Synthesis, molecular docking and molecular dynamic simulation studies of 2-chloro-5-[(4-chlorophenyl)sulfamoyl]-N-(alkyl/aryl)-4-nitrobenzamide derivatives as antidiabetic agents

A series of 2-chloro-5-[(4-chlorophenyl)sulfamoyl]-N-(alkyl/aryl)-4-nitrobenzamide derivatives (5a–5v) has been synthesized and confirmed by physicochemical(Rf, melting point) and spectral means (IR, 1HNMR, 13CNMR). The results of in vitro antidiabetic study against α-glucosidase indicated that compound 5o bearing 2-CH3-5-NO2 substituent on phenyl ring was found to be the most active compound against both enzymes. The electron donating (CH3) group and electron withdrawing (NO2) group on a phenyl ring highly favoured the inhibitory activity against these enzymes. The docking simulations study revealed that these synthesized compounds displayed hydrogen bonding, electrostatic and hydrophobic interactions with active site residues. The structure activity relationship studies of these compounds were also corroborated with the help of molecular modeling studies. Molecular dynamic simulations have been done for top most active compound for validating its α-glucosidase and α-amylase inhibitory potential, RMSD analysis of ligand protein complex suggested the stability of top most active compound 5o in binding site of target proteins. In silico ADMET results showed that synthesized compounds were found to have negligible toxicity, good solubility and absorption profile as the synthesized compounds fulfilled Lipinski’s rule of 5 and Veber’s rule.


Introduction
Diabetes mellitus (DM) is a complex metabolic disorder resulting either due to relative or absolute deficiency of pancreatic insulin secretion or insensitivity to insulin action, ensuing in postprandial hyperglycemia and assorted diabetic complications [1,2]. According to World Health Organization reports, at present around 250 million peoples are living with diabetes and this number is expected to be more than 366 million by 2030 [3] and these statistics are predicted to reach 592 million by 2035 of which 46% may still remain undiagnosed. The reduction of postprandial hyperglycemia by inhibiting carbohydrate hydrolyzing enzymes in gastrointestinal tract is one of the promising approaches for management of diabetes [4,5]. α-Amylase is involved in hydrolyzing long chain of starch and α-glucosidase release glucose into the small intestine by breaking down oligosaccharides and disaccharides [2,6]. α-Glucosidase and α-amylase inhibitors reduced postprandial blood glucose level by delaying the hydrolysis of carbohydrate by inhibiting the digestive enzymes [7]. Acarbose, Miglitol Thakral et al. BMC Chemistry (2020) 14:49 and Voglibose are currently available drugs used as α-glucosidase and α-amylase inhibitors, but due to their deleterious side effects such as abdominal distention, diarrhoea and bloating, flatulence [8][9][10] there is need to explore and synthesize new drug candidates for the management of type-II diabetes mellitus with no or low risk of side effects.
The structure of 2-chloro-5-[(4-chlorophenyl) sulfamoyl]-N-(alkyl/aryl)-4-nitrobenzamide compounds was elucidated by IR, 1 H NMR and 13 C NMR spectral analysis. The stretching frequency due to NH and carbonyl of amide bond were obtained at 3294-3524 cm −1 and 1614-1692 cm −1 respectively. The bands around 1302-1398 cm −1 and 1127-1183 cm −1 were assigned to asymmetric and symmetric stretching of SO 2 of sulfonamide group respectively. The IR spectrum of synthesized compounds exhibits a band around 1506-1587 cm −1 to 1302-1378 cm −1 assignable to asymmetric and symmetric stretching of NO 2 . In the 1 H NMR spectra of compound, singlet for NH protons of SO 2 NH and CONH appeared at δ 3.37-4.08 ppm and δ 10.19-10.81 ppm, respectively. The two aromatic protons of 2-chloro-4-nitro benzoic acid appeared around at δ 8.50 ppm and δ 7.50 ppm. The aromatic protons showed the chemical shift values in region of δ 6.58-8.58 ppm based on their chemical structure. In 13 C NMR, signals for various carbons appeared in the region of δ 17.72 to 168.51 ppm.

In vitro antidiabetic evaluation α-Glucosidase inhibitory activity
All the synthesized compounds were tested for their in vitro α-glucosidase inhibitory activity and revealed their varying degree of inhibitory potential with IC 50 values of 10.75 ± 0.52 to 130.90 ± 2.42 μM (Table 2) as compared to reference acarbose (IC 50 = 39.48 ± 0.80 μM). The compound 5o (R = 2-CH 3 -5-NO 2 ) was found to be most active among this series of synthesized compounds. Most of the compounds exhibited good inhibitory potential with significant IC 50 as compared to positive reference.

Structure activity relationship
The compound 5o (R = 2-CH 3 -5-NO 2 ) was the most active compound (IC 50 = 10.75 ± 0.52 μM; 0.90 ± 0.31 μM) which may be due to the presence of electron withdrawing and electron donating group which generate an uniform electron flow, leading the compound to be more active and potent inhibitor against both enzymes. This fact is supported by the similar results of Adegboye et al. [38]. In compounds 5m (R = 2-CH 3 -3-NO 2 ) and 5p (R = 2-CH 3 -4-NO 2 ) difference in inhibitory potential was mainly affected by position of NO 2 substituent.

Molecular docking
In silico molecular docking study was performed to investigate binding interactions and to explore binding modes of synthesized compounds with their respective targets. The binding affinities of all the synthesized compounds are reported in Table 2.

α-Glucosidase enzyme
The docking results revealed that all the synthesized compounds displayed binding energy ranging from − 9.7 to − 8.0 kcal/mol and depicted various types of significant binding interactions like hydrogen bonding, electrostatic and hydrophobic interactions with the amino acid residues of active site of enzyme. The binding mode of most active compound 5o and modeled protein is presented in Fig , 5n, 5o, 5p displayed more hydrophobic interactions with Phe:177, Arg:312, Val:108, His:279, Phe:157, His:348, Tyr:344, Phe:298 amino acid residues of modeled protein which may have resulted in their higher inhibitory potential. The binding interaction between compounds 5c (R = 2-CH 3 ) and residues of modeled protein was nearly same as 5a (R = 4-CH 3 ) and 5b (R = 3-CH 3 ). The difference was that ortho methyl substituted phenyl ring maintained pi-pi stacked, pialkyl and pi-pi T interactions (hydrophobic interactions) with Try:344, His:348, Phe:298, Phe:177, Phe:158, Tyr:344 amino acid residue that made 5c more active than 5a and 5b.
The compound 5e (R = 2-OCH 3 ) formed less number of hydrogen bonding, electrostatic and hydrophobic interactions as compared to compound 5d (R = 4-OCH 3 ), resulting in decreased inhibitory potential of compound 5e. The binding of compound 5i (R = 3-Cl) facilitated one more pi-alkyl interaction with other hydrogen bonding, hydrophobic and electrostatic interactions same as that of compound 5j (R = 2-Cl), which may be contributing to better potential of compound 5i. Considering the moderately active compound 5r (R = 3-NO 2 ), additional hydrophobic interaction such as pi-pi interactions with amino acid residues were observed as compared to compounds 5k (R = 2-NO 2 ) and 5q (R = 4-NO 2 ). In comparison to compounds bearing aromatic anilines, a decrease in inhibitory potential was observed in compounds 5s (R = n-propyl), and 5t (R = n-butyl), due to less pi-pi interactions between the inhibitory compounds and amino acid residues. The binding interactions of compound 5u (R = C 4 H 3 O-CH 2 (2-furfuryl)) with residues of modeled protein were nearly same as that of 5v (R = C 5 H 5 N-(pyridine-2-yl)) but the difference was that 2-furfuryl ring exhibited pi-pi T shaped interaction with Trp:177 residue and four hydrogen bond interaction with Asp:329, Arg:376, His:90, Trp:93 residues of α-glucosidase with other interactions while compound 5v formed three hydrogen bond interactions, which made 5u more active than 5v against α-glucosidase enzyme.

α-Amylase enzyme
The docking results revealed that all the synthesized compounds displayed binding energy ranging from − 9.8 to − 7.9 kcal/mol. The binding mode of most active compound 5o and 1qho is presented in Fig. 2. The oxygen of 2-CH 3 -5-NO 2 established hydrogen bonding interaction with His:90 amino acid residue at a distance of 3.01 Å whereas His:232 amino acid was found to engage in hydrogen bond interactions with both oxygen of NO 2 of 2-Cl-4-NO 2 substituted phenyl ring with bond lengths of 2.04 Å and 1.86 Å. The nitrogen of 2-CH 3 -5-NO 2 displayed charge-charge interaction with Asp:372 amino acid residue (4.82 Å) while the protonated nitrogen of 2-CH 3 -5-NO 2 presented salt bridge charge-charge interaction with Asp:190 amino acid residue (3.14 Å). The charge-charge interaction was also found between the nitrogen of 2-Cl-4-NO 2 substituted phenyl ring and Glu:256 amino acid residue with bond length of 5.09 Å. The 2-CH 3 -5-NO 2 substituted phenyl ring created pianion interaction with residue Asp:372 of α-amylase Fig. 2 a 3D Binding confirmation of compound 5o with active site residues of α-amylase. b 2D binding confirmation of compound 5o with amino acid residue of nearby active site while nitrogen of 2-CH 3 -5-NO 2 substituted phenyl ring formed pi-cation interaction with His:90 residue. It was shown that His:90 residue (4.88 Å) formed pi-pi T shaped interaction with 2-CH 3 -5-NO 2 substituted phenyl ring and para chloro substituted phenyl ring displayed pi-pi stacked interaction with Trp:177 residue (4.60 Å). In addition 2-Cl-4-NO 2 substituted phenyl ring created pi-pi stacked and pi-pi T shaped interaction with Tyr:258, Phe:188 amino acid residues with bond lengths of 5.3 Å and 5.07 Å, respectively. The pi-alkyl interactions were established by chlorine of 2-Cl-4-NO 2 substituted phenyl ring and methyl of 2-CH 3 -5-NO 2 substituted phenyl ring with Phe:188 and His:328 residues at a distance of 5.48 Å and 4.50 Å, respectively. The CH 3 of 2-CH 3 -5-NO 2 substituted phenyl ring was found to engage in forming pi-sigma interaction with Tyr:92 residue (3.56 Å) while oxygen of NO 2 created pi-donor hydrogen bond with His:90 residue at a distance of 4.01 Å.
The involvement of 2-CH 3 -5-NO 2 substituted phenyl ring in forming more hydrophobic interactions may be contributing to better activity of compound 5o as compared to compounds 5n (R = 2-CH 3 -3-NO 2 ) and 5p (2-CH 3 -4-NO 2 ).The comparison of compound 5c , 5n, 5o, 5p displayed more electrostatic and hydrophobic interactions with Asp:372, Asp:190, Glu:256, His:90, Trp:177, Tyr:258, Phe:188, His:328 and Tyr:92 residues of α-amylase enzyme, which may have resulted in increase in inhibitory potential. The binding interactions between compound 5b (R = 3-CH 3 ) and residues of α-amylase were nearly same as 5a (R = 4-CH 3 ) and 5c (R = 2-CH 3 ). The difference was that meta methyl substituted phenyl ring and methyl group maintained pi-pi stacked, pi-alkyl and pi-sigma interactions (hydrophobic interactions) with Trp:177 amino acid residue which made 5b more active than 5a and 5c. The compound 5e (R = 2-OCH 3 ) formed less number of hydrogen bonding, electrostatic and hydrophobic interactions as compared to compound 5d (R = 4-OCH 3 ), resulting in decrease in inhibitory potential of compound 5e. The binding of compound 5f (R = 4-Br) facilitated two pi-pi T shaped and one pi-pi stacked interaction of para bromo substituted phenyl ring with Tyr:258, Phe:188, Trp:177 amino acid residues and two pi-pi stacked interactions of para chloro substituted phenyl ring with phe:188, Tyr:92 amino acid residues with other interactions, which made compound 5f more active than compounds 5g (R = 3-Br) and 5h (R = 2-Br). Considering the moderate active compound 5q (R = 4-NO 2 ), additional hydrophobic interaction such as pi-pi T shaped, pi-pi stacked interactions with Tyr:258 and Trp:177 residues were observed as compared to compounds 5k (R = 2-NO 2 ) and 5r (R = 3-NO 2 ). In comparison to compounds bearing aromatic anilines, a decrease in inhibitory potential was observed in compounds 5s (R = n-propyl), and 5t (R = n-butyl), due to less pi-pi interactions between the inhibitory compounds and amino acid residues. The binding interaction of compound 5u (R = C 4 H 3 O-CH 2 (2-furfuryl)) with residues of α-amylase was nearly same as 5v(R = C 5 H 5 N-(pyridine-2-yl))but the difference was that 2-furfuryl ring exhibited pi-pi T shaped interaction with Trp:177 residue and four hydrogen bond interactions with Asp:329, Arg:376, His:90, Trp:93 residues of α-amylase with other interactions while compound 5v formed three hydrogen bond interactions, which made 5u more active than 5v against α-amylase.

Molecular dynamics study
A stable protein backbone atoms RMSD vs time is an indication of the near-equilibrium system. As shown in Fig. 3a and b, the protein backbone in both systems attains a constant phase after an initial surge. Whereas, due to the extensive involvement of water molecules (− 500 kJ/mol) the ligand-bound protein backbone has higher RMSD fluctuations compared to the naked protein as represented in Fig. 3a. Figure 4 represent that electrostatic interactions are dominated between the ligand 5o and protein.
The results obtained from the MD simulations demonstrated that water molecules are predominately involved in ligand-protein interactions (Fig. 5). As shown in the figure, the fall in electrostatic energy that corresponds to the ligand-protein interactions is compensated by the water molecules.

In silico ADMET properties prediction
Lipinski's rule of five, topological polar surface area, aqueous solubility and number of rotatable bonds, these calculated parameters are presented in Additional file 1: Table S1. The human intestinal absorption values were found in range of 93.10 to 95.93% which established the moderate to good absorption capacity of synthesized compounds and supported their interaction with target cell.
The in vitro Caco-2 cell permeable property in the range of 0.36-0.55 nm/s, in vitro MDCK cell permeability in range of 0.01-0.97 nm/s designated low permeability of target compounds with the concerned cell line. The synthesized compounds displayed values in range of 95.75-100% confirmed their strong binding capacity with proteins. The in vivo blood brain barrier penetration ranges from 0.01 to 0.32 supported their low to moderate distribution in vivo with medium to good penetration capacity (Additional file 1: Table S2). Bioactivity and toxicity risk values of synthesized compounds are illustrated in Additional file 1: Table S3.

Conclusion
A series of 2-chloro-5-[(4-chlorophenyl)sulfamoyl]-N-(alkyl/aryl)-4-nitrobenzamide derivatives (5a-5v) has been synthesized and all the compounds were found to possess potent to moderate inhibitory potential against α-glucosidase and α-amylase. Compound 5o (2-chloro-5-[(4-chlorophenyl) sulfamoyl]-N-(2-methyl-5-nitrophenyl)-4-nitrobenzamide) was found to be highly active having fourfold inhibitory potential against α-glucosidase and around six times inhibitory activity against α-amylase in comparison to standard drug  acarbose. Molecular docking results of antidiabetic study showed reasonable dock score and binding interactions of synthesized molecules with their respective targets. Analysis of RMSD of ligand protein complex during molecular dynamic simulations suggested stability of the most active compounds 5o in binding site of respective target proteins i.e. α-glucosidase and α-amylase enzymes. Prediction of computational drug like properties showed that most of synthesized compounds are safe with acceptable ADMET and druggable properties.

Chemicals
The analytical grade chemicals and reagents were used as such in experiments without any purification. Decibel melting point apparatus was used for checking the melting point of the synthesized compounds and are reported as uncorrected. The silica gel-precoated aluminum sheets for thin-layer chromatography (TLC) were employed to keep a vigil of the reaction progress. FT-IR (Diffuse Reflectance Method (DRS) -8000A, Shimadzu, Japan) spectrophotometer was utilized for recording infrared spectra and the Bruker Avance III, 400 MHz NMR spectrometer was employed for nuclear magnetic resonance spectra ( 1 H NMR, 13 C NMR; Chemical shift δ valuesppm). α-Glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20, Sigma Aldrich) and α-amylase from malt (232-588-1, HiMedia) have been used for in vitro studies.

In vitro antidiabetic studies α-Glucosidase inhibitory assay
The method adopted for performing α-glucosidase inhibitory assay was similar to our prevenient study, Thakral and Singh [32]. Graph Pad Prism program, version 5 was employed for calculation of the 50% inhibitory concentration (IC 50 ) of all compound [32,42,43].

α-Amylase inhibitory assay
Xiao et al., and Yoshikawa et al., illustrated a method, with little modification this method has been adopted for measuring the activity [32,44].

Homology modeling
The 3D model for α-glucosidase is developed by comparative homology modeling technique using SWISS-MODEL web server (https ://swiss model .expas y.org/) [45] and then the quality of modeled structure was validated by Ramachandran plot (RAMPAGE) (http://mordr ed.bioc.cam.ac.uk/~rappe r/rampa ge.php). The details are available in our previous report [32].

Molecular docking
Ligand molecules were prepared as per reported method [32] using MarvinSketch and AutoDock tools. The crystal structures of α-amylase, 1qho [32,46] from Bacillus sterothermophilus, maltose/acarbose complex downloaded from the protein data bank (http://www.rcsb.org) and α-glucosidase modeled structure [32] was used for docking in antidiabetic evaluation. Docking studies were carried out as reported in our previous study and literature using AutoDock Vina program [32,47].

Molecular dynamic simulations
The respective structures placed in the center of the cubic box, the remaining volume of the box was filled by SPCe [48] water molecules. The whole box is then neutralized by adding the respective number of positive and negative ions using GROMACS 5.4 [49] by replacing the equal number of water molecules. Further energy minimization followed by 10 ns equilibration performed by using OPLS [50] force fields integrated into GROMACS 5.4 package to represent the potential energy of the system.