Substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acetamides as alkaline phosphatase inhibitors: Synthesis, computational studies, enzyme inhibitory kinetics and DNA binding studies

Article abstract of DOI:10.1016/j.bioorg.2019.103108

Substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acetamides 9a-j were synthesized as alkaline phosphatase inhibitors. Phenyl acetic acid 1 through a series of reactions was converted into 5-benzyl-1,3,4-oxadiazole-2-thione 4. The intermediate oxadiazole 4 was then reacted with chloroacetyl derivatives of phenols 6a-f and anilines derivatives 8a-d to afford the title oxadiazole derivatives 9a-j. All of the title compounds 9a-j were evaluated for their inhibitory activity against human alkaline phosphatise (ALP). It was found that compounds 9a-j exhibited good to excellent alkaline phosphatase inhibitory activity especially 9h displayed potent activity with IC₅₀ value 0.420 ± 0.012 μM while IC₅₀ value of standard (KH₂PO₄) was 2.80 μM. The enzyme inhibitory kinetics of most potent inhibitor 9h was determined by Line-weaever Burk plots showing non-competitive mode of binding with enzyme. Molecular docking studies were performed against alkaline phosphatase enzyme (1EW2) to check the binding affinity of the synthesized compounds 9a-j against target protein. The compound 9h exhibited excellent binding affinity having binding energy value (?7.90 kcal/mol) compared to other derivatives. The brine shrimp viability assay results proved that derivative 9h was non-toxic at concentration used for enzyme assay. The lead compound 9h showed LD₅₀ 106.71 μM while the standard potassium dichromate showed LD₅₀ 0.891 μM. The DNA binding interactions of the synthesized compound 9h was also determined experimentally by spectrophotometric and electrochemical methods. The compound 9h was found to bind with grooves of DNA as depicted by both UV–Vis spectroscopy and cyclic voltammetry with binding constant values 7.83 × 10³ and 7.95 × 10³ M^?1 respectively revealing significant strength of 9h-DNA complex. As dry lab and wet lab results concise each other it was concluded that synthesized compounds, especially compound 9h may serve as lead compound to design most potent inhibitors of human ALP.

Full text of DOI:10.1016/j.bioorg.2019.103108

Accepted Manuscript
Substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acet-
amides as alkaline phosphatase inhibitors: synthesis, computational studies,
enzyme inhibitory kinetics and DNA binding studies
Zafar Iqbal, Zaman Ashraf, Mubashir Hassan, Qamar Abbas, Erum Jabeen
PII:
S0045-2068(19)30802-8
https://doi.org/10.1016/j.bioorg.2019.103108
103108
DOI:
Article Number:
Reference:
YBIOO 103108
Bioorganic Chemistry
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Accepted Date:
18 May 2019
1 July 2019
2 July 2019
Please cite this article as: Z. Iqbal, Z. Ashraf, M. Hassan, Q. Abbas, E. Jabeen, Substituted phenyl[(5-benzyl-1,3,4-
oxadiazol-2-yl)sulfanyl]acetates/acetamides as alkaline phosphatase inhibitors: synthesis, computational studies,
enzyme inhibitory kinetics and DNA binding studies, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103108
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Substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acetamides as alkaline
phosphatase inhibitors: synthesis, computational studies, enzyme inhibitory kinetics and
DNA binding studies
Zafar Iqbala, Zaman Ashrafa,*, Mubashir Hassanb, Qamar Abbasc, Erum Jabeena
aDepartment of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan.
bInstitute of Molecular Biology and biotechnology, The University of Lahore, Pakistan.
cDepartment of Physiology, University of Sindh, Jamshoro, Pakistan.
Keywords
Alkaline phosphatase inhibitors, Enzyme kinetics, DNA binding studies, Molecular docking
* Address for Correspondence
Zaman Ashraf
Chemistry Department, Allama Iqbal Open University, Islamabad, Pakistan
Email: mzchem@yahoo.com
Contact number: 0092 3215194461, 0092519057182
Fax: 0092512891471
1

Abstract
Substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acetamides 9a-j were
synthesized as alkaline phosphatase inhibitors. Phenyl acetic acid 1 through a series of reactions
was converted into 5-benzyl-1,3,4-oxadiazole-2-thione 4. The intermediate oxadiazole 4 was
then reacted with chloroacetyl derivatives of phenols 6a-f and anilines derivatives 8a-d to afford
the title oxadiazole derivatives 9a-j. All of the title compounds 9a-j were evaluated for their
inhibitory activity against human alkaline phosphatise (ALP). It was found that compounds 9a-j
exhibited good to excellent alkaline phosphatase inhibitory activity especially 9h displayed
potent activity with IC50 value 0.420 0.012µM while IC50 value of standard (KH2PO4) was
2.80µM. The enzyme inhibitory kinetics of most potent inhibitor 9h was determined by Line-
weaever Burk plots showing non-competitive mode of binding with enzyme. Molecular docking
studies were performed against alkaline phosphatase enzyme (1EW2) to check the binding
affinity of the synthesized compounds 9a-j against target protein. The compound 9h exhibited
excellent binding affinity having binding energy value (-7.90 kcal/mol) compared to other
derivatives. The brine shrimp viability assay results proved that derivative 9h was non-toxic at
concentration used for enzyme assay. The lead compound 9h showed LD50 106.71µM while the
standard potassium dichromate showed LD50 0.891µM. The DNA binding interactions of the
synthesized compound 9h was also determined experimentally by spectrophotometric and
electrochemical methods. The compound 9h was found to bind with grooves of DNA as depicted
by both UV-Vis spectroscopy and cyclic voltammetry with binding constant values 7.83 x
103 and 7.95 x 103 M-1 respectively revealing significant strength of 9h-DNA complex. As dry
lab and wet lab results concise each other it was concluded that synthesized compounds,
especially compound 9h may serve as lead compound to design most potent inhibitors of human
ALP.
2

1. Introduction
Azoles are a class of compounds having five membered heterocyclic rings that contains one
nitrogen atom and at least one other non carbon atom in the ring. The widespread use of azoles in
medicinal chemistry have been reported and is being treated as privileged structured class
exhibiting wide range of biological activities as antifungal, analgesic, hypolipidemic,
ulcerogenic, anti tubercular, antiviral, antimicrobial, antineoplastic, anticancer, inhibition of
tyrosinase, cathepsin K, anti-inflammatory, hypnotic, genotoxic, muscle anticonvulsant, lipid per
oxidation inhibitor [1-4] and vasodilator. Some of the oxadiazole derivatives act as muscle
relaxants, hypnotic, sedatives, and show antimitotic activities [5]. Azole heterocycles show
strong hydrogen bonding interactions with the receptors that increase their pharmacological
activity. The well-known examples of drugs having the azoles nucleus include nesapidil as
antihypertensive, raltegravir as antiretroviral, furamizole as antibiotic drug and zibotenten as
anticancer drug. Well known azole derivatives theophylline and pyrazole are widely used
alkaline phosphatase inhibitors [6].
Alkaline phosphatase (ALP, E.C.3.1.3.1.) is a non-specific phosphomonoester hydrolase that
catalyzes the hydrolysis and transphosphorylation of a wide variety of organic monophosphates
and regulates the functions of many biological systems [7-9]. Cell regulation is an important
biological process and is being controlled by phosphatase enzyme due to which phosphatase are
the area of immense interest in the field of pharmaceutical research [10]. ALP has very complex
structure both in human and Escherichia coli. Substrate specificity is an area of interest in
research because there are different active sites in the substrates and dephosphorylation at
particular site is very much important for the proper cellular functioning [11]. Tissue nonspecific
alkaline phosphatase is articulated in developing spinal and brain which shows its physiological
role in the development of CNS [12]. Proliferation and migration in CNS is also associated with
TNAP. Wide spectrum of monoesters of phosphoric acid is being hydrolyzed by tissue
nonspecific alkaline phosphatase [13]. If the level of alkaline phosphatase exceeds the reference
range with in body it is termed as elevated alkaline phosphatase. Homeostasis within the body is
being maintained by alkaline phosphatase [14]. Elevated level of ALP in body may result in a
number of diseases as metabolic disorder, dysregulation in CNS, mineralization disorders and
obesity, osteoblastic bone tumors, osteomalacia, osteoporosis, bone conditions, biliary
obstruction, hepatitis, myelofibrosis, myocardial infarction, leukemoid reaction, lymphoma,
3

sarcoidosis, hyperparathyroidism and hyperthyroidism are associated with elevated level of the
alkaline phosphatase enzyme [15]. It is evident from literature that azoles represent imperative
pharmacophores and have a wide range of therapeutic properties [16]. Keeping in view the
importance of this moiety the present work describe the synthesis of substituted phenyl [(5-
benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acetamide 9a-j as alkaline phosphatase inhibitors.
The computational studies and enzyme inhibitory kinetics was determined to compare the wet
lab results with dry lab. The DNA binding studies was also determined to check its mode of
binding with DNA which in turn helps us to predict its antiproliferative properties. The brine
shrimp cytotoxicity test was also performed and Herein we selected 1,3,4-oxadiazole as alkaline
phosphatase inhibitors due to their widespread use as therapeutic drugs.
2. Results and Discussion
2.1 Synthesis
Methyl 2-phenylacetate 2 and 2-phenylacetohydrazide 3 were synthesized according to the
already reported methods [17-18]. The Scheme I depicts the synthesis of oxadiazole 4 employing
slight modifications in already reported method. FTIR spectrum confirmed the formation of
oxadiazole compound 4 having peak at 1635cm-1 indicated the presence of C=N absorption and
SH at 2853cm-1. The chloroacetyl derivatives of phenols 6a-f were synthesized by reacting
substituted phenols 5a-f with chloroacetyl chloride in the presence of triethyl amine as base. The
anilines derivatives 8a-d were obtained by reacting anilines 7a-d with chloroacetyl chloride by
following the same procedure used in the preparation of derivatives 6a-f.The title compounds 9a-
j was obtained in good to excellent yield by reacting oxadiazole 4 with substituted phenol
derivatives 6a-f and aniline derivatives 8a-d. This involves the alkylation of mercapto group of
1
13
oxadiazole 4. The formation of the final products was ascertained by FTIR, H NMR and C
NMR spectroscopy.
4

Scheme I Synthetic route for substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]
acetates/acetamides 9a-j.
2.2 Bioassay for alkaline phosphatase inhibitory activity
In the present work oxadiazole based derivatives 9a-j were designed based upon our
previous investigations. Oxadiazole based ALP inhibitors analogues have been designed to
evaluate their inhibitory activity against ALP enzyme. The azole derivatives 9a-j was
5

synthesized by following the already reported method with slight modification shown in Scheme
I. New analogues 9a-j has been synthesized by substituting hydrophilic and hydrophobic groups
on phenyl ring in order to check ALP inhibitory activity. We have evaluated the role of floro,
chloro, alkyl, acetyl, methoxy and aldehydic functional groups on benzene ring in order to check
ALP inhibitory activity. KH2PO4 a well known clinically used ALP inhibitor was used as
reference drug for comparison purpose. The synthesized compounds 9h, 9b, 9c, 9i, and 9a
showed good to excellent inhibition of ALP with IC50 ranged from 0.42µM to 2.37µM higher
activities compared to standard KH2PO4 having IC50 2.80µM. Table 1 presented the IC50 values
of the synthesized azole derivatives 9a-j and it was observed that compound 9h exhibited the
most potent alkaline phosphatase inhibitory activity with IC50 0.420µM. The presence of the
trifloromethyl moiety in compound 9h might be playing very important role in the alkaline
phosphatase inhibitory activity. The derivatives 9b and 9c possess methoxy and alkyl
substitution respectively having IC50 1.23µM and showed activity lower than compound 9h but
higher than rest of the analogues. The compound 9i possesses 4-bromo on phenyl ring having
IC50 1.79µM showed excellent activity. Similarly compound 9a in which phenyl ring was
unsubstituted having IC50 2.37µM showed activity better than the standard KH2PO4. The
compound 9d, 9e, 9f, 9g and 9j having methylene 1,3-dioxal moiety, 4-allyl-2methoxy, 4-
formyl-2-methoxy, phenyl and 4-chloro respectively showed less activity compared to other
derivatives and as compared to standard. It has now been verified that these substitutions does
not play any important role in alkaline phosphatase inhibitory activity. On the basis of these
results we propose that derivative 9h having trifloro methyl substituent may serve as a structural
model for the design and development of novel ALP inhibitors.
Table 1 Alkaline phosphatase inhibitory activity of synthesized compounds 9a-j
Compounds
Alkaline Phosphatase
IC50 SEM (µM)
2.374 0.071
Free
radical
%
scavenging (100µg/mL)
9a
9b
9c
10.13 0.01
10.00 0.03
4.00 0.02
1.233 0.036
1.233 0.036
6

9d
5.012 0.091
4.844 0.145
4.801 0.310
0.420 0.012
3.765 0.112
1.793 0.053
8.348 0.250
2.80 0.065
-------
8.32 0.08
12.35 0.6
35.50 1.06
13.67 0.31
2.5 0.065
15.0 0.35
12.45 0.27
-------
9e
9f
9h
9g
9i
9j
KH2PO4
Vitamin C
96.91 3.0
Values are presented as Mean SEM Standard error of mean
2.3 Kinetic study
On the basis of IC50 value we selected the most potent inhibitor 9h to understand the
inhibitory mechanism of synthesized compound. The results were presented in table 2 and Fig.1.
The kinetic studies showed inhibition mechanism of compound 9h to be non-competitive
inhibitor of calf IALP. The inhibition constant Ki (0.360) was determined from fig B. The value
of 1/Vmax is increased to a new value while that of Km remain same which indicated that
compound 9h simply lowers the concentration of enzyme by a non-competitive binding mode
(Fig. A and B).
Table 2 Kinetic analysis of compound 9h
Entry
Concentration 1/Vmax
Km
Ki
Inhibition type
(µM)
(ΔA /Min
mM
1.0952
(µM)
0.360
1
2
3
4
0.00
200.00
363.63
436.36
500.00
0.100
0.200
0.400
1.0952
1.0952
1.0952
0.360
0.360
0.360
Non-competitive
7

1/Vmax is the inverse of reaction velocities, Km is the Michaelis-Menten constant, Ki is the
inhibition constant.
Fig. 1. (a) Lineweaver–burk plots for the inhibition of compound 9h on Calf alkaline
phosphatase; concentrations were 0.00, 0.100, 0.200 and 0.400 mM. The (b) represents the
secondary plot of 1/Vmax versus concentration of compound 9h to determine the inhibition
constant (Ki).
2.4 Cytotoxicity studies
The viability assay of derivatives 9a-j was carried out on brine shrimp in order to check the
cytotoxicity of the synthesized compounds. The brine shrimp cytotoxicity results showed that
synthesized compounds are no-toxic to the shrimps at concentration range 70µM to 125µM. As
higher is the LD50 values lower is the cytotoxicity and vice versa. LD50 values were calculated
and are shown in Fig. 2 and the results showed that compound 9j showed highest LD50
125.29µM and hence the least cytotoxicity. The lead compound 9h also showed LD50 106.71µM
and hence the much less cytotoxicity than the standard KH2PO4. The concentration of compound
9h selected for other bioassay is less than the LD50 value.
8

Fig. 2. The graphical depiction of LD50 (µM) values of synthesized compounds 9a-j.
2.5 DFT calculations
The spatial charateristics, nodal patterns and involvement of individual atom in charge
transfer process was assayed by computing their forntier molecular orbitals. The DFT
calcultaions revealed that HOMO orbital as well as LUMO orbitals were solely spread over S-
alkylated moiety of the molecule which revealed that this part of molecule will actively
participate in any (oxidation or reduction) charge transfer process Fig.3.
Fig. 3 HOMO and LUMO of the compounds (DFT/B3LYP/6-311G**(++)).
9

The molecular potential energy surface (MPES) was computed to define nucleophilic and
electrophilic regions in the molecule. The Fig. 4 shows the computed MPES model at
DFT/B3LYP/6-311G**(++). It is interesting to find that the nucleophilic character was
dominating in the molecule as revealed by dominance of reddish shade in MPES. This is due to
presence of pi-electron excessive oxadiazole ring and electron attracting -CF3 group at side chain
phenyl. It can be predicted that there is merely any probability of developing electrostatic
interactions between 9h molecule and negatively charged phosphate backbone of DNA as 9h is
deficient in significant electrophilic character. Further, electronic charge spread over the
molecule predicts that stacking of such electron rich structure between DNA base pairs may
generate repulsion with π electron cloud of DNA base pairs. This may create hindrance to
intercalation of 9h through its planner part.
Fig. 4 The computed molecular electrostatic potential energy surface (MPES) of 9h at
DFT/B3LYP/6-311G**(++) level of theory.
2.5.1 Mechanism of Charge transfer process
The cyclic voltammograms of the 9h exhibited reversible redox peak with oxidation at
0.389 V and reduction peak at 0.314 V at 100mV/s Fig. 5. The Epa-Epc was > 60 mV which
revealed that the oxidation and reduction peaks does not correspond to the reversible behavior
rather both reduction and oxidation peaks corresponded to independent charge transfer processes.
This was further confirmed through recording the cyclic voltammograms at different scan rates.
10

Fig. 5 Cyclic voltammograms of 9h in 50% methanol-PBS (pH-7.4) at different scan rates vs.
SEC scanned form 0 to 0.6V. Inset: Cyclic voltammogram under same conditions scanned from
0.6 to 0 V at 100 mV/s.
For a reversible redox couple, the increasing scan rate increases the current without
changing the Ep value while an irreversible oxidation or reduction process will suffer a peak shift
towards more positive or negative value respectively upon increasing scan rate [19]. It was
observed that the increasing scan rates form 100 mV/s to 700 mV/s, the oxidation peaks suffered
a positive shift in peak potential and an increase in current. This showed that raising the scan rate
increases the oxidation, so, it was a fast oxidative process. On the other hand, the response of
reduction peak was interesting as it suffered a shift in peak potential towards positive value
(negative shift in reduction peak) along with an increase in peak current. This revealed that the
reduction process was facilitated by the increase in oxidation process. Therefore, it can be
inferred that the reduction process followed oxidation process.
This was further confirmed by reversing the direction of scan where neither oxidation
peak nor reduction peak was recorded (Fig.5 inset). Thus, the reduction process was following
irreversible oxidation process and was not coupled with it reversibly. Further, the increasing scan
rate did not reduce the peak current of any peak, eradicating the association of any coupled
chemical reaction with oxidation process. Moreover, the two successive scan at each scan rate
resulted in reproduction of both voltammograms (data not shown here) revealing that couple
chemical reaction was not associated with any of charge transfer process. Therefore, the EoEr
11

(electron transfer oxidation followed by electron transfer reduction) was suggested as charge
transfer mechanism.
2.5.2 DNA binding studies of 9h
The UV-Vis spectrum for 9h exhibited two peaks at 245nm and 205nm Fig. 6. When
DNA was added to 9h, the absorbance decreased without any shift in λmax. The successive
addition DNA resulted in successive decrease in absorbance which was then used to obtain
formation constant through Benesi-Hildebrand equation [20]. As there was no shift in λmax, the
electrostatic binding and intercalation stamped out. Based on observed hypochromism, the
groove binding was suggested for 9h-DNA interactions.
Fig. 6 UV-Vis spectra of 9h (20 μM) in 50% methanol-PBS (pH-7.4) in the presence of different
concentrations of DNA.
When the DNA was added to the 9h, the peak current decreased and the Ep did not shift
for oxidation and reduction peaks. The incremental addition of DNA resulted in successive peak
current decrease without effecting the position of any peak Fig. 7. The intact Ep depicts that 9h
charge transfer process was not disturbed by DNA addition, rather only diffusion was troubled.
Further, the diffusion co-efficient was calculated to be 6.5 x 10-4cm2/s for 9h and 0.54 x 10-
4cm2/s for 9h-DNA mixture which revealed the significant reduction in diffusion coefficient of
9h through its binding to DNA. Thus, the decrease in peak current can be attributed to decrease
in diffusion coefficient due to 9h-DNA complex formation. Such cyclic voltammetric responses
often stand for fitting of drug into grooves of DNA [21]. Therefore, the cyclic voltammetry
confirmed the groove binding of 9h with DNA as was indicated by UV-Vis spectroscopy.
12

Fig. 7. Cyclic voltammograms of 9h (30 mM) in 50% methanol-PBS (pH-7.4) at 0.1 V/s vs SCE
in the presence of different concentrations of DNA.
The binding constants calculated from UV-Vis spectroscopy and cyclic voltammetry (Fig. 8)
were 7.83 x 103 and 7.95 x 103 M-1respectively which revealed significant strength of 9h-DNA
complex.
Fig. 8. (a) UV-visible data (b) Cyclic voltammetric data of 9h plots for calculation of kf.
13

2.6 Molecular docking analysis
2.6.1 Binding energy evaluation of synthesized compounds
Molecular docking experiment is best approach to study the binding conformation of
ligands against target protein [22,23]. For the prediction of the best conformational position
within the active region of targeted protein, all the synthesized ligands (9a-j) were docked
against ALP. All the generated docked complexes were analyzed on the basis of minimum
energy values (kcal/mol) and hydrogen/hydrophobic interactions. From the docking results it was
concluded that all the ligands possessed good binding energy values and interestingly no huge
energy difference was being observed among all docked complexes. The docking results
predicted compound 9h to be most active that exhibited good energy value (-7.90 kcal/mol) in
comparison to other derivatives. Moreover, 9a and 9f also exhibited good binding energy values
(-7.70 and-7.60 kcal/mol).Whereas, 9d, 9e and 9i possessed common energy value (-7.50
kcal/mol energy). The other compounds also presents significance energy values against receptor
molecule. Although, the basic chemical nucleus of all the synthesized compounds was same,
therefore most of ligands showed good efficient energy values (Fig 9).
Fig. 9 The graphical depiction of docking energy values of synthesized compounds 9a-j.
2.6.2 Binding pocket analysis of 9j against alkaline phosphatase
9h-docked complex was selected based on in vitro (IC50) and docking results, to deeply
recognize the binding interaction behavior of ligand against target protein. Docking analysis
14

depicted that compound 9h was actively confined within active binding region of receptor
molecule (Fig 10 A, B).The phenolic ring structure showed their intrusion within the inner part
of binding pocket, whereas, the bulky trifloromethyl moiety remain outside in the opening region
of binding pocket. These trifloromethyl may cause a little steric hindrance which may restrict
this part to gain entry inside the binding pocket.
Fig. 10 (A, B) Binding pocket depiction of 9h within active region of target protein
2.6.3 Hydrogen Binding analysis
In detail, the binding analysis study showed that two hydrogen bonds were observed in
9h-docking complex. The carbonyl oxygen atom of 9h forms hydrogen bond with Thr 431 and
fluorine incorporated moiety also showed hydrogen bond with Val89 having bond lengths 2.57
and 2.95 Å, respectively (Fig. 11 A, B). Research data also ensured the importance of these
residues in bonding with other ALP inhibitors which strengthen our docking result [24,25]. The
comparative binding energy and SAR analysis showed the significance of 9h compound and may
consider as potent inhibitor by targeting ALP.
15

Fig. 11 (A) Docking interactions of 9h with alkaline phosphate. (B) the basic skeleton of 9h is
represented in brown, whereas, their functional moieties are justified in red, blue, yellow and
light green colours. The interacted residues are represented in yellow. Amino acids involved in
hydrogen bonding are labelled with red colour, having distances mentioned in angstrom (Å).
3. Conclusions
Substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acetamides 9a-j were
efficiently synthesized in good yields. The alkaline phosphatise inhibitory kinetics revealed that
compound 9h displayed most potent activity with IC50 value 0.420 0.012µM while IC50 value of
standard (KH2PO4) was 2.80µM and showing non-competitive mode of binding with enzyme.
Molecular docking studies performed against alkaline phosphatase enzyme (1EW2) showed that
9h exhibited excellent binding affinity having binding energy value (-7.90 kcal/mol) compared
to other derivatives. The brine shrimp viability assay results proved that derivative 9h was non-
toxic at concentration used for enzyme assay. The lead compound 9h showed LD50 106.71µM
while the standard potassium dichromate showed LD50 0.891µM. The DNA binding interactions
of the synthesized compound 9h was also determined experimentally by spectrophotometric and
electrochemical methods. The compound 9h was found to bind with grooves of DNA as depicted
by both UV-Vis spectroscopy and cyclic voltammetry with binding constant values 7.83 x
103 and 7.95 x 103 M-1 respectively revealing significant strength of 9h-DNA complex. As dry
lab and wet lab results concise each other it was concluded that synthesized compounds,
especially compound 9h may serve as lead compound to design most potent inhibitors of human
ALP.
4. Materials and Methods
4.1 Chemistry
The necessary chemicals and reagents were purchased from Sigma Aldrich Company and no
further purification was needed. Melting point was determined through Digital Gallenkamp
(SANYO) apparatus. Furthermore, FTIR spectrum of synthesized compounds was determined
1
13
through Perkin Elmer spectrophotometer. H NMR and C NMR spectra were determined in
CDCl3 solutions at 300 MHz via Brukner AM-300 spectrophotometer. Elemental Analysis (C, H,
N) were carried out on a Flash 2000 series elemental analyzer with TCD detector system and
results are with 0.3%.
16

4.1.1 Synthesis of 5-benzyl-1,3,4-oxadiazole-2-thiol
Phenyl acetic acid 1 was being esterified by refluxing methanol (20mL), Phenyl acetic acid (5g)
and few drops of sulphuric acid in a round bottom flask for 6 hours. Phenyl acetic acid
hydrazide3 was being synthesized by refluxing Phenyl acetic ester 2 (2mmol), ethanol (20mL)
and hydrazine mono-hydrate (3mmol) for 6hours. TLC technique was being used to determine
the progress of the reaction and the reaction mixture was concentrated under low pressure in
order to obtain solid phenylacetohydrazide 3 and was also being recrystallized in ethanol [26]. 5-
benzyl-1,3,4-oxadiazole-2-thiol 4 was synthesized by mixing phenylacetohydrazide 3 (10mmol),
KOH (6mmol) and dry ethanol (20mL) in a round bottom flask. Then CS2 was added in reaction
mixture in portions (20mmol) and was being refluxed for 12 hours. Completion of reaction was
as confirmed through TLC, was concentrated under the reduced pressure and was then acidified
with HCl to afford oxadiazole 4 as yellow ppts. Ethanol was being used for recrystalization of
oxadiazole 4; Yield: 79.9%; FTIR (KBr, νmax cm-1): 2951 (sp3 C-H stretch), 1617 (C=N), 1511
(C=C aromatic), 1231 (C-O).
4.1.2 Synthesis of phenyl 2-chloroacetate derivatives 6a-f
Phenyl 2-chloroacetate derivatives 6a-f were synthesized by reacting 5a-f (2mmol) with
chloroacetylchloride (2mmol), triethyl amine (2mmol) and dichloromethane as solvent (20mL),
the reaction mixture was continued for stirring up to 6 hours. Finally, under the maintained low
pressure the reaction mixture was being concentrated and product was extracted in ethylacetate.
Synthesis of 2-chloro-N-phenylacetamide derivatives 8a-d
2-chloro-N-phenylacetamide derivatives 8a-d were synthesized by reacting 7a-d (2mmol) with
chloroacetylchloride (2mmol), (triethyl amine 2mmol) and dichloromethane (20mL), the reaction
mixture was kept on stirring for 6 hours. The reaction mixture was being concentrated on low
pressure and product was extracted in ethyl acetate.
4.1.3 Synthesis of 1,3,4 oxadiazole derivatives 9a-f
5-benzyl-1,3,4-oxadiazole-2-thiol 4 (5mmol), acetone (20mL) and K2CO3 (10mmol) was added
in a round bottom flask followed by continuous stirring. Furthermore, chloroacetyl chloride
derivatives 6a-f (6 mmol) were added in above reaction mixture and heated under reflux for 12
hours. The progress of the reaction was being monitored through TLC. After the completion of
17

reaction; the obtained reaction mixture was placed under low pressure to get concentrated solid
oxadiazole derivatives 9a-f which was purified by applying column chromatography [27].
4.1.4 Synthesis of 1,3,4 oxadiazole derivatives 9g-j
5-benzyl-1,3,4-oxadiazole-2-thiol 4 (5mmol), acetone (20mL) and K2CO3 (10mmol) was added
in a round bottom flask followed by continuous stirring. Furthermore, chloroacetyl chloride
derivatives 8a-d (6 mmol) were added in above reaction mixture and heated under reflux for 12
hours. The progress of the reaction was monitored through TLC. After the completion of reaction
the obtained reaction mixture was placed under low pressure to get concentrated solid oxadiazole
derivatives 9g-j which was purified by applying column chromatography [28, 29].
phenyl 2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)acetate 9a Yield: 68.5% FTIR (KBr, νmax cm-1):
3211 (sec. amide NH), 2988 (sp2 C-H stretch), 2943 (sp3 C-H stretch), 1690 (C=O), 1592(C=N),
1489 (C=C aromatic), 1224 (C-O). 1HNMR (CDCl3, δ ppm): 7.39 (1H, m, H-15 ), 7.34 (2H, m,
H-13,17), 7.24 (2H, m, H-14,16), 7.11 (2H, m, H-2,4), 7.07(2H, m, H-1,5), 6.81 (1H, m, H-3),
4.08(2H, s, H-10) 3.07(2H, s, H-7); 13CNMR (CDCl3, δ ppm): 167.7 (C-11), 166.5(C-9),
163.5(C-8), 155.8(C-12), 130.1(C-13,17), 129.5(C-15), 124.4(C-3), 121.4(C-14,16), 120.3(C-6),
116.0(C-1,5), 115.4(C-2,4), 34.2(C-10), 30.9(C-7); Anal Calcd For C17H14N2O3S: C, 62.57; H,
4.29; N, 8.58 Found C, 62.50; H, 4.23; N, 8.51.
2-methoxyphenyl 2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)acetate 9b Yield: 75.7%; FTIR (KBr,
νmax cm-1): 2954 (sp2 C-H stretch), 2837 (sp3 C-H stretch), 1713 (C=O), 1611 (C=C aromatic),
1
1511 (C=N). HNMR (CDCl3, δ ppm): 7.26(2H, d, J=8.4 H-1,5), 7.12(3H, m, H-2,3,4),
.
6.96(2H, m, H-15,16), 6.96(1H, d, J=7.2 H-14), 6.77(1H, d, J=8.3 H-3), 4.30 (2H, s, H-10),
4.11(2H, s, H-7) 3.91(3H, s, H-18). 13C NMR (CDCl3, δ ppm):167.2(C-11), 155.2(C-9),
150.7(C-8), 130.1(C-14,17), 129.6(C-12), 127.3(C-13),125.2(C-15) 122.5(C-16), 120.6(C-2,4),
115.8(C-6), 110.9(C-1,5), 112.4(C-3), 105.4(C-18), 33.9(C-10), 31.0 (C-7); Anal Calcd For
C18H16N2O4S: C, 60.67; H, 4.49; N, 7.86 Found C, 60.61; H, 4.43; N, 7.79.
2-isopropyl-5-methylphenyl 2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)acetate 9c Yield: 70.2%;
m.p.220 °C FTIR (KBr, νmax cm-1): 2989 (sp2 C-H stretch), 2944 (sp3 CH stretch), 1760 (C=O),
1
1686 (C=C aromatic), 1488 (C=N). HNMR (CDCl3, δ ppm)δ 7.83 (1H, s, H-17),7.33(3H, m, H-
2,3,4), 7.06(2H, d, J=8.8 H-1,5), 6.70(1H, d, J=6.8 H-14), 6.28(1H,d, J=7.2 H-15), 4.18(2H,
s, H-10), 4.11(3H,s,H-21), 4.02(2H, s, H-7), 3.60(1H, m, H-18), 1.18(6H, d, J=3.3 H-19,20)
NMR (CDCl3, δ ppm):168.2(C-11), 166.5(C-8),164.4(C-9), 138.8(C-12), 137.6(C-17), 136.0(C-
18

15), 133.9(C-2,4), 128.8(C-1,5), 127.8(C-13) 126.6(C-6), 122.9(C-3), 119.9(C-16), 118.8(C-14),
39.2.(C-10), 31.2(C-7), 30.8(C-18), 26.2(C-19,20), 23.4(C-21); Anal Calcd For C21H22N2O3S: C,
65.96; H, 5.75; N, 7.32 Found C, 65.88; H, 5.69; N, 7.26.
4-allyl-2-methoxyphenyl 2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)acetate 9d Yield: 80.2%;
m.p.74 °C FTIR (KBr, νmax cm-1): 2936 (sp2 C-H stretch), 2944 (sp3 CH stretch), 1755 (C=O),
1
1637 (C=C aromatic) 1507 (C=N). HNMR (CDCl3, δ ppm): 7.34(1H, s, H-14), 7.33(1H, d,
J=7.7 H-16), 7.32(1H, d, J=7.2 H-17), 6.95(2H, d, J=8.4 H-1,5), 6.75(3H, m, H-2,3,4),
5.92(1H, m, H-20), 5.18(2H,m, H-21),4.29(2H,d, J=15 H-19), 4.21(2H,s, H-10), 3.97(3H,s, H-
18), 3.19(2H,s, H-7). 13C NMR (CDCl3, δ ppm):167.3(C-11), 165.8(C-9), 161.2(C-8), 139.7(C-
12), 137.5(C-13), 133.7(C-14), 130.5(C-1,5),129.7(C-17), 129.0(C-6), 128.8(C-15), 127.7(C-3),
124.2(C-2,4), 122.8(C-20), 114.4(C-16), 108.8(C-21), 56.1(C-18) 40.2(C-19), 30.9(C-10),
21.1(C-7); Anal Calcd For C21H20N2O4S: C, 63.63; H, 5.05; N, 7.07 Found C, 63.57; H, 4.98; N,
7.01.
4-formyl-2-methoxyphenyl 2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)acetate 9e Yield: 85.5 FTIR
(KBr, νmax cm-1): 2983 (sp2 C-H stretch), 2942 (sp3 C-H stretch), 1692 (C=O), 1588 (C=C
1
aromatic), 1507 (C=N). HNMR (CDCl3, δ ppm): 9.75(1H, s H-19), 7.38(1H, s, H-14), 7.36(1H,
d, J=7.4 H-16), 7.32(1H, d, J=7.2 H-17), 7.30(3H, m, H-2,3,4), 6.95(2H,d, J=8.4 H-1,5),
13
4.11(2H ,s, H-10), 4.06(2H, s,H-7), 3.89(3H,s,H-18). C NMR (CDCl3, δ ppm):191.3(C-19),
157.8(C-11), 151.8(C-9), 147.2(C-8), 139.7(C-12), 136.5(C-13) 129.7(C-14), 129.5(C-17),
129.0(C-16), 128.8(C-15), 128.5(C-2,4), 127.7(C-1,5), 114(C-3), 108.8(C-6), 56.1(C-18),
33.9(C-10), 31.7(C-7); Anal Calcd For C19H16N2O5S: C, 59.37; H, 4.16; N, 7.29 Found C, 59.31;
H, 4.09; N, 7.22.
benzo[d][1,3]dioxol-5-yl 2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)acetate 9f Yield: 84.5%; FTIR
(KBr, νmax cm-1): 2954 (sp2 C-H stretch), 2893 (sp3 C-H stretch), 1755 (C=O), 1636 (C=C
aromatic), 1580 (C=N). 1HNMR (CDCl3, δ ppm): 7.46(2H, m,H-13,17), 7.44(1H, d, J=7.3 H-
16), 7.36(3H, m, H-2,3,4), 7.04(2H, d, J=9.0 H-1,5), 6.02(2H, s, H-18), 4.05(2H,s, H-10),
3.98(2H,s, H-7). 13C NMR (CDCl3, δ ppm):167.2(C-11), 165.5(C-9), 165.4(C-8), 149.7(C-15),
147.4(C-14), 137.6(C-12), 133.0(C-14), 129.0(C-16), 129.0(C-13), 128.8(C-17), 127.8(C-3),
120.6(C-2,4), 110.9(C-6), 105.4(C-18), 38.2(C-10), 33.4 (C-7); Anal Calcd For C18H14N2O5S: C,
58.37; H, 3.78; N, 7.56 Found C, 58.30; H, 3.70; N, 7.47.
19

2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)-N-phenylacetamide 9g Yield: 75.7%; m.p.142 ̊C FTIR
(KBr, νmax cm-1): 3064 (sp2 C-H stretch), 2968 (sp3 C-H stretch), 1661 (C=O amide), 1597 (C=C
1
aromatic), 1495 (C=N). HNMR (CDCl3, δ ppm): 9.17(1H, NH), 7.39(2H, d, J=7.2 H 13,17),
7.35(2H, d, J=8.7 H-1,5),7.32(3H, m, H-14,15,16), 6.87 (3H, m, H-2,3,4), , 4.20( 2H,s, H-10),
13
3.98(2H,s,H-7). C NMR (CDCl3, δ ppm): 167.2(C-11), 165.5(C-8), 165.4(C-9),137.0(C-12),
133.0(C-6), 129.5(C-13,17), 128.5(C-2,4),127.6(C-1,5),126.7(C-14,16), 124.6(C-15), 119.9.7(C-
3), 36.2(C-10), 31.8(C-7); Anal Calcd For C17H15N3O2S: C, 62.76; H, 4.61; N, 12.92 Found C,
62.69; H, 4.55; N, 12.85.
2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)-N-(3-(trifluoromethyl)phenyl)acetamide 9h Yield:
78.3%; m.p.75 °C FTIR (KBr, νmax cm-1): 3218 (sec. amide NH), 3031(sp2 C-H stretch),
1
2911(sp3 C-H stretch), 1686(C=O amide), 1660(C=C aromatic), 1601(C=N). - HNMR (CDCl3, δ
ppm): 9.20(1H, s, NH), 7.93(2H, s, H-13), 7.29(1H, d, J=7.8 H-15), 7.18(1H, m, H-16),
7.15(1H, d, J=8.2 H-17), 7.14(2H, d, J=7.4 H-1,5), 6.95(3H, m, H-2,3,4), 3.89(2H, s, H-10),
3.41(2H, s, H-7).13C NMR (CDCl3, δ ppm): 169.2(C-11), 166.6(C-8),165.4(C-9), 137.7(C-14),
134.4(C-13), 129.3(C-15), 129.0(C-16), 129.4(C-12), 129.1(C-17), 128.9(C-2,4), 127.3(C-1,5),
127.3(C-3), 126.7(C-6), 118.5(C-18), 39.1(C-10), 32.8(C-7); Anal Calcd For C18H14N2O2SF3: C,
54.96; H, 3.56; N, 10.68 Found C, 54.90; H, 3.49; N, 10.61.
2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)-N-(4-bromophenyl)acetamide 9i Yield: 72.5%; m.p.
248°C FTIR (KBr, νmax cm-1): 3362 (sec. amide NH), 3137(sp2 C-H stretch), 2976(sp3 C-H
1
stretch) 1677(C=O amide), 1608(C=C aromatic), 1556(C=N). HNMR (CDCl3, δ ppm):
9.24(1H, NH), 7.33(2H, d, J=7.5Hz, H-14,16), 7.29 (2H, d, J=7.2Hz, H-13,17),7.33(2H, m, H-
2,4), 7.32(2H, m, H-1,5), 6.72(1H, m, H-3), 3.99(2H, s, H-10), 3.29(2H, s, H-7). 13C NMR
(CDCl3, δ ppm): 167.2(C-11) ,165.5(C-8), 165.4(C-9), 137.6(C-12), 133.1(C-14,16), 129.0(C-
13,17), 128.0(C-1,5), 127.8(C-2,4), 124.8(C-3),119.6(C-15), 119.9(C-6), 36.2(C-10), 31.8(C-7);
Anal Calcd For C17H14N3O2SBr: C, 50.49; H, 3.46; N, 10.39 Found C, 50.43; H, 3.39; N, 10.32.
2-((5-benzyl-1,3,4-oxadiazol-2-yl)thio)-N-(4-chlorophenyl)acetamide 9j Yield: 88.3%; FTIR
(KBr, νmax cm-1): 3250 (sec. amide NH), 3197(sp2 C-H stretch), 2967(sp3 C-H stretch),
1674(C=O amide), 1647(C=C aromatic), 1587(C=N). 1HNMR (CDCl3, δ ppm): 9.24(1H, s, NH),
7.33(2H, d, J=7.5 H-14,16), 7.28(2H, d, J=8.2 H-13,17), 7.20 (2H, m, H-1,5), 7.19(2H, d,
J=7.2 H-2,4), 6.78(1H, m, H-3), 3.99(2H, s, H-10), 3.29(2H, s, H-7). 13C NMR (CDCl3, δ
ppm): 167.3(C-11), 165.6(C-8),165.4(C-9), 136.2(C-12), 132.9(C-14,16),129.8(C-15), 129.5(C-
20

6), 129.2(C-13,17), 128.8(C-1,5), 129.8(C-3) 129.2(C-2,4), 36.1(C-10), 31.8(C-7); Anal Calcd
For C17H14N3O2S: C, 56.74; H, 3.89; N, 11.68 Found C, 56.68; H, 3.83; 11.61.
4.2 Alkaline phosphatase inhibition assay
Previously described method was being used to measure the activity of calf IALP by
spectrophotometric assay [30]. The reaction mixture was pre-incubated for 10 min by adding 5
µL of CIALP (0.025 U/mL), 50 mMTris-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. After this 10 µL of substrate para
nitrophenylphosphate disodium salt was being added to initiate the reaction. At 37 °C the assay
mixture was incubated again for 30 min. OPT MAX, Tunable USA, 96-well micro plate reader
was being used to check the change in absorbance of released p-nitrophenolate and was being
monitored at 405 nm. Experiments were repeated in triplicate manner and potassium dihydrogen
phosphate was used as a reference inhibitor of CIALP.
4.3 Kinetic studies
The most potent inhibitor 9h was being selected for determination of the mechanism of
enzyme inhibition on the basis of IC50 value. We took inhibitor concentrations in the range of
0.0, 0.100, 0.200 and 0.400 µM. Similarly the substrate p-NPP was taken in concentrations of 10,
5, 2.5, 1.25 and 0.625mM. Same conditions were being maintained for pre-incubation time as
described in ALP inhibition assay. Enzyme was being added at per minute’s interval and
maximal initial velocities were also determined from initial linear portion of absorbance up to 10
minutes. The enzyme inhibition type was assayed by Lineweaver-Burk plot. The graph was
being plotted as 1/V (inverse of velocities) versus1/[S] (inverse of substrate concentration) mM-1.
The secondary plot of 1/V versus concentration of inhibitor was being determined by EI
dissociation constant Ki.
4.4 Free radical scavenging assay
2,2-diphenyl-1 picrylhydrazyl (DPPH) assay was being used to determine radical
scavenging activity using already reported method with slight modifications [31,32]. 100 µL of
DPPH (150 µM), 20 µL test compounds was used as assay solution and the volume was attuned
to 200 µL in each well with DMSO. At room temperature the reaction mixture was then
incubated for 30 minutes. For radical scavenging Vitamin C (Ascorbic acid) was used as a
reference inhibitor. The assay measurements were carried out by using OPTIMax, Tunable micro
21

plate reader at 517nm. The percent inhibition caused by the presence of tested inhibitors was
being calculated by comparing the reaction rates. Each concentration was analyzed in three
independent experiments run in triplicate.
4.5 Cytotoxicity evaluation using brine shrimp assay
4.5.1 Culturing and harvesting of Artemiasalina
Two uneven compartments were being made by rectangular dish with a plastic divider
with several holes in which Artemiasalina cysts were incubated for hatching. The container was
filled with 3.3% solution of artificial sea water. The dry yeast sprinkled into the larger darkened
compartment while the smaller compartment was illuminated with light and temperature was
maintained at 28°C. Artificial light and aeration was being used for incubation and after 24
hours, hatched A. salina cysts were transferred to fresh artificial seawater. It was again
incubated for 24 hours. From the lighted compartment the phototropic nauplii were being
collected using pipette.
4.5.2 Brine shrimps assay
Pasteur pipette was being used for counting macroscopically A. salinanauplii (20) against
a lighted background. It was then transferred into each sample vial and the solutions were diluted
with brine solution to 5mL with test compound. A drop of dry yeast suspension was added to
each vial as food and the vials were maintained under light. The surviving nauplii were counted
with the aid of a magnifying glass after 24 hours. The mean mortality at the three dose levels for
compound was determined and repeated in triplicate. Potassium dichromate was used as
reference standard. After 24 the LD50 were calculated by Probit analysis.
4.6 Molecular docking study
4.6.1 Retrieval of alkaline phosphate structure from PDB
The 3D structure of ALP from human placenta was being accessed from PDB having
PDBID 1EW2. UCSF Chimera 1.10.1 tool was being used to minimize the selected target
protein structure [33]. Stereo-chemical properties of ALP were generated by Protparam tool [34].
Discovery Studio 4.1 Client tool was being used for hydrophobicity and Ramachandran plot of
ALP [35]. Online server VADAR 1.8 was being used for prediction of protein architecture and
statistical percentage values of receptor proteins helices, beta-sheets, coils and turn [36].
22

4.6.2 Designing of ligands and molecular docking
ACD/Chem Sketch tool were used to sketch and draw synthesized ligands and were being
minimized by UCSF Chimera 1.10.1and then were access in mol format. Furthermore, UCSF
Chimera 1.10.1 tool was used for energy minimization of each ligand separately. 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, to obtain the good
structure conformation, Gasteiger charges were added using Dock Prep in ligand structure [37].
PyRx virtual screening tool with AutoDock VINA Wizard approach was employed for molecular
docking experiment on all the synthesized ligands against ALP [38]. Active region of the
targeted protein was being selected for better conformational position by using 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). The ligands 9a-j were docked separately against
ALP with default exhaustiveness value =8. The predicted docked complexes were evaluated on
the basis of lowest binding energy (Kcal/mol) values. Discovery Studio (2.1.0) and UCSF
Chimera 1.10.1 tool were used for 3D graphical depictions of all the docked complexes.
4.7 DFT calculations
The geometric and electronic computations were performed at DFT/B3LYP/6-
311G**(++)basis sets [39] for DFT/B3LYP/6-311G level optimized geometry using the Gaussian
03/ Gauss view 09 software.
4.8 DNA binding studies
The 0.1M NaH2PO4 and 0.1M Na2HPO4were mixed to prepare sodium phosphate buffer of
pH-7.4 which also acted as supporting electrolyte in electrochemistry. Calf thymus DNA was
extracted through standard Falcon method. The DNA sample with absorbance ratio(A260/A280) >
1.8 was considered sufficiently pure to be stored at -4°C for DNA interaction experiments and its
molarity was established from its absorbance at 260 nm against ε = 6600 M−1cm−1[40]. All the
reagents used were of analytical grade and every experiment was performed in triplicate. A 3
mM stock solution of the 9h in 50% methanol-phosphate buffer mixture was diluted to required
concentrations. The 9h (20 μM) solution was titrated against variable concentration (5, 10.......35
μM) of DNA and differential spectra was analyzed through Benesi-Hildebrand relation for Kb
calculation. The cyclic voltammograms of 9h (30mM in 50% methanol-phosphate buffer)
23

titration with DNA was recorded at 0.1 V/s vs. SCE at 310 K. The current change was exploited
through the following equation [41].
ͳ
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Conflict of Interest
The authors declare no conflict of interest.
24

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