Synthesis and Biological Evaluation of Arylamide Sulphonate Derivatives as Ectonucleotide Pyrophosphatase/Phosphodiesterase-1 and -3 Inhibitors.

Aberrant level of ectonucleotide pyrophosphatase/phosphodiesterase-1 and -3 is linked with numerous disorders, for instance, diabetes, cancer, osteoarthritis, chondrocalcinosis, and allergic reactions. These disorders may be cured or minimized by blocking the activity of ENPP1 and ENPP3 isozymes. In this study, arylamide sulphonates were synthesized, characterized, and evaluated for their capability to affect the activity of isozymes ENPP1 and ENPP3. Among the selective inhibitors of ENPP1, compounds 4f and 4q exhibited sub-micromolar IC50 values of 0.28 ± 0.08 and 0.37 ± 0.03 μM, respectively, followed by 7a, with IC50 equal to 0.81 ± 0.05 μM, whereas out of the selective inhibitors of isozyme ENPP3, 4t and 7d preferably lessened the activity to half of the maximal inhibitory concentration of 0.15 ± 0.04 and 0.16 ± 0.01 μM alternatively. In addition, many structures including 4c, 4g, 4k, 4l, 4n, 4o, 4r, 4s, 7b, 7c, and 7e inhibited the activity of both isozymes to a significant level. Enzyme kinetic study of compound 4j revealed an uncompetitive mode of inhibition of ENPP1 isozyme, while 7e competitively blocked the activity of ENPP3. Cell viability analysis revealed the compound 4o as a cytotoxic agent against MCF7 (human breast cancer cell line) with a percentage inhibition of 63.2 ± 2.51%, whereas compounds 4c, 4d, 4n, and 7d decreased the HeLa cell viability (human cervical cancer cell line) to more than 50%. The tested compounds were non-cytotoxic against HEK293 (a human embryonic kidney cell line). Molecular docking analysis of selected inhibitors of both isozymes produced optimistic interactions with the influential amino acids, such as Leu290, Lys295, Tyr340, Asp376, His380, and Pro323 of ENPP1, whereas residues Asn226, His329, Leu239, Tyr289, Pro272, Tyr320, and Ala205 of ENPP3 crystallographic structure formed interactions with the potent inhibitors.

Introduction

The members of the ENPP family are conserved and ubiquitous eukaryotic enzymes with an extracellular active site and exist as membrane-bounded glycoproteins.[1] The ENPP family contains seven fundamental isozymes (ENPP1–7), which are numbered as per their cloning order.[2] ENPP1, ENPP2, ENPP3, and ENPP4 can hydrolyze a variety of nucleotides together with dinucleoside polyphosphates, nucleotide sugars, and cyclic (di-)nucleotides and release corresponding nucleoside 5′-monophosphates; for example, ATP is hydrolyzed into AMP and pyrophosphate (PPi).[3] However, among the nucleotide hydrolyzing potential of isozymes (ENPP1, ENPP2, ENPP3, and ENPP4), the nucleotide metabolizing activity of ENPP2 is weak. ENPP2 prefers phospholipids as a substrate, whereas ENPP6 and ENPP7 are alkaline sphingomyelinases; hence, instead of nucleotidases, the subtypes (ENPP2, ENPP6, and ENPP7) may be identified as phospholipases.[4−8]

ENPP1 was initially found as the surface sign for B-lymphocytes that secrete antibodies; therefore, it was named plasma-cell differentiation antigen-1 or PC-1.[9] ENPP1 exists in several tissues such as in the bone (osteoblast) and cartilage (chondrocytes), where it performs an essential part in the mineralization process.[10] ENPP1 is the transmembrane glycoprotein of homodimeric type II, accompanied by an N-terminal transmembrane domain, a catalytic domain, a C-terminal nuclease-like domain, and two somatomedin-B-like domains.[11] The subcellular location of ENPP1 is determined by the transmembrane domain, which is essential for the dimerization of monomers by multiple disulfide bonds. It is also noteworthy that the SMB2 domain of the ENPP1 is assumed to be the residue for the insulin receptor interaction. The ENPP1 catalytic domain amino acid residues make 24–60% likeness among the various isozymes of other human ENPPs (ENPP2–7) and similarity to the alkaline phosphatase family (APs).[12] Like the APs, two Zn2+ cations are strongly linked by six preserved Asp/His residues at the active site. In addition, the nuclease-like domain is connected to the catalytic domain via a linker region called the “lasso loop”.[13] If any mutation occurs in this linker region, the catalytic activity of the isozyme is eliminated. The nuclease-like domain does not itself reveal catalytic activity, but it is required to translocate ENPPs from the endoplasmic reticulum to the Golgi apparatus, necessary for the correct folding of the ENPPs. Moreover, this domain includes a perceived “EF hand” Ca2+ binding motif, which is significant for the catalytic activity of ENPP1. Though PPi is essential for the prevention of ectopic mineralization, its over-supply contributes to the mineral aggregation of calcium pyrophosphate dihydrate (CPPD) in joints. This situation is usually associated with age-related osteoarthritis, known as chondrocalcinosis. The role of ENPP1 was also found for insulin receptor signaling. The over-expression of ENPP1 was found to be linked with defective insulin-stimulated autophosphorylation in patients with type 2 diabetes. Moreover, ENPP1 was also identified and associated with tumor gradation in human astrocytic brain tumors.[14]

The isozyme ENPP3 (a glycoprotein) is associated with the plasma membrane of the cell. ENPP3 (CD203c) is expressed on surfaces of various body organs such as epithelial and mucosal, particularly on mast cells and basophils.[15−17] ENPP3 is actively involved in fluid homeostasis, modulation of bile formation, and cerebral spinal fluid secretion. ENPP3 is actively involved against the synchronization of nucleotide sugar glycosylation of brain-specific proteins and involves intracellular as well as extracellular polyphosphate hydrolysis. The molecular structure of ENPP3 contains parallel β-sheets of eight trapped catalytic phosphodiesterase (PDE) domain in the molecular structure of ENPP3 that hold five active N-glycosylation sites and eight α-helices on each side, representing a major component of alkaline phosphatase superfamily.[12,18] This PDE domain is linked to the nuclease-like region (NUC) through the linker L2, two somatomedin B (SMB1 and SMB2 domains) with the L1 link attached to the NUC.[19] An elevated proportion of ENPP3 on the cell surface was detected in comparison to other inflammatory mediators, which stimulated basophils with antigen-bound IgE. ATP emissions are thus impaired by ENPP3 and served as a sign of recognition that basophils of patients have allergic responsiveness.[20]

Several structures with sulphonate moiety are reported to possess therapeutic importance such as suramin (Figure ) with a complex structure and poly sulphonate groups, being widely used as a positive control of choice in different biological studies.[21] In the previous study, we have reported pyrrolo[2,3-b]pyridine derivatives such as (N-(pyrrolo[2,3-b]pyridine-1-carbonyl)benzenesulfonamide), which was found as a selective inhibitor of ENPP1 with an IC50 value of 4.50 ± 0.16 μM and carbohydrazide-based derivative, which selectively blocked the activity of ENPP3 to half of the maximal value of 0.15 μM.[22,23] Various previously reported structures with sulphonate scaffolds including raloxifene sulphonates, benzofuran, and benzothiophene sulphonates exhibited significant enzyme inhibitory activity to sub-micromolar levels, for example, raloxifene sulphonate derivative 3-(4-(2-(piperidin-1-yl)ethoxy)benzoyl)-2-(4-(tosyloxy)phenyl)benzo[b]thiophen-6-yl-4-methylbenzenesulfonate tabulated selective inhibition of isozyme ENPP1 to an IC50 value of 0.45 μM, and benzothiophene derivative (4-(benzo[b]thiophen-5-yl)phenyl cyclohexanesulfonate) was endowed with IC50 = 0.12 μM against ENPP1 and 1.89 μM toward ENPP3.[24,25] We have also reported sulphonate derivatives mainly based on non-aromatic or saturated cyclic hydrocarbons. These structures exhibited considerable inhibitory activity but limited preferable selectivity among the three isozymes ENPP1, ENPP2, and ENPP3.[26]

Figure 1

Previously reported structures of ENPP inhibitors and the target compounds.

The selected structural determinants in the target compounds are deemed unique scaffolds due to their importance in medicinal chemistry (Figure ). The compounds with carboxamide moiety have been reported as antimycobacterial agents and antibacterial drugs.[27−31] The compounds bearing the sulphonate and sulphamoyl group have shown anticancer, anti-hyperglycemic, and antimicrotubule properties.[32−35] Similarly, 4-aminophenol has been reported as a pharmacophore of the drug “Paracetamol”.[36] Considering the importance of the structural scaffolds of the reported structures such as the carboxamide moiety, sulphonate group, and 4-aminophenol, we moved toward the synthesis of arylamide-based sulphonates. The combination of various structural components referred to the compound as multiple choices of selectivity toward isozymes ENPP1 and ENPP3.

Figure 2

Structure–activity relationship of the designed compounds.

Results and Discussion

Chemistry

The synthetic pathways to design arylamide sulphonate derivatives 4a–t and 7a–f are illustrated in Schemes and 2, respectively. The required sulphonate derivatives (4a–t) were synthesized by a 2-step synthetic pathway. In the first step, the base-catalyzed nucleophilic-electrophilic substitution reaction of 4-aminophenol (1) with 4-methyl benzoyl chloride (2a) or benzoyl chloride (2b) led to the formation of structure 3a or 3b, respectively. In the second step, sulphonate derivatives 4a–l were produced by treating 3a with substituted benzene sulfonyl chloride; similarly, sulphonate derivatives from 4m to 4t were obtained by reaction of 3b with substituted benzene sulphonate chlorides under the same conditions. The second series of sulphonate derivatives (7a–f) were synthesized by a reaction of 1 with 9-methyl-9H-fluorene-9-carbonyl chloride (5) to yield the phenolic intermediate 6, which was further used with various sulfonyl chlorides to get the final products.

Scheme 1

Synthesis of Sulphonate Derivatives (4a–t)

Scheme 2

Synthesis of Sulphonate Derivatives (7a–f)

Enzyme Inhibition Assay

The enzymatic inhibitory potential of the synthesized molecules was determined against the hydrolytic activity of ENPP1 and ENPP3 isozymes. A number of compounds manifested selective and significant inhibition of the activity of both isozymes. The results of the enzymatic evaluation of the compounds 4a–t and 7a–f are depicted in Tables and 2, respectively.

Table 1

ENPP1 and ENPP3 Isozyme Inhibition at 100 μM Compound Concentration, Presented in IC50 ± SEM (μM) Values or Percentage Inhibition ±SD

			IC50 ± SEM (μM)a or % inhibition ± SDb
codes	R1	R2	ENPP1	ENPP3
4a	CH3	4-Me(C6H4)	26.2 ± 2.11b	17.0 ± 3.24b
4b	CH3	phenyl	45.2 ± 1.53b	29.8 ± 2.11a
4c	CH3	4-I(C6H4)	14.41 ± 0.54a	4.23 ± 0.38a
4d	CH3	4-OCH3(C6H4)	0.18 ± 0.01a	45.7 ± 2.35b
4e	CH3	4-Cl(C6H4)	44.8 ± 1.52b	0.82 ± 0.12a
4f	CH3	4-n-propyl(C6H4)	0.28 ± 0.08a	8.4 ± 3.11b
4g	CH3	4-OCF3(C6H4)	0.45 ± 0.07a	0.19 ± 0.02a
4h	CH3	biphenyl	1.73 ± 0.03a	27.1 ± 4.54b
4i	CH3	CH2(C6H4)	0.13 ± 0.01a	46.7 ± 1.54b
4j	CH3	4-n-butyl(C6H4)	0.23 ± 0.03a	42.1 ± 3.43b
4k	CH3	4-F(C6H4)	7.61 ± 0.02a	0.37 ± 0.08a
4l	CH3	2-F(C6H4)	0.15 ± 0.01a	3.48 ± 0.34a
4m	H	4-OCH3(C6H4)	0.82 ± 0.05a	22.6 ± 2.12b
4n	H	CH2(C6H4)	3.96 ± 0.12a	4.12 ± 0.05a
4o	H	4-n-propyl(C6H4)	1.71 ± 0.04a	2.12 ± 0.08a
4p	H	biphenyl	1.51 ± 0.02a	33.5 ± 1.92b
4q	H	8-quinolinyl	0.37 ± 0.03a	19.5 ± 3.53b
4r	H	4-I(C6H4)	2.01 ± 0.34a	0.53 ± 0.19a
4s	H	4-F(C6H4)	5.76 ± 0.57a	2.95 ± 0.25a
4t	H	4-OCF3(C6H4)	41.4 ± 3.24b	0.15 ± 0.04a
Suramin			7.80 ± 0.09a	0.89 ± 0.16a

The results are obtained by means of a triplicate assay; IC50 values are calculated for compounds showing inhibition of enzyme activity to more than 50%; SEM = standard error mean.

Percentage inhibition of the compounds exhibiting <50% inhibition; SD = standard deviation.

Table 2

ENPP1 and ENPP3 Isozyme Inhibition at 100 μM Compound Concentration, Presented in IC50 ± SEM (μM) Values or %Age Inhibition ±SD

		IC50 ± SEM (μM)a or % inhibition ± SDb
codes	R	ENPP1	ENPP3
7a	2-F(C6H4)	0.81 ± 0.05a	28.2 ± 3.22b
7b	CF3	0.90 ± 0.16a	4.16 ± 0.22a
7c	4-F(C6H4)	0.50 ± 0.03a	6.69 ± 1.89a
7d	methyl	9.2 ± 4.13b	0.16 ± 0.01a
7e	4-CH3(C6H4)	18.87 ± 0.54a	0.38 ± 0.13a
7f	phenyl	8.45 ± 0.25a	42.1 ± 2.44b

The results are obtained by means of a triplicate assay; IC50 values are calculated for compounds showing inhibition of enzyme activity to more than 50%; SEM = standard error mean.

Percentage inhibition of the compounds exhibiting <50% inhibition; SD = standard deviation.

Study of Structure–Activity Relationship of Compounds Measured through Enzyme Inhibition Assay

The effect of different structural variations at position “R1, R2” for compounds 4a–t and 7a–f was studied in terms of the structure–activity relationship. Comparison of results of compounds 4a, 4b, 4f, and 4j by the structures indexed interesting results. Compound 4a with 4-methyl phenyl linkage inhibited both isozymes’ activity to a lesser extent, merely 26.2% in the case of ENPP1 and only 17.0% for ENPP3. These results were slightly modified when the p-methyl group was removed with the phenyl ring only (4b). Again structure 4b was devoid of ENPP1 inhibitory potential but blocked the activity of ENPP3 to an IC50 value of 29.76 ± 2.13 μM. Modification of phenyl ring substitution to 4-methoxy phenyl group (4d) inversed the inhibitory potential more selectively to ENPP1 (IC50 = 0.18 ± 0.01 μM) and blocked the ENPP3 isozyme to only 45.7%.

In compliance with the modification at phenyl ring with methyl linkage, elongation of this linkage to three carbons atoms (4f) and four carbon atoms (4j) significantly and selectively turned the activity toward ENPP1, that is, compound 4f with a propyl linkage blocked the ENPP1 to IC50 = 0.28 ± 0.08 μM, whereas 4j with a butyl linker inhibited the isozyme ENPP1 with an IC50 value of 0.23 ± 0.03 μM. These three structures with hydrocarbon linkers (4d, 4f, 4j) poorly blocked the hydrolytic activity of ENPP3 to very low or nearly 50% percent inhibition values such as 45.7, 8.4, and 42.1%, respectively. The outcomes of 4d, 4f, and 4j suggested that the methoxy group or long hydrocarbon side chain with the phenyl ring was favorable to ENPP1 inhibition. A study of SAR for compounds 4b and 4h showed selective but opposite behavior toward isozymes ENPP1 and ENPP3. The molecule with a single phenyl ring attachment at position “R2” possessed the selective potential to ENPP3, whereas the biphenyl attachment at the same position turned the selectivity more specifically to isozyme ENPP1. The structures possessing halogens with phenyl moiety produced variable but promising outcomes. Comparison of SAR for 4c, 4e, and 4k substituted with 4-iodophenyl, 4-chlorophenyl, and 4-fluorophenyl at the “R1” position showed almost non-selective behavior without significant inhibitory difference among the two isozymes, except inhibitor 4e, which exhibited comparatively more affinity for ENPP3 with IC50 equal to 0.82 ± 0.12 μM. The relative study of SAR of 4k and 4l revealed that the presence of fluorine atom at the para position was much favorable for ENPP3, whereas fluorine atom at the meta position shifted the potency toward ENPP1.

The presence of the methoxy group on the phenyl ring rendered compound 4m toward the selectivity of ENPP1 (IC50 = 0.82 ± 0.05 μM). The same selectivity pattern was observed in the case of 4p with biphenyl attachment at the R2 position, reducing the activity of ENPP1 to half of the maximal inhibitory concentration (IC50) of 1.51 ± 0.02 μM and only 33.5% inhibition to ENPP3 isozyme. This pattern of selectivity suggested the −OCH3 or phenyl group linkage at the phenyl ring, which was favorable for ENPP1 inhibition. Elongation of the side chain to three carbon atoms (4o) abolished the selectivity pattern for either of the isozymes. A careful comparative study of structures 4f and 4o highlighted the point that the n-propyl linkage is much beneficial toward the selective inhibition of ENPP1 activity in the presence of methyl group on the other side of the structure. The presence of 8-quinolinyl moiety produced the most potent and selective structure (4q) that blocked the ENPP1 activity to IC50 value equal to 0.37 ± 0.03 μM, suggesting the heterocyclic attachment favorable for the inhibition of ENPP1 isozyme activity. The halogenated structures 4r–t revealed compound 4t possessing −OCF3 as a selective inhibitor for ENPP3 (0.15 ± 0.04 μM), contributing to the effect of −OCF3 toward inhibition of ENPP3 isozyme activity. Moreover, the methoxy group substitution was found preferable for only ENPP1 (4d, 4m), and the results of 4h and 4p demonstrated selective behavior toward ENPP1, suggesting the importance of the biphenyl group for ENPP1 inhibition (Table ).

In the next series of compounds (7a–f), aromatic and aliphatic sulfonyl chlorides were used to produce complex but promising structures. Interestingly, among the six compounds (7a–f), two compounds selectively blocked the activity of ENPP1, 7a with 2-fluorophenyl substitution (IC50 = 0.81 ± 0.05 μM) and 7f (IC50 = 8.45 ± 0.25 μM) possessing a single phenyl ring at position “R”, and the molecule 7d carrying a methyl group preferably inhibited the isozyme ENPP3 illustrating an IC50 value of 0.16 ± 0.01 μM. The trifluoromethane substitution produced structure 7b, which presented dual inhibition of both isozymes (ENPP1, IC50 = 0.90 ± 0.16 μM; ENPP3, IC50 = 4.16 ± 0.22 μM), whereas a single fluorine atom as a 4-fluorophenyl attachment preferably blocked the ENPP1 activity over ENPP3.

Pattern of Enzyme Inhibition of Compounds 4j and 7e

The pattern of enzyme inhibition for a single active compound against each isozyme was assessed by constructing the Lineweaver–Burk graphs (Figure ). The mode of inhibition for the comparatively selective and potent inhibitor of ENPP1 (4j) and ENPP3 (7e) was determined by performing enzyme kinetic studies. Compound 4j blocked ENPP1 uncompetitively, whereas 7e competitively inhibited ENPP3 activity.

Figure 3

Presentation of Lineweaver–Burk plots of enzyme kinetics of 4j (left) and 7e (right). The experiments were performed in triplicates.

Cell Cytotoxicity Analysis (MTT Assay)

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was executed to find out the cytotoxic effect of the compounds on human breast cancer cell line (MCF7), human cervical cancer cell line (HeLa), and human embryonic kidney cell line (HEK293). The compounds were found cytotoxic against cancer cell lines, but none of the molecules were toxic on normal cell lines. The results of the cytotoxicity assay are tabulated in Table . The cytotoxic potential of the compounds was evaluated on the three cancerous cell lines: (1) MCF7, a human breast cancer cell line; (2) HeLa, a human cervical cancer cell line; and (3) HEK293, a human embryonic kidney cell line. Neither of the tested compounds manifested a significant effect on MCF7 cancer cell viability except for 4o with 63.2% inhibition checked at 100 μM compound concentration. The molecules 4c, 4d, 4n, and 7d decreased the viability of HeLa cancer cells to above 50%. None of the compounds affected the viability of normal cell lines to a significant level.

Table 3

Cytotoxicity of 100 μM Compound on MCF7, HeLa, and HEK293 Cell Lines

codes	MCF7 (% inhibition ±SD)a	HeLa cell line (% inhibition ±SD)a	HEK293 (% inhibition ±SD)a
4a	1.1 ± 0.12	16.4 ± 0.92	5.3 ± 0.81
4b	7.4 ± 0.04	44.9 ± 1.81	6.4 ± 0.22
4c	33.2 ± 1.32	57.8 ± 2.72	15.2 ± 0.83
4d	31.5 ± 2.43	53.4 ± 1.93	13.1 ± 0.73
4e	24.2 ± 1.73	32.4 ± 3.82	8.3 ± 0.35
4f	25.7 ± 0.92	49.2 ± 2.12	4.2 ± 0.12
4g	11.7 ± 0.84	17.7 ± 0.72	3.2 ± 0.11
4h	9.1 ± 0.71	48.2 ± 1.43	4.8 ± 0.24
4i	5.7 ± 0.06	38.5 ± 1.14	11.2 ± 0.92
4j	13.3 ± 0.73	19.1 ± 1.11	27.6 ± 2.55
4k	11.2 ± 0.52	7.7 ± 0.73	2.1 ± 1.22
4l	0.5 ± 0.03	14.3 ± 3.52	17.4 ± 0.83
4m	4.1 ± 0.01	7.1 ± 0.81	2.3 ± 1.12
4n	7.3 ± 0.03	51.3 ± 0.82	5.8 ± 0.23
4o	63.2 ± 2.51	38.7 ± 2.43	12.6 ± 0.74
4p	8.4 ± 0.75	5.3 ± 0.07	4.3 ± 0.15
4q	11.2 ± 1.04	24.2 ± 1.44	4.6 ± 0.32
4r	10.2 ± 0.86	12.6 ± 0.73	11.3 ± 0.71
4s	2.1 ± 0.08	16.5 ± 1.12	20.6 ± 1.14
4t	29.6 ± 1.22	49.7 ± 2.53	8.6 ± 0.14
7a	12.7 ± 0.62	10.7 ± 0.72	14.6 ± 1.12
7b	14.1 ± 0.63	32.4 ± 2.63	24.5 ± 1.16
7c	12.4 ± 1.22	23.5 ± 1.64	9.2 ± 0.42
7d	0.2 ± 0.06	56.3 ± 1.17	5.5 ± 0.36
7e	0.3 ± 0.15	33.8 ± 1.56	12.4 ± 1.42
7f	5.6 ± 1.11	28.1 ± 1.33	6.2 ± 1.17
cisplatin	N.D	84.3 ± 2.72	65.5 ± 1.48
doxorubicin	88.8 ± 2.74	N.D	N.D

Cytotoxicity of compounds at 100 μM, calculated by taking a mean of the triplicate assays; % inhibition ± standard deviation; ND = not determined.

Molecular Docking Studies

The putative mode of structural component’s interaction with the amino acid residues of ENPP1 and ENPP3 isozymes was found by applying molecular docking analysis. The in silico findings produce reliable outcomes following the in vitro results. The 2D and 3D modes of interaction are shown in Figures –8.

Figure 4

Illustration of 3D plausible binding modes for selective inhibitor 4f with ENPP1 isozyme.

Figure 8

Presentation of 3D binding interaction of dual inhibitor 4g; (a) 3D binding modes with ENPP1 and (b) 3D binding modes with an ENPP3 crystallographic structure.

Molecular Docking Studies at the Crystal Structure of ENPP1 Isozyme

The most active synthesized compounds were consigned to molecular docking analysis to elaborate the inhibitory results against the ENPP1 isozyme. The reported crystallographic structure of human ENPP1, docked with reference ligand “N-{4-[(7-methoxyquinolin-4-yl)oxy]phenyl}sulfuric diamide” (TZV), was downloaded from Protein Data Bank (PDB) with PDB ID = 6wew.[37] The bound ligand TZV was found in contact with several amino acid residues of the catalytic site; such as the quinoline nucleus was stacked between Tyr340 and Phe257 and through π–π T-shaped interaction with Pro323 and Leu290. Moreover, TZV showed H-bond interaction with Lys338, Lys295, Asp376, and His380. A π-alkyl and π-anion connection was found with Leu290, Pro323, and Asp218.[37] The most selective and relatively potent inhibitor of ENPP1 4f showed H-bonding linkage between the oxygen atom of sulphonate group and amino acid residue Thr256. The inhibitor 4f rested itself through an array of π-linkages, that is, π–π stacked, π–π T-shaped, amide-π stacked, and π-alkyl bonds with residues His380, Tyr371, Tyr340, Trp322, and Leu290. Moreover, 4f was coordinated to zinc metal ion, Asp376, through π-anion and π-cation bonds (Figure ).

The docking studies of another selective inhibitor of isozyme ENPP1 (7a) manifested a wide range of occupancy of amino acids, attributing to its selectivity. Residues Lys255, Asn277, and Thr256 coordinated through hydrogen bonds, predominately with sulphonate group of 7a. Similar to 4f, compound 7a stabilized itself via π-connections such as a π-anion link to Asp376, π–π stacked connection Tyr340, π-alkyl interaction to residues Leu290, and Tyr340. Moreover, Zn2+ chelation connected to the oxygen atom of sulphonate moiety was observed as depicted in Figure .

Figure 5

Illustration of 3D binding interactions of selective inhibitor 7a with residues of ENPP1 isozyme.

Molecular Docking Studies at the Crystal Structure of ENPP3 Isozyme

The selective inhibitor of ENPP3 4t stabilized itself through an array of several amino acid residues. H-bond interactions were observed between oxygen atoms of sulphonate group and residues Ala205, Asn226, and Lys204 and hydrogen of amide moiety and residue Asn482. Several π-interactions contributed to the stability and activity of 4t in the active site. These linkages include π-anion connection with Asp167: π-alkyl stick bridging Ala205; Leu239: π–π stacked connection with His329: π-sulfur attachment with His483: π-anion linkage with His483; and Asp167: π-lone pair link among the lone pair of asparagine residue (Asn477) and phenyl ring of phenyl acetamide moiety. A π-cation linkage was noticed between the zinc ion and sulphonate group oxygen. More noticeably, an unfavorable bond was observed among Asp325 and oxygen atoms of sulphonate moiety, contributing to the high affinity and stability of the molecule 4t (Figure ).

Figure 6

Presentation of 3D binding modes of inhibitor 4t with ENPP3 isozyme residues.

The molecular docking study of another selective structure for ENPP3 (7d) revealed promising interactions with amino acid residues. The structure 7d balanced itself through hydrogen bonding of sulphonate group oxygens with Asn226 and His329. The three aromatic rings and the methyl group connected to the heterocyclic scaffold formed an array of π–π stacked and π-alkyl linkages with residues Tyr289, Pro272, Leu239, Tyr320, and Ala205. In addition, one π-anion interaction was found between the phenyl ring of phenyl methanesulfonate attachment and Asp325 as shown in Figure .

Figure 7

Illustration of 3D binding modes of inhibitor 7d with ENPP3 isozyme residues.

Molecular Docking Studies at the Crystal Structure of ENPP1 and ENPP3 Isozymes

The observation of the plausible binding mode of the relatively potent but dual inhibitor 4g gave insights into the binary nature or non-selective behavior of the structure. In the case of ENPP1, inhibitor 4g formed an array of π-connections; Tyr371 (π–π stacked, π-sigma bond), Tyr340 (π–π T shaped), Trp322 (π–π stacked), and Pro323 and Phe321 (π-alkyl linkage). Similar to inhibitors 4f and 7a, compound 4g was also chelated with zinc metal ions. 4g blocked the catalytic activity of ENPP3 to a low micromolar level, tabulated as the second active inhibitor to ENPP3 (0.19 ± 0.02 μM). The binding modes gave the results in obedience to experimental data. As a result of docking into the ENPP3 crystalline structure, 4g was linked via four H-bonds: two with Lys204 and Asn226 through the oxygen of sulphonate scaffold and two with Asn482 and His329. The whole structure was surrounded by Pi-linkages of several amino acid residues such as π–π bonds with residues Tyr289, Tyr320, Phe206, and His329 and π-alkyl and alkyl bonds with Leu239, Ala205, and Pro272. In addition, the important connections were observed with His483 (π-lone pair bond) and Asp167 (π-anion bond). Zinc ion chelation was also observed through the oxygen atom of the sulphonate group (Figure ).

Conclusions

A new series of compounds possessing specifically sulphonate group was synthesized, characterized, and examined for the affinity and preference to block the activity of ENPP1 and ENPP3 isozymes of the ectonucleotidase pyrophosphatase/phosphodiesterase (ENPP) family. Among the series, 4a–t compounds 4d, 4f, 4h, 4i, 4j, 4m, 4p, and 4q selectively blocked the activity of ENPP1 to less or high preferentially, whereas in the group of structures 7a–f, structure 7a and 7f preferably inhibited the activity of ENPP1. Among both series, compounds 4f, 4h, 4m, 4p, 4q, and 7a were found as more potent and particular inhibitors of ENPP1 with IC50 values of 0.28 ± 0.08, 1.73 ± 0.03, 0.82 ± 0.05, 1.51 ± 0.02, 0.37 ± 0.03, and 0.81 ± 0.05 μM, alternatively. Similarly, compounds 4b, 4e, 4t, and 7d showed discriminatory selective behavior for ENPP3 with most active structures 4t (0.15 ± 0.04 μM) and 7d (IC50 = 0.16 ± 0.01 μM). In the same way, compounds 4c, 4g, 4k, 4l, 4n, 4o, 4r, 4s, 7b, 7c, and 7e exhibited dual inhibitory behavior to ENPP1 and ENPP3, particularly 4g as a more potent structure that tabulated IC50 = 0.45 ± 0.07 μM against ENPP1 and IC50 = 0.19 ± 0.02 μM toward ENPP3. The cancer cell line (MCF7, HeLa) and normal cell line (HEK293) viability assay revealed that compounds 4c, 4d, 4n, and 7d were cytotoxic against HeLa cancer cell lines. The structure 4o significantly decreased the MCF7 cancer cell line viability to 63.2%; however, none of the molecules showed cytotoxicity toward normal cell lines and placed the compounds within a safety profile. The plausible pattern of interactions of selective and non-selective structures with salient amino acids comprising both proteins ENPP1 and ENPP3 complied with in vitro results. In summation, we found several selective and non-cytotoxic inhibitors for both isozymes ENPP1 and ENPP3. These compounds may be further evaluated as therapeutic agents for the ailments associated with ENPP1 and ENPP3 isozymes.

Experimental Section

Reagents, Solvents, and Apparatus

The chemicals and solvents used in this study were obtained from commercial sources. Chemicals were used as such, whereas solvents were dried or distilled where required. 1H NMR and 13C NMR spectra were obtained by a Bruker spectrometer (AV 250, AV 300, and AV 500) in solvents CDCL3 and DMSO. FTIR spectra were detected by an ATR apparatus. The high resonance mass spectra (HRMS) ESI was detected on a device Finnigan MAT 95 XP with an HP-5 capillary column using helium as carrier gas (Thermo Electron Corporation). The melting point of the compounds was obtained with the use of the Büchi apparatus. Column chromatography was carried out by using silica gel 60 A with a mesh size of 60–200. Analytical thin-layer chromatography was performed on silica gel plates (0.20 mm, 60 A), and spots were detected through a UV absorbance lamp 254 nm/366 nm.

General Procedure of Synthesis

Synthesis of N-(4-Hydroxyphenyl)-4-methylbenzamide (3a) and N-(4-Hydroxyphenyl)benzamide (3b)

The synthesis of compounds 3a and 3b was carried out by treating 4-aminophenol (1) (4.12 mmol; 0.450 g) with 4-methyl benzoyl chloride (2a) (2.50 mmol; 0.386 g) or benzoyl chloride (2b) (2.50 mmol; 0.350 g; 0.423 mL), respectively. The reactants were combined with stirring at 0 °C in acetone (50 mL) in the presence of anhydrous potassium carbonate (3.62 mmol; 0.500 g). After that, the reaction mixture was stirred for 4 h at room temperature and filtered. The filtrate proceeded to dryness, the residue was dissolved in ethyl acetate (40 mL), and extraction was carried out by using dilute HCl (30 mL). The organic layer was separated and washing was performed with a saline solution (2 × 20 mL). The separated organic layer was dried of water molecules by using anhydrous sodium sulfate (Na2SO4) and filtered. The organic solvent was evaporated to dryness, and obtained compounds were used for the next step.

Synthesis of Arylamide Sulphonates (4a–l, 4m–t)

The targeted compounds 4a–l were synthesized by the reaction of 3a (0.485 mmol; 0.110 g) with various benzene sulfonyl chloride derivatives (0.985 mmol), and compounds from 4m–t were obtained by treating 3b (0.485 mmol; 0.103 g) with appropriate benzene sulfonyl derivatives. The separate reactions were performed in dry tetrahydrofuran (THF) (12 mL), and triethylamine (2.10 mmol) was added dropwise to the reaction mixture at 0 °C. Each reaction mixture was stirred at room temperature till completion and quenched up to three times with water and ethyl acetate in equal volumes (3 × 20 mL). The organic layer was dried with anhydrous Na2SO4 followed by two times washing with a saline solution (2 × 30 mL), filtered, and subjected to dryness on the rotary evaporator. The crude residue was purified by recrystallization with absolute ethanol.

4-(4-Methylbenzamido)phenyl 4-Methylbenzenesulfonate (4a)

Yield: 74%; colorless solid; mp: 213–216 °C; FT-IR (υ̅, cm1, neat): 3352 (N–H, stretching), 2921, 2851 (C–H, stretching), 1652 (C=O, stretching), 1528, 1506, 1343, 1172, 1143, 1092; 1H NMR (300 MHz, DMSO, δ = ppm): 10.26 (s, 1H, CONH), 7.85 (d, J = 8.2 Hz, 2H, Ar–H), 7.74 (dd, J = 8.8, 4.3 Hz, 4H, Ar–H), 7.48 (dd, J = 8.6, 0.6 Hz, 2H, Ar–H), 7.33 (d, J = 7.9 Hz, 2H, Ar–H), 6.99 (d, J = 9.1 Hz, 2H, Ar–H), 2.43 (s, 3H, CH3), 2.38 (s, 3H, CH3); 13C NMR (75 MHz, DMSO, δ = ppm): 165.39 (C=O), 145.69 (Ar–C), 144.38 (Ar–C), 141.74 (Ar–C), 138.23 (Ar–C), 131.70 (Ar–C), 131.33 (Ar–C), 130.16 (Ar–CH), 128.89 (Ar–CH), 128.23 (Ar–CH), 127.65 (Ar–CH), 122.22 (Ar–CH), 121.28 (Ar–CH), 21.13 (CH3), 20.97 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C21H19NO4S, 382.1113; found, 382.1111.

4-(4-methylbenzamido)phenyl Benzenesulfonate (4b)

Yield: 66%; brown solid; mp: 195–196 °C; FT-IR (υ̅, cm1, neat): 3358 (N–H, stretching), 1652 (C=O, stretching), 1603, 1528, 1343, 1172, 1143, 1092; 1H NMR (500 MHz, DMSO, δ = ppm): 10.26 (s, 1H, CONH), 7.90–7.80 (m, 5H, Ar–H), 7.75 (d, J = 9.0 Hz, 2H, Ar–H), 7.68 (t, J = 7.8 Hz, 2H, Ar–H), 7.33 (d, J = 8.0 Hz, 2H, Ar–H), 7.00 (d, J = 9.0 Hz, 2H, Ar–H), 2.38 (s, 3H, CH3); 13C NMR (126 MHz, DMSO, δ = ppm): 165.91 (C=O), 144.81 (Ar–C), 142.26 (Ar–C), 138.82 (Ar–C), 135.47 (Ar–C), 134.72 (Ar–C), 132.20 (Ar–C), 130.27 (Ar–CH), 129.41 (Ar–CH), 128.72 (Ar–CH), 128.17 (Ar–CH), 122.74 (Ar–CH), 121.79 (Ar–CH), 21.48 (CH3); HRMS (EI, 70 eV), [C20H17O4N1S1], 367.08728; found, 367.08709.

4-(4-methylbenzamido)phenyl 4-Iodobenzenesulfonate (4c)

Yield: 82%; off-white solid; mp: 223–226 °C; FT-IR (υ̅, cm1, neat): 3340 (N–H, stretching), 2921, 2851 (C–H, stretching), 1650 (C=O, stretching), 1522, 1504, 1349, 1195, 1176, 1090; 1H NMR (250 MHz, DMSO, δ = ppm): 10.26 (s, 1H, CONH), 8.04 (d, J = 8.6 Hz, 2H, Ar–H), 7.79 (dd, J = 18.9, 8.7 Hz, 4H, Ar–H), 7.57 (d, J = 8.6 Hz, 2H, Ar–H), 7.31 (d, J = 8.0 Hz, 2H, Ar–H), 7.01 (d, J = 9.1 Hz, 2H, Ar–H), 2.36 (s, 3H, CH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.46 (C=O), 144.24 (Ar–C), 141.81 (Ar–C), 138.73 (Ar–C), 138.47 (Ar–C), 133.80 (Ar–C), 131.73 (Ar–C), 129.69 (Ar–CH), 128.94 (Ar–CH), 127.71 (Ar–CH), 122.31 (Ar–CH), 121.39 (Ar–CH), 104.10 (Ar–CH), 21.02 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C20H16NO4S1, 493.9923; found, 493.9924.

4-(4-Methylbenzamido)phenyl 4-Methoxybenzenesulfonate (4d)

Yield: 61%; light-brown solid; mp: 193–195 °C; FT-IR (υ̅, cm1, neat): 3377 (N–H, stretching), 2981 (C–H, stretching), 1660 (C=O, stretching), 1596, 1498, 1362, 1168, 1149, 1094; 1H NMR (250 MHz, DMSO, δ = ppm): 10.26 (s, 1H, CONH), 7.85 (d, J = 8.2 Hz, 2H, Ar–H), 7.76 (dd, J = 9.1, 4.0 Hz, 4H, Ar–H), 7.38–7.28 (m, 2H, Ar–H), 7.17 (d, J = 9.1 Hz, 2H, Ar–H), 6.99 (d, J = 9.1 Hz, 2H, Ar–H), 3.87 (s, 3H, OCH3), 2.38 (s, 3H, CH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.42 (C=O), 163.97 (Ar–C), 144.47 (Ar–C), 141.77 (Ar–C), 138.22 (Ar–C), 131.75 (Ar–C), 130.65 (Ar–CH), 128.92 (Ar–CH), 127.69 (Ar–CH), 125.45 (Ar–C), 122.32 (Ar–CH), 121.29 (Ar–CH), 114.92 (Ar–CH), 55.91 (OCH3), 21.00 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C21H19NO5S, 398.1062; found, 398.1062.

4-(4-Methylbenzamido)phenyl 4-Chlorobenzenesulfonate (4e)

Yield: 77%; brown solid; mp: 196–197 °C; FT-IR (υ̅, cm1, neat): 3354 (N–H, stretching), 1652 (C=O, stretching), 1524, 1506, 1376, 1176, 1151, 1090; 1H NMR (250 MHz, DMSO δ = ppm): 10.28 (s, 1H, CONH), 7.91–7.81 (m, 4H, Ar–H), 7.77 (dd, J = 9.0, 4.7 Hz, 4H, Ar–H), 7.34 (d, J = 7.9 Hz, 2H, Ar–H), 7.04 (d, J = 9.1 Hz, 2H, Ar–H), 2.39 (s, 3H, CH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.44 (C=O), 144.20 (Ar–C), 141.80 (Ar–C), 140.03 (Ar–C), 138.49 (Ar–C), 133.02 (Ar–C), 131.72 (Ar–C), 130.21 (Ar–CH), 130.00 (Ar–CH), 128.93 (Ar–CH), 127.70 (Ar–CH), 122.31 (Ar–CH), 121.38 (Ar–CH), 21.00 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C20H16NO4SCl, 402.0567; found, 402.0569.

4-(4-Methylbenzamido)phenyl 4-Propylbenzenesulfonate (4f)

Yield: 67%; light-brown solid; mp: 179–180 °C; FT-IR (υ̅, cm1, neat): 3369 (N–H, stretching), 2965, 2930, 2860 (C–H, stretching), 1658 (C=O, stretching), 1607, 1524, 1504, 1364, 1174, 1149, 1090; 1H NMR (250 MHz, DMSO, δ = ppm): 10.26 (s, 1H, CONH), 7.85 (d, J = 8.2 Hz, 2H, Ar–H), 7.75 (dd, J = 8.8, 1.8 Hz, 4H, Ar–H), 7.48 (d, J = 8.6 Hz, 2H, Ar–H), 7.36–7.30 (m, 2H, Ar–H), 6.98 (d, J = 9.1 Hz, 2H, Ar–H), 2.74–2.61 (m, 2H, CH2), 2.38 (s, 3H, Ar–CH3), 1.62 (m, 2H, CH2), 0.88 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.41 (C=O), 150.02 (Ar–C), 144.38 (Ar–C), 141.77 (Ar–C), 138.29 (Ar–C), 131.73 (Ar–C), 131.60 (Ar–C), 129.61 (Ar–CH), 128.92 (Ar–CH), 128.31 (Ar–CH), 127.69 (Ar–CH), 122.26 (Ar–CH), 121.26 (Ar–CH), 36.92 (CH2), 23.51 (CH2), 21.00 (CH3), 13.41 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C23H23NO4S, 410.1426; found, 410.1426.

4-(4-Methylbenzamido)phenyl 4-(Trifluoromethoxy)benzenesulfonate (4g)

Yield: 71%; colorless solid; mp: 196–198 °C; FT-IR (υ̅, cm1, neat): 3348 (N–H, stretching), 1652 (C=O, stretching), 1524, 1508, 1353, 1193, 1141, 1094; 1H NMR (250 MHz, DMSO, δ = ppm): 10.28 (s, 1H, CONH), 8.01 (d, J = 9.1 Hz, 2H, Ar–H), 7.81 (dd, J = 16.5, 8.7 Hz, 4H, Ar–H), 7.66 (dd, J = 9.0, 1.0 Hz, 2H, Ar–H), 7.33 (d, J = 7.9 Hz, 2H, Ar–H), 7.03 (d, J = 9.1 Hz, 2H, Ar–H), 2.38 (s, 3H, CH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.41 (C=O), 152.45 (Ar–C), 144.12 (Ar–C), 141.76 (Ar–C), 138.50 (Ar–C), 132.90 (Ar–C), 131.67 (Ar–C), 131.12 (Ar–C), 128.88 (CF3), 127.66 (Ar–CH), 122.26 (Ar–CH), 121.78 (Ar–CH), 121.61 (Ar–CH), 121.31 (Ar–CH), 20.95 (CH3); 19F NMR (282 MHz, DMSO) δ −56.73 (s); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C21H16NO5SF3, 452.0779; found, 452.0780.

4-(4-Methylbenzamido)phenyl [1,1′-Biphenyl]-4-sulfonate (4h)

Yield: 71%; colorless solid; mp: 243–244 °C; FT-IR (υ̅, cm1, neat): 3360 (N–H, stretching), 1654 (C=O, stretching), 1531, 1508, 1347, 1193, 1174, 1098; 1H NMR (250 MHz, DMSO, δ = ppm): 10.23 (s, 1H, CONH), 7.99–7.86 (m, 4H, Ar–H), 7.81 (d, J = 8.2 Hz, 2H, Ar–H), 7.78–7.69 (m, 4H, Ar–H), 7.52–7.39 (m, 3H, Ar–H), 7.32 (dd, J = 12.7, 7.6 Hz, 2H, Ar–H), 7.07–6.98 (m, 2H, Ar–H), 2.34 (s, 3H, CH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.43 (C=O), 146.13 (Ar–C), 144.37 (Ar–C), 141.78 (Ar–C), 138.37 (Ar–C), 137.82 (Ar–C), 132.95 (Ar–C), 131.73 (Ar–C), 129.23 (Ar–CH), 129.04 (Ar–CH), 128.93 (Ar–CH), 127.79 (Ar–CH), 127.69 (Ar–CH), 127.24 (Ar–CH), 125.98 (Ar–CH), 122.32 (Ar–CH), 121.37 (Ar–CH), 21.00 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C26H21NO4S, 444.1270; found, 444.1265.

4-(4-Methylbenzamido)phenyl Phenylmethanesulfonate (4i)

Yield: 73%; light-brown solid; mp: 205–206 °C; FT-IR (υ̅, cm1, neat): 3360 (N–H, stretching), 2940 (C–H, stretching), 1650 (C=O, stretching), 1524, 1506, 1341, 1195, 1143, 1102, 1073; 1H NMR (250 MHz, DMSO, δ = ppm): 10.31 (s, 1H, CONH), 7.86 (dd, J = 8.6, 7.6 Hz, 4H, Ar–H), 7.51 (dd, J = 6.8, 3.2 Hz, 2H, Ar–H), 7.47–7.40 (m, 3H, Ar–H), 7.38–7.31 (m, 2H, Ar–H), 7.22 (d, J = 9.1 Hz, 2H, Ar–H), 4.95 (s, 2H, CH2), 2.39 (s, 3H, CH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.43 (C=O), 144.40 (Ar–C), 141.78 (Ar–C), 138.15 (Ar–C), 131.77 (Ar–C), 131.07 (Ar–C), 128.94 (CH), 128.81 (CH), 128.65 (CH), 128.11 (C), 127.72 (CH), 122.32 (CH), 121.48 (CH), 55.73 (CH2), 21.01 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C21H19O4NS, 382.1113; found, 382.1116.

4-(4-Methylbenzamido)phenyl 4-Butylbenzenesulfonate (4j)

Yield: 80%; colorless solid; mp, 187–188 °C; FT-IR (υ̅, cm1, neat): 3371 (N–H, stretching), 2954, 2934, 2858 (C–H, stretching), 1660 (C=O, stretching), 1524, 1504, 1366, 1174, 1149, 1092; 1H NMR (250 MHz, DMSO, δ = ppm): 10.26 (s, 1H, CONH), 7.84 (d, J = 8.2 Hz, 2H, Ar–H), 7.79–7.68 (m, 4H, Ar–H), 7.48 (d, J = 8.5 Hz, 2H, Ar–H), 7.41–7.24 (m, 2H, Ar–H), 6.98 (d, J = 9.2 Hz, 2H, Ar–H), 2.78–2.61 (m, 2H, CH2), 2.38 (s, 3H, CH3), 1.69–1.46 (m, 2H, CH2), 1.28 (m, 2H, CH2), 0.89 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.37 (C=O), 150.23 (Ar–C), 144.34 (Ar–C), 141.73 (Ar–C), 138.25 (Ar–C), 131.69 (Ar–C), 131.51 (Ar–C), 129.51 (Ar–C), 128.88 (Ar–C), 128.28 (Ar–C), 127.65 (Ar–C), 122.22 (Ar–C), 121.21 (Ar–C), 34.59 (CH2), 32.43 (CH2), 21.58 (CH2), 20.96 (CH3), 13.65 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C24H25NO4S, 424.1582; found, 424.1580.

4-(4-Methylbenzamido)phenyl 4-Fluorobenzenesulfonate (4k)

Yield: 69%; colorless solid; mp: 185–187 °C; FT-IR (υ̅, cm1, neat): 3360 (N–H, stretching), 1652 (C=O, stretching), 1527, 1508, 1491, 1347, 1191, 1172, 1092; 1H NMR (300 MHz, DMSO, δ = ppm): 10.27 (s, 1H, CONH), 7.93 (dd, J = 9.0, 5.0 Hz, 2H, Ar–H), 7.84 (d, J = 8.2 Hz, 2H, Ar–H), 7.77 (d, J = 9.1 Hz, 2H, Ar–H), 7.51 (t, J = 8.9 Hz, 2H, Ar–H), 7.32 (d, J = 7.9 Hz, 2H, Ar–H), 7.01 (d, J = 9.1 Hz, 2H, Ar–H), 2.37 (s, 3H, CH3); 13C NMR (75 MHz, DMSO, δ = ppm): 167.20 (C=O), 165.41, 163.83, 144.22 (Ar–C), 141.75 (Ar–C), 138.41 (Ar–C), 131.86–131.39 (m, Ar–C), 130.47 (d, J = 3.0 Hz, Ar–CH), 128.89 (Ar–CH), 127.66 (Ar–CH), 122.29 (Ar–CH), 121.32 (Ar–CH), 117.12 (d, J = 23.1 Hz, Ar–CH), 20.96 (CH3); 19F NMR (282 MHz, DMSO, δ = ppm): −102.54 (s); HRMS (EI, 70 eV), [C20H16O4N1F1S1], 385.07786; found, 385.07785.

4-(4-Methylbenzamido)phenyl 2-Fluorobenzenesulfonate (4l)

Yield: 71%; off-white solid; mp: 178–181 °C; FT-IR (υ̅, cm1, neat): 3354 (N–H, stretching), 1654 (C=O, stretching), 1599, 1524, 1502, 1479, 1353, 1188, 1123, 1075; 1H NMR (250 MHz, DMSO, δ = ppm): 10.26 (s, 1H, CONH), 7.89 (ddd, J = 7.5, 2.5, 1.4 Hz, 1H, Ar–H), 7.86–7.72 (m, 5H, Ar–H), 7.62 (ddd, J = 10.5, 8.4, 1.0 Hz, 1H), 7.47–7.37 (m, 1H, Ar–H), 7.36–7.25 (m, 2H, Ar–H), 7.12–7.01 (m, 2H, Ar–H), 2.36 (s, 3H, CH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.41 (C=O), 144.05 (Ar–C), 141.76 (Ar–C), 138.56 (Ar–C), 138.14 (d, J = 8.9 Hz, Ar–C), 131.66 (Ar–C), 131.17 (Ar–C), 128.88 (Ar–CH), 127.66 (Ar–CH), 125.49 (d, J = 3.7 Hz, Ar–CH), 122.31–121.04 (m, Ar–CH), 118.02 (Ar–CH), 117.70 (Ar–CH), 20.96 (CH3); 19F NMR (282 MHz, DMSO, δ = ppm): −108.09 (ddd, J = 10.6, 7.1, 5.2 Hz); HRMS (EI, 70 eV), [C20H16O4N1F1S1], 385.07786; found, 385.07770.

4-Benzamidophenyl 4-Methoxybenzenesulfonate (4m)

Yield: 60%; colorless solid; mp: 197–199 °C; FT-IR (υ̅, cm1, neat): 3365 (N–H, stretching), 1660 (C=O, stretching), 1524, 1500, 1362, 1261, 1149, 1094; 1H NMR (250 MHz, DMSO, δ = ppm): 10.35 (s, 1H, CONH), 7.93 (dd, J = 8.1, 1.6 Hz, 2H, Ar–H), 7.82–7.71 (m, 4H, Ar–H), 7.63–7.47 (m, 3H, Ar–H), 7.23–7.13 (m, 2H, Ar–H), 7.05–6.95 (m, 2H, Ar–H), 3.88 (s, 3H, OCH3); 13C NMR (63 MHz, DMSO, δ = ppm): 165.59 (C=O), 163.93, 144.51 (Ar–C), 138.10 (Ar–C), 134.62 (Ar–C), 131.67 (Ar–C), 130.62 (Ar–C), 128.37 (Ar–CH), 127.60 (Ar–CH), 125.40 (Ar–CH), 122.31 (Ar–CH), 121.28 (Ar–CH), 114.89 (Ar–CH), 55.88 (OCH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C20H17NO5S, 384.0905; found, 384.0906.

4-Benzamidophenyl Phenylmethanesulfonate (4n)

Yield: 76%; light-yellow solid; mp: 179–181 °C; FT-IR (υ̅, cm1, neat): 3288 (N–H, stretching), 2936 (C–H, stretching), 1650 (C=O, stretching), 1524, 1504, 1351, 1184, 1147, 1100, 1073; 1H NMR (300 MHz, DMSO, δ = ppm): 10.40 (s, 1H, CONH), 7.96 (dd, J = 8.2, 1.5 Hz, 2H, Ar–H), 7.86 (d, J = 9.1 Hz, 2H, Ar–H), 7.60 (dd, J = 5.1, 3.5 Hz, 1H, Ar–H), 7.56 (d, J = 7.3 Hz, 2H, Ar–H), 7.51 (dd, J = 5.2, 1.8 Hz, 2H, Ar–H), 7.45 (dd, J = 5.0, 1.8 Hz, 3H, Ar–H), 7.24 (d, J = 9.1 Hz, 2H, Ar–H), 4.96 (s, 2H, CH2); 13C NMR (75 MHz, DMSO, δ = ppm): 165.63 (C=O), 144.48 (Ar–CH), 138.07 (Ar–CH), 134.67 (Ar–C), 131.72 (Ar–C), 131.07 (Ar–C), 128.82 (Ar–CH), 128.65 (Ar–CH), 128.42 (Ar–CH), 128.10 (Ar–CH), 127.67 (Ar–CH), 122.35 (Ar–CH), 121.51 (Ar–CH), 55.73 (CH2); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C20H17NO4S, 368.0956; found, 368.0955.

4-Benzamidophenyl 4-Propylbenzenesulfonate (4o)

Yield: 78%; off-white solid; mp: 192–195 °C; FT-IR (υ̅, cm1, neat): 3362 (N–H, stretching), 2957, 2926, 2860 (C–H, stretching), 1656 (C=O, stretching), 1522, 1502, 1407, 1370, 1174, 1151, 1092; 1H NMR (300 MHz, CDCl3, δ = ppm): 10.35 (s, 1H, CONH), 7.96–7.89 (m, 2H, Ar–H), 7.76 (dd, J = 8.8, 2.1 Hz, 4H, Ar–H), 7.64–7.56 (m, 1H, Ar–H), 7.51 (dd, J = 14.2, 7.9 Hz, 4H, Ar–H), 6.99 (d, J = 9.1 Hz, 2H, Ar–H), 2.72–2.59 (m, 2H, CH2), 1.62 (m, 2H, CH2), 0.88 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (75 MHz, DMSO, δ = ppm): 165.60 (C=O), 150.01 (Ar–C), 144.44 (Ar–C), 138.18 (Ar–C), 134.62 (Ar–C), 131.68 (Ar–C), 131.57 (Ar–C), 129.59 (Ar–CH), 128.38 (Ar–CH), 128.28 (Ar–CH), 127.62 (Ar–CH), 122.27 (Ar–CH), 121.27 (Ar–CH), 36.90 (CH2), 23.49 (CH2), 13.39 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C22H21NO4S, 396.1270; found, 396.1271.

4-Benzamidophenyl [1,1′-Biphenyl]-4-sulfonate (4p)

Yield: 66%; off-white solid; mp: 220–222 °C; FT-IR (υ̅, cm1, neat): 3367 (N–H, stretching), 1658 (C=O, stretching), 1605, 1524, 1502, 1405, 1347, 1312, 1193, 1143, 1098; 1H NMR (300 MHz, DMSO, δ = ppm): 10.36 (s, 1H, CONH), 7.98 (d, J = 8.8 Hz, 2H, Ar–H), 7.92 (m, 4H, Ar–H), 7.80 (d, J = 2.0 Hz, 2H, Ar–H), 7.77 (m, 2H, Ar–H), 7.56 (m, 3H, Ar–H), 7.50 (m, 3H, Ar–H), 7.07 (d, J = 9.1 Hz, 2H, Ar–H); 13C NMR (75 MHz, DMSO, δ = ppm): 165.65 (C=O), 146.14 (Ar–C), 144.45 (Ar–C), 138.29 (Ar–C), 137.82 (Ar–C), 134.65 (Ar–C), 132.95 (Ar–C), 131.71 (Ar–C), 129.23 (Ar–CH), 129.03 (Ar–CH), 128.93 (Ar–CH), 128.40 (Ar–CH), 127.80 (Ar–CH), 127.65 (Ar–CH), 127.23 (Ar–CH), 122.35 (Ar–CH), 121.40 (Ar–CH); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C25H19NO4S, 430.1113; found, 430.1113.

4-Benzamidophenyl Quinoline-8-sulfonate (4q)

Yield: 69%; yellowish-white solid; mp: 173–175 °C; FT-IR (υ̅, cm1, neat): 3329 (N–H, stretching), 1646 (C=O, stretching), 1607, 1524, 1504, 1366, 1316, 1174, 1158, 1075; 1H NMR (300 MHz, DMSO, δ = ppm): 10.29 (s, 1H, CONH), 9.25 (dd, J = 4.2, 1.8 Hz, 1H, Ar–NH), 8.63 (dd, J = 8.4, 1.7 Hz, 1H, Ar–H), 8.45 (dd, J = 8.2, 1.4 Hz, 1H, Ar–H), 8.35 (dd, J = 7.4, 1.4 Hz, 1H, Ar–H), 7.88 (m, 2H, Ar–H), 7.79 (m, 2H, Ar–H), 7.66 (d, J = 9.1 Hz, 2H, Ar–H), 7.56 (d, J = 7.1 Hz, 1H, Ar–H), 7.50 (dd, J = 8.0, 6.4 Hz, 2H, Ar–H), 6.96 (d, J = 9.1 Hz, 2H, Ar–H); 13C NMR (75 MHz, DMSO, δ = ppm): 165.57 (C=O), 152.43, 144.77, 143.11, 137.99 (Ar–C), 137.15 (Ar–C), 136.24 (Ar–C), 134.64 (Ar–C), 134.01, 131.66, 131.36, 128.76 (Ar–CH), 128.37 (Ar–CH), 127.61 (Ar–CH), 125.64 (Ar–CH), 123.07 (Ar–CH), 122.05 (Ar–CH), 121.37 (Ar–CH); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C22H16N2O4S, 405.0909; found, 405.0914.

4-Benzamidophenyl 4-Iodobenzenesulfonate (4r)

Yield: 62%; off-white solid; mp: 211–216 °C; FT-IR (υ̅, cm1, neat): 3365 (N–H, stretching), 1660 (C=O, stretching), 1520, 1502, 1368, 1172, 1149, 1085; 1H NMR (300 MHz, CDCl3, δ = ppm): 10.37 (s, 1H, CONH), 8.07 (d, J = 8.6 Hz, 2H, Ar–H), 7.97–7.89 (m, 2H, Ar–H), 7.78 (d, J = 9.1 Hz, 2H, Ar–H), 7.60 (d, J = 8.6 Hz, 3H, Ar–H), 7.53 (dd, J = 8.0, 6.3 Hz, 2H, Ar–H), 7.04 (d, J = 9.1 Hz, 2H, Ar–H); 13C NMR (75 MHz, CDCl3, δ = ppm): 165.64 (C=O), 144.28 (Ar–C), 138.70 (Ar–CH), 138.36, 134.62, 133.77, 131.70 (Ar–C), 129.65 (Ar–CH), 128.39 (Ar–CH), 127.63 (Ar–CH), 122.30 (Ar–CH), 121.39 (Ar–CH), 104.08 (Ar–C); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C19H14NO4SI, 479.9766; found, 479.9761.

4-Benzamidophenyl 4-Fluorobenzenesulfonate (4s)

Yield: 73%; light-brown solid; mp: 190–192 °C; FT-IR (υ̅, cm1, neat): 3373 (N–H, stretching), 1658 (C=O, stretching), 1590, 1524, 1491, 1407, 1378, 1242, 1176, 1151, 1090; 1H NMR (500 MHz, DMSO, δ = ppm): 10.37 (s, 1H, CONH), 7.94 (dd, J = 12.0, 6.2 Hz, 4H, Ar–H), 7.78 (d, J = 9.0 Hz, 2H, Ar–H), 7.59 (d, J = 7.3 Hz, 1H, Ar–H), 7.52 (m, 4H, Ar–H), 7.03 (d, J = 9.0 Hz, 2H, Ar–H); 13C NMR (126 MHz, DMSO, δ = ppm): 166.58 (C=O), 165.67 (Ar–C), 164.55 (Ar–C), 144.34 (Ar–C), 138.38 (Ar–C), 134.65 (Ar–C), 131.85–131.46 (m), 130.49, 128.42 (Ar–CH), 127.66 (Ar–CH), 122.37 (Ar–CH), 121.39 (Ar–CH), 117.18 (d, J = 23.1 Hz, Ar–CH); 19F NMR (471 MHz, DMSO, δ = ppm): −102.52 (s); HRMS (EI, 70 eV), [C19H14O4N1F1S1], 371.06221; found, 371.06190.

4-Benzamidophenyl 4-(trifluoromethoxy)benzenesulfonate (4t)

Yield: 78%; off-white solid; mp: 184–186 °C; FT-IR (υ̅, cm1, neat): 3365 (N–H, stretching), 1658 (C=O, stretching), 1607, 1526, 1407, 1382, 1151, 1090; 1H NMR (300 MHz, DMSO, δ = ppm): 10.38 (s, 1H, CONH), 8.03 (d, J = 8.9 Hz, 2H, Ar–H), 7.93 (m, 2H, Ar–H), 7.79 (d, J = 9.1 Hz, 2H, Ar–H), 7.67 (dd, J = 8.9, 1.0 Hz, 2H, Ar–H), 7.60 (d, J = 7.0 Hz, 1H, Ar–H), 7.54 (m, 2H, Ar–H), 7.06 (d, J = 9.1 Hz, 2H, Ar–H); 13C NMR (75 MHz, DMSO, δ = ppm): 165.67 (C=O), 152.49 (Ar–C), 144.24 (Ar–C), 138.46 (Ar–C), 134.63, 132.94 (Ar–C), 131.73 (Ar–CH), 131.16 (CF3), 128.41 (Ar–CH), 127.66 (Ar–CH), 122.34 (Ar–CH), 121.68 (Ar–CH), 121.38 (Ar–CH); 19F NMR (282 MHz, DMSO, δ = ppm): −56.73 (s); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C20H14F3NO5S; 438.0623; found, 438.0616.

Synthesis of N-(4-Hydroxyphenyl)-9-methyl-9H-fluorene-9-carboxamide (6)

Compound 6 was synthesized by the reaction of 4-aminophenol (1) (4.12 mmol; 0.450 g) with 9-methyl-9H-fluorene-9-carbonyl chloride (5) (2.50 mmol; 0.605 g) in acetone (50 mL) and base anhydrous potassium carbonate (3.62 mmol; 0.500 g). The reaction proceeded as per conditions followed for the synthesis of 3a and 3b.

Synthesis of Arylamide Sulphonates 7a–F

The desired sulphonate derivatives 7a–f were produced by the reaction of N-(4-hydroxyphenyl)-9-methyl-9H-fluorene-9-carboxamide (6) (0.485 mmol; 0.152 g) with alkyl or aryl sulfonyl chloride derivatives (0.980 mmol) in dry tetrahydrofuran (THF) (12 mL). Triethylamine (2.10 mmol) was added dropwise to the stirring mixture at 0 °C. The reaction proceeded as per the protocol used for the synthesis of compounds 4a–l and 4m–t.

4-(9-Methyl-9H-fluorene-9-carboxamido)phenyl 2-Fluorobenzenesulfonate (7a)

Yield: 77%; dark-brown solid; mp: 146–147 °C; FT-IR (υ̅, cm1, neat): 3053 (N–H, stretching), 2833 (C–H, stretching), 1691 (C=O, stretching), 1479, 1460, 1413, 1221, 1092; 1H NMR (300 MHz, CDCl3, δ = ppm): 7.75–7.68 (m, 2H, Ar–H), 7.64–7.48 (m, 4H, Ar–H), 7.37 (td, J = 7.5, 1.3 Hz, 2H, Ar–H), 7.29 (td, J = 7.4, 1.3 Hz, 2H, Ar–H), 7.19–7.06 (m, 4H, Ar–H), 6.83 (d, J = 9.0 Hz, 2H, Ar–H), 6.63 (s, 1H, CONH), 1.73 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3, δ = ppm): 171.63 (C=O), 161.13, 157.69, 147.21, 145.23, 140.33, 136.87 (d, J = 8.1 Hz), 131.58, 128.85, 128.46, 124.62 (d, J = 4.0 Hz), 124.18, 123.41 (d, J = 13.7 Hz), 122.47, 120.84 (d, J = 5.3 Hz), 117.46 (d, J = 20.7 Hz), 59.04, 23.07 (CH3); HRMS (ESI-TOF) m/z: calcd [M + H]+ for C27H20FNO4S, 474.1175; found; 474.1178.

4-(9-Methyl-9H-fluorene-9-carboxamido)phenyl Trifluoromethanesulfonate (7b)

Yield: 68%; off-white solid; mp: 140–141 °C; FT-IR (υ̅, cm1, neat): 3290 (N–H, stretching), 2965, 2928 (C–H, stretching), 1667 (C=O, stretching), 1508, 1419, 1207, 1133, 1013; 1H NMR (500 MHz, CDCl3, δ = ppm): 7.82 (d, J = 7.6 Hz, 2H, Ar–H), 7.63 (d, J = 7.5 Hz, 2H, Ar–H), 7.48 (td, J = 7.5, 0.9 Hz, 2H, Ar–H), 7.40 (td, J = 7.5, 0.9 Hz, 2H, Ar–H), 7.34 (d, J = 9.1 Hz, 2H, Ar–H), 7.09 (d, J = 9.1 Hz, 2H, Ar–H), 6.76 (s, 1H, CONH), 2.17 (s, 3H, CH3); 13C NMR (126 MHz, CDCl3, δ = ppm): 171.84 (C=O), 147.19, 145.35, 140.40, 137.79, 128.96, 128.57, 124.24, 121.89, 121.18, 120.89, 59.14, 23.04 (CH3); HRMS (EI, 70 eV), [C22H16O4N1F3S1], 447.07466; found 447.07454.

4-(9-Methyl-9H-fluorene-9-carboxamido)phenyl 4-Fluorobenzenesulfonate (7c)

Yield: 79%; colorless solid; mp: 200–201 °C; FT-IR (υ̅, cm1, neat): 3303 (N–H, stretching), 3060 (C–H, stretching), 1669 (C=O, stretching), 1491, 1405, 1347, 1240, 1193, 1151, 1092; 1H NMR (500 MHz, CDCl3, δ = ppm): 7.81 (d, J = 7.6 Hz, 2H, Ar–H), 7.76 (dd, J = 8.9, 5.0 Hz, 2H, Ar–H), 7.62 (d, J = 7.5 Hz, 2H, Ar–H), 7.47 (td, J = 7.5, 0.9 Hz, 2H, Ar–H), 7.39 (td, J = 7.5, 0.9 Hz, 2H, Ar–H), 7.22–7.11 (m, 4H, Ar–H), 6.83–6.75 (m, 2H, Ar–H), 6.72 (s, 1H, CONH), 1.83 (s, 3H, CH3); 13C NMR (126 MHz, CDCl3, δ = ppm): 171.65 (C=O), 167.12, 165.07, 147.25, 145.38, 140.36, 136.79, 131.51 (d, J = 9.5 Hz), 131.21 (d, J = 2.9 Hz), 128.87, 128.49, 124.21, 122.79, 120.83, 116.66 (d, J = 22.9 Hz), 59.08, 23.09 (CH3); HRMS (EI, 70 eV), [C27H20O4N1F1S1], 473.10916; found 473.10858.

4-(9-Methyl-9H-fluorene-9-carboxamido)phenyl Methanesulfonate (7d)

Yield: 55%; off-white solid; mp: 124–125 °C; FT-IR (υ̅, cm1, neat): 3369 (N–H, stretching), 3008, 2926 (C–H, stretching), 1679 (C=O, stretching), 1603, 1504, 1360, 1149; 1H NMR (300 MHz, CDCl3, δ = ppm): 8.28 (ddd, J = 7.6, 1.2, 0.7 Hz, 2H, Ar–H), 8.08 (ddd, J = 7.4, 1.2, 0.7 Hz, 2H, Ar–H), 7.92 (td, J = 7.5, 1.3 Hz, 2H, Ar–H), 7.84 (td, J = 7.4, 1.3 Hz, 2H, Ar–H), 7.73 (d, J = 9.1 Hz, 2H, Ar–H), 7.53 (d, J = 9.1 Hz, 2H, Ar–H), 7.26 (s, 1H, CONH), 3.47 (s, 3H, CH3), 2.29 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3, δ = ppm): 171.58 (C=O), 147.11, 145.03, 140.22, 136.72, 128.74, 128.35, 124.03, 122.33, 121.14, 120.73, 58.89, 37.03 (CH3), 23.01 (CH3); HRMS (EI, 70 eV), [C22H19O4N1S1], 393.10293; found 393.10295.

4-(9-Methyl-9H-fluorene-9-carboxamido)phenyl 4-Methylbenzenesulfonate (7e)

Yield: 81%; yellowish solid; mp: 145–147 °C; FT-IR (υ̅, cm1, neat): 3311 (N–H, stretching), 2973, 2928 (C–H, stretching), 1671 (C=O, stretching), 1502, 1405, 1345, 1195, 1153, 1092; 1H NMR (300 MHz, CDCl3, δ = ppm): 7.80 (ddd, J = 7.6, 1.2, 0.7 Hz, 2H, Ar–H), 7.65–7.58 (m, 4H, Ar–H), 7.46 (td, J = 7.5, 1.3 Hz, 2H, Ar–H), 7.38 (td, J = 7.5, 1.3 Hz, 2H, Ar–H), 7.26 (d, J = 0.6 Hz, 1H. Ar–H), 7.23 (d, J = 0.7 Hz, 1H, Ar–H), 7.19–7.11 (m, 2H, Ar–H), 6.84–6.74 (m, 2H, Ar–H), 6.70 (s, 1H, CONH), 2.41 (s, 3H, Ar–CH3), 1.82 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3, δ = ppm): 171.60 (C=O), 147.29, 145.62, 145.47, 140.36, 136.56, 132.25, 129.86, 128.85, 128.65, 128.47, 124.22, 122.88, 120.82, 120.72, 59.08, 23.11 (CH3), 21.81(CH3); HRMS (EI, 70 eV), [C28H23O4N1S1], 469.13423; found 469.13413.

4-(9-Methyl-9H-fluorene-9-carboxamido)phenyl Benzenesulfonate (7f)

Yield: 85%; off-white solid; mp: 193–196 °C; FT-IR (υ̅, cm1, neat): 3301 (N–H, stretching), 3062 (C–H, stretching), 1669 (C=O, stretching), 1500, 1446, 1405, 1347, 1195, 1153, 1090; 1H NMR (250 MHz, CDCl3, δ = ppm): 7.86–7.70 (m, 4H, Ar–H), 7.67–7.58 (m, 3H, Ar–H), 7.47 (ddd, J = 8.9, 4.9, 1.2 Hz, 4H, Ar–H), 7.38 (td, J = 7.4, 1.3 Hz, 2H, Ar–H), 7.22–7.12 (m, 2H, Ar–H), 6.84–6.75 (m, 2H, Ar–H), 6.72 (s, 1H, CONH), 1.83 (s, 3H, CH3); 13C NMR (63 MHz, CDCl3, δ = ppm): 171.60 (C=O), 147.24, 145.50, 140.34, 136.64, 135.24, 134.31, 129.22, 128.84, 128.59, 128.46, 124.19, 122.81, 120.81, 120.74, 59.06, 23.09 (CH3); HRMS (EI, 70 eV), [C27H21O4N1S1], 455.11858; , found 455.11860

Biological Evaluation

ENPP1 and ENPP3 Transfection

Transfection was done in COS-7 cells using plasmids expressing human ENPP1 (GenBank accession no. NM_006208) or ENPP3 (GenBank accession no. NM_005021) and “Lipofectamine” as transfection reagent.[38,39] In a 10 cm culture dish plate, COS-7 cells (70–80% confluent cells) were incubated with DMEM/F-12 (lacking FBS) including plasmid DNA (6 μg) and 24 μL of Lipofectamine reagent and incubated for 5 h at 37 °C; then an equal volume of DMEM/F-12 supplemented with 20% FBS was added to stop the process of transfection. The transfected cells were isolated from the media after 48–72 h.[40]

Preparation of Protein Aliquots

The transfected cells were washed with cold Tris-saline buffer (4 °C) and collected in the presence of a harvesting buffer that was composed of 95 mM NaCl and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 45 mM Trisbase, pH 7.5. The scrapped cells were subjected to washing through centrifugation twice at 300g for 5 min at 4 °C.[30] The cells were sonicated after re-suspension in the harvesting buffer with aprotinin (10 μg/mL) and centrifuged at 850g at 4 °C for 5 min. The supernatant was collected, and aliquots were preserved at −80 °C after adding glycerol (7.5%). For estimation of protein, Bradford microplate assay was applied using bovine serum albumin as ref (41).

Enzyme Inhibition Assay

The effect of synthesized molecules on the activity of isozymes ENPP1 and ENPP3 was evaluated by using the previously reported method with minor changes.[22,38] Initial screening of the compounds was performed at 100 μM concentration per well in triplicate in the buffer: 5 mM MgCl2, 0.1 mM ZnCl2, and 50 mM Tris–HCl, adjusted to pH 9.5–9.6. Briefly, 30 ng ENPP1 and 32 ng ENPP3 proteins per well were added followed by the addition of 100 μM synthesized compounds dissolved in 10% dimethyl sulfoxide (DMSO). The reaction was initiated by adding a substrate (thymidine 5′-monophosphate para-nitrophenyl ester) 300 μM per well and allowed to incubate for 35 min at 37 °C. The reading was taken with the help of a microplate reader (BioTek FLx800, Instruments, Inc., USA) at a wavelength of 405 nm. The compounds presenting more than 50% inhibition of either isozyme were subjected to serial dilutions, and the IC50 values were calculated through the non-linear regression analysis curve fitting program PRISM 5.0 (GraphPad, San Diego, CA, USA).

Enzyme Kinetic Study

The enzyme kinetic study was performed by using a serial concentration of substrate 0, 1.25, 2.5, 5.0, 7.5, and 10 mM. The compound concentrations for 4j were 0.0, 0.5, 1.0, and 1.5 μM, and for inhibitor 4e, the used concentrations were 0.0, 0.3, 0.6, and 1.2 μM. The assay plate was prepared by adding the concentrations of enzymes (ENPP1 = 30 ng, ENPP3 = 32 ng) per well to the assay buffer. After adding the serial concentrations of compounds and substrates, the assay mixture was incubated at 37 °C, and the reading was noted for up to 35 min with 5 min intervals by using a microplate reader (BioTek FLx800, Instruments, Inc., USA). The data were analyzed by PRISM 5.0 (Graph Pad, San Diego, CA, USA), and Lineweaver–Burk graphs were plotted.

Cytotoxicity Assay (MTT Assay)

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was applied to check the cytotoxic effect of the compounds with minor modifications in the already reported method.[42] The cells were seeded in 96-well plates at a quantity of about 4×104 cells per well and incubated for 24 h in a CO2 incubator. After incubation for 24 h, serum-free media were added to replace the existing media of cell culture, and the cells were treated with test compounds along with positive control at 100 μM concentrations per well. Afterward, the incubation was given for the next 24 h; MTT reagent (0.5 mg/mL) was added and further incubated at 37 °C for 4 h. Dimethylsulfoxide was added to solubilize the violet crystals and a stopping reagent composed of isopropanol (50%) and sodium dodecyl sulfate (10%) was added. The absorbance was measured at 570 nm with the help of a microplate reader (FLUOstar Omega Microplate Reader, Germany), and results were compiled.

Molecular Docking Analysis

For the in silico observations, the available ligand-bounded human crystallographic structures of ENPP1 (PDB ID = 6wew) and ENPP3 (PDB ID = 6C02) were downloaded from PDB.[37,43] The minimization of energy and molecule database of the docked molecules was generated with the help of the Molecular Operating Environment.[44] The protein preparation such as energy minimization, selection of pocket site, removal of water molecules, protonation, and docking studies was performed by using the default system of LeadIT (BioSolveIT GmbH, Germany).[45] Among the selected number of poses for each compound, poses that possessed low binding energy and considerable affinity with the amino acids residues were selected for further visualization by using Discovery Studio Visualizer DS.[46]