In Silico Design, Synthesis and In Vitro Antidiabetic Activity of Novel 5-Furyl-1,3,4-Thiadiazolimines

- Postprandial hyperglycemia plays an important role in the development of T2DM. The promising therapeutic approach is to decrease hyperglycemia by decreasing the postprandial rise in blood glucose concentration. In our study, a series of novel 5-furyl- 1,3,4-thiadiazol-2-imine derivatives were designed and those with good Physico-chemical properties were synthesized by oxidative cyclization between aryl acid and thiosemicarbazide using phosphorous oxychloride and subsequent treatment with aromatic aldehydes with varying substituents. Docking studies with the human pancreatic alpha-amylase enzyme in complex with myricetin (PDB ID: 4GQR) and peroxisome proliferator-activated receptor gamma (PPARgamma) (PDB ID: 1FM6) were done to predict the protein-ligand binding modes. The compounds SA07, SA03 and SA04 showed significantly high docking scores while SA01, SA05 and SA06 showed moderate scores. All derivatives were subjected to in vitro anti-diabetic screening using chromogenic dinitro salicylic acid method (α amylase inhibition assay). Significant percentages of inhibition were reported for SA03, SA07 and SA04 with acarbose as standard. Molecular docking revealed that synthesized derivatives and target proteins were actively involved in binding before and after systemic absorption and had a significant correlation with their biological activity. of the heterodimer of the retinoid X proliferator-activated receptors gamma (PPARgamma) ligand binding domains respectively bound with 9-cis retinoic and rosiglitazone and co-activator peptides retrieved from PDB with PDB with a resolution 2.1A 0 . The active site selected for the are Phe 282, Cys285, Ser289, Leu330, Met364, Tyr473, binding of

In silico approaches are being used today in drug discovery to assess the ADMET properties of compounds at the early stages of discovery and development. ACD Lab Chemsketch, PASS (Prediction of Activity Spectra for Substances), Osiris property calculator, Pre-ADMET prediction, Molinspiration and SwissADME are some of the software which helps pharmaceutical scientists to select the best candidates for development as well as to reject those with a low probability of success [19]. The three-dimensional structure of a protein or protein-ligand complex is helpful in lead identification using molecular modeling. Discovery studio, AUTODOCK, Glide, and SwissDOCK are some of the software providing docking analysis. Discovery Studio is a suite of software for simulating small molecule and macromolecule systems. It is developed and distributed by Dassault Systemes BIOVIA (formerly Accelrys) [20]. Our present study includes the in silico screening of a series of 5-furyl-1,3,4-thiadiazole-2-imine compounds, their docking studies with targets like the human pancreatic alpha-amylase enzyme in complex with myricetin (PDB ID: 4GQR), peroxisome proliferator-activated receptor gamma (PPARgamma) (PDB ID: 1FM6) and in vitro screening for anti-diabetic activity by α-amylase inhibition assay.

EXPERIMENTAL METHODS
The in silico modeling of all proposed compounds were carried out by using Molinspiration (21), ACDLAB Chemsketch (22), Pre ADMET, Osiris property calculator (24), and PASS (Prediction of Activity Spectra for Substances) (25) to predict the physiological and biological parameters. All the chemicals used for synthesis were of laboratory reagent grade and were obtained from Sigma-Aldrich and S.D. fine chemicals Ltd. Melting points were determined in open capillaries in the electrical melting point apparatus and are uncorrected. Analytical thin-layer chromatography was performed on precoated silica gel plates (Merck) to establish the identity of reactants and products monitored in-between reactions as well as at the end for completion of the reaction. The spots were visualized by iodine vapours in an enclosed chamber. Infra-Red spectra of compounds were recorded on 1) Perkin Elmer Spectrum Two FT-IR spectrometer in the range of 4000-200 cm-1. Proton (1H) Nuclear Magnetic Resonance Spectra of compounds were recorded on Bruker Advance II 400 NMR Spectrophotometer using DMSO solvent. Step 1 Synthesis of 2-amino-5 furyl-1,3,4-thiadiazole [26]. An equimolar amount of mixture of 2-furoic acid (0.1mole) and thiosemicarbazide (0.1mole), in POCl 3 (excess), was heated for half an hour, water (90ml) added and reaction mixture refluxed till the completion of reaction (TLC), cooled to room temperature, poured to ice-cold water, neutralized with saturated KOH solution, and recrystallized from 95% ethanol.
To the compound 1(0.01M) in 20ml ethanol, selected aldehyde (0.01M) in 15ml ethanol was added and refluxed for 5-6hr. The volume of resultant solution was reduced to half by distillation under reduced pressure. The resulting solution was poured on crushed ice and kept overnight below 20˚c for crystallization. The solid which got separated was dried and recrystallized from ethanol.

Molecular docking
Docking Analysis was performed using docking software Discovery studio 2018 for identifying the binding affinity of proposed compounds with two targets by Library docking method The human pancreatic alpha amylase enzyme in complex with myricetin with PDB id: 4GQR were retrieved from Protein Data Bank (PDB) with a resolution of 1.2A 0 [28]. The protein consists of a single polypeptide chain with sequence length of 496 amino acids. The binding sites of protein interaction with its native ligand are Trp 59, Gln63, Asp197 and Glu233. Docking analysis were performed by selecting binding site from receptor cavities in the target protein using charmm/charmm36 as force field. The crystal structure of the heterodimer of the human retinoid X receptor alpha and peroxisome proliferatoractivated receptors gamma (PPARgamma) ligand binding domains respectively bound with 9-cis retinoic acid and rosiglitazone and co-activator peptides were retrieved from PDB with PDB ID: 1FM6 with a resolution of 2.1A 0 . The active site selected for the studies are Phe 282, Cys285, Ser289, Leu330, Met364, His449, Tyr473, the binding site of rosiglitazone.
In vitro anti-diabetic activity (α-Amylase Inhibition assay) [29]. Porcine pancreatic α amylase (PPA) was used for the preliminary screening of α amylase inhibitors from the compounds using the chromogenic dinitro salicylic acid (DNSA) method. A total of 500µL of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing 20 mg/mL of α-amylase and varying concentration (25, 50, 75, & 100μg/ml) of extract as inhibitor were pre-incubated at 25 o C for 10 min. After the pre-incubation, 500 µL of a 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added to each tube at timed intervals. The reaction was stopped using 1.0 mL of DNSA colour reagent. The test tubes were incubated for 5 min, cooled., diluted by adding 10ml distilled water and the absorbance was measured at 540nm. Varying concentrations of acarbose (1mg/ml stock) were treated as standard. % ℎ = B−A * 100 (B-C) A-OD of test sample B-OD of blank with starch and alpha amylase C-OD of Control with starch only, RESULTS AND DISCUSSION Seven 1,3,4-thiadiazole derivatives were screened for best physico-chemical parameters using different software. All derivatives were screened for RO5 compliance using Chemsketch and Molinspiration software and were found to obey Lipinski's rule of 5 (RO5). The results were detailed in Table 1. Molecular parameters of proposed derivatives were further evaluated using Ghose filter and Viber filter. Ghose filter rule stated that compounds with Partition coefficient log P in (−0.4 to +5.6) range, Molar refractivity from 40 to 130, Molecular weight from 180 to 480 and Number of atoms from 20 to 70 (includes H-bond donors [e.g. OHs and NHs] and H-bond acceptors [e.g. Ns and Os]) give more druglikeness character. While Veber rule stated that for a compound predicted to have good oral bioavailability then the rotatable bond count should be less than or equal to 10 and polar surface area should be less than or equal to 140. All the proposed derivatives obeyed the Ghose and Veber filter rules. All the proposed derivatives have TPSA value less than140 angstroms. This parameter is significant to correlate proposed derivatives with the human intestinal absorption, Caco-2 monolayer's permeability, and blood brain barrier penetration. Percentage of absorption (% ABS) was estimated using the equation: % ABS=109−(0.345×TPSA), according to Zhao et al. ADME parameters were shown in Table 2. Seven derivatives were synthesized conventionally and their structures were confirmed by IR and 1H-NMR spectra. The details of different substitution, melting point, % yield and spectral details were given in Table 3. Docking studies were performed with human pancreatic alpha amylase enzyme (4GQR) to find out alpha amylase inhibiting activity and with peroxisome proliferator-activated receptor gamma (PPARgamma) (1FM6) to find out the antidiabetic activity after systemic absorption. Docking analysis report as shown in Table 4 revealed that except SA02 all others have good interaction with the binding site of human pancreatic alpha amylase enzyme. The compounds SA07, SA03 and SA04 showed good docking score while SA01, SA05 and SA06 moderate score. Docking with PPARgamma showed that compound SA07 and SA04 have maximum docking score.
In vitro anti-diabetic screening results as given in Table 5 showed maximum percentage of inhibition for SA03and SA07 which have maximum docking score also. Thus in vitro anti-diabetic analytical report compliments docking analysis results. Percentage of inhibition shown by various derivatives were compared with standard acarbose, shown in figure 5.

CONCLUSION
The designed and synthesized 5-furyl-1,3,4-thiadiazol-2-imine derivatives showed good docking score and in vitro anti-diabetic activity. In silico physico chemical prediction studies confirmed that the majority of the title compounds possessed the druglikeness character. In vitro screening results suggested that compounds in which phenyl ring with electron donating groups at 2, 4th position (SA03) and that with furyl ring (SA07) attached to imine linkage exhibited significant alpha-amylase inhibitory activity. The docking studies with PPAR gamma receptor proved that the proposed compound SA07 and SA04 have best docking score, thus giving insight to antidiabetic activity after systemic absorption also. Compounds namely SA03, SA07 and SA04 having significant docking score and percentage of inhibition, can be selected for further optimisation and can be explored for in vivo activity in the mere future thus resulting in the development of novel anti-diabetic drugs with better pharmacological profile.