 131.5, 130.2, 129.8,
δ 8.15–8.06 (m, 6H), 7.90–7.70
δ
128.2, 127.3, 127.3, 126.8, 126.8, 125.6, 125.2, 124.2, 123.6, 123.1, 116.0, 104.3; HR-EI-MS: m/z Calcd for
C23H13N3O5, [M]+ 411.0855; found 411.0833.
4.15. 2-(4-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (13)
Solid; Yield 88%; m.p. 307–308 ◦C; 1H-NMR (DMSO-d6, 500 MHz):
(m, 3H), 7.33 (s, 1H), 7.18 (t, J = 7.0 Hz, 1H), 7.01 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 7.5 Hz, 1H), 6.88 (t,
J = 7.5 Hz, 1H), 3.80 (s, 3H); 13C-NMR (125 MHz, DMSO-d6):
177.4, 164.4, 164.4, 163.5, 160.5, 156.1,
δ 8.08 (br. s, 4H), 7.75–7.70
δ
135.1, 130.3, 129.0, 127.2, 127.2, 126.8, 126.8, 125.6, 125.2, 123.8, 123.2, 116.0, 115.8, 115.8, 114.6, 114.6,
104.4, 56.1; HR-EI-MS: m/z Calcd for C24H16N2O4, [M]+ 396.1110; found 396.1095.
4.16. 2-(4-(5-(3-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (14)
Solid; Yield 84%; m.p. 299–300 ◦C; 1H-NMR (MeOD-d4, 500 MHz):
(d, J = 7.0 Hz, 2H), 7.72 (d, J = 6.0 Hz, 1H), 7.60 (s, 1H), 7.36–7.32 (m, 2H), 7.24–7.20 (m, 2H), 6.98 (d,
J = 7.0 Hz, 1H), 6.95–6.93 (m, 2H), 3.92 (s, 3H); 13C-NMR (125 MHz, DMSO-d6):
177.5, 164.3, 164.3,
δ 8.11 (d, J = 7.0 Hz, 2H), 8.03
δ
163.5, 161.0, 156.0, 135.0, 130.2, 130.1, 130.0, 127.2, 127.2, 126.6, 126.6, 125.6, 125.2, 123.7, 123.2, 119.6,
116.0, 114.2, 111.2, 104.3, 55.4; HR-EI-MS: m/z Calcd for C24H16N2O4, [M]+ 396.1110; found 396.1093.
4.17. 2-(4-(5-(2-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (15)
Solid; Yield 84%; m.p. 286–287 ◦C; 1H-NMR (DMSO-d6, 500 MHz):
4H), 7.72 (d, J = 7.5 Hz, 1H), 7.53 (d, J = 7.0 Hz, 1H), 7.34 (s, 1H), 7.30 (t, J = 7.0 Hz, 1H), 7.15 (t,
J = 7.0 Hz, 1H), 6.95–6.90 (m, 4H) 3.94 (s, 3H); 13C-NMR (125 MHz, DMSO-d6):
177.3, 164.3, 163.5,
δ 8.64 (s, 1H), 8.05–8.00 (m,
δ
162.5, 157.2, 156.0, 135.1, 130.2, 130.0, 129.6, 127.2, 127.2, 126.4, 126.4, 125.1, 125.6, 123.5, 123.1, 121.3,
116.5, 116.0, 112.5, 104.2, 56.0; HR-EI-MS: m/z Calcd for C24H16N2O4, [M]+ 396.1109; found 396.1090.
4.18. 2-(4-(5-(Pyridin-2-yl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (16)
Solid; Yield 80%; m.p. 270–271 ◦C; 1H-NMR (MeOD-d4, 500 MHz):
δ 8.30 (d, J = 7.0 Hz, 1H),
8.10–8.02 (m, 3H), 7.99–7.96 (m, 2H), 7.90 (t, J = 7.0 Hz, 1H), 7.70 (d, J = 7.0 Hz, 1H), 7.42 (d, J = 7.0 Hz,
1H), 7.24–7.20 (m, 2H), 6.96–6.92 (m, 2H); 13C-NMR (125 MHz, DMSO-d6): δ 177.2, 164.3, 164.3, 163.3,
157.2, 156.0, 149.1, 137.1, 135.0, 130.1, 127.2, 127.2, 126.4, 126.4, 125.6, 125.1, 124.4, 124.0, 123.5, 123.2,
116.0, 104.2; HR-EI-MS: m/z Calcd for C22H13N3O3, [M]+ 367.0957; found 367.0941.
4.19. 2-(4-(5-(Pyridin-3-yl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (17)
Solid; Yield 82%; m.p. 280–281 ◦C; 1H-NMR (MeOD-d4, 500 MHz):
8.44–8.40 (m, 2H), 8.05–8.00 (m, 4H), 7.75 (d, J = 6.0 Hz, 1H), 7.52 (d, J = 6.0 Hz, 1H), 7.23–7.20 (m, 2H),
6.94–6.90 (m, 2H); 13C-NMR (125 MHz, DMSO-d6):
177.3, 164.3, 164.3, 163.3, 156.0, 152.6, 147.8, 135.0,
δ 8.58 (d, J = 5.0 Hz, 1H),
δ
134.0, 130.1, 127.2, 127.2, 126.4, 126.4, 125.6, 125.1, 124.3, 124.0, 123.5, 123.2, 116.0, 104.2; HR-EI-MS: m/z
Calcd for C24H16N2O4, [M]+ 367.0957; found 367.0942.
4.20. 2-(4-(5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (18)
Solid; Yield 84%; m.p. 288–289 ◦C; 1H-NMR (MeOD-d4, 500 MHz):
(d, J = 6.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 6.0 Hz, 2H), 7.70 (dd, J = 7.0 Hz, 1.5 Hz, 1H), 7.22
(s, 1H), 7.18 (t, J = 7.0 Hz, 1H), 6.96–6.94 (m, 2H);13C-NMR (125 MHz, DMSO-d6):
177.4, 164.2, 164.2,
δ 8.60 (d, J = 6.0 Hz, 2H), 8.04
δ
163.2, 156.1, 149.7, 149.7, 143.5, 135.1, 130.2, 127.2, 127.2, 126.4, 126.4, 125.4, 125.1, 123.6, 123.2, 121.1,
121.1, 116.0, 104.2; HR-EI-MS: m/z Calcd for C24H16N2O4, [M]+ 367.0957; found 367.0931.

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4.21. 2-(4-(5-Phenyl-1,3,4-oxadiazol-2-yl)phenyl)-4H-chromen-4-one (19)
Solid; Yield 80%; m.p. 280–281 ◦C; 1H-NMR (DMSO-d6, 500 MHz):
δ
8.39 (s, 1H), 8.8–803 (m, 6H),
7.72 (s, 1H), 7.23 (t, J = 7.0 Hz, 1H), 7.22–7.18 (m, 2H), 7.09 (d, J = 7.0 Hz, 1H), 6.94 (d, J = 7.0 Hz, 1H),
6.90 (t, J = 7.0 Hz, 1H), 6.80 (d, J = 7.0 Hz, 1H); 13C-NMR (125 MHz, DMSO-d6):
δ
177.2, 164.1, 164.1,
163.2, 156.0, 135.0, 130.1, 129.0, 129.0, 128.5, 127.3, 127.3, 127.1, 127.1, 126.3, 126.3, 125.4, 123.4, 123.1,
121.3, 116.0, 112.5, 104.2; HR-EI-MS: m/z Calcd for C23H14N2O3, [M]+ 366.1004; found 366.0990.
4.22. β-Glucuronidase Assay
β
-Glucuronidase activity was determined in accordance to method used by Taha et al. [43
by measuring absorbance at 405 nm of the p-nitrophenol formed substrate by a spectrophotometric
method. The total reaction volume was 250 Ls. Reaction mixtures containing 5 L of test compound
solution, 185 L of 0.1M acetate buffer and 10
L of enzyme solution were incubated for 30 min at 37 ◦C.
At 405 nm the plates were recorded on a multiplate reader (SpectaMax, Molecular Devices, Sunnyvale,
CA, USA) after the addition of 50 L of 0.4 mM p-nitrophenyl- -d-glucuronide. Experiments were
performed in triplicate [45]. To avoid precipitation, compound concentrations were decreased, and
,44]
µ
µ
µ
µ
µ
β
the volume of reaction was increased (200
detergents was not needed.
µL). Precipitation probability was less thus addition of
4.23. Molecular Docking Studies Assay
In silico binding interactions of synthesized derivatives against human-
inspected using the Autodock Vina software [46]. The X-ray crystal structure of human-
β
-glucuronidase were
-glucuronidase
β
(PDB ID: 1bhg) was retrieved from the Protein Data Bank [http://www.rcsb.org/]. Water molecules,
ligands and Chain-B were removed from the downloaded PDB file by means of Discovery Studio
Visualizer [47]. Two-dimensional structures of synthesized compounds were sketched and their
energies were optimized using Chembio3D Ultra software (Version; 14.0.0.117) (Cambridge Soft,
PerkinElmer Inc., Boston, MA, USA). Both receptor and ligand files were changed into pdbqt format
by adding charges “q” and autodock type “t” using Autodock Tools (ADT) [48]. Other parameters
contained within configurational file were defined as follow; Grid Box of size 19 × × 19 Å centered at
X = 81.25, Y = 85.15 and Z = 87. 35 so that the active site [49] is entirely enclosed, maximum number of
modes were set 50, energy difference between finest and poorest binding mode was set 4 Kcal/mol and
exhaustiveness = 32. Conformations with lowest free energy of binding were evaluated and visualized
using Discovery Studio Visualizer.
Author Contributions: Conceptualization, M.T. and F.R.; methodology, M.T.; software, M.A.A., M.G. and Y.A.B.
validation, M.G., M.A.A. and Y.A.B.; formal analysis, M.A.; investigation, M.N.K.; resources, R.K.F.; data curation,
R.K.F. and S.A.A.S.; Writing—Original Draft preparation, M.T. and Z.A.Z.; Writing—Review and Editing, Q.U.A.
and Z.A.Z.; visualization, S.A.A.S.; supervision, M.T.; project administration, Z.A.Z. and M.T.; funding acquisition,
M.T. and Q.U.A.
Funding: This research was funded by Deanship Scientific Research Imam Abdulrahman Bin Faisal university for
this study; grant number [2018-066-IRMC].
Acknowledgments: The authors would like to Knowledge Institute for Research and Medical Consultations
(IRMC), Imam Abdulrahman Bin Faisal University, for providing excellent lab facilities for research.
Conflicts of Interest: The authors declare no conflict of interest.
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