Design and evaluation of chalconeimine derivatives as α-amylase inhibitors

Alpha-amylase is a known target for type II diabetes. Therefore, it is of interest to design α-amylase inhibitors based on hydrazone scaffold. The structure of these hybrids was confirmed by spectroscopic analysis (IR, 1H-and 13C NMR). All the compounds have potential inhibitory properties as shown by in vitro α-amylase inhibition activity. The compound 5-((1Z,3Z)-3-(benzo[d][1,3]dioxol-5-yl)-3-((2-chloropyridin-3- yl)imino)prop-1-en-1-yl)-2-(difluoromethoxy)phenol(4a) in 100 µg/mL concentration showed a high inhibition of 85.23%. In vitro α-amylase inhibition was further supported by docking studies of compound against the active site of pig pancreatic α-amylase (PDB ID: 3L2M). Docking studies revealed that the bonding interactions found between the compound and human pancreatic α-amylase are similar to those responsible for α-amylase inhibition by acarbose.

Chalcone is a class of open-chain flavonoids that is not only biosynthesized by plants but also can be prepared synthetically. The simplest chalcone can be prepared by an aldol condensation between a benzaldehyde and an acetophenone in the presence of base [22][23][24]. Hydrazones of chalcones have shown a wide variety of pharmacological effects, including anti-inflammatory and anticancer activities [25][26][27][28][29]. Despite the comprehensive biological studies on chalcones, reports on their anti-diabetic activity are limited [30]. Significant advances have been made in the past few years in the isolation and preparation of several hydrazones of chalcones derivatives.

Material and methods: Chemistry:
Thin layer chromatography (TLC) was used to examine the progress of the reaction. Open glass vessels were used to make a decision for the dissolving on outstanding softening mechanical assembly and were uncorrected. H1 and 13C atomic enticing reverberation (H1 proton magnetic resonance and 13CNMR) 524 ©Biomedical Informatics (2019) spectra were recorded on Bruker Avance II four hundred proton magnetic resonance spectroscope (400 MHz) at 298K, in correct deuterated dissoluble. Concoction move were accounted for as δ (ppm) with relation to tetra methyl silane (TMS) within allowable limit. Infrared spectra (IR) were recorded as KBr pellet on Shimadzu FT-IR spectroscope.
Docking studies: X-ray crystal structures of pig pancreatic alpha-amylase (PDB Id: 3L2M) were retrieved from the Protein Data Bank [31]. To put together the receptor for docking studies, co-crystallized ligand and water molecules were eliminated. At the same time polar hydrogen atoms and Kollman-united costs have been protected by the DNA Gyrase receptor. The essential pdb and pdbqt documents of ligands and Pig pancreatic alpha-amylase receptor were prepared for the       Table 3. The singlet observed in the range 6.30-6.60ppm is due -CH2 methylene proton of 3',4'methylenedioxy acetophenone moiety proton. The singlet observed at 7.41-7.49ppm is due -CH proton of -CHF2 moeity. The signals appearing 7.14-8.38ppm are obviously due to aromatic protons. The five chalconeimine derivatives (4a-e) shown in Figure 1a were taken for docking studies. These compounds are synthesized and their structures have been determined by IR, 1 H and 13 CNMR spectroscopy.

In vitro α-amylase inhibition:
All the synthesized compounds (4a-e) and standard drug were explored for their in vitro α-amylase inhibition studies at different concentrations (50, 100, 200µg/mL) as shown in the Table 4. All the compounds showed good % inhibition of α-amylase when compared with standard drug acarbose. Compound 4b and 4d were found to be more potent among all the synthesized compounds when explored at the concentration of 50µg/mL. Compound 4d shows 76.58% inhibition followed by 4b with 77.18% inhibition. There was a significant rise in % inhibition when concentration has been changed to 100µg/ml from 50µg/mL. Among all, 4b shows 81.35% inhibition followed by 4a which showed 85.23% inhibition at 100µg/ml. Inspired by the results obtained at 100µg/mL concentration, all the synthesized compounds were further screened for there in vitro α-amylase inhibition at 200µg/mL. All compounds exhibited a linear rise in % inhibition.

Docking studies:
Interactions between inhibitors and active site of the target protein can be explored using molecular docking studies. The above results showed that all the synthesized molecules were stronger inhibitors of alpha-amylase as compared to acarbose. Therefore, for ascertaining the binding conformation and interactions responsible for the activity, docking simulation of compound 4a and 4d was performed against active site of pig pancreatic alpha-amylase (PDB ID: 3L2M). Ligands taken for the docking studies are shown in Figure 1a. Pig pancreatic alpha-amylase protein is considered as target protein for this study. Its structure was taken from RCSB Protein Data Bank (PDB) with PDB ID: 3L2M as shown in Figure 2.   Table 4. Docking studies reveled that these amino acids present in the target proteins pocket involves in the binding interaction with the selected compounds.
These complex structures reveal essential interactions between the inhibitor and the protein and these interactions are taken as the reference for the hydrazone derivative (4a-e). The co-crystallized ligand are forms hydrogen bond interaction with the residues GLY 309, GLN 302, ARG 346, ASP316, ARG 267) ( Figure 5) which are present within the ATP binding pocket. The ligand is also further stabilized by a number of hydrophobic contacts with the residues. The five hydrazone derivatives (4a-e) shown in Figure 1a were taken for docking studies. These compounds are synthesized and their structures have been determined by IR, 1 H and 13 CNMR spectroscopy. The docking studies clearly reveal that some of these compounds bind efficiently to the enzymes of pig pancreatic alphaamylase. Binding score of autodock 4.2 varies between -7.8 to -8.9 for compounds 3a-g tested for pig pancreatic alpha-amylase ( Table  5) Out of the five hydrazone derivatives analyzed, compound 4b and 4d forms the best interaction with pig pancreatic alphaamylase.
The compound 4a and 4d has the highest binding score of -8.9 and -8.7. The fluorine, oxgen atom on hydrazone compound forms hydrogen bond with the hydrogen atom of ALA 198, ARG 195, and HIS 299 of pig pancreatic alpha-amylase (Figure 3 and Table 6).
Compound 4d having a binding score of -8.9 makes hydrogen bonds with the active site residue ASP 300, GLU 233, LYS200 and ILE 235 of enzyme (Figure 4). Re-docking of the inhibitor from the co-crystallized complex structure ( Figure 5) of pig pancreatic alphaamylase resulted in a binding score of -7.8, which is comparable to the scores found for compound 4b and 4d ( Table 5). The re-docked conformation of co-crystallized ligand (Figure 2) resembles the conformation of the hydrazone derivative (compound 4b and 4d respectively).

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