Synthesis and computational studies of highly selective inhibitors of human recombinant tissue non-specific alkaline phosphatase (h-TNAP): A therapeutic target against vascular calcification

B i o o r g an i c C h e m i s tr y 1 0 1 (2 020 )1 03999

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Bioorganic Chemistry

journal homepage: www.elsevier.com/locate/bioorg

Synthesis and computational studies of highly selective inhibitors of human

recombinant tissue non-speciﬁc alkaline phosphatase (h-TNAP): A

therapeutic target against vascular calciﬁcation

⁎

Hina Andleeb^a,b,, Muzammal Hussain^c,d, Syeda Abida Ejaz^e, Jean Sevigny^f,g,

⁎

Muhammad Farman^a, Masoom Yasinzai^b, Jiancun Zhang^c,d, Jamshed Iqbal^e,, Shahid Hameed^a,

^aDepartment of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan

^bSulaiman Bin Abdullah Aba Al-Khail – Centre for Interdisciplinary Research in Basic Science (SA-CIRBS), Faculty of Basic and Applied Sciences, International Islamic

University, Islamabad, Pakistan

^cGuangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Science Park, Guangzhou 510530, China

^dUniversity of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China

^eCentre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus 22060, Pakistan

^fDepartement de microbiologie-infectiologie et d'immunologie, Faculte de Medecine, Universite Laval, Quebec, QC G1V 0A6, Canada

^gCentre de Recherche du CHU de Quebec – Universite Laval, Quebec, QC G1V 4G2, Canada

A R T I C L E I N F O

A B S T R A C T

Keywords:

In this study, we have discovered small druglike molecules as selective inhibitors of human tissue-nonspeciﬁc

alkaline phosphatase (h-TNAP), an enzyme critical for the regulation of extracellular matrix calciﬁcation. The

upregulation of h-TNAP is associated with various pathologies particularly the vascular calciﬁcation (VC).

Selective inhibition of h-TNAP over h-NPP1 may serve as a useful therapeutic strategy against vascular calci-

ﬁcation. A series of novel triazolyl pyrazole derivatives (10a-y) in which thiol bearing triazole moiety as the zinc

binding functional group was introduced to a pyrazole based pharmacophore was synthesized and evaluated as

potent and selective inhibitors of h-TNAP over h-NPP1. The biological screening against h-TNAP, h-IAP, h-NPP1

and h-NPP3 showed that many of the synthesized compounds are selective inhibitors of TNAP. Particularly, the

compounds 10a-h, 10j, 10m-q, 10u, 10w and 10x displayed high potency and complete selectivity towards h-

TNAP over h-NPP1. Compound 10q emerged as a highly potent inhibitor (IC₅₀= 0.16 µM or 160 nM) against h-

TNAP with 127-fold increased inhibition compared to levamisole. On the other hand, compound 10e was found

Pyrazoles

Triazoles

Schiﬀ bases

Alkaline phosphatase

Nucleotide pyrophosphatase

Vascular calciﬁcation

to be most selective inhibitor against the tested APs and NPPs (IC₅₀= 1.59

0.36 µM). Binding sites archi-

tecture analysis, molecular-docking and molecular dynamics simulations (MDS), revealed the basis for h-TNAP

and h-IAP ligand selectivity as well as selectivity towards h-TNAP over h-NPP1. These newly discovered in-

hibitors are believed to represent valuable lead structures to further streamline the generation of candidate

compounds to target VC.

1. Introduction

coronary death [3]. The development of VC is inﬂuenced by numerous

factors such as disturbed mineral metabolism, diabetes mellitus and

Cardiovascular diseases (CVDs) are a cause of deaths for 17.9 mil-

lion people every year – 31% of all global deaths according to WHO

stats [1]. Vascular calciﬁcation (VC) is one of the causes of CVD [2]. It

refers to the deposition of hydroxyapatite in cardiovascular tissues,

such as arteries and heart valves, and is a signiﬁcant risk factor in the

pathogenesis of CVD, being associated with myocardial infarction and

uremia [4]. A disturbed mineral metabolism is a hallmark of patients

with chronic renal failure, being highly prevalent and characterized as

chronic kidney disease–mineral and bone disorder (CKD-MBD).

Alkaline phosphatases (APs E.C.3.1.3.1) has been reported to play a

role as a useful indicator of bone health [5]. Owing to the upregulation

of alkaline phosphatase, particularly tissue-nonspeciﬁc alkaline

^⁎Corresponding authors at: SA-CIRBS, International Islamic University, Islamabad, Pakistan. Tel.: 051 9019051 (H. Andleeb); Department of Chemistry, Quaid-i-

Azam University, Islamabad, Pakistan. Tel.: +92 51 9064 2133; fax: +92 51 9064 2241 (S. Hameed); COMSATS University Islamabad, Abbottabad campus. Tel.:

+92-992-383591/96; fax: +92-992-383441 (J. Iqbal).

E-mail addresses: hina.andleeb@iiu.edu.pk (H. Andleeb), drjamshed@ciit.net.pk (J. Iqbal), shameed@qau.edu.pk (S. Hameed).

https://doi.org/10.1016/j.bioorg.2020.103999

Received 12 February 2020; Received in revised form 2 June 2020; Accepted 3 June 2020

A v a i l a b l e o n l i n e 0 8 J un e 2 020

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

generated by nucleotide pyrophosphatase/phosphotransferase-1

(NPP1) from nucleotide triphosphate and also from the transfer of in-

tracellular pyrophosphate to extracellular pyrophosphate by the PPi

transporter progressive ankylosis protein (ANK) (Fig. 1) [13]. This ex-

tracellular pyrophosphate ePPi is cleaved by a membrane bound ec-

toenzyme, tissue-nonspeciﬁc alkaline phosphatase (TNAP), to phos-

phate (Pi) (Fig. 1). Soluble phosphate enters intracellularly via sodium-

dependent phosphate co-transporter 1 (Pit-1) [13]. Vascular smooth

muscle cells are sensitive to changes in inorganic phosphate (Pi) levels.

This is followed by the binding of Pi to calcium resulting in calcium-

phosphate (Hydroxyapatite) crystal. Calcium-phosphate crystal pro-

motes matrix vesicles and ultimately vascular calciﬁcation (Fig. 1).

Conversely, PPi is an inhibitor of calcium phosphate precipitation,

however TNAP is continuously hydrolyzing this mineralization in-

hibitor. In parallel, apoptotic bodies provide a nidus for vascular cal-

ciﬁcation (Fig. 1). Therefore, all these all factors contribute as promo-

tors of vascular calciﬁcation.

It has been observed that the normalization of extracellular pyr-

ophosphate (ePPi) levels by inhibition of TNAP in NPP1 null and ANK-

Fig. 1. Pathways showing promotors of calciﬁcation in a vascular smooth

muscle cell [13]. (NPP1 = nucleotide pyrophosphatase-1, ANK = progressive

ankylosis protein, Pit-1 = phosphate co-transporter 1). PPi is generated by NPP1

from NTP. PPi is cleaved by TNAP. Soluble phosphate (Pi) enters intracellularly

and binds to calcium and creates calcium-phosphate crystal which promotes

matrix vesicles and vascular calciﬁcation.

deﬁcient

mice,

improves

their

calciﬁcation

abnormalities

[10–12,14–16]. The presence of TNAP enriched matrix vesicles (MVs)

in human atherosclerotic lesions suggests an active role in the promo-

tion of the accompanying vascular calciﬁcation [17–20]. Thus, there is

an ample evidence warranting exploration of the therapeutic potential

of TNAP inhibition at sites of arterial calciﬁcation [21,22] to increase

local concentration of PPi thereby reducing abnormal mineralization. A

recent study found that pyrophosphate (PPi) hydrolysis was 51% higher

in post-dialysis than pre-dialysis plasma [23]. Therefore, to improve the

quality of life of CKD patients there is a need of therapeutic approaches

to prevent reduction of plasma pyrophosphate levels during dialysis.

Discovery of potent and selective TNAP inhibitors could facilitate

these explorations. TNAP has been shown to be inhibited by a limited

number of small molecule compounds including ^L-homoarginine [24],

levamisole [25] and theophylline [26]. However, these inhibitors are

not selective for TNAP [24], have low binding aﬃnity [26]. New se-

lective inhibitors of APs are therefore required for better selectivity.

Among non-nucleotide ectonucleotidase inhibitors (NNEIs), pyrazole

and triazole derivatives [27–29] have shown inhibition against alkaline

phosphatases (APs) (Fig. 2 A–D). Pyrazoles has been reported recently as

phosphatase (TNAP) in the vasculature [6] leads to vascular stiﬀening

and heart failure. APs catalyze the hydrolysis of phosphomonoesters

with release of inorganic phosphate (Pi) [7]. A major role for TNAP in

bone tissue is to hydrolyze extracellular pyrophosphate (ePPi) to

phosphate, which then combines with calcium to form hydroxyapatite

leading to bone mineralization under normal conditions [8,9] (Fig. 1).

However, if the TNAP enzyme is overexpressed, phosphate-pyropho-

sphate ratio is disturbed leading to pathologies as vascular calciﬁcation

in blood vessels and ectopic calciﬁcation in tissues [10]. In animal

models, upregulation of TNAP expression in vascular smooth muscle

cells or in endothelial cells leads to pathological calciﬁcation

[6,10–12].

Fig. 1 shows some promotors of vascular calciﬁcation in a vascular

smooth muscle cell. Basically, extracellular pyrophosphate (ePPi) is

Fig. 2. Structures of reported triazole based APIs (A and B) and pyrazole based APIs (C and D). Designed pyrazole integrated triazole Scaﬀold (E).

2

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

NHNH₂

S

a

promising pharmacophore for its activity as NNEIs [28,29]. In order to

explore pharmacotherapies against pathologies such as vascular calci-

ﬁcation at cellular level there is a need for potent inhibitors that are

selective for TNAP over NPP1. A series of novel triazolyl pyrazole (10a-

y) inhibitors in which thiol bearing triazole moiety as the zinc binding

functional group was introduced to a pyrazole based pharmacophore

through a C]N linker was designed (Fig. 2E). As we know that the

binding pocket of alkaline phosphatase contains two zinc ions, it was

envisaged that triazole-3-thiols being active zinc chelators [30] may

serve as important pharmacophoric part of designed inhibitors.

Our major objective was to develop h-TNAP selective and NPP1

inactive candidates in order to target VC. In this regard, synthesized

compounds were ﬁrst tested against alkaline phosphatases (h-TNAP &

h-IAP). After analysis, the highly active compounds against h-TNAP will

be further tested against human nucleotide pyrophosphatases/phos-

phodiesterases (h-NPP1 & h-NPP3). Another objective was to further

reveal the computational basis for absolute h-TNAP ligand selectivity

for the designed inhibitors by performing detailed computational stu-

dies.

NH₂-NH₂.H₂O

S=C=S

+

6

5

NHNH₂

7

O

b

OH

R¹

8a-e

9a : R¹= 2-OCH₃

9b : R¹= 3-OCH₃

9c : R¹= 4-OCH₃

9d : R¹= 2-Cl

N

R¹

SH

N

9e : R¹= 4-Cl

NH₂

9a-e

Conditions: (a) Stirring at 10 - 15 °C followed by heating at 85 °C, 76-85%;

(b) 170-185 °C, oil bath, 1.5 - 2h, 69 - 85%.

Scheme 2. Synthetic pathway to substituted 1,2,4-triazole-3-thiols (9a-e).

N

SH

N

R¹

2. Results and discussion

N

NH₂

9a-e

HS

N

a

O

N

2.1. Chemistry

+

N

H

N

R²

The precursors (4a-e and 9a–e) for the synthesis of 5-aryl-4-(1,3-

disubstituted pyrazolyl)methyl eneamino)-1,2,4-triazole-3-thiols (10a-

y) were synthesized according to the synthetic methods outlined in

Schemes 1 and 2, respectively. The 3-(4-substituted phenyl)-1-phenyl-

1 H -pyrazole-4-carbaldehydes 4a-e) were prepared in two steps. In the

ﬁrst step, substituted acetophenones (2a-e) and phenyl hydrazine (1)

were reacted in the presence of glacial acetic acid in ethanol to aﬀord

the hydrazone derivatives (3a-e). The concomitant formylation and

cyclization of hydrazones (3a-e) to pyrazoles under Vilsmeier–Haack

(DMF–POCl₃) conditions followed by basic hydrolysis furnished the

corresponding 4-carbaldehyde functionalized pyrazole derivatives (4a-

e) (Scheme 1). Electron–rich and electron–deﬁcient substitution on the

ring aﬀorded the desired analogues in good yields.

N

R¹

10a-y

4a-e

10a : R¹= 2-OCH₃, R²= H;

R²

10b : R¹= 2-OCH₃, R²= CH₃

10c : R¹= 2-OCH₃, R²= OCH₃; 10d : R¹= 2-OCH₃, R²= NO₂

10e : R¹= 2-OCH₃, R²= Cl ;

10g : R¹= 3-OCH₃, R²= CH₃

10f : R¹= 3-OCH₃, R²= H

;

10h : R¹= 3-OCH₃, R²= OCH₃

10i : R¹= 3-OCH₃, R²= NO₂; 10j : R¹= 3-OCH₃, R²= Cl

10k : R¹= 4-OCH₃, R²= H;

10l : R¹= 4-OCH₃, R²= CH₃

10m :R¹= 4-OCH₃, R²= OCH₃; 10n : R¹= 4-OCH₃, R²= NO₂

10o : R¹= 4-OCH₃, R²= Cl;

10p : R¹= 2-Cl,

R²= H

10q : R¹= 2-Cl,

10s: R¹= 2-Cl,

10u : R¹= 4-Cl,

10w : R¹= 4-Cl,

10y : R¹= 4-Cl,

R²= CH₃; 10r : R¹= 2-Cl,

R²= NO₂; 10t : R¹= 2-Cl,

R²= OCH₃

R²= Cl

R²= H;

10v: R¹= 4-Cl,

R²= CH₃

R²= NO₂

R²= OCH₃; 10x : R¹= 4-Cl,

R²= Cl;

The reaction of hydrazine (5) with carbon disulﬁde (6) aﬀorded

thiocarbohydrazide in the presence of water with the evolution of hy-

drogen sulﬁde. The synthesized thiocarbohydrazide was further fused

with substituted benzoic acids to aﬀord the cyclized product: sub-

stituted 1,2,4-triazole-3-thiols (9a-e) (Scheme 2).

Conditions: (a) Glacial acetic acid, absolute ethanol, 3 - 4 hrs, 65 - 90 °C, 69 - 88%.

Scheme 3. Synthesis of 1,3-disubstituted pyrazolyl)methyleneamino-5-aryl-

1,2,4-triazole-3-thiols (10a-y).

Next, 5-aryl-4-(1,3-disubstituted pyrazolyl)methyleneamino)-1,2,4-

triazole-3-thiols (10a-y) were furnished by the condensation of sub-

stituted 1,2,4-triazole-3-thiols (9a-e) with diﬀerent 1,3-disubstituted

pyrazole-4-carbaldehydes (4a-e) in the presence of glacial acetic acid

using absolute ethanol as a solvent (Scheme 3). The products with R²as

electron-withdrawing groups were obtained at lower temperature

(45–65 °C) in high yields (80–88%), however, with R²as electron-do-

nating groups the yields were relatively lower (69–79%) with the re-

quirement of a higher temperature (75–90 °C).

O

The formation of products was conﬁrmed by spectroscopic techni-

ques. In the IR spectra of compounds 10a-y, the absence of absorptions

in the region of 3345–3320 cm⁻¹for NH₂of triazole also indicated

successful desired transformation. The formation of compounds 10a-y

was conﬁrmed through ¹H NMR spectroscopy. The ¹H NMR spectra of

compounds 10a-y exhibited a characteristic singlet in the region

9.93–9.40 ppm corresponding to CH]N of imine linkage, conﬁrming

N

Me

NH₂

Me

a

N

H

N

R²

H

1

2a-e

3a-e

R²

b (i, ii)

desired transformation.

A

singlet observed in the region

R²

4a : R²= H

14.37–14.09 ppm corresponding to SH of triazole moiety indicated that

the compounds existed preferentially in the thiol tautomer as compared

to thione form. The ]CeH of the pyrazole ring resonated in the region

9.32–9.09 ppm. For compounds 10b, 10g, 10l and 10q, the methyl

protons resonated in the region of 2.39–2.37 ppm, while the methoxy

hydrogens in compounds 10a-o, 10r and 10w were spotted as singlets

in the region of 3.82–3.67 ppm.

4b : R²= CH₃

4c : R²= OCH₃

4d : R²= NO₂

4e : R²= Cl

N

CHO

4a-e

Conditions: (a) Glacial acetic acid, absolute ethanol, 25 - 70 °C, 80-95%;

(b) i) DMF, POCl₃, 0 - 70 °C, 3 h ii) NaOH aqueous, 67 - 89%.

¹³C NMR spectral data further aided in the conﬁrmation of cyclized

structures 10a-y showing the presence of distinctive signals for imine

(HC]N) carbon in the range of 162.36–161.72 ppm. The methyl

Scheme 1. Synthetic pathway to 1,3-disubstituted 1H-pyrazole-4-carbalde-

hydes (4a-e).

3

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

carbons resonated in the region of 21.36–20.78 ppm for compounds

10b, 10g, 10l and 10q, while the methoxy carbons in compounds 10a-

o, 10r and 10w were observed in the region of 55.82–55.12 ppm. The

results from the elemental analyses and MS were in good agreement

with the suggested chemical structures of the synthesized compounds.

able to advantageously interact with both Zn ions present in the binding

site(s); our initial docking screens for the design of hybrid triazolyl

pyrazole pharmacophore showed that the SH of 1,2,4-triazole-3-thiol

formed a salt bridge with zinc (ZN500) and arginine (R184) in the

active pocket of h-TNAP, thus facilitating an eﬀective binding with

TNAP (Fig. 2 and S6 & S7). This has been observed in docking for all the

mentioned compounds (Figs. S6 & S7); however, majority remained

selective towards TNAP and some of them even completely failed to

bind either of the proteins (the ones with no activity; Table 1). Al-

though these analogues are structurally similar, they represent distinct

chemical modiﬁcations in substitutions at diﬀerent positions of aryl

ring that diﬀer in their physicochemical properties. Thus, this structural

similarity may not necessarily translate into similar binding modes and

speciﬁcity within the h-TNAP and h-IAP binding site(s).

3. Computational studies and bioevaluation

3.1. Modeling of h-TNAP and h-IAP and structural comparison of their

binding site(s) architecture

The diﬀerence between inhibitory activities of analogues studied in

this work, as well as their selectivity for h-TNAP and/or h-IAP, led us to

make structural comparisons in terms of active site(s) architecture (size,

shape, physicochemical properties, etc.) and the intermolecular inter-

actions that mediate protein–ligand binding. The h-TNAP and h-IAP are

closely related by sequence (Fig. S1). Their binding pockets are even

more similar, possessing mainly conserved residues [29]. Yet there is

remarkable diﬀerence in small molecule recognition. The development

of a structural hypothesis underlying these diﬀerences has been limited

by the lack of experimental structures of h-TNAP and h-IAP. Homology

modeling can therefore serve as a valuable approach to develop struc-

tural insights in these enzymes. Our ﬁrst step was to generate two

comparable models of h-TNAP and h-IAP based on the crystal structure

of h-PLAP (PDB id: 1ZED) [29]. Comparative sequence alignments of

the template with modeled proteins are shown in Supplementary Figs.

S2 & S3. Both models were shown to have high quality stereochemical

proﬁles (evaluated by Ramachandran analysis; Supplementary Figs. S4

& S5) and to be structurally similar to the template protein (backbone

root-mean square deviation, RMSD, of 0.76 Å and 0.27 Å for the h-

TNAP and h-IAP models, respectively).

As we know that the target compounds 10a-y may exist in two

tautomeric forms (thiol and thione), therefore we conducted docking

studies using both the tautomers of pyrazolyl triazoles, however it was

found that Thiol-Zinc-Arginine salt bridge was stabilized by thiol tau-

tomer to a greater extent as compared to thione tautomer (Fig. S9). The

thiol tautomer showed better docking scores and binding interactions

than thione form (Fig. S9). This can further explained by measuring the

distance between non-covalent interaction between Zn500 and Sulphur.

It has been observed that the distance between S and Zn500 was found

to be in the range of 2.53–2.73 Å in thiol tautomers leading to stronger

interactions in thiol form (Fig. S9). However no such Zinc-S ineractions

were observed in thione form as the distance between Zn and S is found

to be greater (5.21–6.71 Å). This may be attributed to the fact that lone

pairs in sp³orbitals (S of thiol) are better electron donars as compared

to those in sp²orbitals (S of thione). The stability of the thiol tautomers

of 10a-y was further explained by quantum chemical calculations of

frontier orbitals of these molecules. It was observed that ΔE (E_LUMO

-

E

_HOMO) was found to be higher in case of thiol tautomer as compared to

Fig. 3 highlights some key diﬀerences in the binding pocket archi-

tecture determined by structural comparison and the alignment of the

active site(s) residues of h-TNAP and h-IAP models. In the lower section,

the binding pockets mainly comprise structurally conserved charged

and polar-neutral residues, while the upper section (hereafter referred

as pocket “opening/outlet”) varies in a blend of hydrophobic, charged

and polar-neutral residues (see Fig. 3 legend). The main diﬀerence that

emerged between the two pockets is the presence of a—groove-

shaped— charged cluster (E125, R136, D183 and R184) in the left lip of

the TNAP outlet (Fig. 3A), which is substituted by the hydrophobic

(F126 and F137) and polar-neutral (Q127 and N184) residues in IAP

(Fig. 3B). This also results in a diﬀerence in the overall binding pocket

(s) volume, shape, and the physicochemical character (as mentioned in

the table in Fig. 3). For instance, the IAP opening becomes obstructed/

contracted because of substituting hydrophobic (F126 and F137) and

polar-neutral (Q127) residues (Fig. 3B & C). Similarly, Q127 and N184,

together with the neighboring N186, constitute a polar-neutral cluster

in IAP opening, possessing diﬀerent physicochemical character than the

charged shallow groove in TNAP. Another major diﬀerence is the re-

placement of the extended-chain, positively-charged, arginine (R450)

and the aromatic histidine (H451); their overlapping-ﬂanked side-

chains delineate a cleft-like structure of TNAP outlet with the corre-

sponding short-chain, acyclic, polar-neutral serine residues (S447 and

S448) in the IAP opening, which is likely to make it less attractive for

the aromatic ring systems (of ligands) reaching this area.

thione tautomer depicting higher stability of thiol form in pyrazolyl

triazoles (10a-y) (Fig. S10), this may be attributed to the formation of

intramolecular hydrogen bond of thiol hydrogen with imine nitrogen

forming a ﬁve membered ring (Fig. S11). Also the presence of SH

protons in ¹HNMR spectra and absence of thiocarbonyl carbon signal in

¹³CNMR spectra showed the existence of predominantly thiol form,

therefore the rest of the discussion regarding computational studies was

based on thiol form of 5-aryl-4-(1,3-disubstituted pyrazolyl)methyle-

neamino)-1,2,4-triazole-3-thiols (10a-y).

3.2. Enzyme inhibition against human alkaline phosphatases

Initially, 5-aryl-4-(1,3-disubstituted pyrazolyl)methyleneamino)-

1,2,4-triazole-3-thiols (10a-y) were tested for inhibition of human al-

kaline phosphatases (h-TNAP and h-IAP) (Table 1).

When 5-aryl-4-(1,3-disubstituted pyrazolyl)methyleneamino)-1,2,4-

triazole-3-thiols (10a-y) were tested against h-TNAP, compounds 10a-f,

10h, 10j, 10m-q, 10u, 10w and 10x displayed excellent activity

compared to the standard, levamisole. However, compounds 10i, 10k,

10l, 10r-t, 10v and 10y were inactive.

Structure Activity Relationship. Among the evaluated series, 5-(2-

chlorophenyl)-4-((1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)methylenea-

mino)-4H-1,2,4-triazole-3-thiol (10q) emerged as a highly potent in-

hibitor against h-TNAP with an IC₅₀value of 0.16

0.01 µM (Fig. 4).

The diverse architecture of h-TNAP and h-IAP binding pockets;

particularly the spatial constraints of the polar-neutral and the charged

clusters in the upper region(s) of the two pockets, could be the main

deﬁner of ligand speciﬁcity and the inhibitory potency (vide infra).

Presumably, the IAP pocket would be restrictive to accommodate more

rigid and perhaps larger ligands, while the TNAP pocket would prob-

ably be more friendly (than IAP) to the ligands bearing either electro-

philic or nucleophilic substitutions. This assumption is further corro-

borated by the fact that all the analogues studied in this work share the

common 4H-1,2,4-triazole-3-thiol core (a known zinc chelator [30])

This candidate with 127-fold increased inhibition as compared to le-

vamisole, possesses an ortho-chlorophenyl moiety at triazole ring and a

para-tolyl ring at pyrazole skeleton. Keeping para-tolyl ring at pyrazole

skeleton constant, when we replaced inductively electron-withdrawing

chloro substituent with electron-donating methoxy substituent at the

ortho position of the phenyl ring of triazolyl moiety, compound 10b

showed good activity (IC₅₀= 4.12

0.76 µM) with complete se-

lectivity towards TNAP over IAP, however, with meta methoxy sub-

stituent the compound 10g showed comparable activity with levami-

sole. With the same skeleton, the compounds 10l and 10v bearing para-

4

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Fig. 3. Comparative analysis of h-TNAP (A) and h-IAP (B) binding pockets. The inset table provides a comparison of pocket architectures in terms of physicochemical

properties, volume and shape. (C) highlights the structural alignment of the binding site residues of h-TNAP (green label) and h-IAP (cyan label). The binding pockets

contain two zinc ions (Zn1 and Zn2) and a magnesium ion (Mg) and are aligned by residues that have a charged side-chain (arginine, glutamate and aspartate);

contain an aromatic ring (phenylalanine, tyrosine and histidine), and are neutral yet polar (asparagine, glutamine, serine and threonine). The aromatic histidines and

neutral yet polar residues are here collectively termed as “polar-neutral”. The basic charged residues are colored sky-blue, acidic charged as red, polar-neutral as

magenta, and hydrophobic as grey. There is slightly large prevalence of charged residues (both positive and negative) in or in the vicinity of the pocket outlet of h-

TNAP relative to h-IAP (arginine residues, 5::3; glutamate, 4::3; aspartate, 4::2 in h-TNAP and h-IAP, respectively). The structural comparison also revealed the larger

prevalence of polar-neutral residues in the upper vicinity of h-IAP pocket compared to h-TNAP (7::2, respectively). The groove-shaped charged cluster and cleft-like

areas are encircled in h-TNAP model. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

methoxy and para-chloro substituent on triazolyl phenyl ring com-

pletely lost their activity towards TNAP which indicates that substitu-

tion at ortho position, and not the para position of triazolyl phenyl ring,

is crucial for activity, keeping para-tolyl ring at pyrazole skeleton

constant. The replacement of 4-methyl group in 10q with an in-

ductively electron-withdrawing 4-chloro moiety and replacement of

ortho-chloro with an electron donating ortho-methoxy group produced

compound 10e, which was the next strongest inhibitor of h-TNAP with

10o and 10u were found to be active and selective candidates towards

TNAP owing to pi-cation (10j, 10n and 10u) interactions in the cleft-

like area; with histidine (H451) and positively charged arginine (R450).

Among the derivatives tested against human intestinal alkaline phos-

phatases (h-IAP), compounds 10d, 10i, 10k, 10m, 10p-r and 10v-x

exhibited pronounced activity. Intriguingly, 10m bearing a para-

methoxy phenyl moiety both at triazole and pyrazole skeletons showed

highest inhibition of h-IAP with an increase of activity up to 217 folds

complete selectivity of TNAP over IAP (IC₅₀value of 1.59

0.36 µM)

(IC₅₀value of 0.46

0.03 µM) in comparison to L-phenylalanine. The

(Table 1). Keeping 4-chlorophenyl on pyrazole skeleton constant, it was

observed that electron-donating groups at ortho, meta or para position

of triazolyl phenyl ring in 10e, 10j, and 10o, respectively, made them

active and completely selective towards TNAP over IAP owing to in-

crease in pi-cation interactions with the aromatic histidine (H451) and

positively charged arginine (R450), respectively. However, electron-

withdrawing R¹groups made them inactive towards both alkaline

phosphatases. The replacement of inductively electron-withdrawing

chloro group as R²in 10e (Fig. 4) with a highly polar and strong

electron-withdrawing nitro functionality produced compound 10d

(Fig. 4), which was found to be a dual inhibitor of TNAP and IAP.

When the R²group was a H-atom, compounds 10a and 10f (R¹= 2-

methoxy and 3-methoxy, respectively) showed good activity and se-

lectivity against TNAP. However, compound 10k (R¹= 4-methoxy)

was found to be inactive towards TNAP inhibition and was completely

selective towards IAP. Compound 10p (R¹= 2-chloro) showed dual

inhibition against TNAP and IAP. Compounds 10a, 10e, 10f, 10h, 10n,

next active compound 10k, possessing a para-methoxyphenyl moiety at

the triazole ring and two phenyl rings at pyrazole skeleton, was iden-

tiﬁed

as

a

potent

and

selective

inhibitor

of

h-IAP

(IC₅₀= 0.53

0.04 µM) with 189-fold stronger inhibition than L-

phenylalanine (Table 1).

3.3. Docking studies explain, in part, the basis for h-TNAP and h-IAP ligand

selectivity

Indeed, molecular docking is an important tool for obtaining pos-

sible binding poses for ligands [31]. Therefore, ﬁrst Glide docking [32]

was used to obtain the most possible binding pose for each compound.

Fig. 5 shows an overlay of the docked poses within the h-TNAP and

h-IAP binding sites. Additional illustrations of the detailed binding in-

teractions of each compound are provided in the Supplementary Figs.

S6 and S7, respectively.

The compounds are overall well-aligned with the binding pockets,

5

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Table 1

their scoring functions [31]. Yet the comparative analysis of docked

poses could potentially explain the phenomenon of selectivity or dual

activity of the studied analogues.

Inhibitory potential for 5-aryl-4-(1,3-disubstituted pyrazolyl)-methylenea-

mino)-1,2,4-triazole-3-thiols (10a-y) against alkaline phosphatases.

Compd.

R¹

R²

h-TNAP IC₅₀(µM)^a

h-IAP IC₅₀(µM)^a

3.4. Binding hypotheses derived from docked poses for selective and dual-

active analogues against h-TNAP and h-IAP

10a

2-OMe

3-OMe

4-OMe

2-Cl

H

Me

OMe

NO₂

Cl

3.45

4.12

2.68

1.85

1.59

7.99

21.3

17.1

0.35

0.76

0.57

0.17

0.36

1.47

2.47

2.08

> 100

10b

10c

10d

2.21

0.48

To better understand the binding poses, the docked orientations

were analyzed with particular reference to the structural diﬀerences

observed in the pocket outlets of the two protein models. In case of h-

TNAP selective analogues, the terminal moieties reaching toward the

groove-shaped or cleft-like areas in the pocket outlet, at least in part,

determine selectivity. For instance, some of the analogues (10a, 10f,

10h, 10j, 10n, and 10o) adopt conformations that are more spatially

demanding (Fig. 6A), and probably are not able to adapt to the spatial

constraint of the h-IAP pocket imposed by F126 and Q127. Another

general trend observed for h-TNAP selective analogues is the formation

of Pi-Pi (10a, 10b, 10c, 10e, 10f, 10j, 10n, and 10u) and pi-cation (10j,

10n and 10u) interactions in the cleft-like area; formed by the 2-

methoxyphenyl (on the 4H-1,2,4-triazole-3-thiol core) and 1-phenyl or

4-methoxyphenyl (on the 1H-pyrazol) moieties with aromatic histidine

(H451) and positively charged arginine (R450), respectively (Fig. 6A

and supplementary Fig. 6). The replacement of R450 and H451 with the

corresponding S447 and S448 (which cannot form Pi-Pi and pi-cation

interactions) in h-IAP can further rationalize the speciﬁcity of h-TNAP

selective analogues.

10e

> 100

10f

H

> 100

10g

Me

OMe

NO₂

Cl

10h

10i

> 100

0.92

0.07

10j

7.45

0.87

> 100

0.53 0.04

> 100

10k

H

> 100

10l

Me

OMe

NO₂

Cl

> 100

10m

7.54

1.34

0.48

0.97

0.37

0.01

0.46

0.03

10n

7.86

8.99

5.46

0.16

> 100

10o

10p

H

1.95

0.11

0.31

0.47

10q

2-Cl

Me

OMe

NO₂

Cl

12.37

5.94

10r

2-Cl

> 100

10s

2-Cl

> 100

10t

2-Cl

> 100

10u

10v

4-Cl

H

16.7

1.45

4-Cl

Me

OMe

NO₂

Cl

> 100

8.81

1.32

0.67

0.17

10w

4-Cl

5.42

5.94

0.32

0.41

1.72

1.25

10x

4-Cl

10y

4-Cl

> 100

Levamisole^b

L-Phenylalanine^b

20.21 1.9

100

3

The analogues selective (even the dual-active ones) toward h-IAP,

however, adopt similar planarity in their binding orientations per-

taining to narrower/deep pocket geometry (Fig. 6B). The position of R¹

and the size of the R²substitutions were fundamental to determine the

ﬁtness and selectivity. For instance, comparing the binding orientations

of compound 10i vs 10d (Fig. 6B and 5), meta-methoxy as R¹(in

compound 10i) perhaps could not be a good ﬁt for the cleft-like sub-

pocket area in h-TNAP as compared to ortho-methoxy (in compound

10d). Similarly, the spatial constraint to para-methoxy as R¹substituent

in 10k could render it unﬁt for the cleft-like or groove-shaped areas of

h-TNAP, compared to binding orientations of compounds 10a (ortho-

methoxy) and 10f (meta-methoxy), respectively (Fig. 6A and B). Finally,

comparing the binding orientations of 10r vs 10q and 10w vs 10v

(Fig. 6B and 7), the inability of larger size methoxy substitution at 4

position of phenyl ring (of 10r and 10w) to ﬁt in groove-shaped pocket

of h-TNAP could be the key to deﬁne selectivity.

a

b

IC₅₀values shown are the mean

Used as positive control.

SD of triplicate measurements.

though many of them manifested a diﬀerent binding orientation that

was anticipated either due to diﬀerence in pockets’ architecture and/or

the size and nature of substitutions at R¹and R²positions of the primary

scaﬀold. The core triazole-thiol moiety in all the docked analogues

reaches down into the lower section of the pockets and maintains a

coordination distance to Zn1 (ZN500; in case of both TNAP and IAP) or

Zn2 (ZN501; only in case of IAP with exception of 10i and 10m) and a

structurally conserved arginine (R184:R185), albeit with diﬀerent co-

ordination patterns (Supplementary Figs. S6 and S7). The polar contacts

with the structurally conserved histidine (H171:H172; H338:H336, and

H341:H339) and serine (S110:S111) residues further stabilize the co-

ordination patterns of triazole-thiol moiety in the lower section of the

pockets (Supplementary Figss. S6 and S7). The comparison of the pre-

dicted docking score and the IC₅₀value for each compound are sum-

marized in Table 2. It was observed that the ranking of the predicted

docking scores was not in agreement with the experimental IC₅₀values

(Table 2). This is perhaps not surprising in lieu of the diversity amongst

the contemporary docking algorithms and the known deﬁciencies in

The binding orientations of dual-active analogues appear to be

characterized by a combination of all the trends observed for both h-

TNAP and h-IAP selective analogues. For instance, comparing the two

most potent analogues, 10q (for h-TNAP) and 10m (for h-IAP), the role

of the size, position, and nature of the terminal moieties in determining

binding orientations looks obvious. The electronegative ortho-chloro

(R¹) and para-methyl (R²) terminal substitutions in case of compound

Fig. 4. Structures of top three active compounds against h-TNAP with their IC₅₀Values.

6

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Fig. 5. An overlay of the docked orientations of all the active analogues against h-TNAP (A) and h-IAP (B).

Table 2

nearby pyrazole moiety enhancing pi-pi interactions with Histidine

(HIE338).

The docking scores of compounds calculated by Glide.

Similarly, in case of compound 10m, the steric hindrance and spa-

tial constraints imposed by groove-shaped and cleft-like areas to larger

size para-methoxy substitutions at R¹and R²terminals make it to adopt

a more-strained and less planar (to pocket geometry) “twisted” or-

ientation within h-TNAP pocket as compared to h-IAP, where it adopts

less-strained and more-planar conformation which seems to be well

aligned with pocket geometry. Similar trends were observed for the

other dual-active analogues. For examples: the electrostatic and pi-ca-

tion interactions of para-nitro (R²) substitution (R136 in the groove-

shaped area of h-TNAP) in compound 10d are compromized by spatial

and steric hindrance caused by F126 in h-IAP (less potent against h-

IAP); and the narrower pocket geometry of h-IAP because of the spatial

constraint imposed by F126 and Q127 (and the absence of proximal

steric hindrance that is caused by R450 and H451 in h-TNAP) may

render compounds 10p, 10w, and 10x a more better ﬁtness and

alignment than for h-TNAP (thus more potent against h-IAP).

Compd No.

h-TNAP

h-IAP

IC₅₀(µM)^a

Docking score

IC₅₀(µM)^a

Docking score

TNAP

IAP

10a

10b

10c

10d

10e

10f

3.45

0.35

0.76

0.57

0.17

0.36

1.47

2.08

−5.50

−4.88

−5.30

−4.83

−4.88

−4.37

−5.35

ND

4.12

2.68

1.85

1.59

7.99

17.1

ND

2.21

0.48

−6.19

ND

10h

10i

ND

> 100

0.92

0.07

−6.05

ND

10j

7.45

0.87

−4.85

> 100

−5.93

−4.26

−4.88

−5.97

−5.39

> 100

−5.16

> 100

−4.2

ND

10k

10m

10n

10o

10p

10q

10r

10u

10v

10w

10x

> 100

0.53

0.46

0.04

0.03

−5.70

−6.56

ND

7.54

7.86

8.99

5.46

0.16

1.34

0.48

0.97

0.37

0.01

ND

1.95

12.37

5.94

0.11

0.31

0.47

−6.05

−6.07

−5.94

ND

> 100

16.7

1.45

ND

3.5. Molecular dynamics (MD) simulations

> 100

8.81

1.72

1.25

1.32

0.67

0.17

−5.96

−6.13

−6.04

5.42

5.94

0.32

0.41

In order to substantiate the reliability of docked poses [31], and to

validate the selectivity and dual-activity of respective analogues against

h-TNAP and h-IAP, a round of MD simulations was conducted for four

parallel systems, each 60 ns long, constructed as follows: (1) A dual-

active analogue was selected that was most potent against h-TNAP

(10q) and subjected its docked complexes with h-TNAP and h-IAP to

simulations, (2) Another dual-active analogue that was most potent

against h-IAP (10m) was selected and subjected its docked complexes

with h-IAP and h-TNAP to simulations, (3) Two selective analogues, one

against h-TNAP (10e) and the other against h-IAP (10k), were picked

and subjected their docked complexes to simulations, and (4) cross-

−4.70

a

IC₅₀values shown are the mean

SD of triplicate measurements. ND: not

determined.

10q make it well-aligned to the cleft-like and groove-shaped areas of h-

TNAP pocket, thus rendering it a good ﬁt and most potent analogue

(Fig. 7 and Table 1). Methyl at para position makes the ring rich in

electron enhancing pi-cation interactions with arginine. The ortho

chloro substituent (though electronegative) makes the ring rich in

electrons through M+ eﬀect thereby increasing the electron density of

7

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Fig. 6. Binding orientations of selective analogues against h-TNAP (A) and h-IAP (B). The analogues which docked with more or less similar orientations were

grouped together.

Fig. 7. Comparative representation of the docked poses of dual active analogues against h-TNAP and h-IAP.

docking of the analogues from step 3 (i.e. 10e + h-IAP and 10k + h-

TNAP) was carried out and subjected their docked complexes to si-

mulations. MD trajectories for the systems 1–3 were evaluated in terms

of structural stability of ligand–protein(s) complexes and the binding

free energy calculated by Molecular Mechanics/Poison Born Surface

Area (MM/PBSA) method [33]. MD trajectories for the system 4 were

only evaluated to determine the stability of respective ligand(s) within

the binding site(s).

the simulation trajectories (Fig. 8B and supplementary Table S1). The

average distance ranges from 2.3 to 2.45 Å for all the complexes in

systems 1–3 (supplementary Table S1), which manifests the stability of

ligands within the respective binding pocket(s). These short distances

further suggested that the core triazole-thiol moiety stayed ﬁrmly or-

iented towards the Zn ions during simulations. Nevertheless, the ligands

in system 4 could not remain stable in the binding pocket(s). From the

visual inspection of MD trajectories, a complete expulsion of the com-

pounds from the respective pockets was witnessed, which is also vali-

dated by larger distance(s) between Zn1 and –SH functionality (Fig. 8B

and supplementary Table S1). This provides an indirect validation for

the absolute selectivity of compounds 10e and 10k for h-TNAP and h-

IAP, respectively.

3.6. Stability of the ligand–protein(s) complexes during MD simulations

As shown in Fig. 8A, the RMSD for the main chain atoms of h-TNAP

and h-IAP, in all the complexes for systems 1–3, reached thermo-

dynamic equilibrium within the initial 20 ns. To monitor the stability of

ligand(s) within the protein binding pocket(s), the distance between

Zn1 and the –SH (of the core triazole-thiol moiety) was measured along

The ligand RMSD for systems 1–3 ﬂuctuated largely, as is evident

from the conformational or positional changes observed for the ligand

in each complex (shown in Fig. 8C). Especially the compounds (10e and

8

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Fig. 8. MD simulations for selective and dual active analogues against h-TNAP and h-IAP. (A) Backbone RMSD of the entire protein (h-TNAP and h-IAP) in complex

with the ligands. (B) Distance between Zn1 and –SH group of the core triazole-thiol moiety. (C) Ligand RMSD plots for systems 1–3.

10k) selective against either of the two proteins ﬂuctuated continuously

along the MD trajectories as compared to the dual-active ones (10m and

10q), which are relatively stable in the last 10 ns of the trajectories

(Fig. 8C). Previously, it has been reported that the diﬀerence of the

conformation of the rotatable ring moieties in the ligand(s), as well as

the limitation of the homology modeling in building ﬂexible loop re-

gions of the protein, could induce large ﬂuctuations in ligand RMSD

values under MD simulations [34]. A similar phenomenon was ob-

served in case of h-TNAP and h-IAP complexes, when we overlaid the

representative MD conformations over the initial docking pose(s)

(Fig. 8) and also listed the RMSD details of the backbone of the protein,

the core residues within 6 Å of ligand, and the ligand-only along the last

6 ns of all MD trajectories (supplementary Table S2).

moieties of the ligands may further contribute to changes in RMSD as

compared to the initial docking pose. These observations suggest that

the stability of triazole-thiol core in the lower section does not ne-

cessarily guarantee the stability of terminal ring moieties of the com-

pound (s) in the upper section(s), given the fact that the side-chain

conformations of the loop regions are presumed to be less reliable in

homology modeling [34].

The supplementary Table S3 provides a comparison of interacting

residues for the ligand binding poses from pre-MD (initially docked

complex), the representative conformations at 55 ns and 57 ns of MD,

and the post-MD. Interestingly, the residues that were predicted to be

crucial for binding (H171:H172; H338:H336; H341:H339 and

S110:S111 in the lower section, and, R450:S447, and H451:S448 in the

upper section of the pocket(s)) from the initial docked pose(s) are also

prominent among the MD poses. This, together with the constantly

observed orientation of triazole-thiol moiety towards Zn ions, manifests

the reliability of docking predictions, also providing the reason for

further analyses of MD trajectories to calculate binding free energy.

As can be seen in Table S2, the RMSD values for the core and ligand-

only are almost similar for all the complexes. This suggests that the

positional changes in ligand(s) RMSD could be corresponding to the

conformational ﬂuctuations of the loop regions; constituting the

binding site residues, under MD simulations. This is further evident

from the overlapping docked and MD poses where the structural drift of

the surrounding loop regions of the binding site(s) is very clear (Fig. 9).

Moreover, the conformational ﬂuctuations of the rotatable ring

3.6.1. Binding free energy calculations

The MM/PBSA method has been widely utilized to study the

9

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Fig. 9. Overlapping docked and MD poses highlighting the structural drifts of the surrounding loop regions of the binding site(s) for h-TNAP (A) and h-IAP (B). The

initial docked complexes are colored green (for h-TNAP) and cyan green (for h-IAP) in (A) and (B), respectively. The color scheme for the representative con-

formations over the last 6 ns of MD is as follows: cyan (55 ns), magenta (56 ns), yellow (57 ns), orange (58 ns), grey (59 ns), and sky blue (60 ns). The small arrows

are pointing the loop regions that undergo large structural drifts, which ultimately result in positional or conformational changes in the initially docked poses of the

ligands. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Table 3

Table 4

Comparison of experimental (ΔG_exp) and predicted (Δ_MM/PBSA) binding free

energies.

Inhibitory potential for h-TNAP active inhibitors against nucleotide pyropho-

sphatases.

Compound ID

TNAP

Correlation

R²: 0.988

R²: 0.851

h-TNAP active

R¹

R²

h-NPP-1 IC₅₀

h-NPP-3 IC₅₀(µM)^a

Inhibitors

(µM)^a

ΔG_exp(kcal/mol)^a

Δ_MM/PBSA(kcal/mol)

10a

2-OMe

H

> 100

1.33

0.13

1.01

0.03

1.23

10q

10e

10m

−9.32

−7.95

−7.03

IAP

−5.9

−4.6

−0.67

10b

10c

2-OMe Me

2-OMe OMe

2-OMe NO₂

2-OMe Cl

9.19

1.99

10d

10e

21.5

> 100

2.91

10m

10k

10q

−8.69

−8.61

−7.72

−6.75

−5.8

10f

3-OMe

H

10g

3-OMe Me

3-OMe OMe

3-OMe Cl

0.65

−2.15

10h

10j

> 100

2.88

0.37

0.89

a

10m

10n

10o

10p

10q

10u

10w

10x

4-OMe OMe

4-OMe NO₂

4-OMe Cl

5.75

ΔG_exp(kcal/mol) were calculated from experimental IC₅₀using the equa-

tion: ΔG_exp≈RTln IC₅₀, where R is ideal gas constant, T is the temperature in K

(300 K is used in this paper) [36].

> 100

0.13

2-Cl

4-Cl

H

0.01

0.23

Me

H

2.15

ligand–protein interactions in many cases [33,35]. Average 1000

snapshots were extracted from the last 10 ns of MD trajectory at 10 ps

intervals, and the relative binding free energy (Δ_MM/PBSA) was de-

termined. The corresponding binding free energies for each ligand–-

protein complex are summarized in Table 3. There was a good corre-

> 100

2.55

OMe

NO₂

0.41

0.8

Suramin^b

8.67

1.3

1.27

a

b

IC₅₀values shown are the mean

Used as positive control.

SD of triplicate measurements.

lation between the experimental IC₅₀derived binding energies (ΔG_exp

)

versus predicted binding energies (Δ_MM/PBSA).

further tested against nucleotide pyrophosphatase/ phosphodiesterases

(h-NPP-1 and h-NPP-3) (Table 4). On evaluation of the bioactivity re-

sults, it was revealed that most of the compounds were highly active

and selective inhibitors against TNAP.

3.7. Enzyme inhibition against human nucleotide pyrophosphatases

All those compounds that were active against human TNAP were

10

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Fig. 10. The ligand (AMP) supported model

of h-NPP1 and its binding site analysis. The

residues within 8 Å of the pocket are colored

by their physicochemical nature (charged

acidic: red (14%); charged basic: sky blue

(14%); polar neutral: magenta (32%), and

hydrophobic: grey (40%). The binding site

analysis further reveals the features that are

important for the nucleotide/nucleoside

binding pattern and the proposed catalytic

mechanism; the adenine ring oriented to-

wards the hydrophobic region of the pocket

and the (mono, di, or tri) phosphate chain

oriented towards the Zn (Zn1 and Zn2) ions.

The inset ﬁgure shows the high prevalence

of positively charged lysine residues in the pocket outlet, which might hinder the recognition of analogues studied in this work. (For interpretation of the references

to color in this ﬁgure legend, the reader is referred to the web version of this article.)

Compound

5-(2-chlorophenyl)-4-((1,3-diphenyl-1H-pyrazol-4-yl)

these analogues adopt diﬀerent binding orientations because of dif-

ferent positions and nature of R¹and R²moieties, while conforming to

wider or narrower pocket(s) geometries of TNAP and IAP, respectively.

This eventually may aﬀect their ﬁtness within the binding pocket(s) and

thus lend selectivity or dual-activity against either of the proteins.

Diﬀerent binding orientations observed for TNAP selective analo-

gues may explain their SAR in terms of enzyme inhibition activities and

ﬁtness to pocket geometry. For instance, in case of 10e, 10j and 10o,

the electronegative (Cl) substitution at R²imparts selectivity towards

TNAP pertaining to its high electrostatic character; however, the

varying position of methoxy group on benzene ring results in diﬀerent

binding orientations and, thus may compromise their ﬁtness within the

binding pocket, which eventually aﬀects their binding aﬃnity (Fig. 7A

and Table 1) displaying ortho-methoxy showing higher activity then

meta-methoxy followed by para-methoxy analogue. Similarly, the spa-

tial constraint of the cleft-like area to the ortho-, meta-, and para-

methoxy groups, as well as para-chloro (10u) at R¹may inﬂuence the

ﬁtness of respective TNAP selective analogues within the binding

pocket, which is also reﬂected by the magnitude of their enzyme in-

hibition potencies (ortho > para > meta). Due to high potency and

selective inhibition of compounds 10a-y against h-TNAP over h-NPP1,

these compounds may serve as promising leads against vascular calci-

ﬁcation.

methyleneamino)-4H-1,2,4-triazole-3-thiol (10p) was 10-fold strong

inhibitor (IC₅₀= 0.13

0.01 µM) against nucleotide pyropho-

sphatase-3

as

compared to the standard suramin

0.8 µM) (Table 4). It was also found to be highly

(IC₅₀= 1.27

selective for NPP-3 as compared to NPP-1. However, no activity was

observed with same pattern of substitution when chlorine is at para

position. Notably, all the TNAP active 5-aryl-4-(1,3-disubstituted pyr-

azolyl)methyleneamino)-1,2,4-triazole-3-thiols (10a-y) were found to

be inactive against NPP-1 (Table 4). The plausible reason for the in-

activity towards NPP-1 arise through the diﬀerence in the number ly-

sine residues in the active pocket of TNAP as depicted in the next

section (Fig. 10). NPP-1 is rich in lysine residue as compared to NPP3

and TNAP. Our major objective was to explore NPP-1 inactive and

TNAP selective compounds, since TNAP active pocket is rich in arginine

residues, which has higher potential for electrostatic interactions as

compared to that of lysine. Therefore, compounds 10a-f, 10g, 10h,

10m-q, 10u, 10w-x were found to be NPP-1 inactive and TNAP se-

lective and may serve as lead compounds against vascular calciﬁcation.

These newly discovered inhibitors are believed to represent valuable

lead structures to further streamline the generation of candidate com-

pounds to target vascular calciﬁcation.

3.8. Modeling of h-NPP1 and its binding site analysis

3.9. Drug likeness and pharmacokinetics evaluation of h-TNAP selective

compounds

All the analogues studied in this work did not inhibit h-NPP1

(Table 4). To gain insight into the origin of this absolute no activity

against h-NPP1, we built a ligand supported model for this protein

based on the crystal structure of mouse NPP1 (PDB: 4GTW; as pre-

viously reported by Lecka et al. [37]. As in the case of h-TNAP and h-

IAP, the h-NPP1 model has good stereochemical quality (Fig. S8) and is

structurally similar to the template protein (RMSD of 0.17 Å). The

structural analysis of the binding site highlighted the prevalence of

positively charged lysine residues in the pocket outlet (Fig. 10), which

have previously been proposed to be crucial for diﬀerentiating the

absolute selectivity of negatively charged ATP-like ligands towards h-

NPP1 vs h-NPP3 [37]. Comparing h-NPP1 to the h-TNAP and h-IAP, the

presence and absence of the lysine residues, respectively, seems to be

the main determinant of ligand(s) speciﬁcity. The h-TNAP possesses

more arginine residues (which can favor pi-cation interactions with the

terminal ring moieties of the ligands studied in this work) in its pocket

outlet. Whereas, the high prevalence of lysine residues (unable to favor

pi-cation interactions) may probably disfavor analogue binding to h-

NPP1 binding site.

The compounds active against h-TNAP were evaluated for their

likelihood as a drug molecule by calculating the ADME (absorption,

distribution, metabolism, and excretion) properties. The ADME prop-

erties evaluated included determination of octanol–water partition

coeﬃcient (cLogP), molecular weight (MW), number of hydrogen bond

donors (nNH), number of hydrogen bond acceptors (nON), topological

polar surface area (TPSA), and number of rotatable bonds (nRot) (as

listed in Table 5a). Typically, compounds having MW

< 500,

nON < 10, nNH < 5, nRot < 10, and cLogP < 5 are considered to

be orally bio-available with a favorable ADME proﬁle [38a]. The cLogP

is a measure of compound’s hydrophilicity [38a]. Low hydrophilicities

(high cLogP values) cause poor absorption or permeation. Topological

polar surface area (TPSA) is another useful molecular property that is

widely used in ADME models for predicting oral absorption of drug-like

molecules [38b]. A molecule is expected to be suﬃciently bio-available

if it has a TPSA < 70 Å; however, TPSA ≥ 140 Å is generally un-

desirable for suﬃcient bio-availability [38b]. Majority of the evaluated

compounds showed favorable ADME properties (Table 5a), except

cLogP that was above 5 for many compounds rendering them to be

highly lipophilic. The most potent compound 10q appeared to have

higher cLogP (5.99) and low TPSA (60.91 Å) values which indicate that

it would be best absorbed by lipophilic membranes [38c]. Given this

Overall, the comparative analysis of docked complexes suggests that

the ligand shape complementarity, with respect to pocket(s) geometry,

is the key determinant of selectivity and dual-activity and, to deﬁne

ligand complementarity, the size, position and nature of R¹and R²

substitutions is of critical value. As can be observed from Figs. 7 and 8,

11

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Table 5a

drug eﬀect and/or toxicity due to enzyme induction [39a]. As shown in

Table 5b, the cytochrome CYPP3A4 isoform (Table 5b) could be in-

hibited by compound 10d, 10e 10n, 10o, 10p , 10q, 10u and 10w

bearing electron withdrawing groups in the scaﬀold. Almost all the

compounds were predicted to have potential against inhibition of

CYP2C9 CYP2C19 (Table 5b). However none of the imines (10a-y) is

predicted to have potential to be the inhibitor of CYP2D6. It has also

been observed that almost all of the pyrazolyl triazoles are not found as

substrates of important cytochromes CYP2C9 and CYP2D6 (Table 5b)

which indicates the metabolic stability of the compounds towards these

cytochromes. Fortunately, all compounds did not show any toxicity and

carcinogenicity.

Drug-like properties of compounds active against h-TNAP.

Compound

MW^a

cLogP^b

nNH^a

nON^a

TPSA^a

nRot. bond^a

10a

10b

10c

10d

10e

10f

452.5

4.51

5.01

4.49

4.30

5.25

5.07

5.57

5.05

5.81

5.05

4.86

5.81

5.49

5.99

5.74

5.71

5.52

0

7

70.14

79.38

115.97

70.14

79.38

70.14

79.38

115.97

70.14

60.91

70.14

106.73

6

7

6

7

6

7

6

5

6

466.57

482.57

497.54

486.99

452.54

466.57

482.57

486.99

482.57

497.54

486.99

456.96

470.99

456.96

486.99

501.96

7

8

10

7

10g

10h

10j

7

8

7

10m

10n

10o

10p

10q

10u

10w

10x

8

10

7

3.10. Chemical stability and frontier molecular orbitals (FMOs)

6

It has been reported that the imines derived from acyclic amines are

usually unstable [39b] and undergo spontaneous nonenzymatic hy-

drolysis to the corresponding amine and aldehyde metabolites [39c].

However imines that contain aryl substituents are substantially more

stable and more readily synthesized, while those which contain alkyl

substituents are relatively unstable. It has also been reported that

imines derived from aliphatic aldehydes are relatively unstable while

those derived from aromatic aldehydes having eﬀective conjugation are

more stable [39d,e]. The imines presented in this work are derived from

highly conjugated heteroaryl aldehyde and heteroaryl amines and are

found as stable imines as observed in quantum chemical calculation of

frontier orbitals

6

7

9

a

b

Calculated using Molinspiration® online service.

Calculated using ChemDraw Ultra 10.0, CambridgeSoft.

fact, further lead-optimization studies for compound 10q are desired to

improve its eﬃciency in terms of safety liability.

3.9.1. Pharmakokinetics (absorptiom, metabolism and toxicity)

The ADMET properties of the human-TNAP selective compounds

were calculated using admetSAR and SwissADME (Table 5b). It was

found that almost all tested compounds could be absorbed by the

human intestine however some of the compounds were predicted to

have low intestinal absorption, i.e. compounds bearing nitro function-

ality. Nevertheless, all of tested compounds were predicted as non-

substrates for Pglycoprotein (P-gp) which eﬄuxes drugs and various

compounds to undergo further metabolism and clearance. Many of the

human microsomal cytochrome P450s catalyze the metabolism of a

wide variety of compounds including xenobiotics and drugs. Drug

metabolism via the cytochrome P450 system has emerged as an im-

portant determinant in the occurrence of several drug interactions that

can result in reduced pharmacological eﬀect, and adverse drug reac-

tions [39a]. Most drugs can exhibit decreased eﬃcacy due to rapid

metabolism, but drugs with active metabolites can display increased

To explore the chemical stability of the synthesized imines theore-

tical calculations were conducted using WEBMO where the quantum

chemical calculations on the optimized structures were performed using

Gaussian 16 with Parametric Method 3 (PM3) and Austin Model 1

(AM1) semi-empirical method. The optimized molecular structures of

the investigated inhibitor are given in Fig. 11A while the frontier mo-

lecular orbital density distributions of a representative inhibitor are

depicted in Fig. 11B. The molecular frontier orbitals play an important

role in determining the electronic properties of the molecular systems

[40]. The chemical reactivity of these compounds depends upon the

frontier molecular orbitals (FMOs), consisting of the highest occupied

molecular orbital, HOMO, and the lowest unoccupied molecular orbital,

LUMO [40]. A higher value of energy gap (ΔE = E_LUMO–E_HOMO) in-

dicates chemically stable molecules. Additionally,

a

larger

Table 5b

Insilico absorptiom, metabolism and toxicity) of compounds active against h-TNAP.

TNAP

Human I.A^aHuman

P-GP

CYP2C9

CYP2D6

CYP3A4

CYP2C9

inh.^#

CYP2C19

CYP

2D6

inh.

CYP

UGT

Carcinog-

enicity

Acute oral

Toxicity

#

e

actve

O.B.^b

subs.^c

subs.*

inh.^#

inh.

inhib.

cat.

#

d

compd.

pro.

10a

high

+

–

+

–

+

–

Cat. III

(Very low)

Very low

10b

10c

10d

10e

10f

high

low

+

–

+

–

+

–

+

–

low

high

low

10g

10h

10j

–

10m

10n

10o

10p

10q

10u

10w

10x

low

–

low

+

–

low

high

low

(+indicates positively active response; - indicates inactive or negative responce). a. Human Intestinal Asorption calculated using SwissADME. Rest of the PK

predictions were calculated using admetSAR, b. Human Oral Bioavailability, c. p-Glycoprotein substrate, subs.* = substrate, inh^#. = Inhibitor, d. CYP inhibitory

promiscuity, e. UGT catalyzed.

12

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Fig. 11. A) Optimized geometry of a representative pyrazolylmethyleneaminotriazole-3-thiol, Color: red for oxygen, white for hydrogen, Dark grey for carbon and

blue for nitrogen. B) Frontier molecular orbital density distribution and energy gap (ΔE = E_LUMO–E_HOMO) of the representative pyrazolylmethyleneaminotriazole-3-

thiol investigated in this work. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)

HOMO–LUMO energy gap is shown by chemically hard molecules, in-

dicating that the molecular systems resist changes in electronic con-

ﬁguration. A molecule having a small frontier orbitals gap (ΔE) is more

polarizable and is generally associated with a high chemical reactivity

and low kinetic stability [41]. From the frontier orbital calculations, it

was observed from the calculated HOMO and LUMO energy gap that

1,3-disubstituted

pyrazolyl)methyleneamino-5-aryl-1,2,4-triazole-3-

thiols may possess higher chemical stability (ΔE = 7.86 eV) as com-

pared to other schiﬀ bases reported in literature (ΔE = 0.27–4.65 eV)

[41–43]

3.11. Mechanism of action

Deﬁning the binding mode and site of action is important for

building a predictive pharmacophore model to facilitate future medic-

inal chemistry eﬀorts. When used for determining the mode of enzyme

inhibition, the Lineweaver–Burk plot can distinguish competitive, non-

competitive and uncompetitive inhibitors. Mechanism of action studies

with 10q at diﬀerent concentrations of phosphate- donor substrate, i.e,

2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′-(5-chlorotricyclo

[3.3.1.1]decan])-4-yl]-1-phenyl disodium phosphate) (CDP-star) was

determined. The artiﬁcial substrate CDP-star is expected to provide an

adequate system for studying the eﬀects of most active compound 10q

on h-TNAP. As shown in Fig. 12, [CDP-star]-dependent curves obtained

in the presence of diﬀerent concentrations of 10q were plotted. Line-

weaver-Burk coordinates yields x-intercepts and diﬀerent slopes be-

tween the two data sets keeping y-intercept same as uninhibited en-

zyme (since V_maxis unaﬀected by competitive inhibitors the inverse of

V_maxalso doesn't change). Therefore 10q may inhibit TNAP through

competitive mode of inhibition.

Fig. 12. Lineweaver-Burk plot for h-TNAP inhibition by compounds 10q where

S is concentration of substrate CDP-star® (μM).

ring and a para-tolyl ring at pyrazole skeleton emerged as a highly

potent inhibitor against h-TNAP with 127-fold increased inhibition

compared to levamisole. Compound 10e was found to be most selective

inhibitor against APs and NPPs. Computational studies, involving

binding site(s) architecture analysis, molecular-docking and -dynamics

simulations (MDS), further revealed the basis for h-TNAP and h-IAP

ligand selectivity. Moreover, the presence of arginine and lysine re-

sidues in the binding pockets of h-TNAP and h-NPP-1, respectively,

appeared to be key determinant of absolute selectivity towards h-TNAP

or no activity towards h-NPP1 for the designed inhibitors. These newly

discovered inhibitors are believed to represent lead structures to further

streamline the generation of candidate compounds to target vascular

calciﬁcation.

3.11.1. Conclusions

In this study, a series of novel 5-aryl-4-(1,3-disubstituted pyrazolyl)

methyleneamino)-1,2,4-triazole-3-thiols (10a-y) was designed and

synthesized as potent and selective inhibitors of TNAP over NPP1. The

selective inhibition of h-TNAP over h-NPP1 may serve as a useful

therapeutic strategy against vascular calciﬁcation. The biological

screening against h-TNAP, h-IAP, h-NPP1 and h-NPP3 showed that

compounds 10a-h, 10j, 10m-q, 10u, 10w, and 10x displayed enhanced

activity and complete selectivity towards h-TNAP over h-NPP1.

Compound 10q bearing an ortho-chlorophenyl moiety at the triazole

4. Experimental notes

4.1. Substrates and reagents

The reagents used were of analytical grade. The substituted acet-

ophenones, benzoic acids, phenyl hydrazine, glacial acetic acid, di-

methylformamide and carbon disulﬁde were purchased form Sigma-

13

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

Aldrich (Steinheim, Germany). Ethylenediamine and phosphorus oxy-

chloride were the products of Merck (Darmstadt, Germany). Ethanol,

chloroform and methanol were supplied by Lab Scan (Patuman,

Bangkok, Thailand). Ethanol and dichloromethane were the products of

Riedel de Haen (Seezle, Germany), while ethyl acetate was obtained

from commercial sources and distilled before use. Solvents were dried

prior to use wherever required.

Thiocarbohydrazide was recrystallized from ethanol.

Synthesis of 5-aryl-1,2,4-triazole-3-thiol: Equimolar amounts of thio-

carbohydrazide and appropriate carboxylic acids (10 mmol of each)

were mixed and heated at 170–185 °C for 1.5–2 h. Boiling water

(20 mL) was added to the solid and the mixture was kept at room

temperature for 24 h. The precipitate was ﬁltered and recrystallized

from ethanol to aﬀord the title compounds. Physical data was reported

in literature [44b].

4.2. Instrumentation

4.5. General procedure for synthesis of arylpyrazolylmethyleneamino

triazole-3-thiols (10a-y)

Thin layer chromatography (TLC) was performed on 0.2 mm DC

kieselgel 60 F₂₅₄precoated aluminium plates by Merck (Darmstadt,

Germany). Compounds were visualized by exposure to UV light at

254 nm. Melting points were recorded in open capillaries using

Gallenkamp melting point apparatus (MP-D) (Ontario, Canada) and are

uncorrected. FTIR spectra were recorded on a Thermoscientiﬁc (USA)

Fourier Transform Infra-Red Spectrophotometer model Nicolet 6700

using attenuated total reﬂectance (ATR) technique. NMR spectra were

obtained at room temperature on Bruker DQX400 (Coventry, UK)

spectrometer. ¹H and ¹³C NMR signals were calibrated using the re-

sidual protonated solvent (¹H) or the solvent itself (¹³C). High-resolu-

tion mass spectra were recorded on a VG Autospec spectrometer (New

Jersey, USA) using chemical ionization or on a Micromass LCT elec-

trospray ionization mass spectrometer (New Jersey, USA) operating at a

resolution of 5000 full width half height. The purity of synthesized

compounds was veriﬁed by an HPLC system equipped with an Eclipse

Plus C8 column (4.6 × 150 mm, 5 μm). For measurement of enzymatic

reaction, ELIZA (Bio-Tek ELx800^TMInstrument, Inc. USA) micro-plate

reader and 96 well-plates were used for the sample analysis.

Micropipettes from Gilson (Middleton, USA) were used for sample

loading. Sodium ^D-glucoronate and D,L-glyceraldehyde were used as

substrates along with a cofactor, i.e. NADPH (nicotinamide adenine

dinucleotide phosphate) from Sigma-Aldrich (London, UK). The IC₅₀

values were calculated using non-linear regression analysis of program

PRISM 5.0 (GraphPad, California, USA).

To a stirred solution of an appropriate triazole (9a-e) (1.0 mmol) in

absolute ethanol (6 mL), was added appropriate 1-phenyl-3-(4-sub-

stituted phenyl)-1H-pyrazole-4-carbaldehyde (4a-g) (1.3 mmol) and

stirring continued at ambient temperature for 10 min. Glacial acetic

acid (2–3 drops) was added and the mixture allowed to stand at am-

bient temperature for 15 min. The temperature was then increased to

45 °C. After completion of the reaction (TLC; (40% acetone in n-

hexane), excess solvent was removed in vacuo to aﬀord solid product.

The crude solid was washed with cold ethanol, ﬁltered, dried and re-

crystallized from ethanol followed by drying under vacuum to yield the

desired compound (10a-y).

4.5.1. 4-((1,3-Diphenyl-1H-pyrazol-4-yl)methyleneamino)-5-(2-

methoxyphenyl)-4H-1,2,4-triazole-3-thiol (10a)

Oﬀ-white solid (85%); m.p.: 285–287 °C; R_f: 0.32 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3009 (]CeH), 1616 (C]N), 1570, 1511

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.16 (1H, s, SH), 9.42 (1H, s,

CH]N), 9.14 (1H, s, ]CH_pyrazolyl), 8.04–7.94 (2H, dd, J = 7.9, 1.6 Hz,

AreH), 7.77–7.66 (2H, dd, J = 7.9, 2.0 Hz, AreH), 7.62–7.57 (1H, m,

AreH), 7.57–7.49 (3H, m, AreH), 7.49–7.34 (4H, m, AreH), 7.17 (1H,

d, J = 7.8 Hz, AreH), 7.12 (1H, t, J = 7.6 Hz, AreH), 3.69 (3H, s,

OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 161.7 (CH]N), 160.0, 157.5,

152.9, 147.6, 138.6, 132.8, 131.3, 131.2, 130.3, 129.6, 128.9, 128.6,

128.5, 127.5, 120.5, 119.2, 114.6, 114.4, 111.9, 55.7 (OCH₃). HRMS

(ESI) exact mass calculated for C₂₅H₂₀N₆OS [M+H]⁺: 453.1419.

Found: 453.1417. Analysis calcd: C, 66.35; H, 4.45; N, 18.57; S, 7.09.

Found: C, 66.40; H, 4.44; N, 18.59; S, 7.10.

4.3. General procedure for the synthesis of 1,3-disubstituted-1H-pyrazole-4-

carbaldehydes (4a-e)

Synthesis of phenyl-3-aryl-1H-pyrazole-4-carbaldehydes (4a-e) re-

ported in our previous work [29]. To a stirred solution of phenyl hy-

drazine (1) (12 mmol) in absolute ethanol (20 mL) was added an ap-

propriate acetophenone (2a-e) (10 mmol) and glacial acetic acid

(0.3 mL). The reaction mixture was stirred at 70 °C. After completion of

the reaction (TLC; 30% ethyl acetate in n-hexane), the excess solvent

was evaporated to yield a crude solid that was ﬁltered, washed with

cold ethanol, dried and recrystallized (ethanol) to yield 1-phenyl-2-(1-

(substituted phenyl)ethylidene) hydrazines (3a-e).

4.5.2. 5-(2-Methoxyphenyl)-4-((1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)

methyleneamino)-4H-1,2,4-triazole-3-thiol (10b)

Oﬀ-white solid (80%); m.p.: 253–256 °C; R_f: 0.35 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3001 (]CeH), 1618 (C]N), 1569, 1506

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.23 (1H, s, SH), 9.54 (1H, s,

CH]N), 9.29 (1H, s, ]CH_pyrazolyl), 8.07–8.00 (2H, m, AreH), 7.76 (2H,

d, J = 7.9 Hz, AreH), 7.56 (2H, t, J = 7.8 Hz, AreH), 7.53–7.37 (4H,

m, AreH), 7.31 (2H, d, J = 7.8 Hz), 7.12 (1H, ddd, J = 8.1, 2.6, 1.1 Hz,

AreH), 3.77 (3H, s, OCH₃), 2.38 (3H, s, CH₃); ¹³C NMR (101 MHz,

DMSO‑d₆): δ 162.2, 161.1, 159.0, 153.1, 148.0, 138.6, 138.4, 129.8

129.8, 129.5, 129.2, 128.3, 127.4, 126.6, 120.5, 119.1, 116.3, 114.3,

114.3, 113.3, 55.1 (OCH₃), 20.7 (CH₃). HRMS (ESI) exact mass calcu-

lated for C₂₆H₂₂N₆OS [M+H]⁺: 467.1576. Found: 467.1571. Analysis

calcd: C, 66.93; H, 4.75; N, 18.01; S, 6.87. Found: C, 66.82; H, 4.73; N,

18.05; S, 6.89.

The appropriate 1-phenyl-2-(1-(substituted phenyl)ethylidene)hy-

drazine (3a-e) (1.0 mmol) was added to a cold solution of di-

methylformamide (4 mL) and phosphorus oxychloride (0.5 g,

3.0 mmol); and the resulting mixture heated at 70 °C for 3 h. After

cooling to room temperature, the reaction mixture was poured onto

crushed ice and neutralized with an aqueous solution of sodium bi-

carbonate. The precipitated solid was ﬁltered and puriﬁed through re-

crystallisation from ethanol.

4.5.3. 5-(2-Methoxyphenyl)-4-((3-(4-methoxyphenyl)-1-phenyl-1H-

pyrazol-4-yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10c)

4.4. General procedure for synthesis of 5-aryl-1,2,4-triazole-3-thiols (9a-e)

Oﬀ-white solid (80%); m.p.: 289–291 °C; R_f: 0.34 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3015 (]CeH), 1617 (C]N), 1571, 1513

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.13 (1H, s, SH), 9.40 (1H, s,

CH]N), 9.11 (1H, s, ]CH_pyrazolyl), 8.01–7.93 (2H, d, J = 8.0 Hz,

AreH), 7.69–7.60 (2H, m, AreH), 7.60–7.47 (4H, m, AreH), 7.43–7.34

(1H, m, AreH), 7.20–7.07 (2H, m, AreH), 6.99–6.90 (2H, m, AreH),

3.80 (3H, s, OCH₃), 3.69 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆):

δ 161.7, 160.1, 159.8, 157.5, 152.7, 147.6, 138.7, 132.8, 131.4, 130.4,

Synthesis of thiocarbohydrazide (modiﬁed procedure [44]): Hy-

drazine hydrate (1) (1.2 mol) was placed in a round bottom ﬂask,

0.3 mol of carbon disulﬁde (2) was added dropwise into the ﬂask while

maintaining the temperature below 10 °C and the temperature was

gradually raised for 1.5 h to 40 °C. The completion of the reaction was

indicated by TLC. The reaction mixture was cooled to room tempera-

ture; the precipitate was ﬁltered and washed with water.

14

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

129.8, 129.6, 127.4, 123.5, 120.5, 119.1, 114.7, 114.1, 114.0, 111.9,

55.7 (OCH₃), 55.2 (OCH₃). HRMS (ESI) exact mass calculated for

18.09; S, 6.83.

C

₂₆H₂₂N₆O₂S [M+H]⁺: 484.1525. Found: 483.1529. Analysis calcd: C,

4.5.8. 5-(3-Methoxyphenyl)-4-((1-phenyl-3-(4-methoxyphenyl)-1H-

pyrazol-4-yl)methylene amino)-4H-1,2,4-triazole-3-thiol (10 h)

64.71; H, 4.60; N, 17.42; S, 6.64. Found: C, 64.71; H, 4.65; N, 17.39; S,

6.68.

Oﬀ-white solid (88%); m.p.: 274–276 °C; R_f: 0.29 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3006 (]CeH), 1623 (C]N), 1577, 1527

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.13 (1H, s, SH), 9.40 (1H, s,

CH]N), 9.11 (1H, s, ]CH_pyrazolyl), 8.01–7.93 (2H, d, J = 8.2 Hz,

AreH), 7.69–7.60 (2H, dd, J = 7.2, 2.4 Hz, AreH), 7.60–7.47 (4H, m,

AreH), 7.43–7.34 (1H, m, AreH), 7.20–7.07 (2H, m, AreH), 6.99–6.91

(2H, m, AreH), 3.80 (3H, s, OCH₃), 3.69 (3H, s, OCH₃); ¹³C NMR

(101 MHz, DMSO‑d₆): δ 161.7, 160.1, 159.8, 157.5, 152.7, 147.6,

138.7, 132.8, 131.4, 130.4, 129.8, 129.6, 127.4, 123.6, 120.5, 119.1,

114.7, 114.1, 114.0, 111.9, 55.8 (OCH₃), 55.2 (OCH₃). HRMS (ESI)

exact mass calculated for C₂₆H₂₂N₆O₂S [M+H]⁺: 483.1525. Found:

483.1528. Analysis calcd: C, 64.71; H, 4.60; N, 17.42; S, 6.64. Found: C,

64.83; H, 4.63; N, 17.41; S, 6.70.

4.5.4. (5-(2-Methoxyphenyl)-4-((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-

4-yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10d)

Oﬀ-white solid (79%); m.p.: 253–256 °C; R_f: 0.22 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3005 (]CeH), 1609 (C]N), 1575, 1515

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.15 (1H, s, SH), 9.71 (1H, s,

CH]N), 9.25 (1H, s, ]CH_pyrazolyl), 8.15–8.06 (2H, m, AreH),

8.03–7.91 (4H, m, AreH), 7.56 (3H, m AreH), 7.49 (1H, dd, J = 7.5,

1.7 Hz, AreH), 7.46–7.36 (1H, m, AreH), 7.16–7.04 (2H, m, AreH),

3.67 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 162.0, 160.4,

160.0, 153.0, 147.4, 138.9, 133.4, 133.0, 133.0, 130.8, 130.1, 129.8,

129.7, 127.7, 127.6, 125.3, 123.8, 119.4, 114.2, 114.2, 55.4 (OCH₃).

HRMS (ESI) exact mass calculated for C₂₅H₁₉N₇O₃S [M+H]⁺

:

498.1270. Found: 498.1275. Analysis calcd: C, 60.35; H, 3.85; N, 19.71;

S, 6.44. Found: C, 60.44; H, 3.89; N, 19.77; S, 6.40.

4.5.9. 5-(3-Methoxyphenyl)-4-((1-phenyl-3-(4-nitrophenyl)-1H-pyrazol-4-

yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10i)

Pale yellow solid (84%); m.p.: 279–281 °C; R_f: 0.26 (40% acetone in

n-hexane); FTIR (ATR, cm⁻¹): 3001 (]CeH), 1615 (C]N), 1569, 1511

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.15 (1H, s, SH), 9.71 (1H, s,

CH]N), 9.25 (1H, s, ]CH_pyrazolyl), 8.15–8.06 (2H, d, J = 8.0 Hz,

AreH), 8.03–7.91 (4H, m, AreH), 7.56 (3H, m, AreH), 7.49 (1H, dd,

J = 7.5, 1.7 Hz, AreH), 7.46–7.36 (1H, m, AreH), 7.16–7.04 (2H, m,

AreH), 3.67 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 162.0,

160.4, 160.0, 153.0, 147.4, 138.9, 133.4, 133.0, 133.0, 130.8, 130.1,

129.8, 127.7, 127.6, 123.1, 125.3, 123.8, 119.4, 114.2, 114.2, 55.4

4.5.5. 5-(2-Methoxyphenyl)-4-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-

4-yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10e)

Oﬀ-white solid (82%); m.p.: 262–266 °C; R_f: 0.25 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3022 (]CeH), 1619 (C]N), 1575, 1519

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.16 (1H, s, SH), 9.50 (1H, s,

CH]N), 9.16 (1H, s, ]CH_pyrazolyl), 8.02–7.93 (2H, dd, J = 7.7, 2.1 Hz,

AreH), 7.75–7.66 (2H, dd, J = 7.7, 2.1 Hz, AreH), 7.63–7.47 (4H, m,

AreH), 7.45–7.35 (3H, m, AreH), 7.19–7.06 (2H, m, AreH), 3.68 (3H,

s, OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 161.7, 159.3, 157.5, 151.3,

147.7, 138.5, 133.6, 132.8, 131.4, 131.2, 130.2, 130.1, 129.6, 128.5,

127.6, 120.5, 119.2, 114.6, 114.5, 111.9, 55.7 (OCH₃). HRMS (ESI)

exact mass calculated for C₂₅H₁₉ClN₆OS [M+H]⁺: 487.1029. Found:

487.1025. Analysis calcd: C, 61.66; H, 3.93; N, 17.26; S, 6.58. Found: C,

61.62; H, 3.90; N, 17.29; S, 6.55.

(OCH₃). HRMS (ESI) exact mass calculated for C₂₅H₁₉N₇O₃S [M+H]⁺

:

498.1270. Found: 498.1274. Analysis calcd: C, 60.35; H, 3.85; N, 19.71;

S, 6.44. Found: C, 60.49; H, 3.84; N, 19.79; S, 6.42.

4.5.10. 5-(3-Methoxyphenyl)-4-((1-phenyl-3-(4-chlorophenyl)-1H-

pyrazol-4-yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10j)

Oﬀ-white solid (86%); m.p.: 255–257 °C; R_f: 0.32 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3017 (]CeH), 1625 (C]N), 1576, 1518

(C]C); ¹H NMR (101 MHz, DMSO‑d₆): δ 14.16 (1H, s, SH), 9.48 (1H, s,

CH]N), 9.25 (1H, s, ]CH_pyrazolyl), 7.99–7.91 (2H, d, J = 8.2 Hz,

AreH), 7.84–7.76 (2H, m, AreH), 7.55–7.44 (4H, m, AreH), 7.44–7.30

(4H, m, AreH), 7.04 (1H, ddd, J = 7.9, 2.7, 1.4 Hz, AreH), 3.68 (3H, s,

OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 162.2, 160.5, 160.1, 153.1,

147.5, 139.0, 133.5, 133.2, 133.1, 130.9, 130.2, 129.9, 127.8, 127.8,

125.4, 123.9, 119.5, 114.4, 114.3, 55.6 (OCH₃). HRMS (ESI) exact mass

calculated for C₂₅H₁₉ClN₆OS [M+H]⁺: 487.1029. Found: 487.1024.

Analysis calcd: C, 61.66; H, 3.93; N, 17.26; S, 6.58. Found: C, 61.61; H,

3.97; N, 17.22; S, 6.54.

4.5.6. 4-((1,3-Diphenyl-1H-pyrazol-4-yl)methyleneamino)-5-(3-

methoxyphenyl)-4H-1,2,4-triazole-3-thiol (10f)

Oﬀ-white solid (81%); m.p.: 267–269 °C; R_f: 0.36 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3009 (]CeH), 1617 (C]N), 1562, 1519

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.23 (1H, s, SH), 9.54 (1H, s,

CH]N), 9.32 (1H, s, ]CH_pyrazolyl), 8.11–7.98 (2H, d, J = 7.6 Hz,

AreH), 7.91–7.79 (2H, m, AreH), 7.60–7.54 (2H, m, AreH), 7.49 (4H,

ddd, J = 5.4, 3.2, 1.4 Hz, AreH), 7.47–7.39 (3H, m, AreH), 7.11 (1H,

ddd, J = 8.1, 2.6, 1.2 Hz, AreH), 3.76 (3H, s, OCH₃); ¹³C NMR

(101 MHz, DMSO‑d₆): δ 162.3, 161.2, 159.1, 153.3, 148.1, 138.7,

131.2, 129.9, 129.8, 129.6, 129.0, 128.7, 128.5, 127.6, 126.7, 120.6,

119.2, 116.4, 114.5, 113.4, 55.2 (OCH₃). HRMS (ESI) exact mass cal-

culated for

C

₂₅H₂₀N₆OS [M+H]⁺

:

453.1419. Found: 453.1416.

4.5.11. 4-((1,3-Diphenyl-1H-pyrazol-4-yl)methyleneamino)-5-(4-

methoxyphenyl)-4H-1,2,4-triazole-3-thiol (10k)

Analysis calcd: C, 66.35; H, 4.45; N, 18.57; S, 7.09. Found: C, 66.33; H,

4.48; N, 18.54; S, 7.07.

Oﬀ-white solid (89%); m.p.: 267–269 °C; R_f: 0.38 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3019 (]CeH), 1619 (C]N), 1573, 1511

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.11 (1H, s, SH), 9.59 (1H, s,

CH]N), 9.31 (1H, s, ]CH_pyrazolyl), 8.07 (2H, d, J = 8.1, AreH),

7.93–7.82 (4H, m, AreH), 7.55 (5H, m, AreH), 7.44 (1H, t, J = 7.4 Hz,

AreH), 7.09 (2H, d, J = 8.8 Hz, AreH), 3.82 (3H, s, OCH₃); ¹³C NMR

(101 MHz, DMSO‑d₆): δ 160.9, 160.7, 153.3, 148.3, 138.7, 131.2,

129.9, 129.7, 129.6, 129.0, 128.7, 128.5, 127.5, 119.3, 117.8, 114.6,

114.1, 100.1 55.3 (OCH₃). HRMS (ESI) exact mass calculated for

4.5.7. 5-(3-Methoxyphenyl)-4-((1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)

methyleneamino)-4H-1,2,4-triazole-3-thiol (10 g)

Oﬀ-white solid (79%); m.p.: 262–265 °C; R_f: 0.27 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3011 (]CeH), 1612 (C]N), 1568, 1509

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.24 (1H, s, SH), 9.55 (1H, s,

CH]N), 9.30 (1H, s, ]CH_pyrazolyl), 8.08–7.99 (2H, d, J = 7.9 Hz,

AreH), 7.77 (2H, d, J = 7.9 Hz, AreH), 7.68–7.51 (3H, m, AreH),

7.51–7.36 (3H, m, AreH), 7.32 (2H, d, J = 7.8 Hz, AreH), 7.13 (1H, m,

AreH), 3.78 (3H, s, OCH₃), 2.39 (3H, s, CH₃); ¹³C NMR (101 MHz,

DMSO‑d₆): δ 162.3, 161.2, 159.1, 153.4, 148.1, 138.7, 138.5, 129.9,

129.6, 129.3, 128.3, 128.4, 127.5, 126.7, 120.6, 119.1, 119.2, 116.4,

114.4, 113.4, 55.2 (OCH₃), 20.8 (CH₃). HRMS (ESI) exact mass calcu-

lated for C₂₆H₂₂N₆OS [M+H]⁺: 467.1576. Found: 467.1573. Analysis

calcd: C, 66.93; H, 4.75; N, 18.01; S, 6.87. Found: C, 66.88; H, 4.78; N,

C

₂₅H₂₀N₆OS [M+H]⁺: 453.1419. Found: 453.1416. Analysis calcd: C,

66.35; H, 4.45; N, 18.57; S, 7.09. Found: C, 66.28; H, 4.40; N, 18.53; S,

7.06.

4.5.12. 5-(4-Methoxyphenyl)-4-((1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)

methyleneamino)-4H-1,2,4-triazole-3-thiol (10l)

Oﬀ-white solid (82%); m.p.: 265–267 °C; R_f: 0.41 (40% acetone in n-

15

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

hexane); FTIR (ATR, cm⁻¹): 3009 (]CeH), 1609 (C]N), 1566, 1519

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.10 (1H, s, SH), 9.57 (1H, s,

CH]N), 9.28 (1H, s, ]CH_pyrazolyl), 8.09–8.01 (2H, d, J = 7.8 Hz,

AreH), 7.90–7.83 (2H, m, AreH), 7.79–7.72 (2H, d, J = 7.8 Hz,

AreH), 7.57 (2H, m, AreH), 7.47–7.38 (1H, m, AreH), 7.36–7.29 (2H,

m, AreH), 7.11–7.04 (2H, m, AreH), 3.81 (3H, s, OCH₃), 2.38 (3H, s,

CH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 162.4, 161.3, 161.2, 153.8,

148.7, 139.2, 139.0, 130.4, 130.1, 130.1, 129.8, 128.9, 128.0, 119.8,

118.3, 115.0, 114.6, 55.8 (OCH₃), 21.3 (CH₃). HRMS (ESI) exact mass

calculated for C₂₆H₂₂N₆OS [M+H]⁺: 467.1576. Found: 467.1571.

Analysis calcd: C, 66.93; H, 4.75; N, 18.01; S, 6.87. Found: C, 67.10; H,

4.77; N, 18.05; S, 6.82.

161.9, 158.2, 150.2, 147.4, 147.4, 138.6, 137.9, 133.3, 132.9, 132.7,

132.6, 127.8, 129.8, 129.8, 127.8, 127.9, 127.5, 125.1, 123.5, 119.4,

115.1. HRMS (ESI) exact mass calculated for C₂₄H₁₇ClN₆S [M+H]⁺

:

457.0924. Found: 457.0928. Analysis calcd: C, 63.08; H, 3.75; N, 18.39;

S, 7.02. Found: C, 63.19; H, 3.74; N, 18.42; S, 7.04.

4.5.17. 5-(2-Chlorophenyl)-4-((1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)

methyleneamino)-4H-1,2,4-triazole-3-thiol (10q)

Oﬀ-white solid (79%); m.p.: 273–276 °C; R_f: 0.28 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3015 (]CeH), 1619 (C]N), 1566, 1517

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.35 (1H, s, SH), 9.63 (1H, s,

CH]N), 9.10 (1H, s, ]CH_pyrazolyl), 8.01–7.95 (2H, d, J = 8.1 Hz,

AreH), 7.73 (1H, m, AreH), 7.71–7.63 (2H, d, J = 7.1 Hz, AreH),

7.63–7.59 (2H, m, AreH), 7.54 (3H, m, AreH), 7.43–7.37 (1H, m), 7.25

(2H, d, J = 7.8 Hz, AreH), 2.37 (3H, s, CH₃); ¹³C NMR (101 MHz,

DMSO‑d₆): δ 162.3, 160.5, 153.5, 147.6, 139.1, 138.9, 133.6, 133.3,

133.2, 130.7, 130.3, 130.0, 129.6, 128.8, 128.8, 127.9, 127.9, 125.5,

119.7, 114.7, 21.3 (CH₃). HRMS (ESI) exact mass calculated for

4.5.13. 5-(4-Methoxyphenyl)-4-((3-(4-methoxyphenyl)-1-phenyl-1H-

pyrazol-4-yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10 m)

Oﬀ-white solid (83%); m.p.: 272–275 °C; R_f: 0.33 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3007 (]CeH), 1621 (C]N), 1559, 1513

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.09 (1H, s, SH), 9.54 (1H, s,

CH]N), 9.26 (1H, s, ]CH_pyrazolyl), 8.07–7.99 (2H, dd, J = 7.9, 2.1 Hz,

AreH), 7.92–7.75 (4H, m, AreH), 7.61–7.51 (2H, d, J = 7.8 Hz,

AreH), 7.46–7.36 (1H, m, AreH), 7.13–7.00 (4H, m, AreH), 3.81 (3H,

s, OCH₃), 3.80 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 161.7,

160.1, 159.8, 157.5, 152.7, 147.6, 138.7, 132.8, 131.4, 130.4, 129.8,

129.6, 127.4, 123.6, 120.5, 119.1, 114.7, 114.1, 114.0, 55.8 (OCH₃),

55.2 (OCH₃). HRMS (ESI) exact mass calculated for C₂₆H₂₂N₆O₂S [M

+H]⁺: 483.1525. Found: 483.1529. Analysis calcd: C, 64.71; H, 4.60;

N, 17.42; S, 6.64. Found: C, 64.88; H, 4.65; N, 17.46; S, 6.65.

C

₂₅H₁₉ClN₆S [M+H]⁺: 471.1080. Found: 471.1084. Analysis calcd: C,

63.75; H, 4.07; N, 17.84; S, 6.81. Found: C, 63.88; H, 4.03; N, 17.89; S,

6.85.

4.5.18. 5-(2-Chlorophenyl)-4-((3-(4-methoxyphenyl)-1-phenyl-1H-

pyrazol-4-yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10r)

Oﬀ-white solid (85%); m.p.: 286–288 °C; R_f: 0.26 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3021 (]CeH), 1622 (C]N), 1575, 1517

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.34 (1H, s, SH), 9.62 (1H, s,

CH]N), 9.10 (1H, s, ]CH_pyrazolyl), 7.97 (2H, d, J = 7.9 Hz, AreH),

7.73 (1H, d, J = 7.6 Hz, AreH), 7.66 (4H, m, AreH), 7.54 (3H, m,

J = AreH), 7.39 (1H, t, J = 7.4 Hz, AreH), 6.95 (2H, d, J = 8.4 Hz,

AreH), 3.81 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 161.7,

159.3, 157.5, 151.3, 147.7, 138.5, 133.6, 132.8, 131.4, 131.2, 130.2,

130.1, 129.6, 128.5, 127.6, 120.5, 119.2, 114.6, 114.5, 111.9, 55.7

(OCH₃). HRMS (ESI) exact mass calculated for C₂₅H₁₉ClN₆OS [M

+H]⁺: 487.1030. Found: 487.1038. Analysis calcd: C, 61.66; H, 3.93;

N, 17.26; S, 6.58. Found: 61.62; H, 3.97; N, 17.30; S, 6.53.

4.5.14. 5-(4-Methoxyphenyl)-4-((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-

4-yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10n)

Oﬀ-white solid (80%); m.p.: 281–283 °C; R_f: 0.21 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3012 (]CeH), 1615 (C]N), 1577, 1514

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.33 (1H, s, SH), 9.61 (1H, s,

CH]N), 9.09 (1H, s, ]CH_pyrazolyl), 7.96 (2H, d, J = 7.9 Hz, AreH),

7.73 (1H, d, J = 7.6 Hz, AreH), 7.66 (4H, m, AreH), 7.53 (3H, m,

AreH), 7.38 (1H, m, AreH), 6.94 (2H, d, J = 8.4 Hz, AreH), 3.81 (3H,

s, OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 162.0, 160.4, 160.0, 153.0,

147.4, 138.9, 133.4, 133.0, 133.0, 130.8, 130.1, 129.8, 127.7, 125.3,

123.8, 119.4, 114.2, 114.2, 55.4 (OCH₃). HRMS (ESI) exact mass cal-

4.5.19. 5-(2-Chlorophenyl)-4-((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-

yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10 s)

culated for

C

₂₅H₁₉N₇O₃S [M+H]⁺

:

498.1270. Found: 498.1278.

Oﬀ-white solid (83%); m.p.: 275–277 °C; R_f: 0.29 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3008 (]CeH), 1615 (C]N), 1514, 1511

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.37 (1H, s, SH), 9.93 (1H, s,

CH]N), 9.23 (1H, s, ]CH_pyrazolyl), 8.13 (2H, d, J = 8.4 Hz, AreH),

7.97 (4H, m, AreH), 7.70 (1H, d, J = 7.6 Hz, AreH), 7.58 (2H, m

AreH), 7.52 (1H, m, AreH), 7.51–7.38 (3H, m, AreH); ¹³C NMR

(101 MHz, DMSO‑d₆): δ 161.9, 158.2, 150.2, 147.4, 147.4, 138.6,

137.9, 133.3, 132.9, 132.7, 132.6, 129.8, 129.8, 127.9, 127.7, 127.5,

125.1, 123.5, 119.4, 115.1. HRMS (ESI) exact mass calculated for

Analysis calcd: C, 60.35; H, 3.85; N, 19.71; S, 6.44. Found: C, 60.42; H,

3.81; N, 19.75; S, 6.40.

4.5.15. 5-(4-Methoxyphenyl)-4-((3-(4-chlorophenyl)-1-phenyl-1H-

pyrazol-4-yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10o)

Oﬀ-white solid (86%); m.p.: 263–266 °C; R_f: 0.27 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3014 (]CeH), 1618 (C]N), 1565, 1501

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.13 (1H, s, SH), 9.61 (1H, s,

CH]N), 9.32 (1H, s, ]CH_pyrazolyl), 8.10–8.01 (2H, d, J = 8.1 Hz,

AreH), 7.94–7.77 (4H, m, AreH), 7.64–7.52 (4H, m, AreH), 7.45 (1H,

d, J = 7.4 Hz, AreH), 7.11–7.03 (2H, m, AreH), 3.82 (3H, s, OCH₃);

¹³C NMR (101 MHz, DMSO‑d₆): δ 162.2, 160.5, 160.1, 153.1, 147.5,

139.0, 133.5, 133.2, 133.1, 130.9, 130.2, 129.9, 127.8, 125.4, 123.9,

119.5, 114.4, 114.3, 55.6 (OCH₃). HRMS (ESI) exact mass calculated

for C₂₅H₁₉ClN₆OS [M+H]⁺: 487.1029. Found: 487.1023. Analysis

calcd: C, 61.66; H, 3.93; N, 17.26; S, 6.58. Found: C, 61.62; H, 3.96; N,

17.20; S, 6.51.

C

₂₄H₁₆ClN₇O₂S [M+H]⁺: 501.0775. Found: 501.0778. Analysis calcd:

C, 57.43; H, 3.21; N, 19.53; S, 6.39. Found: C, 57.55; H, 3.27; N, 19.58;

S, 6.41.

4.5.20. 5-(2-Chlorophenyl)-4-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-

4-yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10 t)

Oﬀ-white solid (85%); m.p.: 289–291 °C; R_f: 0.31 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3015 (]CeH), 1619 (C]N), 1569,

1517(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.35 (1H, s, SH), 9.72

(1H, s, CH]N), 9.16 (1H, s, ]CH_pyrazolyl), 8.00–7.95 (2H, d,

J = 8.0 Hz, AreH), 7.74–7.69 (3H, m, AreH), 7.65 (2H, d, J = 8.1 Hz,

AreH), 7.55 (3H, m, AreH), 7.45–7.39 (3H, m, AreH); ¹³C NMR

(101 MHz, DMSO‑d₆): δ 162.0, 151.7, 138.8, 133.9, 133.4, 133.0,

132.9, 132.7, 131.6, 130.5, 130.3, 130.0, 129.3, 129.8, 128.7, 127.8,

127.6, 125.2, 119.5, 114.6. HRMS (ESI) exact mass calculated for

4.5.16. 5-(2-Chlorophenyl)-4-((1,3-diphenyl-1H-pyrazol-4-yl)

methyleneamino)-4H-1,2,4-triazole-3-thiol (10p)

Oﬀ-white solid (88%); m.p.: 253–256 °C; R_f: 0.29 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3007 (]CeH), 1616 (C]N), 1569, 1511

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.37 (1H, s, SH), 9.93 (1H, s,

CH]N), 9.23 (1H, s, ]CH_pyrazolyl), 8.13 (2H, d, J = 8.4 Hz, 7.97 (4H,

m, AreH), 7.70 (1H, d, J = 7.6 Hz, AreH), 7.66–7.47 (6H, m, AreH),

7.43 (1H, t, J = 7.4 Hz, AreH); ¹³C NMR (101 MHz, DMSO‑d₆): δ

C

₂₄H₁₆Cl₂N₆S [M+H]⁺: 491.0534. Found: 491.0538. Analysis calcd: C,

58.66; H, 3.28; N, 17.10; S, 6.53. Found: C, 58.71; H, 3.30; N, 17.09; S,

6.50.

16

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

4.5.21. 5-(4-Chlorophenyl)-4-((1,3-diphenyl-1H-pyrazol-4-yl)

133.6, 133.2, 133.1, 131.8, 130.7, 130.6, 130.2, 130.0, 128.9, 128.0,

127.8, 125.4, 119.7, 114.8. HRMS (ESI) exact mass calculated for

C₂₄H₁₆Cl₂N₆S [M+H]⁺: 491.0534. Found: 491.0539. Analysis calcd: C,

58.66; H, 3.28; N, 17.10; S, 6.53. Found: C, 58.88; H, 3.31; N, 17.04; S,

6.56.

methyleneamino)-4H-1,2,4-triazole-3-thiol (10u)

Oﬀ-white solid (81%); m.p.: 253–256 °C; R_f: 0.27 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3019 (]CeH), 1622 (C]N), 1576, 1511

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.37 (1H, s, SH), 9.93 (1H, s,

CH]N), 9.22 (1H, s, ]CH_pyrazolyl), 8.12 (2H, d, J = 8.4 Hz, AreH),

7.96 (4H, m, AreH), 7.69 (1H, d, J = 7.6, AreH), 7.65–7.46 (6H, m,

AreH), 7.42 (1H, t, J = 7.4 Hz, AreH); ¹³C NMR (101 MHz, DMSO‑d₆):

δ 161.9, 158.2, 150.2, 147.4, 147.4, 138.6, 137.9, 133.3, 132.9, 132.7,

132.6, 129.8, 129.8, 127.4, 127.9, 127.5, 125.1, 123.5, 119.4, 115.1.

4.6. Enzyme inhibition studies

4.6.1. Cell transfection and Preparation of membrane fractions

The transfection of plasmids, expressing human APs (TNAP and IAP)

[45a] or NPPs (NPP-1 and NPP-3) [45a–c], was carried out with COS-7

by using lipofectamine, as discussed previously [45]. The 80–90%

conﬂuent cells were incubated at 37 °C for 5 h in Dulbecco’s modiﬁed

Eagle’s medium (DMEM) containing 6 µg of plasmid DNA and 24 µL of

lipofectamine reagent. After 5 h, the Dulbecco's Modiﬁed Eagle Medium

F-12 (DMEM/F-12) containing 20% FBS was added to stop the trans-

fection and cells were harvested after 40–72 h. The cells were further

processed and stored at −80 °C as reported earlier [46]. At the end,

Bradford microplate assay was used for the estimation of protein con-

centration using bovine serum albumin as a reference standard [47].

HRMS (ESI) exact mass calculated for

C

₂₄H₁₇ClN₆S [M+H]⁺

:

457.0924. Found: 457.0927. Analysis calcd: C, 63.08; H, 3.75; N, 18.39;

S, 7.02. Found: C, 63.18; H, 3.73; N, 18.46; S, 7.07.

4.5.22. 5-(4-Chlorophenyl)-4-((1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)

methyleneamino)-4H-1,2,4-triazole-3-thiol (10v)

Oﬀ-white solid (89%); m.p.: 268–270 °C; R_f: 0.25 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3008 (]CeH), 1614 (C]N), 1570, 1512

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.26 (1H, s, SH), 9.62 (1H, s,

CH]N), 9.26 (1H, s, ]CH_pyrazolyl), 8.08–8.00 (2H, d J = 8.0 Hz,

AreH), 7.98–7.89 (2H, d J = 8.1 Hz, AreH), 7.78–7.71 (2H, m, AreH),

7.64–7.52 (4H, m, AreH), 7.47–7.38 (1H, m, AreH), 7.31 (2H, d,

J = 7.9 Hz, AreH), 2.37 (3H, s, CH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ

162.3, 160.7, 153.4, 147.5, 138.8, 138.6, 135.4, 130.1, 129.7, 129.6,

129.3, 128.8, 128.4, 127.5, 124.4, 119.3, 114.4, 20.9 (CH₃). HRMS

(ESI) exact mass calculated for C₂₅H₁₉ClN₆S [M+H]⁺: 471.1080.

Found: 471.1087. Analysis calcd: C, 63.75; H, 4.07; N, 17.84; S, 6.81.

Found: C, 63.85; H, 4.04; N, 17.86; S, 6.83.

4.6.2. Alkaline phosphatase inhibition assay

The alkaline phosphatase (h-TNAP or h-IAP) inhibition was carried

out with slight modiﬁcations of previously reported method [48]. The

synthesized compounds were tested at the ﬁnal concentration of

100 µM, prepared in 10% DMSO. Total volume of the assay was 50 µL.

Initially, 10 µL of test compound was treated with 10 µL of h-TNAP

(46 ng/well) or of h-IAP (57 ng/well) in 384-well plate. The reaction

mixture was incubated for 5 min at 37 °C and the luminescence signals

were measured using microplate reader (BioTek FLx800, USA). After-

wards, 20 µL of CDP-star® substrate was added and reaction was al-

lowed to incubate again for 15 min at the same temperature. The

change in luminescence signals was measured and the activity of each

compound was compared with total activity control (without any in-

hibitor). Each experiment was carried out in triplicate. The compounds

exhibited 50% inhibition of either h-TNAP or h-IAP isozymes were

further evaluated for their IC₅₀values. For this, a serial dilution method

was used and diﬀerent concentration of solutions (20 nM − 200 nM)

were prepared and tested. The inhibitory values (IC₅₀values) were

calculated using the non-linear regression analysis of program PRISM

5.0 (GraphPad, San Diego, California, USA).

4.5.23. 5-(4-Chlorophenyl)-4-((1-phenyl-4-methoxyphenyl-1H-pyrazol-4-

yl)methyleneamino)-4H-1,2,4-triazole-3-thiol (10w)

Oﬀ-white solid (89%); m.p.: 256–258 °C; R_f: 0.28 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3015 (]CeH), 1618 (C]N), 1569, 1512

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.35 (1H, s, SH), 9.63 (1H, s,

CH]N), 9.11 (1H, s, ]CH_pyrazolyl), 7.98 (2H, d, J = 7.9 Hz, AreH),

7.74 (1H, d, J = 7.6 Hz, AreH), 7.67 (4H, m, AreH), 7.55 (3H, m,

AreH), 7.40 (1H, t, J = 7.4 Hz, AreH), 6.96 (2H, d, J = 8.4 Hz, AreH),

3.82 (3H, s, OCH₃); ¹³C NMR (101 MHz, DMSO‑d₆): δ 162.1, 160.5,

160.1, 153.1, 147.5, 139.0, 133.5, 133.1, 133.1, 130.9, 130.2, 129.9,

127.7, 125.4, 123.9, 119.5, 114.3, 114.3, 55.5 (OCH₃). HRMS (ESI)

exact mass calculated for C₂₅H₁₉ClN₆OS [M+H]⁺: 487.1030. Found:

487.1035. Analysis calcd: C, 61.66; H, 3.93; N, 17.26; S, 6.58. Found:

61.71; H, 3.95; N, 17.31; S, 6.55.

4.6.3. Nucleotide pyrophosphatase inhibition assay

The inhibitory eﬀect of all the derivatives on nucleotide pyropho-

sphatase (h-NPP-1 or h-NPP-3) was determined as reported earlier [50].

The total assay volume was 100 µL. Initially, 70 µL of 50 mM tris-HCl

(pH 9.5) buﬀer, 10 µL of the test compound solution (at 0.1 mM ﬁnal

concentration) was treated with 10 µL of h-NPP-1 (ﬁnal conc. of 27 ng)

or h-NPP-3 (ﬁnal conc. of 25 ng) in 96-well plate. The reaction mixture

was incubated for 10 min at 37 °C and absorbance was measured at

405 nm using microplate reader (BioTek ELx800, Instruments, Inc.

USA). Substrate (10 µL) and p-nitrophenyl-5′-thymidine monopho-

sphate (p-Nph-5-TMP, 0.5 mM), was added to initiate the reaction and

the mixture was allowed to incubate at 37 °C for 30 min. The change in

absorbance was measured. The compounds which exhibited over 50%

inhibition of enzyme activity were further selected for evaluation of

their IC₅₀values. For this, a serial dilution method was used and dif-

ferent concentration of solutions (100 nM − 1000 nM) were prepared

and tested. All the experiments were carried out in triplicate. The IC₅₀

values were calculated using non-linear regression analysis of program

PRISM 5.0 (GraphPad, San Diego, California, USA).

4.5.24. 5-(4-Chlorophenyl)-4-((1-phenyl-4-nitrophenyl-1H-pyrazol-4-yl)

methyleneamino)-4H-1,2,4-triazole-3-thiol (10x)

Oﬀ-white solid (83%); m.p.: 265–268 °C; R_f: 0.29 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3009 (]CeH), 1618 (C]N), 1575, 1514

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.35 (1H, s, SH), 9.72 (1H, s,

CH]N), 9.16 (1H, s, ]CH_pyrazolyl), 8.00–7.95 (2H, d, J = 8.2 Hz),

AreH), 7.74–7.69 (3H, m, AreH), 7.65 (2H, m, AreH), 7.55 (3H, m,

AreH), 7.45–7.39 (3H, m, AreH); ¹³C NMR (101 MHz, DMSO‑d₆): δ

162.0, 151.7, 138.8, 133.9, 133.4, 133.0, 132.9, 131.6, 130.5, 130.3,

130.0, 129.8, 128.7, 127.8, 127.6, 125.2, 119.5, 114.6. HRMS (ESI)

exact mass calculated for C₂₄H₁₆ClN₇O₂S [M+H]⁺: 502.0775. Found:

502.0771. Analysis calcd: C, 57.43; H, 3.21; N, 19.53; S, 6.39. Found: C,

57.59; H, 3.23; N, 19.50; S, 6.33.

4.5.25. 5-(4-Chlorophenyl)-4-((1-phenyl-4-chlorophenyl-1H-pyrazol-4-yl)

methyleneamino)-4H-1,2,4-triazole-3-thiol (10y)

Oﬀ-white solid (86%); m.p 268–270 °C; R_f: 0.22 (40% acetone in n-

hexane); FTIR (ATR, cm⁻¹): 3017 (]CeH), 1616 (C]N), 1579, 1500

(C]C); ¹H NMR (400 MHz, DMSO‑d₆): δ 14.28 (1H, s, SH), 9.66 (1H, s,

CH]N), 9.31 (1H, s, ]CH_pyrazolyl), 8.05 (3H, d, J = 8.1 Hz, AreH),

7.90 (4H, m, AreH), 7.58 (7H, m, AreH), 7.45 (1H, d, J = 7.4 Hz,

AreH); ¹³C NMR (101 MHz, DMSO‑d₆): δ 162.2, 151.9, 139.0, 134.1,

4.6.4. Mechanism of inhibition

In order to characterize the interaction of most potent inhibitor of

human TNAP, the mode of inhibition was determined using

Michaelis–Menten kinetics. For this purpose, the initial rates of the

17

H. Andleeb, et al.

BioorganicChemistry101(2020)103999

enzyme inhibition were measured at diﬀerent substrate concentrations

in the absence and presence of diﬀerent concentration of selected re-

presentative inhibitor against h-TNAP. The results were depicted as

double reciprocal Lineweaver-Burk plots using PRISM 5.0 (GraphPad,

San Diego, California, USA).

Acknowledgments

H.A. is grateful to the Higher Education Commission of Pakistan for

ﬁnancial support under SRGP project No. 2470, and Oxford University

for spectroscopic analysis. M.H is sponsored by CAS-TWAS President’s

Fellowship for international PhD students. J.I. is thankful to the

Organization for the Prohibition of Chemical Weapons (OPCW), The

Hague, The Netherlands and Higher Education Commission of Pakistan

for the ﬁnancial support through Project No. 20-3733/NRPU/R&D/14/

520.

4.7. Computational studies

4.7.1. Homology modeling

Prime module in the Schrödinger program (Schrödinger, LLC, New

York, NY, 2016) was used for generating and reﬁning the homology

models for h-TNAP, h-IAP, and h-NNP-1. To assess the geometrical

consistency of the generated models, Ramachandran plot analysis was

conducted using Molprobity [49]. An online resource CASTp [50] was

used for predicting pocket volumes for h-TNAP and h-IAP. PyMOL

version 1.8.8.2 was used for molecular graphics, structural alignments

and visualizations.

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.bioorg.2020.103999.

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Synthesis and computational studies of highly selective inhibitors of human recombinant tissue non-specific alkaline phosphatase (h-TNAP): A therapeutic target against vascular calcification

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