Synthesis and docking studies of N-(5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamide analogues as potential alkaline phosphatase inhibitors

Article abstract of DOI:10.1002/ddr.21542

A series of N-(5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamides 6a–i were synthesized as alkaline phosphatase inhibitors. The intermediate 5-substituted 1,3,4-oxadiazole-2-thione 4 was synthesized starting with hippuric acid. Hippuric acid in the first step was converted into corresponding methyl ester 2 which upon reaction with hydrazine hydrate furnished the formation of hydrazide 3. The hippuric acid hydrazide was then cyclized into 5-substituted 1,3,4-oxadiazole-2-thione 4. The intermediate 4 was then reacted with alkyl or aryl halides 5a–5i to afford the title compounds N-(5-(methylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamides 6a–i. The bioassay results showed that compounds 6a–i exhibited good to excellent alkaline phosphatase inhibitory activity. The most potent activity was exhibited by the compound 6i having IC₅₀ value 0.420 μM, whereas IC₅₀ value of standard (KH₂PO₄) was 2.80 μM. Molecular docking studies was performed against alkaline phosphatase enzyme (PDBID 1EW2) to check binding affinity of the synthesized compounds 6a–i against target protein. The docking results showed that three compounds 6c, 6e, and 6i have maximum binding interactions with binding energy values of ?8 kcal/mol. The compound 6i displayed the interactions of oxadiazole ring nitrogen with amino acid His265 having a binding distance 2.13 ?. It was concluded from our results that synthesized compounds, especially compound 6i may serve as lead structure to design more potent inhibitors of human alkaline phosphatase.

Full text of DOI:10.1002/ddr.21542

Received: 13 February 2019
DOI: 10.1002/ddr.21542
Revised: 1 April 2019
Accepted: 13 April 2019
R E S E A R C H A R T I C L E
Synthesis and docking studies of N-(5-(alkylthio)-
1,3,4-oxadiazol-2-yl)methyl)benzamide analogues as potential
alkaline phosphatase inhibitors
Zafar Iqbal1 | Ambreen Iqbal1 | Zaman Ashraf1
Mubashir Hassan3 | Humaira Nadeem4
| Muhammad Latif2
|
1Department of Chemistry, Allama Iqbal Open
Abstract
University, Islamabad, Pakistan
2Department is genetics and Inherited
diseases, College of Medicine, Centre for
Genetics and Inherited Diseases (CGID),
Taibah University, Al-Madinah Al-
Munawwarah, Saudi Arabia
A series of N-(5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamides 6a–i were synthe-
sized as alkaline phosphatase inhibitors. The intermediate 5-substituted 1,3,4-oxadiazole-
2-thione 4 was synthesized starting with hippuric acid. Hippuric acid in the first step was
converted into corresponding methyl ester 2 which upon reaction with hydrazine
hydrate furnished the formation of hydrazide 3. The hippuric acid hydrazide was then
cyclized into 5-substituted 1,3,4-oxadiazole-2-thione 4. The intermediate 4 was then
reacted with alkyl or aryl halides 5a–5i to afford the title compounds N-(5-(methylthio)-
1,3,4-oxadiazol-2-yl)methyl)benzamides 6a–i. The bioassay results showed that com-
pounds 6a–i exhibited good to excellent alkaline phosphatase inhibitory activity. The
most potent activity was exhibited by the compound 6i having IC50 value 0.420 μM,
whereas IC50 value of standard (KH2PO4) was 2.80 μM. Molecular docking studies was
performed against alkaline phosphatase enzyme (PDBID 1EW2) to check binding affinity
of the synthesized compounds 6a–i against target protein. The docking results showed
that three compounds 6c, 6e, and 6i have maximum binding interactions with binding
energy values of −8 kcal/mol. The compound 6i displayed the interactions of oxadiazole
ring nitrogen with amino acid His265 having a binding distance 2.13 Ǻ. It was concluded
from our results that synthesized compounds, especially compound 6i may serve as lead
structure to design more potent inhibitors of human alkaline phosphatase.
3Department of Biology, College of Natural
Sciences, Kongju National University, Gongju,
Republic of Korea
4Department of Pharmaceutical Chemistry,
Riphah Institute of Pharmaceutical Sciences,
Riphah International University, Islamabad,
Pakistan
Correspondence
Muhammad Zaman Ashraf, Chemistry
Department, Allama Iqbal Open University,
Islamabad, Pakistan.
Email: mzchem@yahoo.com
K E Y W O R D S
alkaline phosphatase, molecular docking studies, oxadiazoles
2010). Hydrolytic activity, phosphotransferase activity, and pyro-
phosphatase activity are carried out by ALP (Ardecky et al., 2014;
1
| INTRODUCTION
Alkaline phosphatase (ALP, EC 3.1.3.1) is a hydrolase enzyme occurring in
many organisms (from bacteria to human) that catalyze the process of
hydrolysis of esters containing phosphate groups (al-Rashida et al., 2013;
Chirambo, van Niekerk, & Crowther, 2017; Jihye Park, Helal, Kim, & Kim,
2012). It is responsible for removing phosphate groups from nucleotides,
proteins, and alkaloids. It is effective in an alkaline pH of 8–10
(Kawaguchi, Hanaoka, Komatsu, Terai, & Nagano, 2011; Lanier et al.,
Miliutina et al., 2017; Rawat, Kumar, Sharan, Chattopadhyay, &
Maurya, 2009; Salar et al., 2017).The four isozymes of alkaline phos-
phatase are Intestinal (IAP), Placental (PLAP), Germ cell (GCALP), and
Tissue-nonspecific (TNAP). First, three are tissue-specific which show
their expression in intestine, placenta, and germ cells, whereas the fourth
is tissue nonspecific (Millán, 2006b) expressed in bone, liver, kidney, and
the central nervous system (Jo et al., 2019; Kawaguchi et al., 2011;
Drug Dev Res. 2019;1–9.
wileyonlinelibrary.com/journal/ddr
© 2019 Wiley Periodicals, Inc.
1

2
IQBAL ET AL.
Marquès, Buchet, Popowycz, Lemaire, & Mebarek, 2016). IAP is involved
in intestinal absorption of lipids, detoxification of lipopolysaccharide and
in intestinal homeostasis and occurs in small intestine epithelial cells.
PLAP is expressed primarily in the placenta. It is used as tumor marker,
especially in seminomas and ovarian cancer (Millán, 2006a). GCAP is also
known as placental like ALP. It sometimes also appears in placenta but in
lesser amount than PLAP. Tissue-nonspecific ALP is not expressed in any
particular tissue therefore, called as the tissue-nonspecific. It is a mem-
brane bound glycosylated enzyme, a potent inhibitor of calcification, and
its role is to hydrolyze inorganic pyrophosphate therefore this enzyme is
essential for the skeletal mineralization (Desai, Bhatt, Somani, & Trivedi,
2013; Lanier et al., 2010; Miao, Bai, Panda, Karaplis, Goltzman, & McKee,
2004; Millán, 2006b). Elevated level of ALP occurs in bones diseases,
hepatic or liver diseases, pregnancy, thyrotoxicosis, intestinal disease,
cancerous growth (tumors), the intake of anticonvulsant drugs, and rheu-
matoid arthritis. If IAP gets elevated then it causes problems such as obe-
sity caused by inappropriate deposition of fats within the cell (Buchet,
Millán, & Magne, 2013), neurological disorders (Fonta, Barone,
Martinez, & Négyessy, 2015; Pan et al., 2017; Sebastian-Serrano et al.,
2015), chronic kidney disease (Debray et al., 2013), breast cancer, pros-
tate cancer (Lin, Huang, Zeng, & Wu, 2019; Miao et al., 2004; Ooi,
Shiraki, Morishita, & Nobori, 2007), congestion or obstruction of the
biliary tract, also damage kidney, and liver. Hyper alkaline phosphates
level causes sepsis (risk factors of cancer), whereas diffuse metastatic
liver cancer (Ardecky et al., 2014; Jeesook Park & Kim, 2013). ALP iso-
zymes potent inhibitors are involved to cure the bones disease; athero-
sclerosis, renal osteodystrophy, osteoarthritis, rickets (Millán, 2013;
Yang, Oh, & Pandher, 2011), cholecystitis (inflammation of the gall blad-
der), and myocardial infarction. Few reported inhibitors of alkaline phos-
phatase include L-Phe, L-Trp, L-Leu, L-homoarginine, L-leucinamide,
levamisole (Rosenthal et al., 1977), the L-stereoisomer of tetramisole
(Miller et al., 1980), and theophylline (a 1,3-dimethyl derivative of
xanthine) (Bondock, Adel, Etman, & Badria, 2012). These inhibitors of
the reported drug are poorly selective, and the reported IC50 value is
above 5 μM for all three ALPs, and their inhibition is of rare uncom-
petitive type (Chang et al., 2011).
(Xu et al., 2012), antineoplastic, fungicidal, anticancer, inhibition of
tyrosinase cathepsin K (Munawar et al., 2015), analgesic, antimicrobial,
anticancer (El-Din, El-Gamal, Abdel-Maksoud, Yoo, & Oh, 2015), antiox-
idant activities (Ma et al., 2013), anti-inflammatory (Ramaprasad,
ꢀ
Kalluraya, Kumar, & Hunnur, 2010; S¸ahin, Palaska, Ekizoglu, & Özalp,
2002), hypnotic, genotoxic, serve as muscle anti-epileptic and anti-sei-
zure, inhibitor of oxidative degradation of lipid (Zawawi et al., 2015),
and vasodilator. Some act as muscle relaxant, sedative and are also
involved in the inhibition of cell division activity. Some of the reported
1,3,4-oxadiazoles nucleus containing drugs include the antiretroviral
named raltegravir, anti-hypertensive drug (nesapidil), and the antibiotic
Furamizole (Figure 1). Zibotenten is clinically used for the treatment of
cancers also contains oxadiazole moiety. There are many drugs in the
market to regulate ALP but serious side effects have been reported
with them. Levamisole may cause allergic reactions (skin rashes), blood
problems (agranulocytosis), nervous system problems, and theophylline
can cause nausea, diarrhea, increase in heart rate, abnormal heart
rhythms, and CNS excitation; therefore, their therapeutic use is pres-
ently limited (Li et al., 2009). One of the main problems of the ALP
inhibitors is related to their selectivity. Because of the great homology
between PLAP, GCAP, and IALP, no selective inhibitors of the PLAP
enzymes have been reported and also selective inhibitors of IALP
and other isozymes not available which inhibit them completely.
1,3,4-Oxadiazoles are known to be associated with a broad spectrum of
biological activities (Salahuddin, Elbarbary, Allam, & Hashim, 2018).
1,3,4-Oxadiazole derivatives have received considerable attention
of medicinal chemists owing to their wide spectrum of useful chemical,
biological, and pharmacological activities (Khan, Ahuja, & Husain, 2017).
Therefore, the present work is planned to synthesize oxadiazole deriva-
tives which may possess potent alkaline ALP inhibitory.
2
| MATERIALS AND METHODS
2.1 | Chemistry
All the chemicals were purchased from Sigma Aldrich Company and
were used as such without further purification. Digital Gallenkamp
(SANYO) apparatus was used to record the melting points, and Perkin
Elmer spectrophotometer was used to record FTIR spectrum of synthe-
sized compounds. 1H NMR and 13C NMR spectra were determined as
DMSO solutions at 300 MHz on Brukner AM-300 spectrophotometer.
1,3,4-Oxadiazoles have great significance in the heterocyclic com-
pounds, and their derivatives show a wide range of biological activities
such as painkillers, act as anti-hyperlipidemic agents, produce or
develop ulceration, anti-tubercular (Jha et al., 2010), antiviral (Llinas-
Brunet et al., 2004; Wei et al., 2013), hypoglycemic, antimicrobial
FIGURE 1 Chemical structures of oxadiazole-containing drugs

IQBAL ET AL.
3
132.2 (C-4), 129.4 (C-3,5) 128.99 (C-14,16), 128.91 (C-13,17), 128.1
(C-2,6), 127.8 (C-15), 36.2 (C-8), 34.7 (C-11).
2.1.1 | Synthesis of N-(5-mercapto-1,3,4-oxadiazol-
2-yl)methyl)benzamide 4
Hippuric acid 1 was reacted with anhydrous methanol in the presence
of catalytic amount of concentrated sulfuric acid to afford the
corresponding hippuric ester 2. Hippuric acid hydrazide 3 was synthe-
sized by reacting ester 2 (1 mmol) with hydrazine hydrate (1.5 mmol).
N-(5-Mercapto-1,3,4-oxadiazol-2-yl)methyl)benzamide 4 was synthe-
sized by mixing hydrazide 3 (5 mmol), KOH (6 mmol), and anhydrous
ethanol (10 mL) in a round-bottom flask. The reaction mixture was
then treated with CS2 in portions (10 mmol) and heated under reflux
for 12 hr. After the completion of reaction (as monitored by TLC), the
reaction mixture was concentrated under reduced pressure and acidi-
fied with HCl to afford oxadiazole 4 as yellow solid. The crude
oxadiazole 4 was then purified by recrystalization in ethanol (He et al.,
2016; Pidugu, Yarla, Pedada, Kalle, & Satya, 2016).
N-(5-(2-Chlorobenzylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamide 6c
Yield: 94%; m.p. 93–94ꢀC FTIR (KBr, νmax cm−1): 3336 (sec. amide
NH), 3,062 (sp3
C C H stretch), 1,570 (C N),
H stretch), 2,944 (sp3
1,646 (C O amide), 1,603 (C C aromatic). 1H NMR (DMSO, δ ppm):
9.23 (1H, t, J = 5.4 Hz, H-6), 7.87 (2H, d, J = 7.2, H-1,5), 7.52–7.59
(3H, m, H-2,4), 7.45–7.47 (2H, m, H-10,11), 7.20–7.34 (2H, d, J = 7.2,
H-9,12), 4.70 (2H, d, J = 5.7 Hz, H-7), 4.54 (2H, s, H-8); 13C NMR
(DMSO, δ ppm): 163.2 (C-7), 161.6 (C-9), 158.9 (C-10), 136.4 (C-12),
133.5 (C-17), 131.4 (C-1), 129.6 (C-4), 128.1 (C-16), 128.3 (C-3,5)
127.4 (C-2,6), 126.5 (C-13), 125.8 (C-15), 123.6 (C-14), 31.8 (C-8),
30.5 (C-11), 19.1 (C-18).
N-(5-(4-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)
benzamide 6d
Yield: 51%; m.p. 130ꢀC FTIR (KBr, νmax cm−1): 3325 (sec. amide NH),
2.1.2 | Synthesis of N-(5-(alkylthio)-1,3,4-oxadiazol-
2-yl)methyl)benzamide derivatives 6a–i
3,046 (sp3
C
H stretch), 2,944 (sp3 CH stretch), 1,643 (C O amide),
1,478 (C C aromatic). 1H NMR (DMSO, δ 9.21 (1H, t, J = 5.4 Hz, H-
6), 7.87 (2H, d, J = 1.5, H-10,11), 7.42–7.60 (5H, m, H-1-H-5), 7.31
(2H, d, J = 7.6 Hz, H-7), 4.68 (2H, d, J = 5.7, H-7), 4.46 (2H, s, H-8);
13C NMR (DMSO, δ ppm):166.6 (C-7), 164.5 (C-9), 160.4 (C-10),
138.3 (C-12), 132.8 (C-17), 131.6 (C-1), 130.4 (C-4), 129.6 (C-16),
128.8 (C-3,5) 127.5 (C-2,6), 126.8 (C-13), 124.4 (C-15), 122.5 (C-14),
32.6 (C-8), 31.7 (C-11), 17.5 (C-18).
N-(5-Mercapto-1,3,4-oxadiazol-2-yl)methyl)benzamide 4 (5 mmol), ace-
tone (20 mL), and K2CO3 (10 mmol) were added in a round-bottom flask
by continuous stirring. To this reaction mixture, substituted alkyl/aryl
halides 5a–5i (6 mmol) were added and heated under reflux for 3 hr.
The progress of the reaction was monitored by TLC, and after the com-
pletion of reaction, reaction mixture was concentrated under reduced
pressure to get solid oxadiazole derivatives 6a–6i, purified by rec-
rystalization in ethanol (He et al., 2016).
N-(5-(2-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methyl)
benzamide 6e
Yield: 80%; m.p. 77ꢀC FTIR (KBr, νmax cm−1): 3294 (sec. amide NH),
N-(5-Mercapto-1,3,4-oxadiazol-2-yl)methyl) benzamide 4
Yield: 79.9%; m.p. 196ꢀC FTIR (KBr, νmax cm−1): 3303 (sec. amide NH),
2,980 (sp3 C H stretch), 1,582 (NH), 1,640 (C O amide), 1,469 (C
C
aromatic). 1H NMR (DMSO, δ ppm): 7.85 (1H, t, J = 5.5 Hz, NH), 6.52
(2H, d, J = 7 Hz, H-2,6), 6.19 (1H, t, J = 7.5 Hz, H-4), 6.12 (2H, t,
J = 7.5 Hz, H-3,5), 5.96 (1H, d, J = 7.5 Hz, H-16), 5.81–5.80 (2H, m,
H-14,15), 3.32 (1H, d, J = 6 Hz, H-13), 3.11 (1H, d, J = 5.5 Hz, H-8),
1.96 (1H, s, H-11), 0.97 (3H, s, H-18). 13C NMR (DMSO, δ ppm):165.5
(C-7), 164.5 (C-9), 162.0 (C-10), 135.8 (C-12), 132.8 (C-17), 132.7
(C-1), 130.8 (C-4), 129.5 (C-16), 129.0 (C-3,5) 127.5 (C-2,6), 127.2
(C-13), 126.4 (C-15), 125.1 (C-14), 33.4 (C-8), 33.4 (C-11), 17.8 (C-18).
2,853 (SH thiol), 1,635 (C O amide), 1,485 (C C aromatic), 1,304 (C O).
N-(5-(Butylthio)-1,3,4-oxadiazol-2-yl) methyl)benzamide 6a
Yield: 61.9%; m.p. 52–53ꢀC FTIR (KBr, νmax cm−1): 3278 (sec. amide
NH), 2,961 (sp2
C C H stretch), 1,579 (C N),
H stretch), 2,871 (sp3
1,642 (C O amide), 1,475 (C C aromatic). 1H NMR (DMSO, δ ppm):
9.21 (1H, t, J = 5 Hz, H-13), 7.89 (2H, d, J = 8 Hz, H-2,6), 7.48–7.58
(3H, m, H-3,4,5), 4.69 (2H, d, J = 5.5 Hz, H-8), 3.21 (2H, t, J = 7, H-11),
1.65–1.71 (2H, m, H-12), 1.36–1.41 (2H, m, H-13) 0.86 (3H, t, 7.5,
H-14); 13C NMR (DMSO, δ ppm): 166.9 (C-7), 165.6 (C-9), 164 (C-10),
133 (C-1), 132 (C-4), 128.9 (C-3,5), 127.7 (C-2,6), 14.8 (C-8), 12.1
(C-11), 11.46 (C-12), 21 (C-13), 13 (C-14).
N-(5-(2-((4-Methoxyphenethyl)amino)-2-oxoethyl)thio)-
1,3,4-oxadiazol-2-yl)methyl) benzamide 6f
Yield: 85.5%; m.p. 159–163ꢀC FTIR (KBr, νmax cm−1): 3310 (sec. amide
NH), 2,929 (sp2 C-H stretch), 2,836 (sp3 C-H stretch), 1,638 (C O amide),
1,602 (C C aromatic). 1H NMR (DMSO, δ ppm): 9.22 (1H, t, J = 5.5 Hz,
NH), 8.35 (1H, t, J = 5.5 Hz, NH0), 7.88 (2H, d, J = 7 Hz, H-2,6), 7.56 (1H,
t, J = 7.5 Hz, H-4), 7.50 (2H, t, J = 7.5 Hz, H-3,5), 7.12 (2H, d, J = 8.5 Hz,
H-16,20), 6.85 (2H, d, J = 7, H-17,19), 4.69 (2H, d, J = 5.5 Hz, H-8), 4.01
(2H, s, H-11), 3.72 (3H, s, H-21), 3.26 (2H, q, J = 7, H-13), 2.63 (2H,
t, J = 7 Hz, H-14). 13C NMR (DMSO, δ ppm):166.9 (C-12), 166.1 (C-7),
165.7 (C-9), 163.8 (C-10), 158.1 (C-18), 133.8 (C-1), 132.1 (C-4), 131.5
(C-15), 130.0 (C-20,16), 128.9 (C-3,5) 127.8 (C-2,6), 114.2 (C-17,19), 55.4
(C-21), 41.4 (C-8), 36.2 (C-13), 34.7 (C-11), 34.5 (C-14).
N-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl) methyl)benzamide 6b
Yield: 72.72%; m.p. 114–116ꢀC FTIR (KBr, νmax cm−1): 3316 (sec.
amide NH), 3,059 (sp2
C C H stretch), 1,579
H stretch), 2,871 (sp3
(C N), 1,633 (C O amide), 1,601 (C C aromatic). 1H NMR (DMSO, δ
ppm): 9.22 (1H, t, J = 5 Hz, NH), 7.90 (2H, d, J = 7.5, H-2,6),
7.48–7.59 (3H, m, H-3,4,5), 7.42 (2H, d, J = 7, H-13,17), 7.26–7.28
(3H, m, H-14,15,16), 4.69 (2H, d, J = 5.5, H-8). 13C NMR (DMSO, δ
ppm):166.9 (C-7), 165.8 (C-9), 163.6 (C-10), 137.7 (C-12), 133.7 (C-1),

4
IQBAL ET AL.
The change in absorbance of released p-nitrophenolate was monitored
at 405 nm, using a 96-well micro plate reader (OPTI MAX, Tunable). All
the experiments were repeated three times in a triplicate manner.
KH2PO4 was used as the reference inhibitor of alkaline phosphatase.
N-(5-((2-(5-Isopropyl-2-methylphenyl)amino)-2-oxoethyl)thio)-
1,3,4-oxadiazol-2-yl)methyl)benzamide 6g
Yield: 55.7%; m.p. 157ꢀC FTIR (KBr, νmax cm−1): 3,302 (sec. amide
NH), 2,959 (sp2
C C H stretch), 1,639 (C O
H stretch), 2,926 (sp3
amide), 1,600 (C C aromatic). 1H NMR (DMSO, δ ppm): 9.47 (1H, t,
J = 6.5, NH), 8.74 (1H, s, NH0), 7.87 (2H, d, J = 7.5, H-2,6), 7.45–7.55
(4H, m, H-3,4,5,18), 7.313 (1H, d, J = 5.5, H-15), 7.12 (1H, d, J = 7 Hz,
H-16), 6.97 (2H, d, J = 6.5, H-8), 5.4 (2H, s, H-11), 3.75–4.30 (1H, m,
H-20), 3.55 (3H, s, H-22), 1.19 (6H, d, J = 6.5, H-19,21). 13C NMR
(DMSO, δ ppm): 171.6 (C-7), 166.9 (C-12), 159.85 (C-10), 157.79
(C-9), 147.5 (C-17), 136.3 (C-13), 134.4 (C-1), 132 (C-4), 129.2 (C-3),
128.8 (C-5), 128.7 (C-14), 127 (C-15), 123.6 (C-16), 122.9 (C-18), 50.8
(C-8), 42 (C-11), 36 (C-20), 24.2 (C-19,21), 17.8 (C-22).
2.2 | Molecular docking study
2.2.1 | Retrieval of alkaline phosphate structure
from PDB
The three dimensional (3D) structure of alkaline phosphate from human
placenta was accessed from Protein Data Bank (PDB) (www.rcsb.org)
having PDBID 1EW2. The selected target protein structure was mini-
mized by using UCSF Chimera 1.10.1 tool (Studio, 2008). Furthermore,
Prot param tool was used to generate stereo-chemical properties
of alkaline phosphate (Gasteiger et al., 2005). Hydrophobacity and
Ramachandran plots of alkaline phosphate were accessed from Discov-
ery Studio 4.1 Client tool (Studio, 2008). The protein architecture and
statistical percentage values of receptor proteins helices, beta-sheets,
coils, and turn were predicted from online server VADAR 1.8 (Willard
et al., 2003).
N-(5-(2-((4-Isopropylphenyl)amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-
2yl)methyl)benzamide 6h
Yield: 62.5%; m.p. 243–245ꢀC FTIR (KBr, νmax cm−1): 3412 (sec. amide
NH), 3,212 (sp2
matic), 1,640 (C N), 2,975 (sp3
C
H stretch), 1,686 (C O amide), 1,601 (C C aro-
H stretch). 1H NMR (DMSO, δ
C
ppm): 8.76 (1H, t, J = 4, NH), 8.47 (1H, s, NH), 7.85 (2H, d,7, H-2,6),
7.45–7.55 (3H, m, H-3,4,5), 7.37 (2H, d, J = 7.5, H-14,18), 7.23 (2H, d,
J = 7.5 Hz, H-15,17), 4.18 (2H, s, H-11), 3.92 (2H, d, J = 4.5 H-8), 2.94
(1H, m, H-19), 1.24 (6H, d, J = 6.5, H- 20,21). 13C NMR (DMSO, δ
ppm): 171.7 (C-12), 167 (C-7), 165.9 (C-10), 159.6 (C-9), 149.2 (C-19),
134.5 (C-13), 134.4 (C-1), 133 (C-4), 131 (C-3), 128.7 (C-5), 128.4
(C-2), 128.3 (C-6), 127.7 (C-15), 127.6 (C-17), 127.3 (C-14), 127.2
(C-18), 33.68 (C-8), 33.60 (C-11), 33.3 (C-20), 24.2 (C-21).
2.2.2 | Designing of ligands and molecular docking
The synthesized ligands were sketched in drawing ACD/Chem Sketch
tool and minimized by UCSF Chimera 1.10.1. All the synthesized ligands
were sketched in ACD/Chem Sketch tool and access in mol format. Fur-
thermore, UCSF Chimera 1.10.1 tool was employed to energy minimiza-
tion of each ligand separately having default parameters such as
steepest descent steps 100 with step size 0.02 (Å), conjugate gradient
steps 100 with step size 0.02 (Å), and update interval was fixed at 10.
Finally, Gasteiger charges were added using Dock Prep in ligand struc-
ture to obtain the good structure conformation. Molecular docking
experiment was used on all the synthesized ligands against ALP by using
PyRx virtual screening tool with Auto Dock VINA Wizard approach
(Dallakyan & Olson, 2015). The grid box center values of (center
X = 43.3, center Y = 23.1612, and center Z = 9.1269), and size values
were adjusted as (X = 65.56, Y = 71.79, and Z = 64.64) for better con-
formational position in the active region of target protein. All the
synthesized ligands were docked separately against ALP with default
exhaustiveness value = 8. The predicted docked complexes were evalu-
ated on the basis of lowest binding energy (kcal/mol) values and struc-
ture activity relationship (SAR). The three-dimensional graphical
depictions of all the docked complexes were accomplished by Discov-
ery Studio (2.1.0) and UCSF Chimera 1.10.1 tool.
N-(5-(2-(4-Acetylphenyl]amino)-2-oxoethyl)thio)-1,3,4-oxadiazol-2yl)
methyl)benzamide 6i
Yield: 98.3%; m.p.201 ꢀC FTIR (KBr, νmax cm−1): 3348 (sec. Amide
NH), 3,231 (sp2 C-H stretch) 2,990 (sp3 C-H stretch), 1,679 (C
O
amide), 1,601 (C C aromatic), 1,641 (C N). 1H NMR (DMSO, δ ppm):
10.30 (1H, s, NH0), 8.77 (1H, t, J = 5.5, NH), 8.084 (2H, d, J = 8.5, H-
2,6), 7.87 (2H, d, J = 7.5, H-14,15), 7.83 (2H, d, J = 7.5, H-16,17),
7.45–7.58 (3H, m, H-3,4,5), 4.21 (2H, s, H-11), 3.94 (2H, d, J = 5.5 H-
8), 2.62 (3H, s, H-20). 13C NMR (DMSO, δ ppm): 197.7 (C-19), 171.4
(C-12), 167.0 (C-7), 165.9 (C-10), 158.5 (C-9), 139.3 (C-13), 137.0
(C-18), 134.1 (C-1), 131.8 (C-4), 129.3 (C-3), 129.2 (C-5), 129.1 (C-16),
129.0 (C-17), 128.7 (C-2), 127.8 (C-6), 127.7 (C-14), 127.6 (C-18),
42.1 (C-8), 31.1 (C-11), 27.3 (C-20).
2.1.3 | Alkaline phosphatase inhibition assay
Activity of calf intestinal alkaline phosphatase (CIALP) was measured by
spectrophotometric assay as previously described by (Saeed et al.,
2018). The reaction mixture comprised of 50 mM Tris–HCl buffer
(5 mM MgCl2, 0.1 mM ZnCl2 pH 9.5), the compound (0.1 mM with final
DMSO 1% (v/v), and mixture was pre-incubated for 10 min by adding
5 μL of CIALP (0.025 U/mL). Then, 10 μL of substrate (0.5 mM p-NPP
(para-nitrophenylphosphate disodium salt) was added to initiate the
reaction, and the assay mixture was incubated again for 30 min at 37ꢀC.
3
| RESULTS AND DISCUSSION
3.1 | Chemistry
Hippuric ester 2 and hippuryl hydrazide 3 were synthesized according
to the conventional esterification and hydrazide formation reactions.
The intermediate oxadiazole 4 was synthesized by following the

IQBAL ET AL.
5
O
O
O
H
N
O H
OCH3
NH2NH2
N
H
N
H
N
H
NH2
MeOH
+
H
O
O
O
Esterification
1
2
3
CS2
KOH in ethanol
O
O
O
N
O
N
H
acetone
K2CO3
N
S
SH
Ar/R-Cl
5a-i
H
+
N
R/Ar
N
N
4
6a-i
(R/Ar is same as in 5a-i)
R/Ar=
5b =
5e =
5a = -C4H9
5c =
5d =
Cl
C l
H
N
H
N
H
N
H
N
5f =
O
5i =
O
5h =
O
O
O
5g =
O
SCHEME 1 Synthesis of N-(5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamide derivatives 6a–i
already reported method with slight modifications shown in Scheme 1.
The presence of C N absorption at 1635 cm−1 and SH at 2853 cm−1
in FTIR spectrum confirmed the formation of oxadiazole 4. The title
compounds 6a–i were obtained in good to excellent yield by reacting
oxadiazole 4 with substituted halides 5a–i. This involves the alkylation
of mercapto group of oxadiazole 4 with substituted halides. The for-
mation of the final products was ascertained by FTIR, 1H NMR, and
13C NMR spectroscopy.
ALP inhibitory activity than the compounds with alkyl substituted phe-
nyl rings. We may propose that the major inhibitory activity is related to
some sort of polar interactions with the active site of the enzyme.
3.3 | Docking studies
3.3.1 | Structural analysis and physiochemical
properties of ALP
Human placental alkaline phosphate is a class of hydrolase single
chain protein which consists of 539 amino acids with embedded zinc
3.2 | Alkaline phosphatase inhibitory activity
2,5-Disubstituted1,3,4-oxadiazole analogues 6a–i were designed and
synthesized as ALP inhibitors. Table 1 shows that the major determin-
ing factor of alkaline phosphatase inhibitory activity is oxadizole ring
and chloro, methoxy, or acetyl moieties on benzene ring in side chain.
It shows that acetyl substituted benzene moiety play major role in
alkaline phosphatase inhibitory activity. Interestingly, compound 6i
bearing acetyl group on benzene ring in side chain showed most
potent activity having IC50 0.42 μM than 6b, 6c, and 6f, having IC50
1.72, 1.79, and 1.23 μM, respectively. These derivatives possessed
benzyl, chloro benzyl, and methoxy benzyl moieties attached with the
thiol side chain of oxadiazole ring.
TABLE 1 The inhibitory effects of for N-((5-(methylthio)-1,3,
4-oxadiazol-2-yl)methyl)benzamide derivatives (6a–i) on ALP
inhibition
Compounds
IC50 (μM)
log-p
4
4.84 0.87
2.37 0.26
1.72 0.15
1.79 0.13
8.34 1.54
4.80 1.02
1.23 0.32
3.76 0.28
5.44 1.45
0.42 0.05
2.80 0.204
1.46 0.67
2.35 0.60
2.53 0.60
3.12 0.61
3.12 0.61
2.99 0.60
1.71 0.64
3.56 0.62
3.10 0.62
1.91 0.64
—
6a
6b
6c
6d
6e
The substitution pattern of chloro groups at phenyl ring in com-
pound 6c and 6d is the decisive factor of enzyme inhibitory activity in
these compounds. The derivative 6c with chloro group present at ortho
position of phenyl ring has far better IC50 value than that of compound
6d where chloro group is present at para position of benzene ring. It is
also investigated that alkyl substituents at different positions on the
benzene ring do not have any remarkable effect on the enzyme inhibi-
tory activity. The derivatives with unsubstituted phenyl ring have better
6f
6g
6h
6i
KH2PO4
log-p values were calculated from Chemsketch ACD Lab 2012. (—) Not
calculated.

6
IQBAL ET AL.
FIGURE 2 (a) Hydrophobicity graph of ALP protein 1EW2. (b) Ramachandran graph of protein predicted by Discovery Studio
and magnesium ions. The VADAR analysis showed the overall protein
architecture consists of 31% helices, 26% β sheets, and 42% coils.
Moreover, the Ramachandran plot indicated that 96.4% of residues
were present in favored regions which showed the precision of phi (ϕ)
and psi (ψ) angles among the coordinates of ALP. The hydrophobicity
and Ramachandran graphs are mentioned in Supplementary Data
(Figure 2).
In detail, SAR study showed that four hydrogen bonds were
observed in 6i-docking complex. The oxygen atom of 6i forms two
hydrogens bonds with Arg 314 and Glu 321 having bond lengths 2.45
and 3.17 Å, respectively. Moreover, carbonyl oxygen of 6i forms
another hydrogen bond with bond length 2.30 Å against His 317. Sim-
ilarly, the oxygen atom of oxadiazole ring was involved in hydrogen
bonding with His 432 having bond length 2.49 Å. The importance of
these amino acid residues in binding with other ALP inhibitors has also
been well documented (Khan et al., 2015; Saeed et al., 2018). The
comparative binding energy and SAR analysis showed the significance
of 6i compound and may be considered as potent inhibitor of the
enzyme. The rest of all docking complex are shown in Supplementary
Data (Figures S1–S9). The binding pocket is highlighted in green color
3.3.2 | Binding energy evaluation of compounds 6a–i
To predict the best conformational position within the active region
of target protein, all the synthesized ligands 6a–i were docked against
ALP. All the generated docked complexes were analyzed on the basis
of minimum energy values (kcal/mol) and hydrogen/hydrophobic
interactions. Docking results justified that all of the synthesized com-
pounds exhibited excellent binding affinities with the target protein.
Moreover, 6c, 6e, and 6i have a unique binding energy values
(−8.0 kcal/mol). Whereas, both 6b and 6g also possessed same bind-
ing energy value −7.8 kcal/mol. The remaining compounds showed
binding energies values in the range of −6.9 to −7.7. The basic chemi-
cal nuclei of all the synthesized compounds were same; therefore,
most of ligands showed good binding affinity with target protein
(Figure 3).
3.3.3 | Binding interaction of 5f against alkaline
phosphatase
Based on in vitro (IC50) results, 6i-docked complex was selected to
deeply understand the binding interaction behavior of ligand against
target protein. Docking analysis showed that compound 6i was
actively confined within active binding region of receptor molecule
(Figure 4).
FIGURE 3 The graphical depiction of docking energy values of
compounds 6a–i

IQBAL ET AL.
7
FIGURE 4 (a and b) Binding
pocket of alkaline phosphatase
1EW2 with 6i bound ligand
interaction; (c and d) Binding
interactions of compound 6i with
amino acid residues of target
protein 1EW2. The binding
distance is also presented
while ligand structure is represented in yellow color (C) docking inter-
action of (6i) with ALP. (D) The interacted residues are represented in
red, whereas the protein structure is shown in dark gray color. The
protein is highlighted in ribbon format in gray and purple colors. The
interacted residue is mentioned with green color, whereas amino acid
involved in hydrogen bonding are labeled with red color, having dis-
tances mentioned in Angstrom (Å). Figure 5 depicted the functional
groups responsible for binding interactions with target enzyme ALP
FIGURE 5 (a) Chemical structure of
compound 6i with highlighted functional
groups responsible for binding
interactions with target enzyme
(b) Three-dimensional view of compound
6i electrostatic map taken from ACD labs

8
IQBAL ET AL.
Dallakyan, S., & Olson, A. J. (2015). Small-molecule library screening by
docking with PyRx. In Chemical biology (pp. 243–250). New York, NY:
Humana Press.
1EW2. It has been confirmed that the heterocyclic oxadiazole ring
and acetyl group and amide carbonyl play important role in binding
interactions of the most potent derivative 6i. The electrostatic map
with three dimensional structure of compound 6i taken from ACD Lab
is also given in the figure.
Debray, J., Chang, L., Marquès, S., Pellet-Rostaing, S., Mebarek, S.,
Buchet, R., & Lemaire, M. (2013). Inhibitors of tissue-nonspecific alka-
line phosphatase: Design, synthesis, kinetics, biomineralization and
cellular tests. Bioorganic & Medicinal Chemistry, 21(24), 7981–7987.
Desai, N., Bhatt, N., Somani, H., & Trivedi, A. (2013). Synthesis, antimicro-
bial and cytotoxic activities of some novel thiazole clubbed 1, 3,
4-oxadiazoles. European Journal of Medicinal Chemistry, 67, 54–59.
El-Din, M. M. G., El-Gamal, M. I., Abdel-Maksoud, M. S., Yoo, K. H., &
Oh, C.-H. (2015). Synthesis and in vitro antiproliferative activity of
new 1, 3, 4-oxadiazole derivatives possessing sulfonamide moiety.
European Journal of Medicinal Chemistry, 90, 45–52.
Fonta, C., Barone, P., Martinez, L. R., & Nëgyessy, L. (2015). Rediscovering
TNAP in the brain: a major role in regulating the function and develop-
ment of the cerebral cortex. In Neuronal tissue-nonspecific alkaline
phosphatase (TNAP) (pp. 85–106). Springer.
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4
| CONCLUSIONS
Herein, we reported a simple and straightforward synthesis of N-
(5-(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamides 6a–i by using
the chemistry of 1,3,4-oxadiazole. Molecular docking was carried out
to predict the binding affinity of synthesized compounds with target
protein alkaline phosphatase 1EW2 which assured that most of the
synthesized compounds bound at the active binding site. The calcu-
lated results displayed that compounds 6c, 6e, and 6i exhibited maxi-
mum binding interactions having energy values −8 kcal/mol. The
enzyme inhibitory activity results showed that compound 6i showed
excellent activity with IC50 value 0.420 μM. The compound 6i is more
potent than all other derivatives and standard potassium dihydrogen
phosphate. The wet laboratory results were also coordinated with the
computational studies. It was concluded from our results that synthe-
sized compounds, especially compound 6i may serve as lead com-
pound to design more active and potent inhibitors of human alkaline
phosphatase.
He, H., Wang, W., Zhou, Y., Xia, Q., Ren, Y., Feng, J., & Feng, L. (2016). Ratio-
nal design, synthesis and biological evaluation of 1, 3, 4-oxadiazole
pyrimidine derivatives as novel pyruvate dehydrogenase complex E1
inhibitors. Bioorganic & Medicinal Chemistry, 24(8), 1879–1888.
Jha, K. K., Samad, A., Kumar, Y., Shaharyar, M., Khosa, R. L., Jain, J., &
Singh, P. (2010). Design, synthesis and biological evaluation of 1, 3,
4-oxadiazole derivatives. European Journal of Medicinal Chemistry, 45
(11), 4963–4967.
Jo, S., Han, J., Lee, Y. L., Yoon, S., Lee, J., Wang, S. E., & Kim, T. H. (2019).
Regulation of osteoblasts by alkaline phosphatase in ankylosing spon-
dylitis. International Journal of Rheumatic Diseases, 22(2), 252–261.
Kawaguchi, M., Hanaoka, K., Komatsu, T., Terai, T., & Nagano, T. (2011).
Development of a highly selective fluorescence probe for alkaline phos-
phatase. Bioorganic & Medicinal Chemistry Letters, 21(17), 5088–5091.
Khan, I., Shah, S. J. A., Ejaz, S. A., Ibrar, A., Hameed, S., Lecka, J., & Iqbal, J.
(2015). Investigation of quinoline-4-carboxylic acid as a highly potent
scaffold for the development of alkaline phosphatase inhibitors: Syn-
thesis, SAR analysis and molecular modelling studies. RSC Advances, 5
(79), 64404–64413.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ORCID
Zaman Ashraf
https://orcid.org/0000-0002-6620-9080
https://orcid.org/0000-0003-4858-3324
Muhammad Latif
Khan, S. A., Ahuja, P., & Husain, A. (2017). Oxidative cyclization of isonia-
zid with fluoroquinolones: Synthesis, antibacterial and antitubercular
activity of new 2, 5-disubstituted-1, 3, 4-oxadiazoles. Journal of the
Chinese Chemical Society, 64(8), 918–924.
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Iqbal Z, Iqbal A, Ashraf Z, Latif M,
Hassan M, Nadeem H. Synthesis and docking studies of N-(5-
(alkylthio)-1,3,4-oxadiazol-2-yl)methyl)benzamide analogues as
potential alkaline phosphatase inhibitors. Drug Dev Res. 2019;
1–9. https://doi.org/10.1002/ddr.21542