Synthesis of 1,2,3-benzotriazin-4(3H)-one derivatives as α-glucosidase inhibitor and their in-silico study

α-Glucosidase inhibition is considered as an effective strategy for the treatment of diabetes mellitus. Currently, three α-glucosidase inhibitors are being used as drugs; Acarbose, Voglibose and Miglitol. The side effects of these drugs are forcing researchers to search for new and effective molecules. In this research work, novel 1,2,3-benzotriazin-4(3H)-one sulfonamides were synthesized and investigated for their α-glucosidase inhibition activity. 2,4,6-Trichloro-1,3,5-triazine: N,N-dimethylformamide (TCT : DMF) adduct have been utilized for the direct synthesis of targeted sulfonamides. All reactions were performed at room temperature under mild conditions. In-vitro enzyme inhibition studies led us to discover many potent inhibitors demonstrating good to excellent activity. The compound 5c with dimethyl substituent was found to be a more potent inhibitor than acarbose with the IC50 value of 29.75 ± 0.14 μM. Compounds 5a, 5b, 5d, 5e, 5f, and 5m showed good inhibition results with IC50 value 31.97 ± 0.03, 33.24 ± 0.01, 33.76 ± 1.05, 35.98 ± 0.03, 30.87 ± 0.51, and 37.24 ± 0.04 µM respectively. Further structure activity relationship was analyzed by molecular docking studies. Graphical abstract Graphical abstract


Introduction
Diabetes mellitus is a chronic disorder that is adversely affecting the quality of life of a large population worldwide.
In type 2 diabetes mellitus, the cell secrets insulin but the body becomes irresponsive leading to an uncontrolled blood glucose level. α-Glucosidase inhibitors, Voglibose, Acarbose, and Miglitol are being utilized to treat this health condition. These inhibitors control blood sugar by inhibiting α-glucosidase that is responsible for starch and disaccharides' conversion to glucose. The available αglucosidase inhibitors are associated with a number of side effects and thus, there is an urgent need to discover new drugs for the treatment of diabetes [1]. The sulfonamides based on heterocyclic systems are emerging as potent αglucosidase inhibitors. Various sulfonamide molecules based on indole [2], chalcone [3], Celebrex [4], sulfaguanidine [5], and quinoline [6] have demonstrated effective αglucosidase inhibition activity as compared to the available drug, acarbose.
In general, sulfa compounds are known for their applications in pharmaceuticals and agrochemicals. The sulfadizine (antibacterial), darunavir (antiviral drug), and celecoxib (anti-inflammatory drug) are among the wellknown examples of sulfa-drugs [7]. According to a survey, 15% of the most prescribed drugs in cardiovascular, neurological and infectious diseases belong to the family of sulfonamides [8].
In this research work, the synthesis of N-alkyl/aryl-4-(4oxobenzo [1,2,3]triazin-3(4H)-yl)benzenesulfonamides were achieved through one pot, simple and low cost methodology under mild conditions. The most common method for sulfonamide synthesis is by the reaction of sulfonyl chloride and amine under basic conditions. But this reaction is two step and undesired di-sulfonamides are formed [22]. Only a few sulfonyl chlorides are commercially available due to their instability. To avoid these problems, different reagents have been developed to replace sulfonyl chlorides, such as pentafluorophenyl vinylsulfonate and sulfonylbenzotriazole followed by aminolysis [23]. In-addition, triphenylphosphine/pyridine or triethylamine salt, sodium salt sulfonic acid/ 2,4,6-trichloro-1,3,5-triazine [22] and alkylisocyanides have also been reported for the direct conversion of sulfonic acid to sulfonamides [24].
In continuation of our strategy employed for the direct conversion of sulfonic acid to sulfonamides by using 2,4,6-trichloro-1,3,5-triazine (TCT) and N,N-
The reaction conditions and results of all reactants are explained in Table 1. Aliphatic amine, heteroaryl amine and aniline were utilized in reaction. Anilines with electron donating groups showed better yield rather than anilines with electron withdrawing groups. Among all reactants nitro anilines and amino pyridines did not give any result. All products were confirmed by IR, 1 HNMR, and 13 CNMR.

α-Glucosidase inhibition studies
The results of α-glucosidase inhibition studies revealed moderate to excellent activity of newly synthesized compounds 5a-5m ( Table 2). Among the series 5a-5m, compound 5c with two methyl groups showed excellent activity with 94.53 ± 0.42% inhibition assay (IC 50 value 29.75 ± 0.14 µM) that was better than acarbose inhibitory results 92.23 ± 0.16% (IC 50 37.38 ± 0.12 µM). Most probably the presence of two methyl groups on compound 5c enabled it to exhibit the more hydrophobic interactions with enzyme than monosubstituted compounds, 5d (m-methyl) and 5f (pmethyl). Furthermore, the position of methyl substituent on the benzene ring of synthesized compounds also has significant effects and showed distinct potential results. p-Methylated compound 5f with 93.01 ± 0.93% (IC 50 value 30.87 ± 0.51 µM) was found to be a better inhibitor as compared to m-methylated compound 5d with 92.29 ± 1.43% (IC 50 33.76 ± 1.05 µM) value. Compound 5a with one chloro group showed comparable results to di methyl compound with 93.58 ± 0.2 0% inhibition (IC 50 value 31.97 ± 0.03 µM). Because of big size of bromo atom than other substituents, its derivative 5b inhibitory potency was less than 5a compound. Compounds 5a-5f had aryl sulfonamide groups exhibited better results than alkyl sulfonamide compounds 5g, 5h, 5i, 5j, 5k, 5l that just showed moderate activity toward this enzyme.
Nature and position of substituent on the phenyl ring influence the inhibition activity. Aryl sulfonamides make more pi-stacking and pi-cationic interactions with enzyme cavity as compared to alkyl sulfonamide. Compounds 5a and 5b contains chloro and bromo substituent that showed Scheme 1 Synthetic route to target sulfonamide compounds 5a-m better result than methoxy 5e because in halogens more pair of electrons are available for interactions than methoxy group. It is noticeable that compounds 5g, 5h, 5i, 5j, 5k and 5l contain alkyl groups and their enzyme inhibition potential increases as number of carbons atoms increase in alkyl chain i.e., 5g with propyl chain showed 79.33 ± 0.97% (102.16 ± 0.41 µM) while 5l with octyl chain had 88.74 ± 0.17% (50.66 ± 0.05 µM) inhibition. This reason of increase in inhibition from 5g to 5l is due to increase in alkyl chains hydrophobic interactions. However, for the inhibitory activity, one generalization can be postulated that sulfonamide with aryl substitution shows better results as compared to compounds with alkyl groups.

In silico docking studies
In order to define enzyme inhibition, Auto Dock Vina computational studies were applied to explore possible interaction mechanism of α-glucosidase and ligand. In docking studies,  ligand and enzyme best orientation and confirmation was chosen and α-glucosidase counting function algorithm was used to calculate their binding affinity. All synthesized compounds (5a-5m) interacted with the binding sites of α-glucosidase and their inhibition potency was justified. These novel compounds presented good binding energies against α-glucosidase (Cat No. 5003-1KU Type I) ( Table 3). Among all ligands, 5c have highest free binding energy 9.7 kcal mol −1 proves as potent inhibitor of target proteins. Ligands 5a, 5b, 5d, 5f and 5m were also found as good glucosidase inhibitor with binding energy 9.5 kcal mol −1 . Remaining analogues, 5e, 5g, 5h, 5i, 5j and 5k presented less interactions as compared to other compounds with binding energy 8.4-9.2 kcal mol −1 .
For complete docking studies, ligand 5c was selected against α-glucosidase (Cat No. 5003-1KU Type I). 2D and 3D structural interactions were appeared with the several amino acid residues and its picture shown in Fig. 2. These results demonstrated that ligand 5c fits perfectly at the catalytic sites of α-glucosidase with a binding energy of −9.7 kcal/mol.
The interaction studies displayed that ligand bonded to the active site through five H-bonds. There is one hydrogens bond exists in Phe157 with the distance of 2.39 Å (Table 4a). Asn241 interacted through H-bond have distance of 2.77 Å. Two oxygen atoms of SO 2 make H-bonding with the cavity amino acid residues i.e., Arg312 and Asp408 make H-bond with the distance of 2.78 Å and 2.86 Å. His279 form another hydrogen bond with the oxygen atom of benzotriazinone motif with the distance of 3.40 Å.
In ligand there are number of non-polar and aromatic residues that build π-stacking interactions with Phe177 (Table 4c). Pi-cation interactions were also formed because of phenyl rings and these interactions were located at two points with residue His279 (Table 4d).
The analogue structures 5a, 5b, 5d, 5f and 5m demonstrated the second level inhibitory results with αglucosidase binding energy 9.5 kcal/mol. These compounds form one or two hydrogen bonds with enzyme cavity while 5c showed strong interactions because of five H-bonds. These compounds 5a, 5b, 5d, 5f and 5m make same type of hydrophobic interactions with Phe158, Phe300, and Phe177 at two sides of enzyme cavity while Thr215, Glu304 have these interactions at one side Figs. 3-7. 5a, 5b, 5d and 5f form interactions with the active site Glu276 through the hydrogen bond with the distance of 2.34-2.36Å but 5m make its three H-bonds with Glu276 (2.74 Å), His279 (3.20 Å), Arg312 (3.16 Å). His279 in all these compounds bonded to vicinity residues through Pi-cation interaction.
These interactions stabilize the ligand protein complex and forms its strong inhibition effect. From docking studied Table 3 Docking binding energies (kcal mol −1 ) of the docked compounds 5a-m into the active site of α-glucosidase result elucidated that 5c have strong binding within enzyme protein. 5a, 5b, 5d, 5f, 5m have low binding energy than 5c because of less hydrogen bonds. 5g, 5h, 5i, 5j, 5k and 5l are alkyl sulfonamides and have less hydrophobic and pi stacking interactions to amino acid residues that reduce its binding energy as compared to 5c.

Conclusion
TCT: DMF Adduct has been used successfully for the synthesis of N-alkyl or aryl 4-(4-oxobenzo[1,2,3]triazin-3 (4H)-yl)benzenesulfonamides. All reactions were done at the same time under same conditions. This method is simple   one step conversion that provides rapid and good yield product under mild conditions. So, it is efficient method to convert 1,2,3-benzotriazin-4(3H)-one sulfonic acid to their corresponding sulfonamides. In comparison to acarbose all synthesized compounds were screened for their potency to inhibit α-glucosidase. Compound 5c found potent inhibitor (IC 50 = 29.75 ± 0.14 μM) than acarbose (IC 50 = 37.38 ± 0.12) and most of the elaborated benzotriazinone derivatives showed inhibitory activity close to acarbose. Molecular docking studies clearly demonstrate that structures 5a-m bind to the active sites of α-glucosidase and possesses strong inhibitory potential.