Bi-heterocyclic benzamides as alkaline phosphatase inhibitors: Mechanistic comprehensions through kinetics and computational approaches

Article abstract of DOI:10.1002/ardp.201800278

Novel bi-heterocyclic benzamides were synthesized by sequentially converting 4-(1H-indol-3-yl)butanoic acid (1) into ethyl 4-(1H-indol-3-yl)butanoate (2), 4-(1H-indol-3-yl)butanohydrazide (3), and a nucleophilic 5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazole-2-thiol (4). In a parallel series of reactions, various electrophiles were synthesized by reacting substituted anilines (5a–k) with 4-(chloromethyl)benzoylchloride (6) to afford 4-(chloromethyl)-N-(substituted-phenyl)benzamides (7a–k). Finally, the nucleophilic substitution reaction of 4 was carried out with newly synthesized electrophiles, 7a–k, to acquire the targeted bi-heterocyclic benzamides, 8a–k. The structural confirmation of all the synthesized compounds was done by IR, ¹H NMR, ¹³C NMR, EI-MS, and CHN analysis data. The inhibitory effects of these bi-heterocyclic benzamides (8a–k) were evaluated against alkaline phosphatase, and all these molecules were identified as potent inhibitors relative to the standard used. The kinetics mechanism was ascribed by evaluating the Lineweaver–Burk plots, which revealed that compound 8b inhibited alkaline phosphatase non-competitively to form an enzyme–inhibitor complex. The inhibition constant K_i calculated from Dixon plots for this compound was 1.15 μM. The computational study was in full agreement with the experimental records and these ligands exhibited good binding energy values. These molecules also exhibited mild cytotoxicity toward red blood cell membranes when analyzed through hemolysis. So, these molecules might be deliberated as nontoxic medicinal scaffolds to render normal calcification of bones and teeth.

Full text of DOI:10.1002/ardp.201800278

Received: 20 September 2018
DOI: 10.1002/ardp.201800278
Revised: 29 November 2018
Accepted: 5 December 2018
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FULL PAPER
Bi-heterocyclic benzamides as alkaline phosphatase inhibitors:
Mechanistic comprehensions through kinetics and
computational approaches
Muhammad A. Abbasi1,2
Majid Nazir2
Aziz ur-Rehman2
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Sabahat Z. Siddiqui2
Muhammad Shahid4
Mubashir Hassan1
Sung-Yum Seo1
Hussain Raza1
Syed A. A. Shah3
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1 Department of Biological Sciences, College
of Natural Sciences, Kongju National
University, Gongju, South Korea
Abstract
Novel bi-heterocyclic benzamides were synthesized by sequentially converting 4-(1H-
indol-3-yl)butanoic acid (1) into ethyl 4-(1H-indol-3-yl)butanoate (2), 4-(1H-indol-3-
yl)butanohydrazide (3), and a nucleophilic 5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazole-
2-thiol (4). In a parallel series of reactions, various electrophiles were synthesized by
reacting substituted anilines (5a–k) with 4-(chloromethyl)benzoylchloride (6) to afford
4-(chloromethyl)-N-(substituted-phenyl)benzamides (7a–k). Finally, the nucleophilic
substitution reaction of 4 was carried out with newly synthesized electrophiles, 7a–k,
to acquire the targeted bi-heterocyclic benzamides, 8a–k. The structural confirmation
of all the synthesized compounds was done by IR, 1H NMR, 13C NMR, EI-MS, and CHN
analysis data. The inhibitory effects of these bi-heterocyclic benzamides (8a–k) were
evaluated against alkaline phosphatase, and all these molecules were identified as
potent inhibitors relative to the standard used. The kinetics mechanism was ascribed by
evaluating the Lineweaver–Burk plots, which revealed that compound 8b inhibited
alkaline phosphatase non-competitively to form an enzyme–inhibitor complex. The
inhibition constant Ki calculated from Dixon plots for this compound was 1.15 μM. The
computational study was in full agreement with the experimental records and these
ligands exhibited good binding energy values. These molecules also exhibited mild
cytotoxicity toward red blood cell membranes when analyzed through hemolysis. So,
these molecules might be deliberated as nontoxic medicinal scaffolds to render normal
calcification of bones and teeth.
2 Department of Chemistry, Government
College University, Lahore, Pakistan
3 Faculty of Pharmacy and Atta-ur-Rahman
Institute for Natural Products Discovery
(AuRIns), Level 9, FF3, Universiti Teknologi
MARA, Puncak Alam Campus, Selangor Darul
Ehsan, Malaysia
4 Department of Biochemistry, University of
Agriculture, Faisalabad, Pakistan
Correspondence
Dr. Muhammad A. Abbasi, Department of
Chemistry, Government College University,
Lahore-54000, Pakistan.
Email: abbasi@gcu.edu.pk
Prof. Dr. Sung-Yum Seo, Department of
Biological Sciences, College of Natural
Sciences, Kongju National University, Gongju
32588, South Korea.
Email: dnalove@kongju.ac.kr
Funding information
National Research Foundation of Korea,
Grant number: 2017R1D1A1B03034948
K E Y W O R D S
benzamides, bi-heterocyclic, kinetics, molecular docking, phosphatase
pyrrole ring.[1] Indoles are found in various natural products and have
been identified as products of chemical and biological importance.
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1
INTRODUCTION
Indole is composed of a benzo-pyrrole in which the benzene and
pyrrole rings are fused together through the 2- and 3-positions of the
Indole is a highly conserved heterocyclic molecule that acts as a free
radical scavenger and has a broad spectrum of antioxidant activity.
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Arch Pharm Chem Life Sci. 2019;e1800278.
wileyonlinelibrary.com/journal/ardp
© 2019 Deutsche Pharmazeutische Gesellschaft
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Indole core is also an important component in many of recent drugs for
treatment of chemotherapy-induced nausea and vomiting, cluster
headache, or as antihypertensive, antineoplastic, and antimitotic
agents.[2–4]
2
RESULTS AND DISCUSSION
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2.1 Chemistry
1,3,4-Oxadiazoles derivatives have played a significant role in
medicinal chemistry. Considerable attention has been focused on 2,5-
disubstituted-1,3,4-oxadiazole containing compounds due to their
remarkable biological activities. Such compounds have been recog-
nized as antibacterial, anticancer, anti-Parkinson, anti-HIV, and anti-
proliferative agents.[5–8]
The convergent synthesis of targeted N-substituted derivatives was
accomplished in very good yields through several steps. First, the
4-(1H-indol-3-yl)butanoic acid (1) was subjected to esterification by
ethanol in the presence of concentrated sulfuric acid taken in catalytic
amount. Ethanol was used as reactant and also as a solvent in order to
push the equilibrium toward product side, as it was a reversible
reaction. The product was collected by solvent extraction after the
addition of a weak base and excess of water. The base was added to
neutralize the catalytic sulfuric acid and the unreacted carboxylic acid.
The salts of these acids were partitioned to the aqueous layer while the
resulted ester to the organic phase during solvent extraction. Thus,
ethyl 4-(1H-indol-3-yl)butanoate (2) was obtained as brownish liquid
(solid at refrigeration).[19,20] The second step was performed to convert
this ethyl ester to respective carbohydrazide, 3, by the nucleophillic
hydrazine in the presence of an organic solvent like methanol and
refluxed for 14 h. Thus, 4-(1H-indol-3-yl)butanohydrazide (3) was
obtained as a light brown-colored solid.[14,15] The third step was a
cyclization to form heterocyclic ring through reaction with CS2 in a
basic alcoholic medium. The resulting product was 5-[3-(1H-indol-3-yl)-
propyl]-1,3,4-oxadiazole-2-thiol (4) having a mercapto group at its
Benzamides have been reported as muscle relaxants and
activators of potassium channel. These are also exploited as analgesic,
anti-inflammatory, and antihelmintic agents.[9,10] Some heterocyclic
benzamides have shown activity in central nervous system while
others act as anti-psychotics, anti-emetics, and gastric motility
stimulants.[11–13]
Alkaline phosphatase (ALP; E.C.3.I.3.1.) is a ubiquitous mem-
brane-bound glycoprotein that catalyzes the hydrolysis of phosphate
monoesters at basic pH values. Alkaline phosphatase is divided into
four isozymes depending upon the site of tissue expression that are
intestinal ALP, placental ALP, germ cell ALP, and tissue-nonspecific
alkaline phosphatase or liver/bone/kidney (L/B/K) ALP. Mammalian
alkaline phosphatases (ALPs) are zinc containing metalloenzymes
encoded by a multigene family and function as dimeric molecules.
Three metal ions including two Zn+2 and one Mg+2 in the active site
are essential for enzymatic activity. However, these metal ions also
contribute substantially to the conformation of the ALP monomer
and indirectly regulate subunit–subunit interactions. Although ALPs
are present in many mammalian tissues and have been studied for
the last several years, still little is known about them.[14] High
concentrations of tissue-nonspecific alkaline phosphatase (TNAP)
have been found in mineralizing tissues such as bones and teeth,
where role of TNAP is essential for normal bone formation.
Overexpression of TNAP has been known to cause abnormal
calcification including vascular calcification and leads to increased
deposition of mineral hydroxyapatite causing hydroxyapatite depo-
sition disorder (HADD). A decrease in concentration of extracellular
ATP via TNAP inhibition has been recognized to be essential for
second carbon.[14,15] In
a parallel phase of reactions, different
electrophiles were synthesized by reacting substituted anilines
(5a–k) with 4-(chloromethyl)benzoyl chloride (6) to afford 4-(chlor-
omethyl)-N-(substituted-phenyl)benzamides (7a–k).[21] Then in the
last step, acidic proton of 4 was replaced with different electrophiles,
7a–k, in the presence of LiH base using aprotic polar medium to get
required bi-heterocyclic benzamide derivatives, 8a–k, as sketched in
Scheme 1 and different groups are listed in Table 1.
The structural analysis of one of the compounds is discussed
hereby in detail for the benefit of the reader. The molecule 8f was
obtained as yellow-colored amorphous powder. Its molecular formula,
C28H26N4SO2, was established through the molecular ion peak at m/z
482 in its EI-MS spectrum and by CHN analysis data. The count of the
number of protons in its 1H NMR spectrum and number of carbon
resonances in its 13C NMR spectrum was also in agreement with its
deduced molecular formula. Different functionalities in this molecule
were depicted by absorption bands in its IR spectrum at ν 3317 (N-H
neuronal development and synaptic function.
A number of
Alzheimer's disease patients have indicated considerable elevated
TNAP activity in the hippocampus section of brain. TNAP can

therefore be explored as
neurological disorders.[15–18]
a potential target for treatment of
str.), 2955 (C-H aromatic str.), 1654 (CO str.), 1599 (C C aromatic
str.), 1535, 1484, 1449 (str. for oxadiazole), 1155 (C-O-C str.), 1103
(CN str.), 681 (C-S str.) cm−1. With the help of 1H NMR spectrum of this
molecule, the indole heterocyclic core was identified clearly by the
characteristic signals at δ 10.82 (s, 1H, NH-1), 7.51 (br.d, J = 7.6 Hz, 1H,
H-7), 7.35 (br.d, J = 8.0 Hz, 1H, H-4), 7.16–7.14 (m, 3H, H-2, H-2″″ &
H-6″″), 7.06 (br.t, J = 7.2 Hz, 1H, H-6) and 6.97 (br.t, J = 7.2 Hz, 1H, H-
5) ppm. Similarly, the resonance at δ 7.65 (br.d, J = 7.9 Hz, 2H, H-3″″ &
H-5″″), a merged signal at δ 7.16–7.14 (m, 3H, H-2, H-2″″ & H-6″″),
along with one methyl signal at δ 2.09 (s, 3H, CH3-4″″) ppm, were
altogether typical for a 4-methylphenyl group in the molecule. The C
Our group has already reported lead compounds based on
indole and oxadiazole scaffold as enzyme inhibitors,[19,20] and the
current study was expanded to explore new therapeutic
potentials of such hybrid molecules as alkaline phosphatase
inhibitors. In silico studies were also carried out to ascertain
different types of interactions with active pocket of enzyme.
Moreover, their cytotoxicity was also assessed to find their
utility as harmless drug candidates in drug discovery and
development.

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SCHEME 1 Outline for the synthesis of 4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazol-2-yl}sulfanyl)methyl]-N-(substituted-phenyl)-
benzamides (8a–k). Reagents and conditions: (A) EtOH/H2SO4/refluxing for 8 h. (B) MeOH/N2H4 · H2O/refluxing for 14 h. (C) EtOH/CS2/
KOH/refluxing for 16 h. (D) Aq. Na2CO3 soln./pH 9–10/stirring at RT for 20–30 min. (E) DMF/LiH/stirring for 40–50 h
and N-substituted 4-yl-methylbenzamido group was identified by
four signals at δ 10.16 (s, 1H, CONH), 7.91 (br.d, J = 7.9 Hz, 2H, H-2‴
& H-6‴), 7.57 (br.d, J = 7.8 Hz, 2H, H-3‴ & H-5‴) and 4.54 (s, 2H,
CH2-7‴). In the up-field region of spectrum, the signals of three
intervening methylene groups at δ 2.86 (t, J = 7.2 Hz, 2H, CH2-1′), 2.77
(t, J = 7.1 Hz, 2H, CH2-3′), and 2.05 (quint., J = 7.2 Hz, 2H, CH2-2′),
were helpful to ascertain the connectivity of indole moiety from its
3-position to the 5-position of oxadiazole scaffold. The 1H NMR
spectrum of this compound and its expanded zones are shown in
Figure 1(a–c).
The carbon skeleton of this molecule was also fully supported by
its 13C NMR spectrum, shown in Figure 2. The 13C NMR spectrum due
to some symmetrical duplets depicted overall 24 carbon resonances,
where the most downfield quaternary carbons signals at δ 168.09
(C-5″) and 162.29 (C-2″) belonged to the 1,3,4-oxadiazole ring, thus
confirming its cyclization. The other three quaternary carbons
appearing at δ 136.29 (C-8), 127.00 (C-9), and 113.22 (C-3) ppm
were attributes of the indole core. The methine carbon resonances
appearing at δ 122.49 (C-2), 120.86 (C-6), 118.18 (C-7), 118.16 (C-5),
111.35 (C-4) were also coherent with the indole moiety.[19] The dually
substituted 4-yl-methylbenzamido group was confirmed by its discrete
resonances, one for a carbonyl group at δ 164.90 (C-8‴), other four
signals at δ 140.19 (C-4‴), 134.35 (C-1‴), 128.93 (C-3‴ & C-5‴),
127.80 (C-2‴ & C-6‴), and one signal of methylene group at δ 35.34
(C-7‴).[20] The 4-methylphenyl group attached with a nitrogen atom
was thoroughly corroborated by two quaternary signals at δ 136.56
(C-1″″) and 132.57 (C-4″″), two methine signals at δ 128.88 (C-2″″ &
C-6″″) and 120.31 (C-3″″ & C-5″″), along with one methyl signal at δ
20.46 (CH3-4″″). In the up-field region of the spectrum, three signals at
δ 26.48 (C-2′), 24.26 (C-1′), and 23.81 (C-3′) were characteristic for
three connected methylenes, which were joining the indole moiety
with heterocyclic oxadiazole core in the molecule.[19] The salient
connectivities in the carbon skeleton of this molecule were thoroughly
TABLE 1 Different groups (-R1 and -R2) in Scheme 1
Compd.
-R1
-R2
-H
-H
-H
-H
-H
-H
Compd.
-R1
-R2
5a, 7a, 8a
5b, 7b, 8b
5c, 7c, 8c
5d, 7d, 8d
5e, 7e, 8e
5f, 7f, 8f
3-OC2H5
4-OC2H5
2-C2H5
2-CH3
3-CH3
4-CH3
5g, 7g, 8g
5h, 7h, 8h
5i, 7i, 8i
2-CH3
2-CH3
2-CH3
3-CH3
3-CH3
3-CH3
4-CH3
6-CH3
4-CH3
5-CH3
5j, 7j, 8j
5k, 7k, 8k

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FIGURE 2 13C NMR spectrum of 8f molecule
their results are presented in Table 2. All these compounds exhibited
very potent inhibitory activities against this enzyme, as evident from
their lower IC50 (μM) values, relative to standard, KH2PO4, having IC50
value of 5.2421 0.4722 μM. Though the exposed activity is
accumulative of a whole molecule, yet a limited SAR was predictable
by discerning the effect of different aryl entity on the inhibitory
potential, as it was the only varying part and all other parts were similar
in all molecules. The general structural parts of the studied benzamides
are accentuated in Figure 4.
The compounds 8a and 8b both contained an ethoxy group in the
aryl part and exhibited very superb and comparable inhibitory
activities, IC50 = 0.0676 0.0057 and IC50 = 0.0427 0.0167 μM,
respectively. Moreover, 8b bearing 4-ethoxy group was recognized
as the most active compound in the synthetic series. It means, the
presence of a medium-sized polar group in aryl part generally rendered
good inhibitory potentials to these molecules (Figure 5).
Among the following mono-substituted compounds, 8c–f, a
random trend was observed. The molecule 8c (IC50
=
0.3806 0.0103 μM) with a relatively bulky ethyl group at ortho
position was more active than 8d (IC50 = 2.7219 0.1311 μM) having
FIGURE 1 (a) 1H NMR spectrum of 8f. (b) Expanded aromatic
region of 1H NMR spectrum of 8f. (c) Expanded aliphatic region of
1H NMR spectrum of 8f
substantiated by its HMBC spectrum. This spectrum, along with
significant correlations, is shown in Figure 3. So, on the basis of
aforesaid collective evidences, the structure of 8f was confirmed and it
was named as 4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazol-2-yl}-
sulfanyl)methyl]-N-(4-methylphenyl)benzamide.
A similar protocol
was exercised for the structural characterization of other derivatives
in the synthesized series.
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2.2 Alkaline phosphatase inhibition and
structure–activity relationship (SAR)
The synthesized bi-heterocyclic benzamides (8a–k) were evaluated for
FIGURE 3 HMBC spectrum and significant correlations of 8f
their inhibitory potentials against alkaline phosphatase enzyme and
molecule

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TABLE 2 Alkaline phosphatase inhibitory and hemolytic activity of bi-heterocyclic benzamides (8a–k)
Compounds
8a
Aryl part
Alkaline phosphatase activity IC50 SEM (μM)
Hemolysis (%) (mean SEM)
0.0676 0.0057
10.36 0.03
8b
8c
0.0427 0.0167
0.3806 0.0103
3.20 0.01
6.85 0.02
8d
8e
2.7219 0.1311
0.9666 0.0427
7.23 0.03
5.54 0.03
8f
2.8069 0.6172
0.0520 0.0011
8.61 0.01
1.13 0.01
8g
8h
8i
0.4785 0.0497
0.3205 0.0431
11.90 0.04
8.02 0.02
8j
0.2063 0.0111
0.0456 0.0048
3.41 0.02
2.45 0.03
8k
KH2PO4
Triton X
5.2421 0.4722
–
–
95.32 0.01
SEM, standard error of the mean; values are expressed in mean SEM. PBS hemolysis = 2.45 0.01%.
a smaller methyl group at the same position. Interestingly, 8f
(IC50 = 2.8069 0.6172 μM) having the same methyl group but at
para position showed very resembling activity to that of 8d. However,
8e (IC50 = 0.9666 0.0427 μM) bearing a meta-methyl group behaved
as a better inhibitor relative to other two methylated molecules, 8d and
8f. It indicated that an electron donating medium-sized group either at
ortho position or a small group at meta position could be considered as
creditable options for the better inhibitory potential (Figure 6).

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FIGURE 4 General structural features of compounds 8a–k
FIGURE 5 Structure–activity relationship of compounds 8a and 8b
When the inhibitory potential of following di-methylated regio-
isomers was compared, it was observed that the compound 8g having
methyl groups at ortho and meta positions possessed superior
inhibitory activity (IC50 = 0.0520 0.0011 μM) as compared to 8h
(IC50 = 0.4785 0.0497 μM) and 8i (IC50 = 0.3205 0.0431 μM). In 8h,
these methyl groups were attached at ortho and para positions while in
8i a di-ortho substitution was present. It indicated that the molecule
bearing ortho and meta methyl groups had better interactions with the
enzyme relative to other analogues (Figure 7).
meta or para position in aryl part, have superb activity. In addition, the
presence of at least one small-sized methyl group at meta position is
another creditable option for the promising inhibitory potential of
these molecules.
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2.3 Kinetic mechanism
Presently, the most potent compound 8b was studied for the mode of
inhibition against alkaline phosphatase. The potential of the compound
to inhibit the free enzyme and enzyme–substrate complex was
determined in terms of EI and ESI constants, respectively, at 37°C. The
kinetic studies of the enzyme by the Lineweaver–Burk plot of 1/V
versus substrate para-nitrophenyl phosphate disodium salt 1/[S] in the
presence of different inhibitor concentrations gave a series of straight
lines as shown in Figure 9a. The results of compound 8b showed that
compound intersected within the second quadrant. The analysis
showed that Vmax decreased to new increasing doses of inhibitors on
the other hand Km remained the same. This behavior indicated that 8b
The comparison of 8j, having meta and para methyl groups in aryl
part, with 8k, bearing di-meta methyl groups, indicated that the latter
molecule exhibited enhanced inhibitory potential (IC50
=
0.0456 0.0048 μM) relative to former (IC50 = 0.2063 0.0111 μM).
Here, coherently again the molecule with meta substitutions possessed
commendable inhibitory activity and was recognized as the second
most active compound in the synthetic series (Figure 8).
So, it was inferred from the SAR that among such hybrid bi-
heterocycles, the molecules possessing medium-sized polar group at
FIGURE 6 Structure–activity relationships of 8c–f

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FIGURE 7 Structure–activity relationships of 8g–i
inhibited the alkaline phosphatase non-competitively to form an
enzyme inhibitor complex. Secondary plot of slope against the
concentration of inhibitors (Figure 9b) showed enzyme-inhibitor
dissociation constant (Ki). The kinetic results are presented in Table 3.
other designed ligands. Anyhow, among all docking energy values, no
big differences were observed because all compounds have similar
chemical skeletons except variation in aryl part. The docking energy
values are represented in Figure 10.
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2.4 Hemolytic activity
2.5.1 Binding pocket and hydrogen binding analysis
All the synthesized compounds, 8a–k, were also subjected to
hemolytic assay to find out their cytotoxicity profile. Results of
percentage hemolysis (%) are shown in Table 2. Our results showed
that all compounds of this series possessed moderate toxicity toward
red blood cell membrane. Maximum membrane toxicity was shown by
the compound 8h (11.90 0.04%) while minimum toxicity was
recorded in 8g (1.13 0.01%). Precisely, a low toxicity was observed
for molecule 8k (2.45 0.03%), 8b (3.20 0.01%), and 8j
(3.41 0.02%) relative to Triton-X having % hemolysis of 89.11 0.01.
Docking analysis showed that all compounds were confined in the
active binding region of receptor molecule with different conforma-
tional poses. However, the binding of ligands showed their good
interactive behavior within the active region of target protein. The
binding pocket in surface format and all ligands attachment within
active region are depicted in Figure 11(A and B, respectively).
Based on in vitro analysis, 8b was selected to observe the detail
binding interactive behavior within the active region of target protein.
In alkaline phosphatase docking results, single hydrogen bond was
observed in 8b–receptor docking complex. The oxygen molecule of
benzamide interacted with Arg150 with binding distance 1.91 Å. The
detail structural analysis reports showed that Arg150 is a part of core
region of alkaline phosphatase.[26] The 3D and 2D graphical
representation of docking complexes are mentioned in Figures 12
and 13, respectively. Our docking results showed a good correlation
with already published data.[27] All the docking-complexes graphical
2D representations are mentioned in supplementary data (Supporting
Information Figures S1–S10).
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2.5 Docking energy and binding interaction pattern
of synthesized compounds
Molecular docking experiment is best approach to study the binding
conformation of ligands within the active region of target proteins.[22–
25] To predict the best-fitted conformational position, the synthesized
compounds, 8a–k, were docked against alkaline phosphatase. The
generated docked-complexes were examined on the basis of Glide
docking score (kcal/mol) and bonding interaction (hydrogen/hydro-
phobic) pattern. The binding energy values depicted the conforma-
tional positions of ligands within the active region of target protein.
Results showed that 8c, 8d, and 8f possessed low and good energy
values (−4.03, −4.30, and −4.17 kcal/mol), respectively, compared to
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3
CONCLUSION
In conclusion, the newly synthesized bi-heterocyclic benzamides
behaved as promising inhibitors for the calf intestinal alkaline
FIGURE 8 Structure–activity relationship of 8j and 8k

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FIGURE 9 Lineweaver–Burk plots for inhibition of alkaline phosphatase in the presence of compound 8b. (a) Concentrations of 8b were
0.00, 0.0427, and 0.1708 μM. (b) The inset represents the plot of the slope versus inhibitor 8b concentrations to determine inhibition
constant. The lines were drawn using linear least squares fit
phosphatase. The structural corroboration of compounds 8a–k was
performed through spectroscopic and elemental analysis. Among
these derivatives, the molecule 8b, bearing 4-ethoxy group in aryl part,
was identified as the most potent derivative in the series with IC50
value of 0.0427 0.0167 μM. Lineweaver–Burk plot showed that 8b
inhibited alkaline phosphatase non-competitively and possessed Ki
value of 1.15 μM. The binding profile of 8b also showed its good
interactive behavior within the active region of target protein. So, it
was inferred from the current study that 8b might find its utility as an
impressive therapeutic agent for alkaline phosphatase-associated
disorders, particularly, normal calcification of bones and teeth.
mobile phase), initial purity of compounds was detected at 254 nm.
Elemental analyses were performed on a Foss Heraeus CHN-O-Rapid
instrument and were within 0.4% of the theoretical values. IR peaks
were recorded on a Jasco-320-A spectrometer by using KBr pellet
method. EI-MS spectra were measured on
a JEOL JMS-600H
instrument with data processing system. 1H NMR spectra (δ, ppm)
were recorded at 600 MHz (13C NMR spectra, at 150 MHz) in DMSO-
d6 using the Bruker Advance III 600 As-cend spectrometer using BBO
probe. The abbreviations used in interpretation of 1H NMR spectra are
as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; br.t,
broad triplet; q, quartet; quint., quintet; m, multiplet; dist., distorted.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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4
EXPERIMENTAL
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4.1 Chemistry
4.1.2 Synthesis of ethyl 4-(1H-indol-3-yl)butanoate
(2)
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4.1.1 General
4-(1H-Indol-3-yl)butanoic acid (0.2 mol; 1) dissolved in absolute
ethanol (70 mL) and catalytic amount of concentrated sulfuric acid
(20 mL) was taken in a 500 mL round-bottomed (RB) flask and refluxed
for 8 h until the maximum completion of reaction, supervised through
TLC. At the end, reaction mixture was neutralized with 10% aqueous
sodium carbonate (40 mL). The product was isolated by solvent
Chemicals were purchased from Sigma–Aldrich
& Alfa Aesar
(Germany) and solvents of analytical grades were supplied by local
suppliers. By using open capillary tube method, melting points were
taken on Griffin and George apparatus and were uncorrected. By using
thin layer chromatography (with ethyl acetate and n-hexane (30:70) as
TABLE 3 Kinetic parameters of the alkaline phosphatase for para-nitrophenylphosphate disodium salt activity in the presence of different
concentrations of 8b
Conc. (μM)
0.00
Vmax (ΔA/min)
0.007917273
0.00532
Km (mM)
0.25
Inhibition type
Ki (μM)
Non-competitive
1.15
0.0427
0.1708
0.25
0.004646364
0.25
Vmax, the reaction velocity; Km, Michaelis-Menten constant; Ki, EI dissociation constant.

ABBASI ET AL.
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FIGURE 10 Docking energy graph
FIGURE 12 The 3D interactions depiction of 8b against alkaline
phosphatase
extraction using chloroform (50 mL × 3). The solvent was distilled off
and the required ester, 2, was obtained as reddish brown liquid.[19,20]
(0.26 mol) was added subsequently. Mixture was refluxed for 16 h. On
completion of the reaction, excess chilled distilled water and dil.
HCl was added to adjust the pH at 5–6. The precipitates were
filtered, washed, and dried to get desired cyclized bi-heterocyclic
product, 4.[19,20]
|
4.1.3 Synthesis of 4-(1H-indol-3-yl)butanohydrazide
(3)
4-(1H-Indol-3-yl)butanoate (0.15 mol; 2) in 60 mL methanol and
hydrazine monohydrate (80%; 25 mL) was taken in a 500 mL round-
bottomed flask. The reaction mixture was refluxed for 14 h at room
temperature (RT). After absolute conversion, the acid hydrazide was
obtained by distilling methanol off from the reaction mixture. The
precipitates were filtered, washed with cold n-hexane, and air-dried to
get pure 4-(1H-indol-3-yl)butanohydrazide (3).[19,20]
|
4.1.5 Preparation of 4-(chloromethyl)-N-
(substituted-phenyl)benzamides (7a–k)
Preparation of various 4-(chloromethyl)-N-(substituted-phenyl)benza-
mides (7a–k) was carried out by reaction of various substituted anilines
(5a–k) with 4-(chloromethyl)benzoyl chloride (6) in equimolar quanti-
ties (0.001 moles) and shaking manually in 10% aqueous Na2CO3
solution. Solid precipitates were formed after 20–30 min, which were
filtered and washed with cold-distilled water to obtain the desired
electrophiles, 7a–k.[21]
|
4.1.4 Synthesis of 5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-ylhydrosulfide (4)
4-(1H-Indol-3-yl)butanohydrazide (0.13 mol; 3) in absolute ethanol
(30 mL) and KOH (0.13 mol) was taken in a RB flask. Carbon disulfide
FIGURE 11 (A and B) The binding conformational poses of bi-heterocyclic benzamides within active region of alkaline phosphatase in
surface and ribbon format, respectively.

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(CH3CH2O-3″″), 35.32 (C-7‴), 26.47 (C-2′), 24.25 (C-1′), 23.80 (C-3′),
14.63 (CH3CH2O-3″″). Anal. calcd. for C29H28N4SO3 (512.18): C,
67.95; H, 5.51; N, 10.93. Found: C, 67.90; H, 5.45; N, 10.89. EI-MS
(m/z): 512 (C29H28N4SO3)·+ (M)+, 369 (C19H19N3SO3)+, 287
(C16H16NO2S)+, 258 (C13H12N3SO)·+, 186 (C12H12NO)+, 184
(C12H12N2)·+, 151 (C8H9NO2)+, 143 (C10H9N)+, 130 (C9H8N)+, 120
(C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
N-(4-Ethoxyphenyl)-4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-yl}sulfanyl)methyl]benzamide (8b)
Light brown-colored sticky liquid; yield: 78%; mol. formula:
C
29H28N4SO3; mol. weight: 512 g/mol; IR (KBr, υ, cm−1): 3293 (N-H


str.), 2966 (C-H aromatic str.), 1642 (C O str.), 1598 (C C aromatic
str.), 1529, 1488, 1447 (str. for oxadiazole), 1153 (C-O-C str.), 1126
FIGURE 13 The 2D interactions depiction of 8b against alkaline
phosphatase
(C N str.), 687 (C-S str.); 1H NMR (600 MHz, DMSO-d6, δ, ppm):

10.81 (s, 1H, NH-1), 9.92 (s, 1H, CONH), 7.81 (br.d, J = 7.8 Hz, 2H,
H-2‴ & H-6‴), 7.57 (br.d, J = 7.8 Hz, 2H, H-3‴ & H-5‴), 7.51 (br.s, 1H,
H-7), 7.41 (br.d, J = 7.8 Hz, 2H, H-2″″ & H-6″″), 7.35 (m, 1H, H-4), 7.14
(br.s, 1H, H-2), 7.07 (br.t, J = 7.8 Hz, 1H, H-6), 6.97 (br.t, J = 7.8 Hz, 1H,
H-5), 6.84 (br.s, 2H, H-3″″ & H-5″″), 4.55 (s, 2H, CH2-7‴), 3.86 (s, 2H,
CH3CH2O-4″″), 2.87 (t, J = 7.2 Hz, 2H, CH2-1′), 2.75 (t, J = 7.1 Hz, 2H,
CH2-3′), 2.06–2.04 (m, 2H, CH2-2′), 1.16–1.10 (m, 3H, CH3CH2O-
4″″). 13C NMR (150 MHz, DMSO-d6, δ, ppm): 168.10 (C-5″), 165.13
(C-8‴), 162.29 (C-2″), 140.29 (C-4‴), 139.81 (C-4″″), 136.28 (C-8),
133.53 (C-1‴), 130.69 (C-1″″), 128.52 (C-3‴ & C-5‴), 128.09 (C-2″″
& C-6″″), 127.76 (C-2‴ & C-6‴), 126.99 (C-9), 126.58 (C-3″″ & C-5″
″), 122.50 (C-2), 120.86 (C-6), 118.17 (C-5 & C-7), 113.21 (C-3),
111.34 (C-4), 63.06 (CH3CH2O-4″″), 35.32 (C-7‴), 26.47 (C-2′), 24.25
(C-1′), 23.79 (C-3′), 14.10 (CH3CH2O-4″″); Anal. calcd. for
|
4.1.6 Synthesis of 3-[({5-[3-(1H-indol-3-yl)propyl]-
1,3,4-oxadiazol-2-yl}sulfanyl)methyl]-N-(substituted-
phenyl)benzamides (8a–k)
Synthesis of 5-[3-(1H-indol-3-yl)propyl]-1,3,4-oxadiazol-2-ylhydro-
sulfide (0.2 g, 4) was added in DMF (5 mL) contained in a 250 mL
round bottom flask at room temperature, added one pinch of LiH and
stirred for 30 min. 4-(Chloromethyl)-N-(substituted-phenyl)benzamides
(7a–k) were added in equimolar amounts separately in each respective
reaction and stirred for 40–50 h. The completion of the reaction
was monitored by TLC and after its completion, the reaction mixture
was quenched with ice cold water (100 mL). The respective derivatives,
(8a–k), were collected through filtration or solvent extraction according
to nature of the product.
C29H28N4SO3 (512.18): C, 67.95; H, 5.51; N, 10.93. Found: C,
67.90; H, 5.45; N, 10.89. EI-MS (m/z): 512 (C29H28N4SO3)·+ (M)+, 369
(C19H19N3SO3)+, 287 (C16H16NO2S)+, 258 (C13H12N3SO)·+, 186
(C12H12NO)+, 184 (C12H12N2)·+, 151 (C8H9NO2)+, 143 (C10H9N)+,
130 (C9H8N)+, 120 (C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
N-(3-Ethoxyphenyl)-4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-yl}sulfanyl)methyl]benzamide (8a)
Yellow-colored amorphous powder; yield: 73%; mp: 129°C; mol.
formula: C29H28N4SO3; mol. weight: 512 g/mol; IR (KBr, υ, cm−1): 3290
N-(2-Ethylphenyl)-4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-yl}sulfanyl)methyl]benzamide (8c)
Brown-colored amorphous powder; yield: 55%; mp: 140°C; mol.
formula: C29H28N4SO2; mol. weight: 496 g/mol; IR (KBr, υ, cm−1): 3286


(N-H str.), 2966 (C-H aromatic str.), 1644 (C O str.), 1596 (CC
aromatic str.), 1525, 1488, 1445 (str. for oxadiazole), 1152 (C-O-C str.),
1

1125 (C N str.), 688 (C-S str.); H NMR (600 MHz, DMSO-d6, δ, ppm):
10.82 (s, 1H, NH-1), 10.18 (s, 1H, CONH), 7.89 (br.d, J = 7.5 Hz, 2H, H-
2‴ & H-6‴), 7.57 (br.d, J = 7.7 Hz, 2H, H-3‴ & H-5‴), 7.51 (br.d,
J = 7.8 Hz, 1H, H-7), 7.46 (br.d, J = 6.7 Hz, 1H, H-2″″), 7.37 (dist.d,
J = 7.5 Hz, 2H, H-4 & H-6″″), 7.23 (br.t, J = 7.9 Hz, 1H, H-5″″), 7.14 (s,
1H, H-2), 7.06 (br.t, J = 7.1 Hz, 1H, H-6), 6.97 (br.t, J = 7.1 Hz, 1H, H-5),
6.67 (br.d, J = 7.5 Hz, 1H, H-4″″), 4.55 (s, 2H, CH2-7‴), 4.02 (q,
J = 6.7 Hz, 2H, CH3CH2O-3″″), 2.88 (t, J = 7.1 Hz, 2H, CH2-1′), 2.77 (t,
J = 6.9 Hz, 2H, CH2-3′), 2.04 (quint., J = 7.1 Hz, 2H, CH2-2′), 1.34 (t,
J = 6.7 Hz, 3H, CH3CH2O-3″″). 13C NMR (150 MHz, DMSO-d6, δ,
ppm): 168.10 (C-5″), 165.14 (C-8‴), 162.28 (C-2″), 158.63 (C-3″″),
140.34 (C-1″″), 140.25 (C-4‴), 136.28 (C-8), 134.29 (C-1‴), 129.31
(C-5″″), 128.91 (C-3‴ & C-5‴), 127.84 (C-2‴ & C-6‴), 127.00 (C-9),
122.49 (C-2), 120.86 (C-6), 118.17 (C-7), 118.16 (C-5), 113.21 (C-3),
112.36 (C-6″″), 111.21 (C-4), 109.69 (C-4″″), 106.41 (C-2″″), 62.89
(N-H str.), 2957 (C-H aromatic str.), 1656 (CO str.), 1599 (C
C
aromatic str.), 1527, 1481, 1441 (str. for oxadiazole), 1141 (C-O-C str.),
1101 (CN str.), 677 (C-S str.); 1H NMR (600 MHz, DMSO-d6, δ, ppm):
10.82 (s, 1H, NH-1), 9.87 (s, 1H, CONH), 7.93 (br.d, J = 7.3 Hz, 2H, H-
2‴ & H-6‴), 7.57 (br.d, J = 7.3 Hz, 2H, H-3‴ & H-5‴), 7.51 (br.d,
J = 7.9 Hz, 1H, H-7), 7.34 (br.d, J = 7.9 Hz, 1H, H-4), 7.29 (br.d,
J = 7.2 Hz, 1H, H-6″″), 7.23 (br.d, J = 7.3 Hz, 1H, H-3″″), 7.18–7.17 (m,
2H, H-4″″ & H-5″″), 7.14 (br.s, 1H, H-2), 7.06 (br.t, J = 6.7 Hz, 1H, H-6),
6.97 (br.t, J = 6.9 Hz, 1H, H-5), 4.55 (s, 2H, CH2-7‴), 2.87 (t, J = 6.8 Hz,
2H, CH2-1′), 2.77 (t, J = 6.4 Hz, 2H, CH2-3′), 2.62 (quartet, J = 6.8 Hz,
2H, CH3CH2-2″″), 2.05 (q, J = 6.8 Hz, 2H, CH2-2′), 1.13 (t, J = 6.8 Hz,
3H, CH3CH2-2″″). 13C NMR (150 MHz, DMSO-d6, δ, ppm): 168.11 (C-
5″), 165.22 (C-8‴), 162.29 (C-2″), 140.24 (C-4‴), 139.77 (C-1″″),
136.28 (C-8), 135.66 (C-1‴), 133.81 (C-2″″), 128.96 (C-3‴ & C-5‴),

ABBASI ET AL.
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128.55 (C-3″″), 127.78 (C-2‴ & C-6‴), 127.55 (C-4″″), 127.00 (C-9),
126.46 (C-5″″), 125.96 (C-6″″), 122.50 (C-2), 120.86 (C-6), 118.18 (C-
7), 118.16 (C-5), 113.21 (C-3), 111.35 (C-4), 35.32 (C-7‴), 26.47 (C-2′),
24.25 (C-1′), 23.79 (C-3′), 23.92 (CH3 CH2-2″″), 14.08 (CH3CH2-2″″).
Anal. calcd. for C29H28N4SO2 (496.19): C, 69.69; H, 5.43; N, 11.61.
Found: C, 69.67; H, 5.45; N, 11.58. EI-MS (m/z): 496 (C29H28N4SO2)·+
(M)+, 339 (C18H17N3SO2)+, 258 (C13H12N3SO)·+, 224 (C15H14NO)+,
186 (C12H12NO)+, 184 (C12H12N2)·+, 143 (C10H9N)+, 130 (C9H8N)+,
120 (C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
137.69 (C-3″″), 136.28 (C-8), 134.30 (C-1‴), 128.90 (C-3‴ & C-5‴),
128.14 (C-5″″), 127.82 (C-2‴ & C-6‴), 126.99 (C-9), 124.32 (C-4″″),
122.49 (C-2), 120.91 (C-2″″), 120.86 (C-6), 118.17 (C-7), 118.16 (C-5),
117.48 (C-6″″), 113.21 (C-3), 111.34 (C-4), 35.33 (C-7‴), 26.47 (C-2′),
24.25 (C-1′), 23.80 (C-3′), 21.17 (CH3-3″″). Anal. calcd. for
C28H26N4SO2 (482.18): C, 69.69; H, 5.43; N, 11.61. Found: C,
69.67; H, 5.45; N, 11.58. EI-MS (m/z): 482 (C28H26N4SO2)·+ (M)+, 339
(C18H17N3SO2)+, 258 (C13H12N3SO)·+, 224 (C15H14NO)+, 186
(C12H12NO)+, 184 (C12H12N2)·+, 143 (C10H9N)+, 130 (C9H8N)+, 120
(C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
4-[({5-[3-(1H-Indol-3-yl)propyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-
methyl]-N-(2-methylphenyl)benzamide (8d)
4-[({5-[3-(1H-Indol-3-yl)propyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-
methyl]-N-(4-methylphenyl)benzamide (8f)
Yellow-colored amorphous powder; yield: 87%; mp: 168°C; mol.
formula: C28H26N4SO2; mol. weight: 482 g/mol; IR (KBr, υ, cm−1): 3317

Light brown-colored amorphous powder; yield: 63%; mp: 211°C; mol.
formula: C28H26N4SO2; mol. weight: 482 g/mol; IR (KBr, υ, cm−1): 3296

(N-H str.), 2958 (C-H aromatic str.), 1652 (C O str.), 1591 (CC
aromatic str.), 1531, 1489, 1444 (str. for oxadiazole), 1155 (C-O-C str.),
1122 (CN str.), 687 (C-S str.); 1H NMR (600 MHz, DMSO-d6, δ, ppm):
10.81 (s, 1H, NH-1), 9.86 (s, 1H, CONH), 7.93 (br.d, J = 7.5 Hz, 2H, H-
2‴ & H-6‴), 7.57 (br.d, J = 7.7 Hz, 2H, H-3‴ & H-5‴), 7.51 (br.d,
J = 7.9 Hz, 1H, H-7), 7.37 (br.d, J = 7.4 Hz, 1H, H-6″″), 7.33 (m, 1H, H-
4), 7.27 (br.d, J = 7.4 Hz, 1H, H-3″″), 7.21 (br.t, J = 7.5 Hz, 1H, H-5″″),
7.17 (br.t, J = 7.5 Hz, 1H, H-4″″), 7.14 (br.s, 1H, H-2), 7.06 (br.t,
J = 7.2 Hz, 1H, H-6), 6.97 (br.t, J = 7.1 Hz, 1H, H-5), 4.55 (s, 2H, CH2-
7‴), 2.87 (t, J = 7.2 Hz, 2H, CH2-1′), 2.76 (t, J = 7.2 Hz, 2H, CH2-3′),
2.14 (s, 3H, CH3-2″″), 2.04 (quint., J = 7.3 Hz, 2H, CH2-2′). 13C NMR
(150 MHz, DMSO-d6, δ, ppm): 168.10 (C-5″), 164.85 (C-8‴), 162.38
(C-2″), 140.31 (C-4‴), 136.27 (C-8), 135.69 (C-1″″), 134.21 (C-1‴),
130.63 (C-2″″), 130.25 (C-3″″), 128.95 (C-3‴ & C-5‴), 127.82 (C-2‴
& C-6‴), 126.98 (C-9), 126.50 (C-6″″), 125.94 (C-4″″ & C-5″″),
122.50 (C-2), 120.86 (C-6), 118.17 (C-5 & C-7), 113.23 (C-3), 111.34
(C-4), 35.32 (C-7‴), 26.47 (C-2′), 24.49 (C-1′), 23.79 (C-3′), 17.84
(CH3-2″″). Anal. calcd. for C28H26N4SO2 (482.18): C, 69.69; H, 5.43; N,
11.61. Found: C, 69.67; H, 5.45; N, 11.58. EI-MS (m/z): 482
(C28H26N4SO2)·+ (M)+, 339 (C18H17N3SO2)+, 258 (C13H12N3SO)·+,
224 (C15H14NO)+, 186 (C12H12NO)+, 184 (C12H12N2)·+, 143
(C10H9N)+, 130 (C9H8N)+, 120 (C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
(N-H str.), 2955 (C-H aromatic str.), 1654 (CO str.), 1599 (C
C
aromatic str.), 1535, 1484, 1449 (str. for oxadiazole), 1155 (C-O-C str.),
1103 (CN str.), 681 (C-S str.); 1H NMR (600 MHz, DMSO-d6, δ, ppm):
10.82 (s, 1H, NH-1), 10.16 (s, 1H, CONH), 7.91 (br.d, J = 7.9 Hz, 2H, H-
2‴ & H-6‴), 7.65 (br.d, J = 7.9 Hz, 2H, H-3″″ & H-5″″), 7.57 (br.d,
J = 7.8 Hz, 2H, H-3‴ & H-5‴), 7.51 (br.d, J = 7.6 Hz, 1H, H-7), 7.35 (br.
d, J = 8.0 Hz, 1H, H-4), 7.16–7.14 (m, 3H, H-2, H-2″″ & H-6″″), 7.06
(br.t, J = 7.2 Hz, 1H, H-6), 6.97 (br.t, J = 7.2 Hz, 1H, H-5), 4.54 (s, 2H,
CH2-7‴), 2.86 (t, J = 7.2 Hz, 2H, CH2-1′), 2.77 (t, J = 7.1 Hz, 2H, CH2-
3′), 2.09 (s, 3H, CH3-4″″), 2.05 (quint., J = 7.2 Hz, 2H, CH2-2′).
13C NMR (150 MHz, DMSO-d6, δ, ppm): 168.09 (C-5″), 164.90 (C-8‴),
162.29 (C-2″), 140.19 (C-4‴), 136.56 (C-1″″), 136.29 (C-8), 134.35 (C-
1‴), 132.57 (C-4″″), 128.93 (C-3‴ & C-5‴), 128.88 (C-2″″ & C-6″″),
127.80 (C-2‴ & C-6‴), 127.00 (C-9), 122.49 (C-2), 120.86 (C-6),
120.31 (C-3″″ & C-5″″), 118.18 (C-7), 118.16 (C-5), 113.22 (C-3),
111.35 (C-4), 35.34 (C-7‴), 26.48 (C-2′), 24.26 (C-1′), 23.81 (C-3′),
20.46 (CH3-4″″). Anal. calcd. for C28H26N4SO2 (482.18): C, 69.69; H,
5.43; N, 11.61. Found: C, 69.67; H, 5.45; N, 11.58. EI-MS (m/z): 482
(C28H26N4SO2)·+ (M)+, 339 (C18H17N3SO2)+, 258 (C13H12N3SO)·+, 224
(C15H14NO)+, 186 (C12H12NO)+, 184 (C12H12N2)·+, 143 (C10H9N)+,
130 (C9H8N)+, 120 (C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
4-[({5-[3-(1H-Indol-3-yl)propyl]-1,3,4-oxadiazol-2-yl}sulfanyl)-
methyl]-N-(3-methylphenyl)benzamide (8e)
N-(2,3-Dimethylphenyl)-4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-yl}sulfanyl)methyl]benzamide (8g)
Camel brown-colored amorphous powder; yield: 61%; mp: 106°C; mol.
formula: C28H26N4SO2; mol. weight: 482 g/mol; IR (KBr, υ, cm−1): 3274
Light yellow-colored amorphous powder; yield: 89%; mp: 138°C; mol.
formula: C29H28N4SO2; mol. weight: 496 g/mol; IR (KBr, cm−1): υ 3286



(N-H str.), 2953 (C-H aromatic str.), 1703 (C O str.), 1590 (CC
(N-H str.), 2957 (C-H aromatic str.), 1656 (C O str.), 1599 (C C
aromatic str.), 1531, 1489, 1452 (str. for oxadiazole), 1159 (C-O-C str.),
aromatic str.), 1527, 1481, 1441 (str. for oxadiazole), 1141 (C-O-C str.),
1
1


1100 (C N str.), 666 (C-S str.); H NMR (600 MHz, DMSO-d6, δ, ppm):
1101 (C N str.), 677 (C-S str.); H NMR (600 MHz, DMSO-d6, δ, ppm):
10.81 (s, 1H, NH-1), 10.15 (s, 1H, CONH), 7.90 (br.d, J = 7.5 Hz, 2H, H-
2‴ & H-6‴), 7.64 (br.s, 1H, H-2″″), 7.58 (dist.d, J = 7.9 Hz, 3H, H-3‴,
H-5‴ & H-6″″), 7.51 (br.d, J = 7.9 Hz, 1H, H-7), 7.34 (br.d, J = 7.8 Hz,
1H, H-4), 7.23 (br.t, J = 7.8 Hz, 1H, H-5″″), 7.14 (s, 1H, H-2), 7.06 (br.t,
J = 7.4 Hz, 1H, H-6), 6.97 (br.t, J = 7.1 Hz, 1H, H-5), 6.93 (br.d,
J = 7.3 Hz, 1H, H-4″″), 4.54 (s, 2H, CH2-7‴), 2.88 (t, J = 7.2 Hz, 2H,
CH2-1′), 2.76 (t, J = 7.20 Hz, 2H, CH2-3′), 2.31 (s, 3H, CH3-3″″), 2.09–
2.03 (m, 2H, CH2-2′). 13C NMR (150 MHz, DMSO-d6, δ, ppm): 168.10
(C-5″), 164.85 (C-8‴), 162.28 (C-2″), 140.27 (C-4‴), 138.98 (C-1″″),
10.82 (s, 1H, NH-1), 9.92 (s, 1H, CONH), 7.94 (br.dd, J = 7.6, 11.4 Hz,
2H, H-2‴ & H-6‴), 7.57 (br.d, J = 7.8 Hz, 2H, H-3‴ & H-5‴), 7.51 (br.d,
J = 7.8 Hz, 1H, H-7), 7.33 (br.d, J = 7.9 Hz, 1H, H-4), 7.14 (br.s, 1H, H-2),
7.11–7.05 (m, 4H, H-6, H-4″″, H-5″″ & H-6″″), 6.97 (br.t, J = 7.4 Hz,
1H, H-5), 4.55 (s, 2H, CH2-7‴), 2.87 (t, J = 7.3 Hz, 2H, CH2-1′), 2.76 (t,
J = 7.2 Hz, 2H, CH2-3′), 2.28 (s, 3H, CH3-3″″), 2.11–2.03 (m, 5H, CH2-
2′ & CH3-2″″). 13C NMR (150 MHz, DMSO-d6, δ, ppm): 168.10 (C-5″),
164.89 (C-8‴), 162.29 (C-2″), 140.24 (C-4‴), 136.92 (C-3″″), 136.28
(C-8), 136.16 (C-1‴), 133.86 (C-1″″), 132.66 (C-2″″), 128.93 (C-3‴ &

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C-5‴), 127.80 (C-2‴ & C-6‴), 127.50 (C-4″″), 127.00 (C-9), 125.19 (C-
5″″), 124.63 (C-6″″), 122.50 (C-2), 120.86 (C-6), 118.18 (C-7), 118.16
(C-5), 113.20 (C-3), 111.35 (C-4), 35.33 (C-7‴), 26.47 (C-2′), 24.25 (C-
1′), 23.80 (C-3′), 20.09 (CH3-3″″), 14.16 (CH3-2″″). Anal. calcd. for
133.52 (C-1″″), 129.00 (C-3‴ & C-5‴), 128.00 (C-2‴, C-3″″, C-5″″ &
C-6‴), 126.75 (C-4″″), 126.95 (C-9), 122.46 (C-2), 120.92 (C-6),
118.23 (C-7), 118.15 (C-5), 113.24 (C-3), 111.38 (C-4), 35.30 (C-7‴),
26.40 (C-2′), 24.25 (C-1′), 23.77 (C-3′), 17.90 (CH3-2″″ & CH3-6″″).
Anal. calcd. for C29H28N4SO2 (496.19): C, 70.14; H, 5.68; N, 11.28.
Found: C, 70.10; H, 5.65; N, 11.22 EI-MS (m/z): 496 (C29H28N4SO2)·+
(M)+, 353 (C19H19N3SO2)+, 258 (C13H12N3SO)·+, 239 (C16H17NO)+,
186 (C12H12NO)+, 184 (C12H12N2)·+, 143 (C10H9N)+, 130 (C9H8N)+,
120 (C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
C29H28N4SO2 (496.19): C, 70.14; H, 5.68; N, 11.28. Found: C, 70.10;
H, 5.65; N, 11.22. EI-MS (m/z): 496 (C29H28N4SO2)·+ (M)+, 353
(C19H19N3SO2)+, 258 (C13H12N3SO)·+, 239 (C16H17NO)+, 186
(C12H12NO)+, 184 (C12H12N2)·+, 143 (C10H9N)+, 130 (C9H8N)+, 120
(C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
N-(2,4-Dimethylphenyl)-4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-yl}sulfanyl)methyl]benzamide (8h)
N-(3,4-Dimethylphenyl)-4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-yl}sulfanyl)methyl]benzamide (8j)
Yellow-colored amorphous powder; yield: 93%; mp: 177°C; mol.
formula: C29H28N4SO2; mol. weight: 496 g/mol; IR (KBr, υ, cm−1): 3283
Yellow-colored amorphous powder; yield: 77%; mp: 104°C; mol.
formula: C29H28N4SO2; mol. weight: 496 g/mol; IR (KBr, υ, cm−1): 3292


C


(N-H str.), 2957 (C-H aromatic str.), 1654 (C O str.), 1598 (C
(N-H str.), 2949 (C-H aromatic str.), 1666 (C O str.), 1599 (C C
aromatic str.), 1526, 1481, 1441 (str. for oxadiazole), 1142 (C-O-C str.),
aromatic str.), 1528, 1487, 1449 (str. for oxadiazole), 1159 (C-O-C str.),
1109 (CN str.), 688 (C-S str.); 1H NMR (600 MHz, DMSO-d6, δ, ppm):
10.81 (s, 1H, NH-1), 10.09 (s, 1H, CONH), 7.95 (br.d, J = 7.4 Hz, 2H, H-
2‴ & H-6‴), 7.55 (br.d, J = 7.4 Hz, 2H, H-3‴ & H-5‴), 7.88 (br.s, 1H, H-
2″″), 7.51 (br.d, J = 7.8 Hz, 1H, H-7), 7.48 (br.d, J = 7.7 Hz, 1H, H-6″″),
7.34 (br.d, J = 8.1 Hz, 1H, H-4), 7.09 (br.d, J = 7.8 Hz, 1H, H-5″″), 7.13
(br.s, 1H, H-2), 7.06 (br.t, J = 7.2 Hz, 1H, H-6), 6.97 (br.t, J = 7.2 Hz, 1H,
H-5), 4.52 (s, 2H, CH2-7‴), 2.88 (t, J = 7.1 Hz, 2H, CH2-1′), 2.76 (t,
J = 7.1 Hz, 2H, CH2-3′), 2.18 (s, 3H, CH3-4″″), 2.08 (s, 3H, CH3-3″″),
2.04 (quint., J = 7.0 Hz, 2H, CH2-2′). 13C NMR (150 MHz, DMSO-d6, δ,
ppm): 168.12 (C-5″), 164.85 (C-8‴), 162.31 (C-2″), 140.16 (C-4‴),
136.70 (C-3″″), 136.27 (C-8), 136.11 (C-1″″), 134.32 (C-1‴), 131.45
(C-4″″), 128.87 (C-3‴ & C-5‴), 127.93 (C-2‴ & C-6‴), 126.98 (C-9),
122.48 (C-2), 121.60 (C-5″″), 120.88 (C-6), 118.18 (C-7), 118.04 (C-5),
117.96 (C-2″″), 117.91 (C-6″″), 113.23 (C-3), 111.35 (C-4), 35.34
(C-7‴), 26.45 (C-2′), 24.22 (C-1′), 23.77 (C-3′), 19.57 (CH3-4″″), 18.76
(CH3-3″″). Anal. calcd. for C29H28N4SO2 (496.19): C, 70.14; H, 5.68; N,
11.28. Found: C, 70.10; H, 5.65; N, 11.22. EI-MS (m/z): 496
(C29H28N4SO2)·+ (M)+, 353 (C19H19N3SO2)+, 258 (C13H12N3SO)·+,
239 (C16H17NO)+, 186 (C12H12NO)+, 184 (C12H12N2)·+, 143
(C10H9N)+, 130 (C9H8N)+, 120 (C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
1

1100 (C N str.), 677 (C-S str.); H NMR (600 MHz, DMSO-d6, δ, ppm):
10.81 (s, 1H, NH-1), 9.81 (s, 1H, CONH), 7.92 (dist.d, J = 5.8 Hz, 2H,
H-2‴ & H-6‴), 7.56 (dist.d, J = 6.3 Hz, 2H, H-3‴ & H-5‴), 7.51 (br.d,
J = 6.7 Hz, 1H, H-7), 7.34 (br.d, J = 7.4 Hz, 1H, H-4), 7.17 (br.d,
J = 6.6 Hz, 1H, H-6″″), 7.13 (br.s, 1H, H-2), 7.07 (br.s, 2H, H-6 & H-
3″″), 7.01 (br.d, J = 6.6 Hz, 1H, H-5″″), 6.97 (m, 1H, H-5), 4.53 (s, 2H,
CH2-7‴), 2.87 (t, J = 7.4 Hz, 2H, CH2-1′), 2.74 (t, J = 7.3 Hz, 2H, CH2-
3′), 2.27 (s, 3H, CH3-4″″), 2.17 (s, 3H, CH3-2″″), 2.05–2.03 (m, 2H,
CH2-2′). 13C NMR (150 MHz, DMSO-d6, δ, ppm): 168.13 (C-5″),
164.90 (C-8‴), 162.32 (C-2″), 140.23 (C-4‴), 136.27 (C-8), 135.12 (C-
1‴), 133.83 (C-1″″), 133.63 (C-2″″), 133.50 (C-4″″), 130.78 (C-3″″),
128.92 (C-3‴ & C-5‴), 127.78 (C-2‴ & C-6‴), 126.98 (C-9), 126.47
(C-5″″ & C-6″″), 122.48 (C-2), 120.88 (C-6), 118.18 (C-7), 118.17 (C-
5), 113.23 (C-3), 111.36 (C-4), 35.33 (C-7‴), 26.45 (C-2′), 24.22 (C-1′),
23.84 (C-3′), 20.48 (CH3-4″″), 17.73 (CH3-2″″); Anal. calcd. for
C29H28N4SO2 (496.19): C, 70.14; H, 5.68; N, 11.28. Found: C, 70.10;
H, 5.65; N, 11.22. EI-MS (m/z): 496 (C29H28N4SO2)·+ (M)+, 353
(C19H19N3SO2)+, 258 (C13H12N3SO)·+, 239 (C16H17NO)+, 186
(C12H12NO)+, 184 (C12H12N2)·+, 143 (C10H9N)+, 130 (C9H8N)+, 120
(C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
N-(2,6-Dimethylphenyl)-4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-yl}sulfanyl)methyl]benzamide (8i)
Light yellow-colored amorphous powder; yield: 91%; mp: 102°C; mol.
formula: C29H28N4SO2; mol. weight: 496 g/mol; IR (KBr, υ, cm−1): 3298

N-(3,5-Dimethylphenyl)-4-[({5-[3-(1H-indol-3-yl)propyl]-1,3,4-
oxadiazol-2-yl}sulfanyl)methyl]benzamide (8k)
Light brown-colored sticky liquid; yield: 77%; mol. formula:
C
29H28N4SO2; mol. weight: 496 g/mol; IR (KBr, υ, cm−1): 3282 (N-H


(N-H str.), 2961 (C-H aromatic str.), 1653 (CO str.), 1581 (C
C
str.), 2945 (C-H aromatic str.), 1664 (C O str.), 1599 (C C aromatic
aromatic str.), 1527, 1483, 1441 (str. for oxadiazole), 1157 (C-O-C str.),
str.), 1535, 1473, 1456 (str. for oxadiazole), 1155 (C-O-C str.), 1116
1
(C N str.), 689 (C-S str.); 1H NMR (600 MHz, DMSO-d6, δ, ppm):


1121 (C N str.), 689 (C-S str.); H NMR (600 MHz, DMSO-d6, δ, ppm):
10.77 (s, 1H, NH-1), 9.80 (s, 1H, CONH), 7.93 (br.d, J = 7.6, Hz, 2H,
H-2‴ & H-6‴), 7.55 (br.d, J = 7.8 Hz, 2H, H-3‴ & H-5‴), 7.49 (br.d,
J = 7.8 Hz, 1H, H-7), 7.34 (br.d, J = 8.0 Hz, 1H, H-4), 7.10 (m, 3H, H-3″″,
H-4″″ & H-5″″), 7.13 (br.s, 1H, H-2), 7.06 (br.t, J = 7.6 Hz, 1H, H-6),
6.96 (br.t, J = 7.7 Hz, 1H, H-5), 4.50 (s, 2H, CH2-7‴), 2.87 (t, J = 7.2 Hz,
2H, CH2-1′), 2.78 (t, J = 7.3 Hz, 2H, CH2-3′), 2.14 (s, 6H, CH3-2″″ &
CH3-6″″), 2.02 (quint., J = 7.3 Hz, 2H, CH2-2′). 13C NMR (150 MHz,
DMSO-d6, δ, ppm): 168.20 (C-5″), 164.87 (C-8‴), 162.39 (C-2″),
140.31 (C-4‴), 136.25 (C-8), 135.56 (C-2″″ & C-6″″), 135.03 (C-1‴),
10.83 (s, 1H, NH-1), 10.08 (s, 1H, CONH), 7.91 (br.d, J = 8.1 Hz, 2H, H-
2″′ & H-6″′), 7.57 (br.d, J = 8.1 Hz, 2H, H-3‴ & H-5‴), 7.51 (br.d,
J = 7.7 Hz, 1H, H-7), 7.41 (br.s, 2H, H-2″″ & H-6″″), 7.35 (br.d,
J = 8.1 Hz, 1H, H-4), 7.14 (s, 1H, H-2), 7.07 (br.t, J = 7.3 Hz, 1H, H-6),
6.97 (br.t, J = 7.4 Hz, 1H, H-5), 6.74 (br.s, 1H, H-4″″), 4.54 (s, 2H, CH2-
7‴), 2.88 (t, J = 7.4 Hz, 2H, CH2-1′), 2.77 (t, J = 7.2 Hz, 2H, CH2-3′),
2.26 (s, 6H, CH3-3″″ & CH3-5″″), 2.06 (quint., J = 7.3 Hz, 2H, CH2-2′).
13C NMR (150 MHz, DMSO-d6, δ, ppm): 168.09 (C-5″), 164.95 (C-8‴),
162.25(C-2″), 140.21 (C-4‴), 138.91 (C-1″″), 137.46 (C-3″″ & C-5″″),

ABBASI ET AL.
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136.30 (C-8), 134.35 (C-1‴), 128.88 (C-3‴ & C-5‴), 127.81 (C-2‴ &
C-6‴), 127.01 (C-9), 125.15 (C-4″″), 122.49 (C-2), 120.86 (C-6), 118.16
(C-7), 118.16 (C-5), 118.09 (C-2″″ & C-6″″), 113.22 (C-3), 111.35 (C-4),
35.34 (C-7‴), 26.47 (C-2′), 24.25 (C-1′), 23.81 (C-3′), 21.08 (CH3-2″″ &
CH3-6″″). Anal. calcd. for C29H28N4SO2 (496.19): C, 70.14; H, 5.68; N,
11.28. Found: C, 70.10; H, 5.65; N, 11.22. EI-MS (m/z): 496
(C29H28N4SO2)·+ (M)+, 353 (C19H19N3SO2)+, 258 (C13H12N3SO)·+, 239
(C16H17NO)+, 186 (C12H12NO)+, 184 (C12H12N2)·+, 143 (C10H9N)+, 130
(C9H8N)+, 120 (C8H10N)+, 91 (C6H5N)+, 78 (C5H4N)+.
and a suspension was made. Test compound solution (20 μL,
10 mg/mL) was mixed with 180 μL of RBCs suspension and incubate
for 30 min at room temperature. PBS was used as negative control and
Triton 100-X was taken as positive control.[30,31] The % age of
hemolysis was taken as by using formula:
ð%Þ of Hemolysis
Absorbance of Sample ꢀ Absorbance of Negative Control
¼
Absorbance of Positive Control
ꢁ 100
|
4.2 Alkaline phosphatase assay
Activity of calf intestinal alkaline phosphatase (CIALP) was measured
by spectrophotometric assay as previously described.[28,29] The
reaction mixture consisted 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 37°C. The change in absorbance of released p-nitrophenolate was
|
4.5 Statistical analysis
All the measurements were carried out in triplicate and statistical
analysis was performed by Microsoft Excel 2010. The results are
presented as mean SEM with 96% CL.
|
4.6 Computational methodology: Grid generation
and molecular docking
The alkaline phosphatase structure was retrieved from Protein Data
Bank (PDB) (www.rcsb.org) with PDBID 1EW2 in protein preparation
wizard. The selected crystal structure of protein was preprocessed and
minimized using default parameters in Maestro interface. Bond orders
were assigned and hydrogen atoms were added to the protein. All four
structureswere minimized separately to reach the converged root mean
square deviation (RMSD) of 0.30 Å with the OPLS_2005 force field.
Furthermore, docking experiment was performed against all synthe-
sized ligands and target protein by using Glide docking protocol.[32] The
predicted binding energies (docking scores) and conformational
positions of ligands within active region of protein were also performed
using Glide experiment. Throughout the docking simulations, both
partial flexibility and full flexibility around the active site residues are
performedby Glide/SP/XP and inducedfit docking (IFD) approaches.[32]
monitored at 405 nm, using a 96-well microplate reader (OPTIMAX
,
Tunable USA). All the experiments were repeated three times in a
triplicate manner. KH2PO4 was used as the reference inhibitor of
CIALP.
The alkaline phosphatase activities were calculated according to
the following formula:
Alkaline phosphatase activity ð%Þ
À
Á
¼
ODcontrol ꢀ ODsample ꢁ 100 =ODcontrol
where ODcontrol and ODsample represent the optical densities in the
absence and presence of sample, respectively.
|
4.3 Kinetics assay
On the basis of IC50 results, the most potent inhibitor 8b was selected
for determining the mechanism of enzyme inhibition. The inhibitor
concentrations used were 0.0, 0.0427, and 0.1708 μM. Substrate
p-NPP concentrations were 10, 5, 2.5, 1.25, and 0.625 mM at 37°C.
Pre-incubation time and other conditions were same as described in
alkaline phosphatase inhibition assay section. Maximal initial velocities
were determined from initial linear portion of absorbances up to
10 min after addition of enzyme at per minute's interval. The inhibition
type on the enzyme was assayed by Lineweaver–Burk plot of inverse
of velocities (1/V) versus inverse of substrate concentration 1/[S] in
mM−1. The EI dissociation constant Ki was determined by secondary
plot of 1/3 versus inhibitor concentration.
ACKNOWLEDGMENT
The current study was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by
the Ministry of Education (2017R1D1A1B03034948).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ORCID
Muhammad A. Abbasi
http://orcid.org/0000-0003-3439-9286
|
4.4 Hemolytic activity
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saline (0.9% NaCl), and centrifuged at 1000×g for 10 min. Erythrocytes
were separated and diluted in phosphate buffer saline (PBS) of pH 7.4
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How to cite this article: Abbasi MA, Nazir M, ur-Rehman A,
et al. Bi-heterocyclic benzamides as alkaline phosphatase
inhibitors: Mechanistic comprehensions through kinetics and
computational approaches. Arch Pharm Chem Life Sci.
2019;1–14. https://doi.org/10.1002/ardp.201800278
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