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. 2020 Oct 29;11(12):2397–2405. doi: 10.1021/acsmedchemlett.0c00343

Synthesis, Characterization, and In Silico Studies of Novel Spirooxindole Derivatives as Ecto-5′-Nucleotidase Inhibitors

Abida Ashraf †,‡, Zahid Shafiq †, Muhammad Siraj Khan Jadoon §, Muhammad Nawaz Tahir ∥, Julie Pelletier ⊥, Jean Sevigny ⊥,#, Muhammad Yaqub †,*, Jamshed Iqbal §,*
PMCID: PMC7734640  PMID: 33335662

Abstract

graphic file with name ml0c00343_0012.jpg

Ecto-5′-nucleotidase (ecto-5′-NT, CD73) inhibitors are promising drug candidates for cancer therapy. Traditional efforts used to inhibit the ecto-5′-nucleotidase have involved antibody therapy or development of small molecule inhibitors that can mimic the acidic and ionizable structure of adenosine 5′-monophosphate (AMP). Herein, we report an efficient, environment friendly route for the synthesis of non-nucleotide based small molecules, i.e., substituted spirooxindole derivatives 9a–9l and investigated their inhibitory potential on human and rat recombinant ecto-5′-nucleotidase isozymes. These attempts have resulted in the identification of compound 9f (IC50 = 0.15 ± 0.02 μM) inhibitor on h-ecto-5′-NT which showed 280-fold higher inhibition and compound 9h (IC50 ± 0.19 ± 0.03 μM) on r-ecto-5′-NT with 406-fold enhanced inhibition than reference standard sulfamic acid. Moreover, in silico studies were carried out to assess binding interactions of potent compounds within enzyme active sites and demonstrated excellent correlation with the experimental findings.

Keywords: Pryazolo[3,4-b] pyridines; 1-methylisatin; enzyme inhibition; CD73; X-ray crystallography

Ecto-5′-nucleotidase (ecto-5′-NT, EC 3.1.3.5, CD73) is a glycosylphosphatidylinositol (GPI)-linked surface enzyme which is vital for normal cell functioning of the body.1 The ecto-5′-NT can be cleaved off by hydrolysis from its GPI linker and found in serum as a soluble active form.2 The primary structures of human placenta ecto-5′-NT, rat liver, and electric ray fish have shown that the mature enzyme consists of 548 amino acids with a molecular weight of about 61 kDa.3−5 Zn2+ metal ion is essential for its catalytic activity; therefore, it is classified as a metalloenzyme.6 The main enzymatic action of ecto-5′-NT is to hydrolyze 5′-nucleoside monophosphate (5′-AMP, i.e., only the d- but not the l-enantiomer is a substrate) to adenosine stereoselectively; however, it shows no activity toward nucleoside 2′- and 3′-monophosphates.1 The overexpression of ecto-5′-nucleotidase has been observed in different cancers such as leukemia, glioblastoma, bladder, ovarian, thyroid, breast, colon, prostate, esophageal, gastric, and melanoma,7 which increases the extracellular level of adenosine in the tumor microenvironment that inhibits T cells and promotes angiogenesis.8,9 It has been reported that ecto-5′-NT leads to drug resistance and tumor promoting functions.10,11 Therefore, either genetic ablation of enzyme or the use of antibodies against ecto-5′-NT have been revealed to attenuate the cancer growth and its metastasis. In agreement of these observations, there is a need of some potential and specific inhibitors of ecto-5′-NT to fight against cancer progression.12

The literature discloses that there are few small molecule inhibitors reported in the context of ecto-5′-NT which possess potential metabolic liabilities and/or moderate potencies.13−15 Some of the known ecto-5′-NT inhibitors nucleotide derivatives such as AB680,16 adenosine 5′-(α,β-methylene)diphosphate (AOPCP),17 adenosine diphosphate (ADP);18,19 non-nucleotide derivatives, PSB-096312 (anthraquinone derivative), benzotriazole,13 and pyrazolopyridine20 derivatives are shown in Figure 1.

Figure 1.

Figure 1

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Selected inhibitors of ecto-5′-NT and the target inhibitor.

Adenosine triphosphate (ATP), ADP, and their close analogues, adenosine 5′-(α,β-methylene)diphosphate (AOPCP), are competitive inhibitors of ecto-5′-NT in the low micromolar range, but they are not ideal drug candidates due to very limited oral absorption and enzymatic degradation by nucleotide pyrophosphatase (NPPs), nucleoside triphosphate diphosphohydrolases (NTPDases), and alkaline phosphatases.21−23 Recently, the first clinical compound AB680 was announced, which is a phosphonate-based competitive inhibitor of ecto-5′-NT. Different types of sulfonamide and anthraquinone derivatives were also found to display competitive mechanism of inhibition, whereas polyoxometalates (POMs) and polyphenols are noncompetitive inhibitors.17,24 Very recently, benzotriazole based inhibitors have been reported with potency in the nanomolar range.13

Keeping in view the importance of ecto-5′-NT enzyme in metastasis and cancer progressions along with previously reported inhibitor structures, we have decided to synthesize a new class of potent, selective, and non-nucleotide based inhibitors. Spirooxindoles and pyrazolo[3,4-b] pyridines are attractive class of heterocyclic compounds due to their diverse biological applications such as antimicrobial, antimalarial, antimitotic, anti-inflammatory,25−28 antitumor,29,30 and hypotensive.31 Pyrazolo[3,4-b] pyridines have also been evaluated as selective and effective inhibitors of glycogen synthase kinase-3 (GSK-3),32 cyclin-dependent kinase 1 (CDK-1),33 protein kinase,34 and HIV reverse transcriptase.35

The biological importance of such two scaffolds spirooxindoles and pyrazolo[3,4-b] pyridines have promoted our research group to report novel spirooxindole-pyrazolo[3,4-b]pyridine derivatives 9a–9l and screened them against ecto-5′-NT for the first time. The putative binding interactions of these derivatives within the active site of enzymes have been determined by in silico studies. The results of binding interactions showed good connection with experimental findings and provide an exceptional basis for further drug design approaches envisioned at the development of potent ecto-5′-NT inhibitors.

Results and Discussion

Design and Chemistry

To explore the potential of pyrazole based spiroindolines, an effective regioselective green synthetic approach has been established. The one-pot three-component fusion was carried out by taking equimolar amounts of isatins 6, malononitrile 7, and (un)substituted 3-aminopyrazoles 8 at 100 °C on a sand bath in a reaction flask (Scheme 1). The reaction conditions were optimized by using solvents of varying polarity like water, methanol, THF, DMSO, ethanol, and DCM, and optimal conditions were established by a fusion reaction (Table S1). To extend the substrate scope of the reaction, some substituted isatins and (un)substituted 3-aminopyrazoles have been used for regioselective synthesis. The targeted spiroindoline-pyrazolo[3,4-b]pyridines 9a–9l were obtained in excellent yields (87–93%). The substrate scope reaction results are summarized in Figure 2.

Scheme 1. Synthesis of Spiro[indoline-3,4′-pyrazolo[3,4-b]pyridine]-5′-carbonitrile 9a–9l.

Scheme 1

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Reaction conditions: Isatin/1-methylisatin 6 (2 mmol), malononitrile 7 (2 mmol), 3-amino-5-methylpyrazole/3-aminopyrazole 8 (2 mmol), fusion at 100 °C.

Figure 2.

Figure 2

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Structures and percentage yield of different derivatives of spiro[indoline-3,4′-pyrazolo[3,4-b]pyridine]-5′-carbonitrile 9a–9l.

The structures of novel synthesized spiro[indoline-3,4′-pyrazolo[3,4-b]pyridine]-5′-carbonitrile 9a–9l were established on the basis of their spectroscopic data, i.e., IR, NMR, spectroscopy, mass spectrometry, and X-ray crystallography.

The IR spectra of spiro[indoline-3,4′-pyrazolo[3,4-b]pyridine]-5′-carbonitrile 9a–9l showed NH stretching of pyrazole, pyridine, and amino functions in the 3481–3282 cm–1 region. The cyanide C≡N and lactam C=O stretchings were observed in the range from 2236 to 2169 cm–1 and 1716–1679 cm–1, respectively. The 1HNMR spectra showed resonance at δ 3.14–3.29 ppm for N–CH3 and three separate singlets at δ 5.68–5.90 ppm for NH2, δ 9.22–9.50 ppm for pyridine N7′-H and δ 11.86–12.29 ppm for N2′-H protons, respectively, in all the cases. 13CNMR data of compounds 9a–9l also supported the IR and 1HNMR results. The ESI (MS/MS) spectra of these compounds showed the loss of carbon monoxide (CO) as the main fragmentation pathway. The structure of these compounds showed the formation of fragments by cleavage of N–C=O, N–CH3, and C–NH bonds which validate their molecular weights and the formation of respective compounds 9a–9l. The proposed fragmentation pattern of 9b is illustrated in Figure S1. X-ray structure of representative compound 9d (Figure 3) was obtained to validate the proposed structures of synthesized 1-methyl-spiro[indoline-3,4′-pyrazolo[3,4-b]pyridine]-5′-carbonitrile 9a–9l.

Figure 3.

Figure 3

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Molecular structure (ORTEP diagram) of 9d. Thermal ellipsoids are drawn at the 50% probability level with H-atoms as small circles of arbitrary radii.

In 6′-amino-5-chloro-1-methyl-2-oxo-1,2,2′,7′-tetrahydrospiro[indole-3,4′-pyrazolo[3,4-b] pyridine]-5′-carbonitrile (Figure 3, Table S2), the 5-chloro-1-methylindolin-2-one moiety A (C1-C9/N1/O1/CL1), 1H-pyrazole ring B (C10-C12/N2/N3), and 1,4,5,6-tetrahydropyridin-2-amine moiety C (C7/C10/C12-C14/N4/N5) are planar with an RMS deviation of 0.0711, 0.0041, and 0.0864 Å, respectively. The dihedral angle between A/B, A/C, and B/C is 80.9 (5)°, 88.5 (3)°, and 8.12 (7)°, respectively. The C and N-atom of cyano moiety D (C15/N6) are at the distance of 0.3204 (2) Å and 0.5144 Å, respectively, from the mean square plane of 1,4,5,6-tetrahydropyridin-2-amine moiety.

The molecules are connected with each other in the form of dimer through N–H···N bonding to form the R22 (8) loop, where NH is from the 1,2,3,4-tetrahydropyridine ring (six membered ring) and N-atom is from the 1H-pyrazole ring (five membered) as shown in Figure 4 and given in Table S3.

Figure 4.

Figure 4

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Packing diagram of titled compound showing interlinkage of dimers with each other. The H-atoms not involved in H-bonding are not shown for clarity.

The dimers are interlinked through N–H···O bonding to form unsymmetrical bifurcated R32 (9) loops, where NH is from the amino group of the 1,4,5,6-tetrahydropyridin-2-amine moiety and the O-atom is from the 1-methyl-1H-pyrrol-2(3H)-one moiety. The R22 (14) loop are also generated as shown in Figure 4. In this, molecules form an infinite 2D network with base vectors [0 1 0] and [0 0 1] in the plane (1 0 0).

In Vitro Biological Activity of Ecto-5′-NT

The synthesized compounds 9a–9l were tested against purified recombinant isozymes of human and rat ecto-5′-NT, i.e., h-ecto-5′-NT and r-ecto-5′-NT. The reaction was monitored by measuring the release of inorganic phosphate via enzymatic reaction using AMP as a substrate. The synthesized compounds 9a–9l showed excellent inhibitory potential exhibiting submicromolar IC50 values as compared to standard sulfamic acid for both h-ecto-5′-NT and r-ecto-5′-NT. The results are illustrated in Table 1 which showed inhibition of h-ecto-5′-NT with IC50 ± SEM in a range of 0.15 ± 0.02 to 0.69 ± 0.08 μM and inhibition of r-ecto-5′-NT with IC50 ± SEM in range of 0.19 ± 0.03 to 7.99 ± 0.31 μM. Whereas, the unsubstituted compounds (unmethylated at the N-terminal of isatin) 9a and 9g were almost inactive on both of the isozymes.

Table 1. Activity of Spirooxindole Derivatives 9a–9l Expressed as IC50 ± SEM (μM) and % Inhibition against h-ecto-5′-NT and r-ecto-5′-NT.

  h-ecto-5′-NT r-ecto-5′-NT
compound code IC50 ± SEM (μM)a/% inhibition/Ki (μM)b
9a 27.57% 41.70%
9b 0.69 ± 0.08a 41%
9c 0.46 ± 0.009a 7.99 ± 0.31a
9d 39.72% 0.68 ± 0.05a
9e 38.01%a 2.12 ± 0.13a
9f 0.15 ± 0.02a 0.52 ± 0.02a
9g 33.02% 36.50%
9h 36.60% 0.19 ± 0.03a
9i 0.26 ± 0.01a 5.01 ± 0.83a
9j 33.02% 1.56 ± 0.02a
9k 34.27% 45.11%
9l 0.18 ± 0.08a 3.56 ± 0.30a
sulfamic acid(14) 42.1 ± 7.80a 77.3 ± 7.0a
AOPCP(17) 0.08b 0.19b
PSB-0963(12)   0.15b
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a

IC50 is the concentration at which 50% of the enzyme activity was inhibited.

b

Ki value of standards in μM.

Biological SAR and In Silico Study

After the in vitro studies, structure activity relationships (SAR) of compounds 9a–9l for ecto-5′-NT enzymes (h-ecto-5′-NT and r-ecto-5′-NT) have been described by the presence of different functional groups on isatin and pyrazole rings. In order to explain the obtained in vitro biological results, molecular docking studies were also carried out for compounds 9a–9l, which showed putative binding interactions of potent compounds with human ecto-5′-nucleotidase and rat ecto-5′-nucleotidase, respectively, to find a better rationale.

The crystal structure of human ecto-5′-nucleotidase enzyme, having a resolution of 1.55 Å, was acquired from the Protein Data Bank (PDB ID 4H2G).36 The enzyme had a single unique chain of amino acids, predocked with its unique ligand adenosine (ADN) in the residual sequence of the protein. The ligand-binding site of the corresponding enzyme constituted both hydrophobic and hydrophilic amino acids. A group of total eight surface residues (Asn390, Gly393, Arg395, Gly418, Gly419, Thr420, Asn499, and Asp524) makes the hydrophilic region, while the hydrophobic region comprises of Leu389, Leu415, Pro416, Phe417, Phe421, Pro498, and Phe500 residues. Adenosine (ADN) was bound at the active site of the receptor through hydrogen bonds of four different residues via Asn390, Arg395, Asn499, and Asp506 displaying hydrophilic interactions. The crystal structure for rat ecto-5′-nucleotidase is not available in the protein databank, so homology modeled structure was utilized to perform the molecular docking.37 For the docking studies against h-ecto-5′-NT, the structures of compounds 9b, 9c, 9f, 9i, and 9l were prepared in ChemDraw, and their energies were minimized using Chem3D. The receptor was kept rigid for docking protocol while the ligand was flexible.

The comparison of compound 9a with 9b for h-ecto-5′-NT activity showed that inhibitory potential has been increased when the methyl group is present at the N-terminal of isatin. The compound 9f was found to be a more selective and potent inhibitor of h-ecto-5′-NT from the main stream of compounds 9a–9l. This derivative unveiled an inhibitory value of IC50 ± SEM = 0.15 ± 0.02 μM, which showed a 280-fold higher inhibition as compared to the reference standard, i.e., sulfamic acid (IC50 ± SEM = 42.1 ± 7.8 μM). When the structure of 9f was compared with other compounds having the same basic nucleus, it was noticed that the higher inhibitory potential might be due to the presence of the trifluoromethoxy group at the 5-position of 1-methylisatin, which make the nucleus more stable.

The docking study for 9f (Figure 5) suggested that the ligand exhibited hydrogen bonding between the trifluoromethoxy group (fluorine and oxygen of the indolinone ring) and Asn390 and Gly393 residues, whereas Gly392 and Arg395 formed hydrogen bonding with the carbonitrile nitrogen of the pyrazolo-pyridine ring of the ligand. The Gly392 residue also formed a π–π linkage with the indolinone ring of the ligand, and the Asp506 residue of the receptor had a π–anionic interaction with the pyrazolo-pyridine ring of the ligand. The ligand also exhibited van der Waals interactions with the residues of the active pocket. Therefore, the presence of several binding interactions such as hydrogen bonding, π–anionic, and van der Waals with various vital amino acids in the active site of the enzyme make the compound 9f more potent for h-ecto-5′-NT inhibition.

Figure 5.

Figure 5

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Ligand 9f inside the 4H2G receptor’s active pocket showing interaction with important residues.

The detailed SAR study of compounds 9a–9l for h-ecto-5′-NT showed the following decreasing order of inhibition.

graphic file with name ml0c00343_m001.jpg

The comparison of compounds 9b, 9c, and 9f showed distinguished behavior when the trifluoromethoxy group in 9f was replaced with fluoro in 9c and hydrogen in 9b. The compound 9f showed 4.6-fold refinement in inhibition for 9b (IC50 ± SEM = 0.69 ± 0.08 μM) and 3.1-fold refinement for 9c (IC50 ± SEM = 0.46 ± 0.009 μM). The compound 9c has 17.3-fold selectivity in h-ecto-5′-NT vs r-ecto-5′-NT.20,37

In the case of 9b, the best pose (Figure S2) of the ligand exhibited hydrogen bonding between the amino group of the pyrazolo-pyridine ring and the Asp506 residue, while Thr446 formed hydrogen bonding with the heteronitrogen of the pyrazolo-pyridine ring. Aminoacides Gly392 and Gly447 formed hydrogen bonds with the carbonyl oxygen of the indolinone ring of the ligand, and Arg395 residue of the receptor had π–π interaction with the indolinone ring of the ligand. The ligand also exhibited van der Waals interactions with the residues of the active pocket. The most suitable pose for 9c (Figure S3) showed the ligand exhibited hydrogen bonding between the carbonyl oxygen of the indolinone ring and the Asn390 residue, whereas Thr446 and Gly392 formed hydrogen bonding with the pyrazolo-pyridine ring of the ligand. Phe417 and Phe500 residues formed a π–alkyl linkage with the indolinone ring of the ligand, and the Arg395 residue of the receptor had a π–cationic interaction with the indolinone ring of the ligand. The ligand also exhibited van der Waals interactions with the residues of the active pocket. So a docking study of compounds 9b and 9c showed that the binding interactions of the ligand with amino acids have been effected when the trifluoromethoxy group in 9f was replaced with fluoro in 9c and hydrogen in 9b.

The second most potent inhibitor was 9l (IC50 ± SEM = 0.18 ± 0.08 μM) with a trifluoromethoxy group at the 5-position (showing interaction with vital amino acid in docking studies) of 1-methylisatin and methyl group at the pyrazole ring. The replacement of the trifluoromethoxy group in 9l with the fluoro group in 9i resulted in a reduced inhibitory potential of 9i (IC50 ± SEM = 0.26 ± 0.01 μM).

Compound 9l, the most suitable pose (Figure 6) of the ligand exhibited hydrogen bonding between the trifluoromethoxy fluorines of the indolinone ring and Asn390 and Gly393 residues while Phe500 formed hydrogen bonding with the carbonitrile nitrogen of the pyrazolo-pyridine ring of the ligand. Phe417 and Phe500 residues formed a π–π linkage with the indolinone ring, and the Asp47 residue of the receptor had π–anionic interaction with the pyrazolo-pyridine ring. The ligand also exhibited van der Waals interactions with the residues of the active pocket.

Figure 6.

Figure 6

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Ligand 9l inside the 4H2G receptor’s active pocket showing interaction with important residues.

Although the incorporation of fluorine resulted in a good activity of the compound 9i, it was a much lesser extent than the trifluoromethoxy group at the 5-position of compound 9l. The best docking pose for 9i (Figure S4) showed that 5-substituted fluorine of the methylindolin-2-one was involved in the hydrogen bonding with the Asn390 and Gly392 residues, while Arg395, Thr446, and Gly447 residues formed hydrogen bonds with the nitrogen atoms of the pyrazolo-pyridine ring. Phe500 had a π–π interaction, and Asp506 showed a π–cationic interaction with the indolinone ring. The ligand also exhibited van der Waals interactions with the residues of the active pocket.

The SAR and docking studies were also carried out for r-ecto-5′-NT. The SAR of compounds 9a–9l revealed that the compound 9h (IC50 ± SEM = 0.19 ± 0.03 μM; Figure 7) was selective and the most potent inhibitor, and it showed a 406-fold enhanced inhibition in comparison to the reference standard inhibitor sulfamic acid (IC50 ± SEM = 77.3 ± 7.0 μM). The structures of the most potent compounds 9d, 9f, and 9h were prepared in ChemDraw for docking studies, and their energies were minimized using Chem3D. The receptor was kept rigid for the docking protocol, while the ligand was flexible. The structural analysis of 9h with compound 9g suggested that the inhibitory potential has been increased due to methylation of the N-terminal of isatin.

Figure 7.

Figure 7

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Ligand 9h inside r-ecto-5′-NT exhibiting interactions with the vital amino acids.

In docking studies for compound 9h, the amino group of the pyrazolo-pyridine ring formed a hydrogen bond with the Gln446 residue, and the amino acid Arg443 formed hydrogen bonding with the carbonitrile nitrogen of the pyrazolo-pyridine ring of the ligand. Lys181 had a π–alkyl interaction with the indolinone ring. The ligand also exhibited van der Waals interactions with the Glu182, Met227, and Arg443 residues of the active pocket.

The decreasing order of inhibitory potential for r-ecto-5′-NT was

graphic file with name ml0c00343_m002.jpg

The comparative study of compounds 9h, 9j, 9l, and 9i showed that the replacement of the hydrogen atom with chloro, trifluoromethoxy, and fluoro groups at the 5-position of 1-methylisatin resulted in reduced inhibitory potential for r-ecto-5′-NT. The second most potent inhibitor was 9f (IC50 ± SEM = 052 ± 0.02 μM) with the trifluoromethoxy group at the 5-position of 1-methylisatin and no methyl substitution at the pyrzole moiety. With the docking study of compound 9f, the best pose (Figure 8) suggests that the trifluoromethoxy fluorine of the indolinone ring exhibited hydrogen bonding with the His439, Asp508, and Gly509 residues. The nitrogen heteroatom of the pyrazolo-pyridine ring formed hydrogen bonds with the Gln446 residue, while the amino substituent at the pyrazolo-pyridine ring formed hydrogen bonding with the Ser180 residue. Arg443 had a π–cationic interaction with the pyrazolo-pyridine ring of the ligand. The ligand also exhibited van der Waals interactions with the Lys181, Glu182, Phe185, Arg443, and Gly509 residues of the active pocket.

Figure 8.

Figure 8

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Ligand 9f inside r-ecto-5′-NT presenting interaction with the important residues.

In SAR, the comparison of compound 9f with 9d, 9e, and 9c showed a reduced inhibitory potential for r-ecto-5́-NT due to replacement of the trifluoromethoxy group with chloro, nitro, and fluoro groups, respectively. These findings have been supported by the docking studies of the respective compound. In compound 9d, the best pose (Figure S5) showed the amino group of the pyrazolo-pyridine ring formed a hydrogen bond with the Gln446 residue and the amino acid Arg443 formed hydrogen bonding with the carbonitrile nitrogen of the pyrazolo-pyridine ring. His442 had a π–π interaction with the indolinone ring of the ligand, while Pro467 showed a π–alkyl interaction with the chlorine substituent of the indolinone ring. The ligand also exhibited van der Waals interactions with the His442 and Arg443 residues of the active pocket.

From the SAR analysis and docking studies of compounds 9a–9l, it can be concluded that the presence of the trifluoromethoxy and the N-methyl group on isatin increases the inhibitory potential for h-ecto-5′-NT (and so as the interaction of ligand with vital amino acids in the active pocket), while the derivatives possessing a methyl group on the pyrazole moiety and the N-terminal of isatin showed maximal r-ecto-5′-NT inhibition (interactions in the active pocket) as shown in Figure 9.

Figure 9.

Figure 9

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Summary of structure–activity relationships for ecto-5′-NT inhibitory activities of pyrazole based spiroindoline derivatives.

The comparison of some reported newly developed ecto-5′-NT inhibitors is shown in Table S4, and it has been observed that novel spiroindoline-pyrazolo[3,4-b]pyridine derivatives are more potent than benzenesulfonyl hydrazide based,38 pyrazolopyridine based,20 pyridinebenzanilide based,39 pyrrolopyridine based,40 acetylpyrazole based,37 and naphthalene sulfonic acid based14 scaffolds. When we mapped the structure of 1-methylspirooxindole-pyrazolo[3,4-b]pyridines with reported inhibitors, it showed the presence of two moieties, a 1-methylspirooxindole heterocycle and pyrazolo[3,4-b]pyridine ring, with amino and amide functionalities which increased hydrophilicity and had a strong impact on inhibitory activity. Moreover, currently used standard inhibitors of ecto-5′-NT are AOPCP17 (Ki human = 0.08 and Ki rat = 0.19 μM) and PSB-096312 (Ki rat = 0.15 μM). The ecto-5′-NT inhibitory values of newly synthesized compounds 9f (IC50 human = 0.15 μM) and 9h (IC50 rat = 0.19 μM) are equivalent to the above-mentioned standard potent inhibitors.

Mechanism of Enzyme Inhibition

In order to determine the mode of inhibition, the two most potent compounds (9f for h-ecto-5′-NT and 9h for r-ecto-5′-NT) were selected. The results obtained exhibited that compound 9f showed noncompetitive inhibition while compound 9h exhibited an uncompetitive mode of inhibition. In both cases, maximal velocity (Vmax) was found almost constant even after using different concentrations of both inhibitors; however, the other parameter Michaelis–Menten constant (Km) was found in increasing order as depicted in Figure 10.

Figure 10.

Figure 10

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Lineweaver–Burk plot of the most potent compounds: (A) compound 9f against h-ecto-5′-NT, (B) compound 9h against r-ecto-5′-NT. 9f showed an uncompetitive mode of inhibition, while 9h showed a noncompetitive mode of inhibition. S is the substrate concentration used (0, 50, 100, 200, 400 and 600 μM), and the compound concentration is shown as 0, 0.1, 0.3, and 0.6 μM with circle, square, and wedges, respectively.

Conclusion

In summary, a novel series of spirooxindole-pyrazolo[3,4-b]pyridine derivatives 9a–9l was synthesized by an efficient, environmentally friendly protocol with high yields and purity. The synthesized compounds were investigated for their inhibitory potential on human and rat recombinant ecto-5′-NT isozymes. The spirooxindole derived pyrazole based compounds represented an entirely new class of ecto-5′-NT enzyme inhibitors. The structure activity relationship showed a low micromolar range, and compound 9f was found to be most potent (IC50 ± SEM = 0.15 ± 0.02 μM) on h-ecto-5′-NT, and compound 9h (IC50 ± SEM = 0.19 ± 0.03 μM) was most active against r-ecto-5′-NT. Enzyme kinetic studies of 9f indicated a noncompetitive mode of inhibition, while 9h showed an uncompetitive inhibition. Further in silico studies showed a good binding pattern of the most potent compounds in the active pockets of the enzymes.

To the best of our knowledge, this new chemical series represents the non-nucleotide based spirooxindole derivatives for ecto-5′-NT inhibition with good potency in comparison with standard inhibitors (AOPCP and PSB0963) and newly developed pyrazolopyridine based scaffolds; the nonphosphonate nature of the compounds is of particular significance, as poor membrane permeability and the low oral bioavailability associated with some nucleotide phosphonates may obstruct their clinical development.

Acknowledgments

Dr. Waqar Rauf, Principal Scientist, NIBGE, Faisalabad, Pakistan, is acknowledged for performing the ESI-MS analysis. Prof. Dr. Shahid Hameed, Department of Chemistry, QA University, Islamabad, Pakistan, is acknowledged for the NMR analysis.

Glossary

Abbreviations

CD73

ecto-5′-nucleotidase

GPI

glycosylphosphatidylinositol

AMP

adenosine monophosphate

AOPCP

adenosine 5′-(α,β-methylene)diphosphate

ADP

adenosine diphosphate

ATP

adenosine triphosphate

NPPs

nucleotide pyrophosphatase

NTPDases

nucleoside triphosphate diphosphohydrolases

POMs

polyoxometalates

GSK-3

glycogen synthase kinase-3

CDK-1

cyclin-dependent kinase 1

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00343.

  • Optimization of reaction conditions; ESI (MS/MS) fragmentation pattern of compound 9b; experimental details of titled compounds and hydrogen-bond geometry (Å, deg) for 9d; ligands 9b, 9c, and 9i inside the 4H2G receptor’s active pocket showing interaction with important residues and ligand 9d inside rat ecto-5′-nucleotidase exhibiting interactions with the vital amino acids; adenosine redocked with RMSD 0.3970; comparison of inhibitors; general information and procedure; and 1HNMR, 13CNMR, and mass spectra of 9a–9l (PDF)

Author Contributions

A. Ashraf and Z. Shafiq conducted all experiments for the synthesis of spirooxindole derivatives and interpreted the data for characterization of the compounds. M. S. K. Jadoon, J. Pelletier, and J. Sevigny carried out the ecto-5′-nucleotidase inhibition studies and molecular docking. M. N. Tahir did the X-ray analysis of the crystals. M. Yaqub and J. Iqbal developed the concept and wrote the manuscript together with A. Ashraf and Z. Shafiq and discussed the results with all of the authors. All authors have given approval to the final version of the manuscript.

M. Yaqub is thankful to BZ University, Multan, Pakistan, for financial support. Z. Shafiq is thankful to the Higher Education Commission of Pakistan through Project No. 6975/NRPU/R&D for the financial support.

The authors declare no competing financial interest.

Supplementary Material

ml0c00343_si_001.pdf (1.9MB, pdf)

References

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