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

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.

In this study, we report the synthesis and in-vitro bovine carbonic anhydrase-II (bCA-II) and human carbonic anhydrase-II (hCA-II) inhibitory activities of a series of quinazolinone analogs. Furthermore, the mode of inhibition was further explored by kinetic studies of the active analogs. Additionally, molecular docking studies were carried out to predict the main structural features responsible for the anti-CA-II activities of the compounds to predict the structure-activity relationships.

EXPERIMENTAL Chemistry
The chemicals used in this study were purchased from Sigma and Aldrich in extra purified form. Melting points for all compounds were recorded by Büchi 434 melting point apparatus. FT-IR spectra (KBr disks) were measured on a Bruker FT-IR IFS48 spectrophotometer. NMR spectra were recorded on Bruker Avance 400 spectrometers in DMSOd 6 . The chromatograms were visualized under UV light irradiation. The CHN analysis was performed on a Carlo Erba Strumentazione-Mod-1106.
General Procedure for Synthesis of 2-substituted-4H-benzo[d][1,3]oxazin-4-ones (3a-p) (Bari et al., 2020) To an 100 mL round bottom flask, anthranilic acid 1 (0.01 mol) and dry pyridine (30 mL) were added at room temperature with stirring. The solution was cooled to 0 • C, followed by dropwise addition of the corresponding aromatic acid chloride 2a-p (0.02 mol) in 10 mL of dry pyridine with constant stirring. After this addition, the reaction mixture was stirred for a further half an hour at room temperature and set aside for 1 h. The product, obtained as a pasty mass, was diluted with ice cold water (50 mL) and treated with aqueous sodium bicarbonate solution.
When the effervescence ceased, the precipitate obtained was filtered and washed with water. The crude benzoxazine obtained was dried and recrystallized from aqueous ethanol.
General Procedure for the Synthesis of Quinazolinone (4a-p) (Panicker et al., 2010) To a 50 mL round bottom flask fitted with reflux condenser were added 2-substituted-4H-benzo[d] [1,3]oxazin-4-one (0.01 mol) and hydrazine hydrate (0.06 mol) in excess. The reaction mixture was heated under reflux in an oil bath for 10-16 h. The course of the reaction was monitored by thin layer chromatography (TLC). After completion of the reaction as checked by TLC, the reaction mixture was cooled and separated solids were collected by filtration, washed with water/hexane, dried, and purified by column chromatography (methanol/chloroform/water, 10:2:1) to afford the corresponding quinazolinones 4a-p.

In-vitro Assay Protocol
In-vitro bCA-II and hCA-II activities were measured by following the spectrophotometric method described by Pocker and Meany with slight modifications (Pocker and Meany, 1967;Ur Rehman et al., 2020). The spectrophotometric assay was conducted in HEPES-Tris buffer of pH 7.4 (20 mM) at 25 • C. Each inhibitory well-consisted of 140 µL of HEPES-Tris buffer solution, 20 µL of bCA-II enzyme solution (0.1 mg/mL HEPES-Tris buffer), and 20 µL of test compound in HPLC grade DMSO (maintain 10% of the final concentration). The mixture solution was preincubated for 15 min at 25 • C. Substrate p-nitrophenyl acetate (p-NPA) (0.7 mM) was prepared in HPLC grade methanol and the reaction was started by adding 20 µL to a well in a 96well-plate. The amount of product formed was measured for 30 min continuously at 1 min intervals at 400 nm in a 96well-plate using xMARK microplate spectrophotometer, Bio-Rad (USA). The activity of the controlled compound was taken as 100%. All experiments were carried out in triplicates of each used concentration, and results are represented as a mean of the triplicate.

Molecular Docking Protocol
The docking studies were performed on the crystal structures of bCA-II (1V9E, resolution = 1.95 Å) (Saito et al., 2004) and hCA-II (1BN1, resolution = 2.1 Å) (Boriack-Sjodin et al., 1998). The selected target receptors were retrieved from Protein Data Bank (www.rcsb.org) in PDB format. Chain A of each protein (1V9E and 1BN1) was chosen for docking. For the molecular docking simulation, Molecular Operating Environment (MOE) docking suite was used (MOEv2014.09). The crystal structures were prepared using Protein Preparation Wizard implemented in the MOE with the default settings. Hydrogen atoms were added in the MOE using protonate 3D protocol and the protein structures were minimized with an MMFF94x force field until an RMSD gradient of 0.1 kcal·mol −1 Å −1 was achieved. The 3Dstructures of ligands were prepared by Chemdraw and minimized by default option of MOE (Force field = MMFF94x, RMS gradient = 0.1 kcal·mol −1 Å −1 ). For docking, Triangle Matcher placement method and London dG scoring function were applied (Edelsbrunner, 1992;Naïm et al., 2007). After docking, thirty docked poses of each compound were saved and the best-scoring docked pose of each ligand was visualized.

Chemistry
The target compounds were synthesized according to the route depicted in Scheme 1. Anthranilic acid 1 was treated with corresponding acid chlorides (2a-p) in the presence of pyridine to form precursor heterocycle (3a-p) via literature method (Scheme 1) (Bari et al., 2020). The corresponding benzoxazinones were refluxed in excess of hydrazine hydrate to furnish the 3-Amino-2-aryl quinazolin-4(3H)-ones (4a-p) in SCHEME 1 | Synthesis of quinazolinone derivatives.
The structures of compounds (4a-p) were established using microanalysis (CHN) and spectral data, i.e., IR and 1 H NMR. The C=N band in FTIR appeared in the range of 1,489-1,521 cm −1 . Moreover, the carbonyl group of compounds (4a-p) appeared at 1,660-1,689 cm −1 . The amino moiety (-NH 2 ) of compounds (4ap) was verified by the characteristic peak at 3,300-3,319 cm −1 in FT-IR spectra. The amino-moiety of compounds (4a-p) was verified by 1 H NMR spectra which appeared as a singlet for amino protons in a range from δ 4.92-5.52 ppm. The spectral data of other aromatic and aliphatic protons was also in accordance with the structures of anticipated compounds. CHN analysis also supported the anticipated structures (4a-p) and the observed values were in good agreement with the values found.

Kinetics Studies
To investigate the mechanism of action of these compounds, kinetics studies were performed. The kinetics studies were used to discover the type of inhibition and dissociation constant (Ki). Kinetics studies of the most active compound, 4d, against both bCA-II and hCA-II were performed, using different substrate concentrations on one side with different concentrations of 4d on the other side. Compound 4d inhibited both the bCA-II and hCA-II enzymes in a concentration-dependent manner with Ki values of 13.0 ± 0.013 and 14.25 ± 0.017 µM, respectively. From the kinetics studies, it was deduced that the compound 4d is a competitive inhibitor for both bCA-II and hCA-II. The Lineweaver-Burk plots were used for determination of the type of inhibition, in which the reciprocal of substrate concentrations was plotted against the reciprocal of the rate of the reaction to monitor the effect of the inhibitor on both K m and V max . The Lineweaver-Burk plots of 4d against both bCA-II and hCA-II clearly showed that the mode of inhibition of 4d is competitive (Figures 2A, 3A). In competitive inhibition, the K m of enzyme increased, while V max are not affected. The Lineweaver-Burk plots of compounds 4d (Figures 2A, 3A) showed that in the presence of compounds 4d, the K m of both enzymes bCA-II and hCA-II increased significantly on applying compounds 4d, while the V max remain unchanged, which described the competitive behavior of compounds 4d and its interaction in the active site of the enzyme. The K i values of compounds 4d for both enzymes bCA-II and hCA-II were deduced by secondary replots of Lineweaver-Burk plots by plotting the slope of each line in the Lineweaver-Burk  plots against different concentrations of compound 4d (Figures 2B, 3B). The K i values were confirmed by Dixon plot after plotting the reciprocal of the rate of reaction against different concentrations of compound 4d (Figures 2C, 3C).

Computational Studies
Computational medicinal chemistry has expedited the pace of drug design and discovery over the last four decades (Lin et al., 2020). Docking is a computational method which is widely used to study and understand the interaction between two macro-molecules (for e.g., protein-protein or protein-DNA) or between a macromolecule and ligand (e.g., for a drugreceptor, drug-DNA, or drug-RNA) and effectively predict the inhibitory mechanism of drugs. Therefore, we predicted the mode of interactions of all the active compounds though molecular docking. The reference inhibitor (acetazolamide) and quinazolinones were docked into the catalytic active pocket of hCA-II and bCA-II. The binding modes and receptor-ligand interactions in the binding site of bCA-II and hCA-II in the threedimensional and two-dimensional form were carefully examined by visual analysis through MOE. The results are presented in Tables 3, 4. Molecular docking revealed that all the active quinazolinones derivatives were exactly fitted into the active catalytic pocket of both enzymes (hCA-II and bCA-II). The docked orientation of compounds revealed direct interactions of ligands with the zinc ion present in the active site. Moreover, interaction of compounds with the active site residues and water molecule stabilize the compounds in the active site.
The docking process was first validated by re-docking the standard inhibitor acetazolamide in the active site of enzyme and is presented in Figures 4D, 6D.

Binding Interactions of Active Compounds and Their Predictive SAR Against bCA-II
From the docked poses of the all active compounds, it was confirmed that the compounds directly interact with Zn 2+ ion through their quinazolinone-carbonyl moiety. Compounds 4c, 4d, 4e, 4f, 4k, 4l, and 4m were found to be potent inhibitors of bCA-II with IC 50 values in the range of 8.9-19.7 µM. Compound 4m was the most potent inhibitor (IC 50 = 8.9 ± 0.31µM), followed by 4e > 4l > 4c > 4f > 4k > 4d, while compounds 4p > 4n displayed moderate active, and 4g > 4h > 4j >   The docked view of the least active compounds (4g, 4h, 4j, 4a, and 4b) showed that quinazolinone carbonyl of 4g interacted with the side chain of Thr198 via H-bond (2.81 Å) and the side chain of Asn66 offered a hydrophobic interaction to the compound (3.50 Å). Whereas, quinazolinone-amide group of 4h mediated a H-bond with the side chain of Thr198  Frontiers in Chemistry | www.frontiersin.org (2.99 Å), and the amide group of 4j interacted with a water molecule (WAT340) present in the active site. Similarly, the carbonyl oxygen and amide nitrogen of compounds 4a and 4b mediated H-bonds with the side chain of Thr197 and Gln91, respectively. Additionally, 4b formed a bond with a water molecule (WAT435). The binding interactions and docking scores of each docked compound are tabulated in Table 3. Figure 4 shows the binding interactions of the most active (4m), moderate (4n), and the least active (4a) compounds, however, a docked view of the most potent compound (4d) and all the compounds are presented in Figure 5.

Binding Interactions of Active Compounds and Their
Predictive SAR Against hCA-II Docking analysis deduced that the quinazolinone moieties of these compounds are responsible for the formation of metallic interactions with the Zn 2+ atom in the catalytic cavity. Compound 4g (IC 50 = 14.0 ± 0.60 µM) was the most potent and selective inhibitor of hCA-II, as compared to standard acetazolamide (IC 50 = 19.6 ± 1.23 µM), followed by compounds 4c > 4d > 4o and least active 4e > 4f > 4h > 4a. The quinazolinone moiety of 4g interacted with the side chain of Asn62, Thr200, and Wat270 with bond lengths of 3.34, 2.99, and 2.45 Å?, respectively. Similarly, 4c interacted with the side chain of Gln92 and Wat270, whereas 4d was linked with Zn 2+ atom through its nitro-oxygen group. The nitro-oxygen and aminoquinoline moiety of 4o interacted with Zn 2+ atom and the side chains of Gln92 and Thr200 via hydrogen bonds. However, the quinazolinone moiety of 4e interacted with the Zn 2+ ion and Gln92. On the other hand, the least active compounds, 4a, 4f, and 4h, interacted with the side chains of Asn62, Asn67, Thr200, and water molecules within the active site. The detailed binding interactions of active compounds and their docking scores are tabulated in Table 4. The binding interactions of most active (4g), moderate (4c), and least active (4a) compounds in the active site of hCA-II are presented in Figure 6. The docked orientation of all active compounds and the binding interaction of compound 4d are shown in Figure 7.

CONCLUSION
Quinazolinone derivatives (4a-p) were synthesized in search of new therapeutic agents for glaucoma and other diseases associated with hyper activity of CA-II. The in-vitro results showed that these skeletons displayed moderate to significant inhibition of both the enzymes (bCA-II and hCA-II). Compounds 4c, 4e, 4f, 4l, and 4m showed superior activity against bCA-II, while compounds 4g, 4c, 4d, and 4o were found to be significantly active against hCA-II. The structure-activity relationship reflected that the nitro group on phenyl ring at R position plays an important role in the overall inhibitory activities of compounds. Among the tested compounds, 4g and 4o are more selective for hCA-II. Moreover, kinetics studies showed the competitive behavior of inhibition of this series. Additionally, molecular docking predicted that the compounds bind efficiently with Zinc ion and several residues within the active site, therefore, through appropriate fitting and binding, these compounds effectively inhibit both bCA-II and hCA-II enzymes.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
AA-H and ZS conceived and designed the study. ZK synthesized all compounds. AK and MK performed in-vitro assay. MK and SH performed the computational studies and analyzed the data. AK wrote the manuscript with input and comments from MK, SH, ZK, ZS, and AA-H. All authors have read and approved the final version of the manuscript.

FUNDING
The generous support of this project through project number (BFP/RGP/HSS/19/198).