Quinazolinones as Competitive Inhibitors of Carbonic Anhydrase-II (Human and Bovine): Synthesis, in-vitro, in-silico, Selectivity, and Kinetics Studies

Abstract

Carbonic anhydrase-II (CA-II) is associated with glaucoma, malignant brain tumors, and renal, gastric, and pancreatic carcinomas and is mainly involved in the regulation of the bicarbonate concentration in the eyes. CA-II inhibitors can be used to reduce the intraocular pressure usually associated with glaucoma. In search of potent CA-II inhibitors, a series of quinazolinones derivatives (4a-p) were synthesized and characterized by IR and NMR spectroscopy. The inhibitory potential of all the compounds was evaluated against bovine carbonic anhydrase-II (bCA-II) and human carbonic anhydrase-II (hCA-II), and compounds displayed moderate to significant inhibition with IC50 values of 8.9–67.3 and 14.0–59.6 μM, respectively. A preliminary structure-activity relationship suggested that the presence of a nitro group on the phenyl ring at R position contributes significantly to the overall activity. Kinetics studies of the most active inhibitor, 4d, against both bCA-II and hCA-II were performed to investigate the mode of inhibition and to determine the inhibition constants (Ki). According to the kinetics results, 4d is a competitive inhibitor of bCA-II and hCA-II with Ki values of 13.0 ± 0.013 and 14.25 ± 0.017 μM, respectively. However, the selectivity index reflects that the compounds 4g and 4o are more selective for hCA-II. The binding mode of these compounds within the active sites of bCA-II and hCA-II was investigated by structure-based molecular docking. The docking results are in complete agreement with the experimental findings.

Synthesis, Bioactivity Assessment, and Molecular Docking of Non-sulfonamide Benzimidazole-Derived N-Acylhydrazone Scaffolds as Carbonic Anhydrase-II Inhibitors

“…In vitro carbonic anhydrase inhibition activity of the target compounds ( 9–29 ) was evaluated using reported methods. 21 All the target compounds showed inhibition activity against bCA-II except 11 , 13 , 14 , 16 , 19 , and 21 , which were found inactive ( Table 1 ). Furthermore, compounds 10 , 12 , 15 , and 9 showed higher inhibitions than the standard acetazolamide (IC 50 : 18.6 ± 0.43 μM) where compound 9 was the most active inhibitor (IC 50 : 13.3 ± 1.25 μM) followed by 15 (IC 50 : 4.5 ± 1.05 μM), 12 (IC 50 : 14.6 ± 0.62 μM), and 10 (IC 50 : 17.2 ± 1.24 μM).…”

“…The activity of control (in the absence of the inhibitor) was taken as 100%. The measurements were taken in triplicate at each used concentration …”

“…The measurements were taken in triplicate at each used concentration. 21 The % inhibition was calculated using the following formula % Inhibition 100 (OD test well/OD control) 100 = − × Molecular Docking. Two-dimensional (2D) structures of compounds 9−29 were drawn in ChemDraw and then altered to 3D structures, which were then minimized using Chimera.…”

“…To study the mechanism of inhibition for carbonic anhydrase-II and the binding modes of acylhydrazones ( 9–29 ), molecular docking studies were performed. Bovine carbonic anhydrase-II (PDB ID: 1V9E) in complex with zinc metal was downloaded from the Protein Data Bank and used for docking of compounds. , The active site of carbonic anhydrase-II where zinc metal is present lies at the bottom of the deep cleft. The ligand–receptor interactions and binding modes in the binding cavity of bovine carbonic anhydrase-II in 2D and 3D forms were examined carefully by visual evaluation employing GOLD.…”

Bis-pharmacophore of cinnamaldehyde-clubbed thiosemicarbazones as potent carbonic anhydrase-II inhibitors

“…Evaluation of carbonic anhydrase inhibition was done by spectrophotometric technique as narrated by Pocker and Meany with little alteration 69 , 70 as described previously 3 . The process was carried out in HEPES-Tris buffer (20 mM) having pH 7.4 at 25 °C.…”