Potent Alkaline Phosphatase Inhibitors, Pyrazolo-Oxothiazolidines: Synthesis, Biological Evaluation, Molecular Docking, and Kinetic Studies

To develop new alkaline phosphatase inhibitors (ALP), a series of pyrazolo-oxothiazolidine derivatives were synthesized and biologically assessed, and the results showed that all of the synthesized compounds significantly inhibited ALP. Specifically, compound 7g displayed the strongest inhibitory activity (IC50 = 0.045 ± 0.004 μM), which is 116-fold more active than monopotassium phosphate (IC50 = 5.242 ± 0.472 μM) as a standard reference. The most potent compound among the series (7g) was checked for its mode of binding with the enzyme and shown as non-competitively binding with the target enzyme. The antioxidant activity of these compounds was examined to investigate the radical scavenging effect. Moreover, the MTT assay method was performed to evaluate their toxic effects on the viability of MG-63 human osteosarcoma cells, and all compounds have no toxic effect on the cells at 4 μM. Computational research was also conducted to examine the binding affinity of the ligands with alkaline phosphatase, and the results revealed that all compounds showed good binding energy values within the active site of the target. Therefore, these novel pyrazolo-oxothiazolidine derivatives might be employed as promising pharmacophores for potent and selective alkaline phosphatase inhibitors.


Introduction
Alkaline phosphatases [ALP; orthophosphoric monoester phosphohydrolase (alkaline optimum) EC 3.1.3.1], which are widely distributed in nature from prokaryotes and higher eukaryotes to humans [1,2], are known as plasma membrane-bound glycoproteins [3,4]. Alkaline phosphatase belongs to a large family of dimeric enzymes that hydrolyzes various monophosphate esters at a high pH optimum with the release of inorganic phosphate [5,6]. It is often restricted to the cell surface [7,8]. ALP, as a representative mediator of biochemistry, actively participates in most biological processes, including metabolism, signal transduction, molecular transport, and the manifestation of genetic information. ALP has a catalytic function because of its structural composition, which consists of two monomers with five cysteine residues, two zinc atoms (Zn 2+ ), and one magnesium atom (Mg 2+ ) in each monomer [9][10][11]. The ALP monomer's conformation is significantly affected by these metal ions, and they also indirectly adjust interactions between subunits [12]. Human ALPs can be divided into at least four isoenzymes, depending on the specificity of the tissue that will express them. Three of them are tissue-specific enzymes including placental alkaline phosphatase (PLALP or Regan isozyme), intestinal alkaline phosphatase (IALP), and germ cell ALP (GCALP or NAGAO isozyme), while the fourth is tissue nonspecific enzyme as liver/bone/kidney alkaline phosphatase (L/B/K ALP) [13]. By performing inorganic pyrophosphate (PPi) hydrolysis, L/B/K ALP maintains an optimum pyrophosphate level in bone tissues. The intestinal ALPs are Heterocyclic compounds have a cyclic structure in which the ring contains two or more different types of atoms. Also, a large number of heterocyclic compounds are known, and the number is continually growing. Heterocycles are well-studied bioactive compounds and thought to be attractive synthetic targets for the creation of new therapeutics [28]. One important topic for organic chemists is the synthesis of various heterocyclic compounds from straightforward and easily accessible starting materials [29][30][31][32].
Pyrazoles are a five-membered aromatic heterocyclic system from the azole branch with two adjacent nitrogen atoms. They are a significant class of heterocyclic compounds that have a variety of chemical, biological, agrochemical, and pharmacological activities [33].
A modern concept in drug development is the use of pharmacophore hybrids to create compounds that have superior affinity and efficacy [50]. In considering the aforementioned facts, a reasonable and promising area of research in current medicinal chemistry is the design of novel small molecules with drug-like properties based on the pharmacologically attractive scaffolds of pyrazole and thiazolidinones [50]. Some recent synthetic studies of pyrazol-thiazolidines and related hybrids, along with their biological investigations, have been patented as necroptosis inhibitors [51], VHR protein tyrosine phosphatase inhibitors [52], Pin1-modulating compounds [53], and RNA-binding protein modulators [54]. The pyrazole-thiazolidinone hybrids were investigated for their ability to inhibit the TNFa-TNFRc1 interaction [55], histone acetyltransferases inhibitors [56], COX inhibitors [57], and ADAMTS-5 enzymes [58].
As a result, in accordance with previously reported alkaline phosphatase inhibitors (Figure 1), we combined two scaffolds (pyrazole and thiazolidinone) to create a novel scaffold that was used in the ALP inhibition investigation. Moreover, to the best of our knowledge, pyrazolo-oxothiazolidine derivatives have never been researched as alkaline phosphatase inhibitors. Here, we described the design and synthesis of a novel class of ALP inhibitory agents which were also evaluated for antioxidant activity, cytotoxicity, and computational studies.

Chemistry
According to Scheme 1, a new series of pyrazolo-oxothiazolidine derivatives was synthesized in three steps. Initially, the Claisen-Schmidt condensation of acetophenone or para-methoxy acetophenone (1a, b) and aromatic aldehydes (2a-2h) in the presence of sodium hydroxide in ethanol resulted in 1,3-diphenylprop-2-en-1-ones (chalcones, 3a-3n). Then, the intermolecular cyclization of different chalcone derivatives (3a-3n) with thiosemicarbazide (4) produced pyrazole compounds (5a-5n) in refluxing ethanol under basic conditions. Finally, the reaction of compounds 5a-5n and diethyl acetylenedicarboxylate (6) is accomplished under reflux conditions in ethanol, to form pyrazolo-oxothiazolidine derivatives (7a-7n) in reasonable yields (80-92%). The structural confirmation of all synthesized compounds (7a-7n) was verified through the spectroscopic analysis utilizing FT-IR, 1 H NMR, and 13 C NMR. To comprehend the structural identification of the synthesized target compounds, we have presented a structural interpretation of one of the target compounds (7a). Molecule 7a was obtained in 84% yield as a white solid with a melting point of 242-244 • C. FT-IR spectroscopy was used to examine the molecular vibrational studies to confirm various functionalities. The aliphatic C-H stretching vibrations were observed at 2974 cm −1 . The stretching bands for conjugated C=O group appeared at 1694 cm −1 . The other significant absorption bands in the IR spectrum emerged at 1602 (C=C, alkene), 1548, 1498 (C=C, aromatic), 1489 (C=N, stretching), 1218 (C-O, stretching), 1177, 1114, 1073, 1010 (C-N stretching and aromatic C-H out of plane bending), 823, 767, 757 (aromatic C-H out of plane bending), 689 (C-Br stretching), which prove the existence of functional groups such as pyrazole, ester, alkene, and aromatic in this molecule. The number of proton-containing atoms in the produced molecules was measured using 1 H NMR spectroscopic techniques.
There are two phenyl rings in the target compound 7a. In the aromatic region, the two multiplet peaks at δ 7.14-7.18 (m, 2H, H-13 & 17), and 7.83-7.85 (m, 2H, H- 14 & 16) belonged to the aromatic ring which holds the bromo group at the para position, whereas five protons of second phenyl ring represented by multiplet at δ 7.46-7.58 (m, 5H, H-1, 2, 3, 4 & 6). In the up-field region of the spectrum, the signals at δ 1.37 (t, J = 8 Hz, 3H, CH 3 ) and 4.34 (q, J = 7.2 Hz, 2H, CH 2 ), exposed the presence of methyl and methylene groups, which split triplet and quartet, respectively. At δ 6.93 (s, 1H, H-24), there was a distinct singlet signal for vinylic hydrogen. The typical ABX system, which is associated with the CH 2 and CH protons of the pyrazoline ring, was clearly detected in the predicted regions. The H A proton was observed at δ 3.43 (dd, J = 18, 4 Hz, 1H, H-8 A ), the H B proton appeared at δ 4.03 (dd, J = 18, 12 Hz, 1H, H-8 B ), and the H X emerged at 5.85 (dd, J = 11.2, 4 Hz, 1H, H-9), due to vicinal coupling with two magnetically non-equivalent protons of the methylene group at position 4 of the pyrazoline ring. Furthermore, the carbon backbone of the produced target compound 7a was studied using the 13 C NMR technique. Due to some duplets for magnetically equivalent carbons that reduced the total number of resonances in the molecule, the 13 C NMR spectra showed a total of eighteen carbon resonances. Two resonances for the phenyl ring which holds the bromo group at the para position were observed at δ 127.68 (C-13 & C-17) and 132.39 (C-14 & C-16), while another phenyl ring emerged at δ 127.52 (C-4 & C-6), 129.11 (C-1 & C-3), and 129.43 (C-2). The two carbonyl carbons (C-25) and (C-20) appeared at δ 166.59 and 173.81 ppm, whereas two imine carbons (C-7) and (C-18) were observed at δ 160.79 and 178.68 ppm. Four quaternary signals were characterized at δ 122.65 (C-15), 132.16 (C-5), 138.15 (C-12), and 146.52 (C-21). One signal at δ 118.17 ppm belonged to the methine carbon (C-24). There were four peaks in the aliphatic region that were distinguished by the methyl and methylene carbons at positions 14.23 (C-29), 43.30 (C-8), 61.75 (C-28), and 63.38. (C-9) ( Figure 2). at 2974 cm −1 . The stretching bands for conjugated C=O group appeared at 1694 cm −1 . The other significant absorption bands in the IR spectrum emerged at 1602 (C=C, alkene), 1548, 1498 (C=C, aromatic), 1489 (C=N, stretching), 1218 (C-O, stretching), 1177, 1114, 1073, 1010 (C-N stretching and aromatic C-H out of plane bending), 823, 767, 757 (aromatic C-H out of plane bending), 689 (C-Br stretching), which prove the existence of functional groups such as pyrazole, ester, alkene, and aromatic in this molecule. The number of proton-containing atoms in the produced molecules was measured using 1 H NMR spectroscopic techniques. There are two phenyl rings in the target compound 7a. In the aromatic region, the two multiplet peaks at δ 7.14-7.18 (m, 2H, H-13 & 17), and 7.83-7.85 (m, 2H, H- 14 & 16) belonged to the aromatic ring which holds the bromo group at the para position, whereas five protons of second phenyl ring represented by multiplet at δ 7.46-7.58 (m, 5H, H-1, 2, 3, 4 & 6). In the up-field region of the spectrum, the signals at δ 1.37 (t, J = 8 Hz, 3H, CH3) and 4.34 (q, J = 7.2 Hz, 2H, CH2), exposed the presence of methyl and methylene groups, which split triplet and quartet, respectively. At δ 6.93 (s, 1H, H-24), there was a distinct singlet signal for vinylic hydrogen. The typical ABX system, which is associated with the CH2 and CH protons of the pyrazoline ring, was clearly detected in the predicted regions. The HA proton was observed at δ 3.43 (dd, J = 18, 4 Hz, 1H, H-8A), the HB proton appeared at δ 4.03 (dd, J = 18, 12 Hz, 1H, H-8B), and the HX emerged at 5.85 (dd, J = 11.2, 4 Hz, 1H, H-9), due to vicinal coupling with two magnetically non-equivalent protons of the methylene group at position 4 of the pyrazoline ring. Furthermore, the carbon backbone of the produced target compound 7a was studied using the 13 C NMR technique. Due to some duplets for magnetically equivalent carbons that reduced the total number of resonances in the molecule, the 13 C NMR spectra showed a total of eighteen carbon resonances. Two resonances for the phenyl ring which holds the bromo group at the para position were observed at δ 127.68 (C-13 & C-17) and 132.39 (C-14 & C-16), while another phenyl ring emerged at δ 127.52 (C-4 & C-6), 129.11 (C-1 & C-3), and 129.43 (C-2). The two carbonyl carbons (C-25) and (C-20) appeared at δ 166.59 and 173.81 ppm, whereas two imine carbons (C-7) and (C-18) were observed at δ 160.79 and 178.68 ppm. Four quaternary signals were characterized at δ 122.65 (C-15), 132.16 (C-5), 138.15 (C-12), and 146.52 (C-21). One signal at δ 118.17 ppm belonged to the methine carbon (C-24). There were four peaks in the aliphatic region that were distinguished by the methyl and methylene carbons at positions 14.23 (C-29)    The synthesized chemical compounds (7a-7n) were evaluated against the calf intestinal alkaline phosphatase (CIAP) enzyme inhibition for the IC 50 evaluation. Table 1 gives an overview of the CIAP results. Monopotassium phosphate (KH 2 PO 4 ) was employed in this experiment as a positive control to evaluate the performance of our synthetic compounds. Interestingly, all these compounds showed extremely strong inhibitory activity against this enzyme, as evidenced by their lower IC 50 (0.045-2.987 µM) values in comparison to the standard reference KH 2 PO 4 , which had an IC 50 value of 5.242 ± 0.472 µM. Though the observed activity is the resultant of the entire molecule, the effect of various aryl groups on the inhibitory potential allowed for the recognition of a limited structure-activity relationship (SAR). To make the classification of the structure activity relationship (SAR) more understandable, we divide it into three categories. The first five compounds 7(a-d), and 7l, while having the unsubstituted phenyl ring scaffold, differ in their second phenyl ring derivatives. It observed that compounds 7a and 7l, which have halogen groups (Br and Cl) at the para position, showed better activity when compared to other derivatives (7b, 7c, 7d), which have electron-donating groups at the para position. However, a reverse trend was noticed in the second set of seven compounds 7e, 7f, 7g, 7h, 7i, 7k, and 7m, which all possess a para-methoxy phenyl ring, and the second phenyl ring has various derivatives. Among halogenated molecules (7h, 7m, 7f, and 7i), compound 7h with bromo group at para position exhibited less activity probably because a bulky bromo group decreases the inhibitory activity. Compound 7f with chloro group at ortho and para position showed lower inhibitory potential as compared to 7m in which chloro group was at para position, and presumably due to steric crowding prevents the good interaction with the active site of the enzyme. Compounds 7i and 7m, which have fluoro and chloro groups in the para positions, respectively, displayed a similar inhibitory effect. Additionally, the methyl, methoxy-benzyloxy, and methoxy groups found in compounds 7e, 7g, and 7k in this category showed overall the strongest inhibitory potentials against the target enzyme. So, it can be concluded that, when one phenyl ring has a para-methoxy group, the electronwithdrawing groups significantly disrupted the electronic density of the aromatic ring, and more effective interactions are made by electron-donating substances with the enzyme. Surprisingly, the most effective molecule 7g (IC 50 = 0.045 ± 0.004 µM) comprised both benzyloxy and methoxy groups, which showed synergistic effects. This might be caused by the ability of both groups to donate electrons via resonance and created more hydrogen bonds. Finally, between compounds 7n and 7j, compound 7j (IC 50 = 0.434 ± 0.011 µM) with only one methoxy group at para position showed almost six-fold better inhibitory potential compared to 7n (IC 50 = 2.987 ± 0.734 µM) having unsubstituted phenyl rings. It means that compared to an unsubstituted compound, the presence of an electron-donating substituent on the phenyl group makes the molecule a more suitable inhibitor. Overall, all of the derivatives have noticeable inhibitory activity, particularly compound 7g possessing the strongest effect compared to reference standard. As a consequence of these findings, it can be concluded that phenyl rings need to contain electron-rich species like methoxy and benzyloxy groups in order to have alkaline phosphatase inhibitory activity. Therefore, compound 7g could be a perfect choice for medicinal purposes. like methoxy and benzyloxy groups in order to have alkaline phosphatase inhibitory activity. Therefore, compound 7g could be a perfect choice for medicinal purposes. Values are presented as mean ± SEM (Standard error of the mean).

Kinetic Mechanism for CIAP
Presently, the most active compound 7g was considered for the mode of inhibition of CIAP. The capacity of the compound to inhibit the free enzyme and enzyme-substrate complex was determined in terms of EI and ESI constants, respectively. The kinetic research of the enzyme by the Lineweaver-Burk plot of 1/V versus substrate p-NPP 1/[S] in the presence of different inhibitor concentrations gave a series of straight lines as shown in Figure 3A. The results of compound 7g showed that the compound intersected within the second quadrant. The analysis confirmed that Vmax was reduced to new growing doses of inhibitor, on the other hand Km stays the same. This behavior suggests that compound 7g inhibits the alkaline phosphatase non-competitively to shape an enzyme-inhibitor complex. A secondary plot of slope against the concentrations of inhibitor confirmed enzymeinhibitor dissociation constant (Ki) Figure 3B. The kinetic results are presented in Table 2.

No.
Compound R R Alkaline Phosphatase IC 50  Values are presented as mean ± SEM (Standard error of the mean).

Kinetic Mechanism for CIAP
Presently, the most active compound 7g was considered for the mode of inhibition of CIAP. The capacity of the compound to inhibit the free enzyme and enzyme-substrate complex was determined in terms of EI and ESI constants, respectively. The kinetic research of the enzyme by the Lineweaver-Burk plot of 1/V versus substrate p-NPP 1/[S] in the presence of different inhibitor concentrations gave a series of straight lines as shown in Figure 3A. The results of compound 7g showed that the compound intersected within the second quadrant. The analysis confirmed that V max was reduced to new growing doses of inhibitor, on the other hand K m stays the same. This behavior suggests that compound 7g inhibits the alkaline phosphatase non-competitively to shape an enzyme-inhibitor complex. A secondary plot of slope against the concentrations of inhibitor confirmed enzyme-inhibitor dissociation constant (Ki) Figure 3B. The kinetic results are presented in Table 2.

Free Radical Scavenging Activity
All synthesized target compounds were assessed for 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging ability. From our results, all of the compounds showed activity (Figure 4), but the most effective compound (7g) showed a bit of inhibition (29.7%) compared to the others at the concentration of 100 µg/mL. Ascorbic acid (vitamin C) was employed as a reference to evaluate the radical scavenging activity.

Cell Viability
In this experiment, all fourteen pyrazolo-oxothiazolidine derivatives (7a-7n) after confirmation activity against in vitro ALP enzyme inhibition were carried out to check their toxic effect on the human osteosarcoma MG-63 cell lines using the MTT assay technique. At first, four concentrations (0.5, 1, 2, and 4 µM) were chosen for each compound, along with a positive control (erlotinib), and all compounds and the standard drug were treated and incubated for 24 h. Normal control was considered to be 100% viable without any treatment. The viability of the cells was examined after incubation, and the results are presented in ( Figure 5). From these results, it is clear that all compounds have no toxic effect on the cells, and even at the concentration of 4 µM, the cells were viable up to >70% except the compounds 7e, 7j, and 7m. However, at the first three treatments, all the compounds along with the standard drug showed >90% viability. These findings suggest that these compounds could be employed as a safe target for drug design within pharmaceutical approaches.  The biochemical properties of all synthesized compounds (7a-7n) were predicted and analyzed through computational resources. The synthesized target compounds (7a-7n) were validated through RO5 analysis. The standard parameters in RO5 showed that the molecular mass and logP values must be less than 500 (g/mol) and 5, respectively. Moreover, the compounds should possess no greater than 10 HBA and 5 HBD, correspondingly. According to the literature, excessive values of HBA and HBD result in poor permeation [59,60]. The hydrogen-bonding capacity has been considered a significant parameter for drug permeability. Our results demonstrated the synthesized ligands possessed <10 HBA and <5 HBD values, which were comparable to standard values. However, the logP values in most compounds were also comparable with standard values (>5). The molecular weight (g/mol) of all compounds exceeded the standard value (Table 3). However, there are many examples available which deviate RO5 violation amongst the existing drugs [61,62]. Polar surface area (PSA) is also considered a good descriptor for characterizing drug absorption, including intestinal absorption, bioavailability, and blood-brain barrier penetration. Our predicted results showed that all compounds possess <140 Å 2 values, which showed their good lead-like behavior. The drug-likeness score is a mixture of a complex balance of various molecular properties and structural features of chemical compounds, which determine whether the molecule is like as-known drugs [63]. Our results showed that all the synthetic compounds (7a-7n) showed good drug score values. The majority of the compounds exhibited positive drug score values, whereas 7b and 7n possessed negative score values (Table 3).

Structural Analysis and Physiochemical Properties of ALP
Human placental alkaline phosphatase is a class of hydrolase single-chain protein, having 539 amino acids with embedded zinc and magnesium ions at different structural sites. The VADAR analysis showed the overall protein architecture consists of 31% helices, 26% β sheets, and 42% coils. Moreover, the Ramachandran plot indicated that 96.4% of residues were present in favored regions which showed the precision of phi (φ) and psi (ψ) angles among the coordinates of ALP ( Figure 6).

Binding Energy Evaluation of Compounds
To predict the best conformational position within the active region of the target protein, all the synthesized ligands (7a-7n) were docked against ALP. All the generated docked complexes were analyzed based on minimum energy values (kcal/mol) and hydrogen/hydrophobic interactions. Docking results justified that all the synthesized target compounds exhibited good docking binding affinities with ALP. Both 7d (−8.3 Kcal/mol) and 7g (−8.1 Kcal/mol) exhibited higher binding energy values as compared to other synthesized ligands. The remaining compounds showed different binding energy values, and most of the ligands were bound within the active region of the target protein. The basic chemical nuclei of all the synthesized compounds were the same; therefore, most ligands showed a good binding affinity with the target protein (Table 4).

Binding Interaction of 7g against ALP
Based on in vitro (IC 50 ) results, the 7g-docked complex was selected to understand the binding interaction behavior of the compound against ALP. Docking analysis showed that 7g was actively confined within the active binding region of ALP (Figure 7). In detail, the SAR study showed that four hydrogen bonds were observed in the 7g-docking complex. The oxygen atom of 7g forms two hydrogen bonds with His_153 and His_317 having bond lengths 1.78 and 2.55 Å, respectively. The importance of these amino acid residues in binding with other ALP inhibitors has also been well documented [64,65]. The comparative binding energy and SAR analysis showed a significance of 7g and may be considered as a potent inhibitor of the enzyme. The rest of all docking complexes have been shown in Supplementary Data (Figures S43-S56).

Chemicals and Instruments
The MPA 160 apparatus of Fisher Scientific [Waltham, MA, USA] was used to determine the melting points (uncorrected) of the synthesized compounds. The FT-IR spectra was obtained using a Frontier IR spectrophotometer (Perkin Elmer, Waltham, MA, USA). 1 H NMR and 13 C NMR spectra of the compounds were recorded on a Bruker Avance III (Karlsruhe, Germany) at 400 and 100 MHz spectrophotometers using CDCl 3 as solvent. Chemical shifts were reported in parts per million (ppm) (δ). A thin layer chromatography (TLC) method was used to monitor the progress of the reaction. All commercially available chemicals and reagents were utilized without further purification.
3.2. General Procedure for the Synthesis of the Compounds 3.2.1. Procedure for Synthesis of Chalcones (3a-3n) Acetophenone or 4-methoxyacetophenone (1a and 1b, 1 mmol) was dissolved in approximately 10 mL of absolute ethyl alcohol and stirred for 5 min in an ice bath, as shown in Scheme 1. The sodium hydroxide (1.1 mmol) was then added portion-wise to the reaction flask. Following that, various substituted aromatic aldehydes (2a-2h, 1 mmol) were slowly added to the reaction mixture and stirred for 24 h in an ice bath. When the reaction was completed (determined by thin layer chromatography) the reaction mixture was poured into crushed ice and neutralized with hydrochloric acid. The obtained precipitate was stirred for 30 min at room temperature and then filtered, washed with cold water/ethanol, and dried to afford the desired chalcones (3a-3n) [66].

Procedure for Synthesis of 1-Thiocarbamoyl Pyrazole Derivatives (5a-5n)
As displayed in Scheme 1, 1.5 mmol thiosemicarbazide (4) and absolute ethanol were mixed for 10 min. Then, under the nitrogen atmosphere, 1.1 mmol potassium hydroxide was added and stirred for another 5-10 min, followed by the addition of corresponding chalcones (3a-3n, 1 mmol). The reaction was subsequently continued under reflux for 8 h until the reaction was completed (monitored by thin layer chromatography). The product was poured into crushed ice, neutralized with hydrochloric acid, stirred for 30 min, then filtered, washed with cold ethanol, and dried to obtain the 1-thiocarbamoyl pyrazole derivatives (5a-5n) [67].

Procedure for Synthesis of 1-Thiazolyl-2-Pyrazoline Derivatives (7a-7n)
As presented in Scheme 1, to a suspension of 1-thiocarbamoyl pyrazole compounds (5a-5n, 1mmol) in absolute ethanol, diethyl acetylenedicarboxylate (6, 1mmol) was added and followed by refluxing for 4 h. The reaction mixture was cooled at room temperature after completion (as confirmed by thin layer chromatography), and the precipitate was filtered and recrystallized from ethanol to achieve the desired target compounds (7a-7n) in good yields (80-92%). As displayed in Scheme 1, 1.5 mmol thiosemicarbazide (4) and absolute ethanol were mixed for 10 min. Then, under the nitrogen atmosphere, 1.1 mmol potassium hydroxide was added and stirred for another 5-10 min, followed by the addition of corresponding chalcones (3a-3n, 1 mmol). The reaction was subsequently continued under reflux for 8 h until the reaction was completed (monitored by thin layer chromatography). The product was poured into crushed ice, neutralized with hydrochloric acid, stirred for 30 min, then filtered, washed with cold ethanol, and dried to obtain the 1-thiocarbamoyl pyrazole derivatives (5a-5n) [67].

Alkaline Phosphatase Inhibition Assay
As previously mentioned [65,68], spectrophotometric analysis was used to evaluate the activity of calf intestinal alkaline phosphatase (CIAP). The reaction mixture contained 50 mM Tris-HCl buffer (5 mM MgCl 2 , 0.1 mM ZnCl 2 pH 9.5), the 10 µL compound, and it was preincubated for 10 min by adding 5 µL of CIAP (0.025 U/mL). Then, 10 µL of a substrate (0.5 mM p-NPP (para nitrophenyl phosphate disodium salt)) was delivered to initiate the response, and therefore the assay mixture became incubated once more for 30 min at 37 • C. The change in absorbance of released p-nitrophenolate was monitored at 405 nm, using a 96-well microplate reader (Thermo Scientific Multiskan GO, USA). All the experiments were repeated three times in a triplicate manner. KH 2 PO 4 (potassium dihydrogen phosphate) was used as the reference inhibitor of CIAP. The inhibition activity was calculated according to the following equation: For calculation of the IC 50 values, 6-8 serial dilutions were used and the results examined using the non-linear regression analysis of GraphPad Prism 5 [69].

Kinetic Mechanism Analysis
Primarily based on the IC 50 findings, we selected the most effective inhibitor 7g for CIAP to determine the mechanism of enzyme inhibition using our published method [70]. The inhibitor (7g), and substrate p-NPP concentrations were used at 0.00, 0.023, 0.045 and 0.091 µM, 10, 5, 2.5, 1.25, 0.625 and 0.3125 mM, respectively. The pre-incubation time and other conditions were the same as those mentioned in the section on the CIAP assay. Maximal initial velocities were determined from the initial linear portion of absorbances up to 10 min after the addition of enzyme per minute interval. The inhibition type of the enzyme was assayed by the Lineweaver-Burk plot of inverse velocities (1/V) versus the inverse of substrate concentration 1/[S] mM −1 . The EI dissociation constant Ki was determined by a secondary plot of 1/V versus inhibitor concentration.

Free Radical Scavenging Assay
Free radical scavenging activity was determined by modifying the method by DPPH assay [71]. The assay solution composed of the 100 µL of DPPH (150 µM) and 20 µL of the sample (100 µg/mL) was introduced to gain the final volume of each well up to 200 µL with methyl alcohol. Then, it was incubated in the dark at room temperature for the following half-hour. Ascorbic acid (Vitamin C) was used as a reference inhibitor. The measurements were performed by using a microplate reader (Thermo Scientific Multiskan GO, USA) at 517 nm. Each experiment was carried out in triplicate.

Cell Culture and Treatment of Compounds
The human osteosarcoma MG-63 cells were acquired from the Korean Cell Line Bank (Seoul, Korea). The MG-63 cell lines were incubated in the humidified atmosphere at 37 • C containing 5% CO 2 and maintained in the DMEM (Sigma, St. Louis, MO, USA), supplemented with 10% fetal bovine serum (FBS, WELGENE, Korea) and 50 unit/mL penicillin (Sigma) and 50 µg/mL streptomycin (Sigma). The medium was completely renewed every two days. The synthesized compounds were completely dissolved in dimethyl sulfoxide (DMSO) and further diluted to various concentrations (0, 0.5, 1, 2, and 4 µM). The control groups were treated with the same amount of DMSO.

Cell Viability
MTT assay technique was applied to check the toxic effect of our compounds. The MG-63 cell lines were seeded onto 96-well plates at a density of 0.4 × 10 5 cells/well with the medium for 24 h. After reaching 50% density, the cells were exposed to synthesized target compounds at the dose of 0, 0.5, 1, 2, and 4 µM for 24 h in media. We employed erlotinib as a positive control to compare the results of the synthesized derivatives. After 24 h, the cell sustained 100 µL DMEM containing of 0.5 mg/mL 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) at 37 • C for 4 h. Then, the purple formazan crystals were dissolved with 100 µL of buffer (10% SDS, 0.01 N HCl). Finally, the absorbance was measured at 570 nm using a microplate reader (Thermo Fisher, Waltham, MA, USA).

Retrieval of Alkaline Phosphate Structure from PDB
The three-dimensional (3D) structure of alkaline phosphate from the human placenta was accessed from Protein Data Bank (PDB) (www.rcsb.org) having PDBID 1EW2. The selected target protein structure was minimized by using the UCSF Chimera 1.10.1 tool [72,73]. The protein architecture and statistical percentage values of receptor proteins helices, β-sheets, coils, and turn were predicted from the online server VADAR 1.8 [74].

Designing of Ligands and Molecular Docking
The synthesized ligands were sketched in the drawing ACD/Chem Sketch tool and minimized by UCSF Chimera 1.10.1. All the synthesized ligands were sketched in ACD/Chem Sketch tool and accessed in mol format. Furthermore, the UCSF Chimera 1.10.1 tool was employed for energy minimization of each ligand separately having default parameters such as steepest descent steps 100 with step size 0.02 (Å), conjugate gradient steps 100 with step size 0.02 (Å), and update interval was fixed at 10. Finally, Gasteiger charges were added using Dock Prep in ligand structure to obtain the good structure conformation. The molecular docking experiment was used on all the synthesized ligands against ALP by using the PyRx 0.8, virtual screening tool with Auto Dock VINA Wizard v.1.2.0. approach [75]. The grid box center values of (center X = 43.3, center Y = 23.1612, and center Z = 9.1269), and size values were adjusted as (X = 65.56, Y = 71.79, and Z = 64.64) for better conformational position in the active region of the target protein. All the synthesized ligands were docked separately against ALP with a default exhaustiveness value = 8. The predicted docked complexes were evaluated based on the lowest binding energy (kcal/mol) values and structure activity relationship (SAR). The three-dimensional graphical depictions of all the docked complexes were accomplished by Discovery Studio (2.1.0) and UCSF Chimera 1.10.1 tool.

Conclusions
In the current study, we have successfully synthesized and characterized fourteen pyrazolo-oxothiazolidine derivatives using a multistep synthetic process. All the synthesized compounds were evaluated for in vitro alkaline phosphatase inhibitor activity and found to be stronger than monopotassium phosphate. Among them, compound 7g (IC 50 = 0.045 ± 0.004 µM) had the most effective inhibitory effect against alkaline phosphatases compared to the standard reference KH 2 PO 4 (IC 50 = 5.242 ± 0.472 µM). Furthermore, the MTT assay was performed against MG-63 at various concentrations to screen the cell viability potential of synthesized compounds, and they showed no toxic effect on the viability of cells. Molecular docking studies were conducted, to determine the binding affinity of the ligands with the target protein within the active site, and all compounds exhibited good docking binding affinity with alkaline phosphatase and showed good docking scores. According to research findings, these compounds, particularly 7g, might employ as worthwhile therapeutic candidates for alkaline phosphatase-associated disorders in medicinal chemistry.