Bisthioureas of pimelic acid and 4-methylsalicylic acid derivatives as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) and intestinal alkaline phosphatase (IAP): Synthesis and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2020.103996

Alkaline phosphatases (ALPs) are membrane bound metalloenzymes, distributed all over the body. Recent studies have revealed that by targeting ALPs can lead towards the treatment of many deadliest diseases including cardiac, cancerous and brain diseases. Thioureas and their derivatives are of considerable significance and are privileged scaffolds in medicinal chemistry. They show a wide range of pharmacological activities such as antibacterial, antiparasitic, anti-inflammatory and antioxidants etc. On the other hand, salicylic acid and its derivatives are known for its broad spectrum of activities. The work presented comprises of synthesis of N-acyl-N'-aryl substituted bisthioureas of pimelic acid (1–7) and 3,5-dimethyl pyrazole (11), 1-aroyl-3-aryl thiourea (12) and 1,3,4-oxadiazole (13) derivatives of 4-methyl salicylic acid. Structures of all the synthesized compounds were characterized by FT-IR and ¹H NMR spectroscopic analysis. Synthesized compounds were evaluated for their alkaline phosphatases inhibition potential and exhibited high potency as well as selectivity towards h-TNAP and h-IAP. Compound 7 and 12 which were the bisthiourea derivative of pimmelic acid and thiourea derivative of 4-methyl salicylic acid, respectively, showed excellent selectivity against h-TNAP and h-IAP, respectively.

Full text of DOI:10.1016/j.bioorg.2020.103996

Journal Pre-proofs
Bisthioureas of pimelic acid and 4-methylsalicylic acid derivatives as selec-
tive inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) and intesti-
nal alkaline phosphatase (IAP): Synthesis and molecular docking studies
Amara Mumtaz, Kiran Saeed, Abid Mahmood, Sumera Zaib, Aamer Saeed,
Julie Pelletier, Jean Sévigny, Jamshed Iqbal
PII:
S0045-2068(20)31293-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.103996
YBIOO 103996
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
18 April 2020
27 May 2020
2 June 2020
Please cite this article as: A. Mumtaz, K. Saeed, A. Mahmood, S. Zaib, A. Saeed, J. Pelletier, J. Sévigny, J. Iqbal,
Bisthioureas of pimelic acid and 4-methylsalicylic acid derivatives as selective inhibitors of tissue-nonspecific
alkaline phosphatase (TNAP) and intestinal alkaline phosphatase (IAP): Synthesis and molecular docking studies,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103996
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Bisthioureas of pimelic acid and 4-methylsalicylic acid derivatives as selective
inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) and intestinal
alkaline phosphatase (IAP): Synthesis and molecular docking studies
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Amara Mumtaz1#*, Kiran Saeed1#, Abid Mahmood2, Sumera Zaib2, Aamer Saeed3, Julie
Pelletier4, Jean Sévigny4,5, Jamshed Iqbal2*
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1Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-
22060, Pakistan.
2Center for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan
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3Department of Chemistry, Quaid-i-Azam University Islamabad, Pakistan.
4Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada.
5Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, G1V 0A6, Canada.
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*Corresponding author: Dr. Amara Mumtaz, Department of Chemistry, COMSATS University,
Abbottabad Campus Ph No. +923005316570, email: amaramumtaz@ciit.net.pk. Prof. Dr.
Jamshed Iqbal, Centre for Advanced Drug Research, COMSATS University, Abbottabad Campus,
22060,
Pakistan,
Ph
#
No.
+923365101651,
Email:
drjamshed@cuiatd.edu.pk;
jamshediqb@gmail.com. equal contribution: Dr Amara Mumtaz, KiranSaeed, Department of
Chemistry, COMSATS University, Abbottabad Campus Ph No. +923125091628, email:
kiransaeed8310@gmail.com
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Bisthioureas of pimelic acid and 4-methylsalicylic acid derivatives as selective
inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) and intestinal
alkaline phosphatase (IAP): Synthesis and molecular docking studies
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Amara Mumtaz1*, Kiran Saeed1, Abid Mahmood2, Sumera Zaib2, Aamer Saeed3, Julie Pelletier4,
Jean Sévigny4,5, Jamshed Iqbal2*
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1Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-
22060, Pakistan.
2Center for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan
3Department of Chemistry, Quaid-i-Azam University Islamabad, Pakistan.
4Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada.
5Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, G1V 0A6, Canada.
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Abstract:
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Alkaline phosphatases (ALPs) are membrane bound metalloenzymes, distributed all over the body.
Recent studies have revealed that by targeting ALPs can lead towards the treatment of many
deadliest diseases including cardiac, cancerous and brain diseases. Thioureas and their derivatives
are of considerable significance and are privileged scaffolds in medicinal chemistry. They show a
wide range of pharmacological activities such as antibacterial, antiparasitic, anti-inflammatory and
antioxidants etc. On the other hand, salicylic acid and its derivatives are known for its broad
spectrum of activities. The work presented comprises of synthesis of N-acyl-N'-aryl substituted
bisthioureas of pimelic acid (1-7) and 3,5-dimethyl pyrazole (11), 1-aroyl-3-aryl thiourea (12) and
1,3,4-oxadiazole (13) derivatives of 4-methyl salicylic acid. Structures of all the synthesized
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compounds were characterized by FT-IR and H NMR spectroscopic analysis. Synthesized
compounds were evaluated for their alkaline phosphatases inhibition potential and exhibited high
potency as well as selectivity towards h-TNAP and h-IAP. Compound 7 and 12 which were the
bisthiourea derivative of pimmelic acid and thiourea derivative of 4-methyl salicylic acid,
respectively, showed excellent selectivity against h-TNAP and h-IAP, respectively.
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Key Words: Thiourea, pimelic acid, salicylic acid, alkaline phosphatase inhibitors
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1. Introduction:
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Alkaline phosphatases (ALPs) are membrane bound metalloenzymes and found in most of the
living organisms including prokaryotes and eukaryotes. Mammalian ALPs have low sequence
similarities with the E. Coli, but the residues involved in the catalytic activity of active sites were
found to be identical. Therefore, catalytic mechanism of both the enzymes is similar [1].
Mammalian ALPs are classified based on distribution in different tissues and organs. In humans,
expression of some ALPs is specifically confined to certain tissue or organs and designated as
tissue specific alkaline phosphatases [2]. Intestinal ALPs (IAP), placental ALPs (PALP) and germ
cells ALPs (GCAP) are tissue specific isozymes of ALPs. Tissue specific ALPs have different heat
stabilities and competitive mode of inhibition. Tissue specific isozymes of ALPs differ based on
level of post-translational glycosylation which affect their catalytic efficacy [3]. A fourth isozyme
is ubiquitous, expressed in liver, bones and kidneys, known to be tissue non-specific ALP (TNAP)
[4]. ALPs catalyze the nucleotides and liberate inorganic phosphates (Pi). Such cleavage of
nucleotides results in generation of adenosine, an agonist for P1 receptors in purinergic signaling
pathways. Purinergic signaling is known to involve in various physiological and patho-
physiological conditions [5]. Alkaline pH is essential for optimal activity of isozymes. ALPs
exhibit wide range of substrate specificity including inorganic polyphosphates, phosphatidates,
glucose-phosphates and p-nitrophenyl phosphates [6]. TNAP is the most abundant isozyme,
comprising about more than 90% of all other circulating ALPs isozymes. The highest levels of
TNAP has been found in bones, liver and kidneys. Active pocket of TNAP possesses two zinc ions
and a magnesium ion as cofactors as well as a calcium ion in specific calcium binding sites, thus
regulating the activity of enzyme [7]. All isozymes of ALPs are ectoenzymes and anchor to the
cell membrane through glycosylphosphatidylinositol (GPI) moiety. Only the circulation level of
ALPs defines the activity level of enzyme. It has been reported that GPI-specific phospholipase-
D release the anchored enzyme into serum. TNAP causes bone mineralization by providing
inorganic phosphate (Pi) and inactivation of calcification inhibitors [8]. TNAP and IAP detoxify
the circulating endotoxins through dephosphorylation. Under-expression of TNAP results in
genetic disorder, like rickets/osteomalacia, known as hypophosphatasia. Level of circulating
TNAP has been reported to increase in bone diseases, cardiovascular abnormalities, kidney
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dysfunctions and genetic disorders [9]. TNAP seem to be a novel target for treatment of vascular
calcification and diabetes mellitus type 2 and chronic kidney disease (CKD). Intestinal AP
prevents the accumulation of unwanted compounds from small intestine by the absorption of
lipopolysaccharide (LPS). Excess level of IAP may leads to inflammatory bowel disease (IBD)
[10]. Variety of salicylic acid derivatives are known as COX inhibitors and have been reported for
treatment of IBD [11]. Salicylic acid derivatives of thiourea and sulfur containing heterocyclic
compounds have been investigated for biological activities and known to possess good
antimycobacterial activity [12]. Levamisole and theophylline are well known inhibitors of TNAP
with Ki values of 16 µM and 82 µM, respectively [13]. Thiourea derivatives have been well
reported for broad range of biological activities in pharmaceutical industry including antibacterial,
antiparasitic, antifungal, antioxidant, anticancer, anti-HIV activities, urease inhibition, carbonic
anhydrase inhibition, butyrylcholinesterase and acetylcholinesterase inhibition [14]. Several
thiourea derivatives have been synthesized and characterized for their biological activities while
bis(thiourea) are relatively less reported for their biological potential. Incorporation of alkyl chain
in thiourea derivatives was reported to possess enhanced biological activities due to increased
lipophilicity [15]. Presence of free N-H allows the further derivatization of thiourea compounds
with heterocycles. Carbonyl and thiocarbonyl moieties provide hard and soft donors for metal
complexes formation [14].
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In the present study, bis(thiourea) derivatives of pimmelic acid (1-7) and 3,5-dimethyl pyrazole
(11), 1-aroyl-3-aryl thiourea (12) and 1,3,4-oxadiazole (13) derivatives of 4-methyl salicylic acid
were synthesized and evaluated for selective inhibitory potential for h-TNAP and h-IAP.
Moreover, molecular docking studies were carried out to investigate the role of selective
compounds at catalytic site of enzymes.
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2. Results & Discussion:
Chemistry
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The work presented describes the synthesis of bis(thiourea) derivatives of pimmelic acid (1-7),
3,5-dimethyl pyrazole (11), 1-aroyl-3-aryl thiourea (12) and 1,3,4-oxadiazole (13) derivatives of
4-methyl salicylic acid were synthesized and evaluated for selective inhibitory potential for h-
TNAP and h-IAP. For the synthesis of bisthioureas (1-7) pimelic acid was first converted into its
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respective acid chloride by reaction with thionyl chloride which was further reacted with solution
of potassium isothiocyanate in dry acetone followed by reaction with different substituted anilines
for appropriate time till completion of reaction. Progress of reaction was monitored by using TLC.
Thioureas were obtained by adding cold water to reaction mixture and purified using column
chromatography. FT-IR spectra of compounds showed characteristic stretching band for NH
around 3200-3300 cm-1, band for C=O from 1650-1695 cm-1, C=S stretching band at around 1220-
1254 cm-1 and aliphatic C-H stretching band around 2900 cm-1 confirmed the synthesis of
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compounds (1-7). In the HNMR spectrum of compounds 1-7 and 12 appearance of two
characteristic singlets at 12.60 ppm (CONH) and 11.46 ppm (CSNH) confirmed the presence of
two NH groups which are the characteristics of thiourea [14]. 3,5-Dimethylpyrazole (11) and 1,3,4-
oxadiazole (13) derivatives of 4-methylsalicylic acid were synthesized starting from the common
precursor hydrazide (9). Condensation of 9 with acetyl acetone led toward the compound 11 while
reaction of 9 with carbondisulfide, potassium hydroxide followed by acidic workup finished on
compound 13. FT-IR spectrum of compound (11) showed characteristic stretching band for C-O
at 1299 cm-1, appearance of characteristic band of C=O at 1650 cm-1 and =C-H stretching band at
3000 cm-1 confirmed the synthesis of 3,5-dimethyl-1H-pyrazol-1-yl)(4-methyl-2-(4nitro
benzyl)oxy)phenyl)methanone (11), while in the 1HNMR spectrum appearance of a singlet at 6.30
ppm and two singlets at 2.35 ppm and 2.51 due to methyl groups attached to pyrazole ring
confirmed
the
synthesis
of
the
3,5-dimethyl-1H-pyrazol-1-yl)(4-methyl-2-(4nitro
benzyl)oxy)phenyl)methanone (11) [16]. In the FT-IR spectrum of 5-(4-methyl-2-(4-
nitrobenzyl)oxy)phenyl)-1,3,4-oxadiazole-2(3H)-thione (13) absorption band at 3327 cm-1 isthe
characteristic band for N-H stretching while the appearance of C=N bands at 1605 cm-1, C=S
stretching band at 1170 cm-1 and Ar-CH absorption band at 3000 cm-1 confirmed the synthesis of
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compound. In the HNMR spectrum, broad singlet at 14.9 ppm confirmed the synthesis of
compound (13). Doublets appearing at 8.24 ppm and 7.86 ppm showed the presence of protons
attached to benzene ring [17].
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Structure-activity relationship (SAR):
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Thioureas also known as thiocarbanides are rich source of nitrogen. Such derivatives have wide
range of application in medicinal chemistry. These compounds are known to possess antibacterial,
antifungal, anticancer, antioxidant, anti-diabetics as well as DNA binding properties [18]. The data
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presented in the Table 1 provide an overview about the inhibitory potential as well as selectivity
of synthesized compounds. Compounds were found to show selective inhibitory activities for h-
TNAP and h-IAP. Among bis(thiourea) derivatives, compound 7 exhibited higher inhibitory
activity for h-TNAP, represented by IC50 value of 4.63±0.31 µM which is about five times more
than positive control levamisole (IC50 value: 20.2±1.90). Compound 7was also demonstrated
inhibition for h-IAP with an IC50 value of 6.72±0.94 µM. Presence of 4-nitrophenyl moiety at the
both ends of compound 7conferred this potency as well as dual inhibition. Compounds 3, 4and5
demonstrated higher selective inhibitory activity for h-TNAP with an IC50 values of 15.4±0.75
µM, 5.28±0.51 µM and 15.9±0.31 µM, respectively. These compounds possess dichlorophenyl
moieties at their terminal ends that might be responsible potency as well as selectivity for h-TNAP.
It is evident from the structures of 3, 4 and 5 compounds, change in position of substitution of
chloro group on terminal benzene has no influence on the selectivity of compounds towards h-
TNAP. Even reducing the di-substitution to mono-substitution of chloro group has no effect on
the selectivity of these compounds. Thus chloro group substitution on terminal benzene rings
confer a selectivity towards h-TNAP. Bis(thiourea) derivatives containing dimethoxyphenyl
(compound 1) and methoxyphenyl (compound 2) moieties were expressed selectivity towards h-
IAP with IC50 value of 1.01±0.11 µM and 9.46±1.23 µM, respectively. Compound 1 was most
potent and selective compound for h-IAP among all the tested compounds that indicate the
importance of number of methoxy substitutions on phenyl rings. p-tolyl group in bis(thiourea)
derivative (6) was also proved to play significant role to confer selectivity towards h-IAP isozyme.
As replacement of methoxy group in 1 and 2 compounds with methyl moieties diminish the
selectivity of both compounds. IC50 values of compound 6 were 11.6±0.55 µM and 2.80±0.94 µM
for h-TNAP and h-IAP, respectively. 4-methyl salicylic acid derivatives were also expressed
selectivity and potency for both isozymes of alkaline phosphatase. Among salicylic acid
derivatives, 4-methyl-2-((4-nitrobenzyl) oxy)-N-(o-tolylcarbamothioyl)benzamide (12) was found
to possess highest potency and selectivity for h-IAP with IC50 value of 1.50±0.24 µM, in
comparison to positive control (L-Phenylalanine: 100±3.15 µM). Chemical structure of compound
12 possesses all those features that made it selective for h-IAP, including the presence of tolyl-
group, 4-nitrophenyl group and carbamothioylbenzamide moiety. 5-(4-Methyl-2-((4-nitrobenzyl)
oxy) phenyl)-1,3,4-oxadiazole-2(3H)-thione (13) exhibited inhibition for h-TNAP with IC50 value
of 4.89±0.84 which is comparable to inhibitory potential of standard inhibitor levamisole. This
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selectivity of 13might be due to 1,3,4-oxadiazole-2(3H)-thione substitution, based on literature
available. 1,3,4-oxadiazole-2(3H)-thione derivatives were found to possess wide range of
biological application including potent inhibitors of various enzymes. These derivatives have the
ability to inhibit carbonic anhydrase, lipoxygenase, succinate dehydrogenase, monoamine oxidase
and cyclooxygenase [19]. Rest of the synthesized derivatives of 4-methyl salicylic acid were
exhibited less than 50 percent of inhibitory activities for both isozymes of alkaline phosphatase.
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Table 1. Alkaline phosphatase inhibition and docking scores of pimelic acid and 4-
methylsalicylic acid derivatives.
Compound
h-TNAP
h-IAP
h-TNAP
h-IAP
(IC50±SEM)a/ %Inhibition
docking score by FlexX for top pose
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2
3
4
5
6
7
8
9
10
11
12
13
–11.29
–13.32
–21.57
–27.88
–20.16
–24.64
–30.25
–10.09
–10.92
–9.27
–28.54
–21.19
–12.65
–11.38
–10.24
–24.57
–22.80
–9.82
–14.25
–12.56
–11.23
–27.34
–13.61
21.6%
32.5%
1.01 ± 0.11
9.46 ± 0.34
36.3%
28.8%
37.4%
2.80 ± 0.03
6.72 ± 0.02
21.6%
15.4 ± 0.75
5.28 ± 0.51
15.9 ± 0.31
11.6 ± 0.55
4.63 ± 0.31
27.6%
30.2%
25.4%
34.8%
38.3%
23.4%
26.7%
30.3%
–13.21
–12.86
–26.37
1.50 ± 0.24
40.23%
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4.89 ± 0.84
20.2 ± 1.90
Levamisole
L-phenylalanine
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100 ± 3.15
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a IC50 represents concentration at which the 50% of the enzyme activity was inhibited. All the
values were expressed as IC50 ± SEM (standard error mean), n=3.
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Molecular modeling investigation
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For further detailed insight into the activities of synthetic compounds (1-13) for human alkaline
phosphatases (tissue non-specific and intestinal), molecular modeling was performed for
selective and most active analogues. LeadIT software was used for carrying out the modeling
analysis of selected compounds. The crystal structures of the target proteins were unavailable
at RCSB protein databank, hence previously reported homology models were used.
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Levamisole ((S)-6-phenyl-2,3,5,6-tetrahydroimidazo[2,1-b]thiazole) was used as positive
control in the biological assay and was docked inside the binding site of tissue non-specific
APs (Figure 1). The resultant interactions involve conventional hydrogen bonding (1.86 Å) by
nitrogen of imidazole ring with His437 and π-alkyl linkage of the same residue (3.85 Å) with
thiazole ring. Val90 was at alkyl linkage (4.61 Å) with the thiazole ring. Three additional
carbon H-bonds were noticed by Glu108 (2.60 Å), Pro91 (2.76 Å) and Thr436 (2.45 Å).
Compound 4, the most potent and selective inhibitor exhibited several interactions within the
binding pocket of h-TNAP as represented in Figure 1a. The compound contain bis(2,6-
dichlorophenyl)carbamothioylheptanediamide and shown several hydrogen bonds with
important residues of the active site of human tissue non-specific alkaline phosphatase.
Hydrogen bonds were formed by His434 (2.75 Å) with oxygen, His437 (1.64 Å) by amide
group, His437 (2.86 Å) and Thr436 (3.36 Å) with oxygen of carbamothioylheptanediamide
moiety. Moreover, His437 was also involved in π-lone pair interaction with oxygen (2.97 Å)
and π-alkyl linkage with one of the chloro group (4.29 Å). Additionally, Asp320 showed pi-
anion interactions with phenyl ring (4.57 Å). His324 formed 2 π-alkyl linkage with (3.85 Å
and 4.96 Å) with both the chloro groups of same phenyl ring. Amino acid His434 in addition
to hydrogen bond, was found to involve in π-sulfur interaction (5.20 Å) and 2 π-alkyl
interactions one with chloro group (4.29 Å) at one phenyl ring, while second π-alkyl linkage
with chloro group (4.48 Å) of another phenyl ring. When the interaction analysis of compound
13 was performed, it was noted that His324 (4.23 Å) was at π-π stacked interaction with
nitrobenzyl ring and π-π T-shaped interactions at 5.93 Å with 4-methyl phenyl ring. Moreover,
His434 (3.37 Å) also formed π-πstacked with 4-methyl phenyl ring. 2 hydrogen bonds were
formed by Ser93 (3.18 Å and 2.46 Å) with both the oxygens of nitro group. Similarly, Arg167
(2.39 Å) was making H bond with oxygen of nitro group. In addition to these interactions,
metal interactions were noted by nitrobenzyl ring Zn ions (2.89 Å and 3.92 Å) and withMg ion
(4.67 Å).In case of compound 7, His437 exhibited π-π T-shaped 4.24 Å with 1 of the
nirophenyl group and H bond at a distance of 2.63 Å with oxygen atom of the
carbamothioyl)heptanediamide. Similarly, hydrogen bonds were noticed by His434 (1.75 Å),
Arg151 (2.84 Å) and Thr436 (2.50 Å) with oxygen atom of the carbamothioyl)heptanediamide.
Moreover, π-π T-shaped interactions (5.71 Å) were observed by His321 with 1 of the phenyl
rings. Ser93 was at distance of 2.50 Å making H bond with oxygen atom of other nitrophenyl
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ring. π-π stacked interaction was noticed by phenyl ring with His324 (5.03 Å). Additionally,
metal interactions were found with Mg (4.56 Å) and both the Zn ions (3.96 Å and 3.26 Å). The
docking analysis revealed that the most active inhibitor exhibits the important interactions
within the binding pocket of TNAP and maybe responsible for the inhibitory activity of the
compound towards the enzyme. Table 1 showed the docking scores of all the compounds
against TNAP and IAP.
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(a)
(b)
(c)
(d)
Figure 1. Plausible binding modes of compounds 4 (a); 13 (b); 7 (c) and Levamisole (d) inside tissue
non-specific alkaline phosphatase model
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Molecular docking analysis of L-phenylalanine inside the binding pocket of human intestinal
alkaline phosphatase revealed that π-π stacked interactions were shown by phenyl ring with His320
(4.32 Å) and π-π T shaped with His432 (5.55 Å). His358 showed a hydrogen bond (2.77 Å) and a
carbon H bond (2.18 Å) with phenylalanine. Amino acid Ser92 made a hydrogen bond (2.16 Å)
with oxygen of the alanine group and the same oxygen was involved in a metal acceptor interaction
with one of the Zinc ion (2.39 Å). Compound 1 (the most active and selective inhibitor of h-IAP)
exhibited extensive network of hydrogen bonds like in one of the 3,4-dimethoxyphenyl ring
formed H bond with Arg150 (3.09 Å and 2.90 Å), and the other one showed H-bonding with
Arg314 (3.06 Å) by one methoxy group while with His279 (2.98 Å) by second methoxy group.
However, carbamothioylheptanediamide moiety formed 3 hydrogen bonds one with Tyr276 (2.62
Å), second with Glu321 (1.96 Å) and third by sulfur of thiol group with Arg166 (3.79 Å).Other
important interactions were π-sulfur bonds of thiol group with His317 (5.26 Å) and His153 (4.87
Å). Both the phenyl rings showed π-π T shaped interactions with amino acid His153 (4.74 Å) and
π-π stacked interactions with His279 (5.39 Å). Moreover, metal acceptor interactions were also
shown by oxygen (2.40Å) and (3.21 Å) sulfur atoms of carbamothioyl)heptanediamide moiety
with Zinc ion. Moreover, a metal acceptor interaction was observed with zinc ion. Compound 12
showed significant number of hydrogen bonds by Arg150 (2.87 Å and 2.95 Å) with benzamide
group and by Ser92 (2.99 Å and 3.09 Å) with 4-nitrobenzyl group. Moreover, other important
interactions like π-π stacked with His317 (4.02 Å) and π-πT-shaped with His153 (5.80 Å) were
also observed. In case of metal interactions, compound 12 showed interaction with both the Zn
ions (3.12 Å and 3.98 Å). For compound 7showed hydrogen bond by Glu321 (2.16 Å) with
heptanediamide moiety, Ser92 (3.16 Å) with nitro phenyl group, Tyr276 (2.57 Å) and Arg150
(3.04 Å) with oxygen atom. π-sulfur interactions were noticed by His153 (4.81 Å) sulfur atom in
the compound. Moreover, Tyr276 (5.76 Å) showed π-π T-shaped interactions. Additionally, metal
interactions were noticed by Zn ions (3.25 Å and 3.84 Å). The structure-activity relationship and
the docking studies of identified potent inhibitors provide an outstanding platform for further
development of alkaline phosphatase inhibitors. The results of docking studies for the selected
compounds were descriptive of the in-vitro enzyme inhibitory activity results, and the plausible
binding poses elucidated the binding modes of these analogues.
269
10

(a)
(b)
(c)
(d)
Figure 2. Plausible binding modes of compounds 1(a); 12 (b); 7(c) and L-phenylalanine (d) inside intestinal
alkaline phosphatase model
270
271
3. Conclusions:
272
273
274
275
276
277
278
279
Synthesis of bis(thiourea) derivatives of pimmelic acid (1-7), and 3,5-dimethyl pyrazole (11), 1-
aroyl-3-aryl thiourea (12) and 1,3,4-oxadiazole (13) derivatives of 4-methyl salicylic acid were
synthesized and evaluated for selective inhibitory potential for h-TNAP and h-IAP. Among
bis(thiourea) derivatives, 7 exhibited higher inhibitory activity for h-TNAP, represented by IC50
value of 4.63±0.31 µM which is about four times more than positive control levamisole (IC50
value: 19.2±0.01. Compounds 3, 4 and 5 demonstrated higher selective inhibitory activity for h-
TNAP with IC50 values of 15.4±0.75 µM, 5.28±0.51 µM and 15.9±0.31 µM, respectively. Among
salicylic
acid
derivatives,
4-methyl-2-((4-nitrobenzyl)
oxy)-N-(o-
11

280
281
282
283
284
285
286
tolylcarbamothioyl)benzamide(12) was found to possess highest potency and selectivity for h-IAP
with IC50 value of 1.50±0.24 µM, in comparison to positive control (L-phenylalanine: 80.1±0.01
µM). Compound (13), 1,3,4-oxadiazole-2-thione derivative of salicylic acid exhibited inhibition
for h-TNAP with IC50 value of 4.89±0.84 which is comparable to inhibitory potential of standard
inhibitor levamisole. Further insight of these selective and potent compounds were investigated by
molecular docking analysis and it was observed that selected compounds exhibit key interactions
with important residues of active site in the structures of enzymes.
287
288
4. Experimental:
Material & Method:
289
290
291
292
293
294
295
296
297
298
299
300
301
302
Pimelic acid, thionyl chloride, potassium thiocyanate, p-methoxy aniline, 3,4 dimethoxy aniline,
p-chloroaniline, 2,6-dichloroaniline, 2,5-dichloroaniline, p-toluidine, p-nitroaniline, 4-methyl
salicylic acid, p-nitrobenzyl bromide, potassium carbonate, sodium bicarbonate, sodium hydroxide
were purchased from sigma Aldrich. Acetone, ethyl acetate, n-Hexane, methanol, acetonitrile,
ethanol, dichloromethane, carbon disulphide were brands of Riedel-de-Haen. All solvents were
distilled before use. The progress of reaction was observed with the help of chromatography
technique using TLC silica gel 60 F254 (coated on aluminum sheet) produced by MERCK. For
development of chromatogram different solvent systems were used including n-Hexane, ethyl
acetate and methanol in different ratios. Melting point was observed in organic and biological lab
COMSATS University Islamabad, Abbottabad campus on SMP 20 digital Melting point apparatus
OE/Digi(08-09)169/1 DMPA 09-01. Infrared spectra of all the samples were recorded on Hitachi
Infrared spectrometer model 270 in KBr pellets Quaid-e-Azam University Islamabad. 1H NMR δ
(ppm) spectra were recorded in CDCl3 and DMSO- d6 by using Bruker AM-400 NMR
spectrometer at 400 MHz in COMSATS University Islamabad, Abbottabad Campus.
303
Synthesis of N-aryl-N'-acyl-bisthioureas of pimelic acid (1-7)
304
305
306
307
308
The acid chloride was prepared by treating pimelic acid (3 mmol, 0.5 g) with SOCl2 (9 mmol, 0.7
mL) on gentle heating for 3-4 hours. The pimeloyl chloride was reacted drop by drop with the
solution of potassium thiocyanate (6 mmol, 0.6 g) in dry acetone. The resulting mixture was
subjected to stirring for 45 mins accompanied by the addition of solution of substituted anilines
which led to conversion into thioureas on heating under reflux. Upon completion, small amount
12

309
310
311
of ice-cold water was poured to precipitate out the product. Purification of all the products was
carried out by using coloum chromatography. Synthesized compounds were obtained in yield (55-
80%) [14].
O
O
O
O
SOCl2
SO2
HCl
HO
OH
Cl
Cl
2-4 hrs reflux
KSCN
dry acetone
35 min
stirring
O
O
S
O
O
S
R-NH2
dry acetone 1h reflux
S
C N
N C S
HN
N
N
NH
H
H
R
R
1-7
312
313
Scheme 1. Synthesis of N-acyl-N'-aroyl-thioureas of pimelic acid
314
315
N1,N7-bis(3,4-dimethoxyphenyl)carbamothioyl)heptanediamide (1)
316
317
318
319
320
321
Yield: 60 %; Rf=0.3 (n-Hexane/EtOAc/MeOH, 2:7:1); m.p: 200-204 oC; FT-IR (KBr pellets) (ῡ,
cm-1): 3273 (NH stretch), 2941 (CH stretch), 1651 (C=O Stretch), 1233 (C=S), 1171 (CN); (1H
NMR (400 MHz, DMSO-d6) δ (ppm): 12.60 (s, 1H, NH), 11.69 (s, 1H, OH), 11.46 (s, 1H, NH),
7.12 (d, J = 8.7 Hz, 1H, Ar-H-5), 6.80 (dd, J = 8.6, 2.2 Hz, 1H, Ar-H-6), 6.67 (d, J = 2.1 Hz, 1H,
Ar-H-2), 3.80 (s, 3H, OCH3), 3.69 (s, 3H, OCH3), 2.45 (t, J = 7.6 Hz, 2H, H-2"), 2.20 (t, J = 7.3
Hz, 2H, H-6"), 1.49-1.58 (m, 4H, 2CH2,H-3", H-5"), 1.30-1.27 (m, 2H, H-4").
322
N1,N7-bis(4-methoxyphenyl)carbamothioyl)heptanediamide (2)
323
324
325
326
327
Yield: 55 %; Rf =0.3 (n-Hexane/EtOAc/MeOH = 2:7:1); m.p: 182-185 oC; FT-IR (KBr pellets) (ῡ,
1
cm-1): 3273 (NH stretch), 2941 (CH stretch), 1651 (C=O Stretch), 1233 (C=S), 1171 (CN); H
NMR (400 MHz, DMSO-d6) δ (ppm): 12.35 (s, 2H, NH), 11.38 (s, 2H, NH), 7.50 (d, J = 8.6 Hz,
4H, Ar-H-2, Ar-H-6), 6.49 (d, J = 8.6 Hz, 4H, Ar-H-3, Ar-H-5), 3.37 (s, 6H, 2OCH3), 2.21 (t, J =
7.3 Hz, 4H, 2CH2, H-1"), 1.50-1.58 (m, 4H, 2CH2, H-2"), 1.30-1.27 (m, 2H, CH2, H-3").
328
N1,N7-bis (2,5 dichlorophenyl)carbamothioyl)heptanediamide) (3)
13

o
329
330
331
332
333
Yield: 80 %; Rf = 0.5 (n-Hexane/EtOAc, 7:3); m.p: 176-179 C; FT-IR (KBr pellets) (ῡ, cm-1):
3188 (NH stretch), 2948 (CH stretch), 1649 (C=O Stretch), 1242 (C=S), 1171 (CN); 1H NMR (400
MHz, DMSO-d6) δ (ppm): 12.46 (s, 2H, NH), 11.64 (s, 2H, NH), 7.89 (s, 2H, Ar-H-6), 7.48 (d, J
= 8.0 Hz, 2H, Ar-H-3), 7.22 (d, J = 8.0 Hz, 2H, Ar-H-4), 2.29 (t, J = 7.8 Hz, 4H, 2CH2, H-1"),
1.53-1.58 (m, 4H, 2CH2, H-2"), 1.23-1.29 (m, 2H, CH2, H-3").
334
N1,N7-bis (2,6 dichlorophenyl)carbamothioyl)heptanediamide (4)
335
336
337
338
339
Yield: 65 %; Rf = 0.5 (n-Hexane/EtOAc = 7:3); m.p: 175-178 oC; FT-IR (KBr pellets) (ῡ, cm-1):
1
3273 (NH stretch), 2941 (CH stretch), 1651 (C=O Stretch), 1233 (C=S), 1171 (CN); H NMR (
400 MHz, DMSO-d6 ) δ (ppm): 11.80 (s, 2H, NH), 11.60 (s, 2H, NH), 7.50 (d, J = 8.1 Hz, 4H, Ar-
H-3 & Ar-H-5), 7.33 (t, J = 8.0 Hz, 2H, Ar-H-4), 2.21 (t, J = 7.28, Hz, 4H, 2CH2, H-1"), 1.58-1.51
(m, 4H, 2CH2, H-2"), 1.29-1.35 (m,2H, CH2, H-3").
340
N1,N7-bis((4-chlorophenyl)carbamothioyl)heptanediamide (5)
341
342
343
344
345
Yield: 65 %; Rf = 0.4 (n-Hexane/EtOAc = 7:3); m.p: 203-206 oC; FT-IR (KBr pellets) (ῡ, cm-1):
3300 (NH stretch), 2919 (CH stretch), 1665 (C=O Stretch), 1246 (C=S), 1171 (CN); 1H NMR (400
MHz, DMSO-d6) δ (ppm): 12.64 (s, 2H, NH), 11.40 (s, 2H, NH), 7.69 (d, J = 8.0 Hz, 4H ,Ar-H-
3, Ar-H-5), 7.37 (d, J = 8.0 Hz, 4H, Ar-H-2, Ar-H-6), 2.29 (t, J = 7.8 Hz, 4H, 2CH2, H-1"), 1.48-
1.53 (m, 4H, 2CH2, H-2"), 1.29-1.23 (m ,2H, CH2, H-3").
346
N1,N7-bis(p-tolylcarbamothioyl)heptanediamide (6)
347
348
349
350
351
Yield: 60 %; Rf = 0.5 (n-Hexane/EtOAc = 7:3); m.p: 212-214 oC; FT-IR (KBr pellets), (ῡ, cm-1):
1
3304 (NH stretch), 2939, (CH stretch), 1694 (C=O Stretch), 1254 (C=S), 1171 (CN); H NMR
(400 MHz, DMSO-d6) δ (ppm): 12.25 (s, 2H, NH), 10.01 (s, 2H, NH), 7.72 (d, J = 8.2 Hz, 4H, Ar-
H-2 & Ar-H-6), 7.34 (d, J = 8.1, 4H, Ar-H-3 & Ar-H-5), 2.53 (s, 6H, 2CH3), 2.40 (t, J = 7.3 Hz,
4H, 2CH2, H-1"), 1.74-1.87 (m, 4H, 2CH2, H-2"), 1.59-1.51 (m, 2H, CH2, H-3").
352
N1,N7-bis(p-nitrocarbamothioyl)heptanediamide(7)
353
354
355
Yield: 69 %; Rf = 0.4 (n-Hexane/EtOAc = 7:3); m.p: blacken at 222 oC; FT-IR (KBr pellets), (ῡ,
cm-1): 3185 (NH stretch), 2968, (CH stretch), 1692 (C=O Stretch), 1592, 1429, 1264 (C=S),
1171 (CN).
14

356
Synthesis of ester from 4-methyl salicylic acid
357
358
359
360
361
362
Ester of 4-methyl salicylic acid was prepared by refluxing methanolic solution of salicylic acid
(6.57 mmol, 0.5g) with catalytic amount of concentrated sulphuric acid for 12 hours. Excess acid
was quenched using sodium bicarbonate solution (10 %). Ethyl acetate (20 mL × 3) was used to
extract the ester from aqueous layer. Anhydrous MgSO4 was added to remove water from organic
layer. Filtration was performed to remove MgSO4 and filtrate was evaporated to get ester (57) as
an oily product with 85 % yield [20].
363
364
Yield: 85 %; Rf = 0.8 (n-Hexane/EtOAc = 7:3); m.p (oC):Oil.
Alkylation of OH group of 4-methyl salicylic acid(8)
365
366
367
368
369
370
p-Nitrobenzyl bromide (3 mmol, 0.648 g) was dissolved in 10 ml of acetonitrile. 4-Methyl
salicylate (3 mmol, 0.5 g) and potassium carbonate (3.3 mmol, 0.453 g) was added to this solution.
Stirring the mixture for 1.5 hours and then heating at 60 ℃ for 16 hours led to the formation of
product followed by filtration to remove impurities. To remove the salt formed during the reaction
filtration was performed. Filtrate was concentrated to get protected ester (8). Purification of the
products 8 was carried out by using coloum chromatography to get 72 % yield [20].
371
372
373
374
375
Yield: 72 %; Rf =0.5 (n-Hexane/EtOAc = 7:3); m.p: 135-138oC; FT-IR (KBr pellets) (ῡ, cm-1):
2950 (CH stretch), 1692 (C=O stretch), 1539 (NO2 stretch), 1299 (C-O);1H NMR (400 MHz,
DMSO-d6) δ (ppm): 8.31 (d, J = 8.8 Hz, 2H, Ar-H-3', Ar-H-5'), 7.80 (d, J = 8.8 Hz, 2H, Ar-H-2',
H-6'), 7.67 (d, J = 7.8 Hz, 1H, Ar-H-6), 7.09 (s, 1H, H-3), 6.90 (d, J = 7.8 Hz, 1H, H-5), 5.35 (s,
2H, H-7'), 3.82 (s, 3H, OCH3), 2.35 (s, 3H, CH3).
376
Synthesis of 2-(4-nitrobenzyl)oxy)benzohydrazide (9)
377
378
379
380
O-Protected methyl salicylate (8) (0.7 mmol, 0.2 g,) & NH2NH2.H2O (1.3 mmol, 0.1mL) were
dissolved in ethanol and subjected to heating under reflux for 4 hours. Ethanol was evaporated on
rotavapor and solid product (9) was rinsed with cold water, filtered, dried and was purified by
using coloum chromatography to yield 78 % pure compound [20].
381
382
Yield: 78 %; Rf = 0.2 (n-Hexane/EtOAc = 6:4); m.p: 156-158 oC; FT-IR (KBr pellets) (ῡ, cm-1):
3350 (NHstretch), 2990 (Ar CH stretch), 1604 (C=O stretch), 1299 (C-O); 1H NMR: (400 MHz,
15

383
384
385
DMSO-d6) δ (ppm): 9.20 (s, 1H, NH), 8.28 (d, J = 8.7 Hz, 2H Ar-H-3', Ar-H-5'), 7.77 (d, J = 8.7
Hz, 2H, Ar-H-2', Ar-H-6'), 7.50 (d, J = 7.8 Hz, 1H, Ar-H-6), 6.98 (s,1H, H-3), 6.78 (d, J = 7.8 Hz,
1H, H-5), 5.38 (s, 2H, H-7), 4.52 (s, 2H, NH2), 2.35 (s, 3H, CH3).
386
Saponification process (10)
387
388
389
390
391
392
393
O-Protected ester (9) (0.664 mmol, 0.2 g) was dissolved in 10 mL of methanol. To this solution
was added sodium hydroxide (1.5 equiv, 20 % aqueous solution). Solution was heated at 65 ℃ for
8 hours. Progress of reaction was followed using TLC. Addition of 5M solution of HCl to the
reaction mixture on completion of reaction decreased pH = 2. Acid (10) was precipitated out and
precipitates were collected by filtration and washed with water and petroleum ether to remove the
unreacted ester. Purification of the compounds was done by recrystallization using methanol.A
yellow colored product with 78 % yield was obtained [20].
o
394
395
396
397
398
Yield: 78 %; Rf =0.3 (n-Hexane/EtOAc = 6:4); m.p: 175-178 C; FT-IR (KBr pellets) (ῡ, cm-1):
3050 (Ar CH stretch), 1650 (C=O stretch), 1539 (NO2 stretch), 1243 (C-O ), 1HNMR (400 MHz,
DMSO-d6) δ (ppm): 11.20 (s, 1H, OH), 8.39 (d, J = 8.0 Hz, 2H, Ar-H-5', H-3'), 8.15 (d, J = 8.0
Hz, 2H, Ar-H-6', Ar-H-2'), 7.87 (d, J = 8.2 Hz, 1H, H-6), 7.67 (s, 1H, H-3), 6.75 (d, J = 8.2 Hz,
1H, H-5), 5.35 (s, 2H, H-7'), 2.08 (s, 3H, CH3).
399
400
Synthesis of (3,5-dimethyl-1H-pyrazol-1-yl)(4-methyl-2-(4nitro
benzyl)oxy)phenyl)methanone (11)
401
402
403
404
405
Hydrazide (11) (0.83 mmol, 0.2508 g) & acetyl acetone (0.83 mmol, 0.085 mL) were dissolved in
5 mL of MeOH and concentrated HCl was used to catalyse the reaction. Heating of reactants were
carried out at 56oC for 12 hours to achieve the formation of products. By using rotary excess
solvent was removed and solid residue was washed with chilled ethanol to get product (62) in 70
% yield [16].
o
406
407
408
409
410
Yield: 70 %; Rf =0.6 (n-Hexane/EtOAc = 6:4); m.p: 105-107 C; FT-IR (KBr pellets) (ῡ, cm-1):
2990 (Ar CH stretch), 1650 (C=O Stretch), 1299 (C-O); 1H NMR: (400 MHz, DMSO-d6) δ (ppm):
8.24 (d, J = 8.7 Hz, 2H, Ar-H-3', Ar-H-5'), 7.80 (d, J = 8.7 Hz, 2H, Ar-H-2', H-6'), 7.70 (d, J = 8.0
Hz, 1H, H-6), 7.19 (s, 1H, H-3), 7.01 (d, J = 7.8 Hz, 1H, H-5), 6.30 (s, 1H, H-4"), 5.44 (s, 2H,
H-7'), 2.50 (s, 3H, H-6"), 2.35 (s, 3H, H-7"), 2.19 (s, 3H, H-7).
16

411
Synthesis of 1-aroyl-3-arylthiourea of 4-methyl salicylic acid (12)
412
413
414
415
416
417
418
419
420
The acid chloride was prepared by dissolving 4-methyl-2-((4-nitrobenzyl)oxy)benzoic acid (10)
(0.348 mmol, 0.1 g) in toluene. SOCl2 (0.45 mmol, 0.04 mL) was added to convert acid into halide.
Synthesis of acid chloride involved the heating of reactants for 4 hrs. Conversion of acid chloride
into isothiocyanate was achieved by dropwise addition of it to the potassium thiocyanate solution
in dry acetonitrile. The reaction mixture was subjected to stirring for 1.5 hours leading to the
formation of isothiocyanate which was attacked by p-toluidine in dry acetonitrile. Upon
completion of reaction small amount of distilled ice-cold water was poured in reaction mixture to
precipitate out thiourea (12). Purification of the compounds was carried out by recrystallization
using methanol to get 70 % yield [20].
421
422
423
424
425
426
427
Yield: 70 %; Rf =0.6 (n-Hexane/EtOAc = 6:4); m.p: 135 oC; FT-IR (KBr pellets) (ῡ, cm-1): 3300
(NH stretch), 2990 (Ar CH stretch), 1650 (C=O stretch), 1239 (C=S), 1171 (CN); 1H NMR (400
MHz, DMSO-d6) δ (ppm): 12.54 (s, 1H, NH), 11.10 (s, 1H, NH), 8.23 (d, J = 8.6 Hz, 2H, Ar-H-
3', Ar-H-5'), 7.91 (d, J = 8.6, 2H, Ar-H-2', Ar-H-6'), 7.80 (d, J = 7.8 Hz, 1H, H-6), 7.68 (s, 1H,
H-3), 7.53 (d, J = 7.8 Hz ,1H, H-5), 7.20 (d, J = 8.5 Hz, 2H, Ar- H-2", Ar-H-6"), 6.93 (d, J = 8.5
Hz, 2H, Ar -H-3", Ar-H-5"), 5.47 (s, 2H, CH2, H-7'), 2.40 (s, 3H, CH3, H-7), 2.31 (s, 3H, CH3, H-
7").
428
Synthesis of 5-(4-methyl-2-(4-nitrobenzyl)oxy)phenyl)-1,3,4-oxadiazole-2(3H)thione (13)
429
430
431
432
433
434
435
2-(4-Nitrobenzyl)oxy)benzohydrazide (9) (0.3322 mmol, 0.1 g) was dissolved in ethanol (5 mL),
carbon disulfide (0.495 mmol, 0.03 mL,) and potassium hydroxide (0.3322 mmol, 0.002 g) was
added to it. Mixture was refluxed till the required product was obtained. After the reaction was
completed, solid residue obtained by evaporation of solvent was dissolved in water to separate the
water insoluble impurities. Conc. HCl was added to acidify the filtrate. Product (13) was
precipitated out which was washed with water and purified by colum chromatography to obtain in
65 % yield [17].
o
436
437
438
Yield: 65 %; Rf =0.3 (n-Hexane/EtOAc = 6:4); m.p: 200-204 C; FT-IR (KBr pellets) (ῡ, cm-1):
3327 (NHstretch), 3000 (Ar CH stretch), 1605 (C=O stretch), 1299 (C-O), 1170 (C=S); 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 14.9 (s, 1H, NH), 8.24 (d, J = 8.7 Hz, 2H, Ar-H-3', Ar-H-5'), 7.86
17

439
440
(d, J = 8.7 Hz, 2H, Ar-H-2 & H-6'), 7.70 (d, J = 7.8 Hz, 1H, H-6), 7.19 (s, 1H, H-3), 7.00 (d, J =
7.8 Hz, 1H, H-5), 5.40 (s, 2H, H-7'), 2.38 (s, 3H, H-7).
NO2
NO2
i. SOCl2
O
O
O
O
S
ii. KSCN,
p-toulidine
acetone
OH
N
H
N
H
10
NaOH
12
NO2
NO2
NO2
OH
O
O
O
acetyl acetone
i. MeOH/H
i. NH2NH2.H2O
O
OH
N
O
O
N
ii.4-nitrobenzyl bromide
K2CO3, acetonitrile, 60 °C
HCl
O
O
NHNH2
11
9
8
CS2/KOH
12h stirring
NO2
NH
N
O
S
O
13
441
442
Scheme 2. Synthesis of 4-methyl salicylic acid derivatives
443
444
445
Biological Evaluation:
Transfection to express alkaline phosphatases
446
447
448
449
Expression of ALPs (h-TNAP and h-IAP) in COS-7 cells was carried out in 10cm2 plates through
lipofectamine, as previously reported protocol [21]. The plasmids expressing human TNAP and
IAP were already described [22, 23]. Transfection was performed when cells became confluent.
Cells were incubated in serum free DMEM/F-12 containing 24µL of lipofectamine reagent and 6
18

450
451
452
µL of plasmid DNA. After 24 hr, media was replaced with DMAM/F-12 containing 20% fetal
bovine serum (FBS). Transfected cells were harvested after 40-72 hr from the addition of plasmid
and transfecting reagent mixture.
453
Extraction of protein content:
454
455
456
457
458
459
460
461
Desired protein was extracted from the cells as previously reported [21]. Transfected cells were
washed with Tris-saline buffer and were removed from the plate through harvesting buffer (0.1
mM phenylmethylsulfonyl fluoride (PMSF), 95 mM NaCl and 45 mM Tris-buffer, pH 7.5).
Harvested cells were washed twice by centrifugation at 4 oC and 300×g for 5 min. Cells were re-
suspended and sonicated in harvesting buffer containing aprotinin (10 µg/mL). Subsequently,
cellular debris was removed by centrifugation for 10 min at 4 oC and 300×g. Glycerol was added
to the supernatant at a final concentration 7.5% and store at -80 oC. Before use in the assay, protein
concentration was determined by Bradford microplate assay [22].
462
Enzyme inhibition assay for alkaline phosphatases:
463
464
465
466
467
468
469
470
471
472
473
CDP-Star®, a chemiluminescent substrate, was used to determine the inhibitory potential of
synthesized compounds after slight modification in previously reported protocol [24]. Assay buffer
containing 250 mM diethanolamine (DEA), 2.5 mM MgCl2, 0.5 mM ZnCl2 at a pH 9.5 was used.
h-TNAP and h-IAP were added in each well of white 384 wells microplate at a concentration of
47 ng and 56 ng protein/well, respectively. Test compounds were analyzed at a final concentration
of 200 µM with subsequent addition of CDP-Star® substrate. Change in luminescence was
recorded by microplate reader (BioTek FLx800, Instruments, Inc. USA) in reference to positive
control as well as blank. Levamisole is the known inhibitor of h-TNAP and L-phenylalanine was
used as positive control for h-IAP. Data was analyzed PRISM 5.0 (GraphPad, San Diego,
California, USA). IC50 values for those compounds were determined that exhibited inhibitory
potential more than 50%.
474
Molecular docking studies
475
476
477
478
To justify the inhibition caused by potent inhibitors, most plausible binding modes were predicted
using molecular docking studies. Because of unavailability of x-ray crystallographic structure of
human alkaline phosphatases, homology models generated previously by our research group were
used for docking studies [23]. Structures of the tested compounds were drawn by MOE builder
19

479
480
481
482
483
484
485
486
487
488
489
tool [25] and optimization was achieved using MMFF94x forcefield [26]. Afterwards the energy
minimization of the target proteins was carried out by Molecular Operating Environment [25].
LeadIT (BioSolveIT GmbH, Germany) [27] was used to perform docking analysis of the prepared
ligands inside the respective receptors. Load Receptor Utility of the LeadIT software was used to
load the receptor and the metallic ions were selected as part of the protein. Active pocket of the
protein for docking analysis was identified by keeping the amino acid residues in 10.0 Å radius
around zinc ions. Values of the amino acid flips, metal co-ordinates and water handling were kept
as by default. Once docking analysis was completed, the possible interactions of ligands with
receptor proteins were inspected for studying the possible interactions using HYDE assessment
[28]. Discovery Studio Visualizer was used to perform visualize the interactions of ligand and
receptors [29].
490
491
492
493
Conflict of Interest:
The author(s) declare that they have no conflict of interests.
Acknowledgements:
494
495
496
497
498
A.M. is thankful to CUI for providing startup research grant under CIIT. J.I. is thankful to the
Higher Education Commission of Pakistan for the financial support through Project No.Ph-V-MG-
3/Peridot/R&D/HEC/2019. J.S. received support from the Natural Sciences and Engineering
Research Council of Canada (NSERC; RGPIN-2016-05867) and was the recipient of a “Chercheur
National” Scholarship from the Fonds de Recherche du Québec – Santé (FRQS).
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Bisthioureas of pimelic acid and 4-methylsalicylic acid derivatives as selective
inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) and intestinal
alkaline phosphatase (IAP): Synthesis and molecular docking studies
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Amara Mumtaz1*, Kiran Saeed1, Abid Mahmood2, Sumera Zaib2, Aamer Saeed3, Julie Pelletier4,
Jean Sévigny4,5, Jamshed Iqbal2*
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1Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-
22060, Pakistan.
2Center for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan
3Department of Chemistry, Quaid-i-Azam University Islamabad, Pakistan.
4Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada.
5Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, G1V 0A6, Canada.
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Abstract:
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Alkaline phosphatases (ALPs) are membrane bound metalloenzymes, distributed all over the body.
Recent studies have revealed that by targeting ALPs can lead towards the treatment of many
deadliest diseases including cardiac, cancerous and brain diseases. Thioureas and their derivatives
are of considerable significance and are privileged scaffolds in medicinal chemistry. They show a
wide range of pharmacological activities such as antibacterial, antiparasitic, anti-inflammatory and
antioxidants etc. On the other hand, salicylic acid and its derivatives are known for its broad
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spectrum of activities. The work presented comprises of synthesis of N-acyl-N'-aryl substituted
bisthioureas of pimelic acid (1-7) and 3,5-dimethyl pyrazole (11), 1-aroyl-3-aryl thiourea (12) and
1,3,4-oxadiazole (13) derivatives of 4-methyl salicylic acid. Structures of all the synthesized
1
compounds were characterized by FT-IR and H NMR spectroscopic analysis. Synthesized
compounds were evaluated for their alkaline phosphatases inhibition potential and exhibited high
potency as well as selectivity towards h-TNAP and h-IAP. Compound 7 and 12 which were the
bisthiourea derivative of pimmelic acid and thiourea derivative of 4-methyl salicylic acid,
respectively, showed excellent selectivity against h-TNAP and h-IAP, respectively.
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603
Bis thioureas of pimelic acid and 4-methylsalicylic acid derivatives as selective
inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) and Intestinal
alkaline phosphatase (IAP) inhibitors: Synthesis and molecular docking
studies
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Amara Mumtaz1#*, Kiran Saeed1#, Abid Mahmood2, Sumera Zaib2, Aamer Saeed3, Julie
Pelletier4, Jean Sévigny4,5, Jamshed Iqbal2*
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1Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-
22060, Pakistan.
2Center for Advance Drug Research, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan
3Department of Chemistry, Quaid-i-Azam University Islamabad, Pakistan.
4Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada.
5Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, G1V 0A6, Canada.
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Conflict of Interest:
The author(s) declare that they have no conflict of interests.
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623
Bis thioureas of pimelic acid and 4-methylsalicylic acid derivatives as selective
inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) and intestinal
alkaline phosphatase (IAP): Synthesis and molecular docking studies
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Amara Mumtaz1#*, Kiran Saeed1#, Abid Mahmood2, Sumera Zaib2, Aamer Saeed3, Julie
Pelletier4, Jean Sévigny4,5, Jamshed Iqbal2*
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1Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-
22060, Pakistan.
2Center for Advance Drug Research, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan
3Department of Chemistry, Quaid-i-Azam University Islamabad, Pakistan.
4Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada.
5Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, G1V 0A6, Canada.
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Research Highlights
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1. Synthesis of Pimelic acid and 4-methylsalicylic acid derivatives
2. Identification of selective alkaline phosphatases inhibitors
3. Molecular docking studies
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