VIRTUAL SCREENING, SYNTHESIS OF NEWER HETEROCYCLES AS PPAR γ AGONISTS WITH ANTIDIABETIC ACTIVITY

VIRTUAL SCREENING, SYNTHESIS OF NEWER HETEROCYCLES AS PPAR γ AGONISTS WITH ANTIDIABETIC ACTIVITY

R. Priyadarsini ^*, V. Durga and Shahana Ahmed

Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College, Chennai-03, Tamilnadu India.

ABSTRACT: Recent decades have experienced a sharp increase in the incidence and prevalence of diabetes mellitus. The major therapeutic approach is to diabetes is to reduce gastrointestinal glucose production and absorption through the inhibition of carbohydrate digesting enzymes such as α-amylase and α-glucosidase. The agonists of PPAR γ is of great interest to the pharmaceutical industry since they regulate the expression of genes Associated with diseases like cancer, diabetes, atherosclerosis and obesity The aim of the current study was to synthesize newer PPAR γ agonists with alleged antidiabetic properties for α-amylase and α-glucosidase inhibitory activities. Newer PPAR γ agonist ligands containing three parts acidic head, a linker, and a hydrophobic tail as Pharmacophore features were designed. Four different compounds containing thiazole as heterocyclic nucleus have been synthesised and characterised by analytical methods like IR, NMR, spectral data. All the synthesised molecules were Optimizied using Lipinksi’s rule of five and subjected to docking studies using ARGUS LAB 4.0. Docking studies showed the important interactions of lead molecules posses with some of the common active site residues like ARG 288, CYS 285, HIS 323, HIS 449, LEU 353, LYS 367, MET 364, MET 348, PHE 363 of different PPAR γ receptors like 3FEJ, 3V9V. The synthesised compounds were evaluated for their antidiabetic activity by α amylase and α glucosidase inhibitory methods and all were found to exhibit an effective inhibition against both the enzymes. Out of the four synthesised compounds, KPSV 4 was found to be most effective with percentage growth inhibition of 61.92% and 80..46% at 1000μg when compared with other three synthesised compounds like KPSV 1, KPSV2, KPSV3.

PPAR- Peroxisome Proliferator Activated Receptors; PDB –Protein Data Bank;T2DM-Type 2 Diabetes Mellitus; NCE-Newer Chemical Entities; IR-Infra Red spectroscopy; NMR-Nuclear Magnetic Resonance

INTRODUCTION: Diabetes mellitus is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period ¹. Diabetes Mellitus is responsible for 7% of global deaths (WHO, 2015) Type 2 diabetes is caused by a decreased sensitivity of target cells to insulin accompanying serious, potentially life-threatening complications like atherosclerosis, retinopathy, neuropathy, foot problems, nephropathy ².

Peroxisome Proliferator-Activated Receptors (PPARs) are a group of receptor proteins that function as transcription factors regulating the expression of genes. PPARs play essential roles in the regulation of cellular differentiation, development, and metabolism (carbohydrate, lipid, protein), and tumorigenesis of higher organisms ³. Three types of PPARs have been identified: alpha, gamma, and delta (beta): γ (gamma): Two types 1. γ₁ - expressed in heart, muscle, colon, kidney, pancreas, and spleen. 2. γ₂ - expressed mainly in adipose tissue (30 amino acids longer), macrophages, large intestine ⁴.

The course of the study and research has been to identify novel molecule for Anti- diabetic activity PPAR γ receptors, so following steps were performed. Identification of common Pharmacophore features responsible for inhibiting PPAR γ from the literature review which is carried out as part of the current research ⁵. Designing of a series of compounds that selectively modulate the activities of Peroxisome Proliferator- Activated Receptors PPAR γ for exhibiting anti diabetic activity. The current study, the binding mechanism of PPAR γ receptors and newly designed  ligands  were studied  using molecular docking using docking tools like ARGUS LAB 4.0.Finally the newly designed ligands of high dock value were selected  and optimised using Lipinski’ rule. Based on the synthetic feasibility, certain compounds containing thiazoleheterocycles were synthesised and purified. All the synthesised molecules were characterised by analytical methods like IR, NMR, spectral data. In vitro anti -diabetic screening was carried out for all the four synthesised compounds by α -Amaylase and α-Glucosidase inhibitory method.

FIG. 1: 3D STRUCTURE OF PPARg

MATERIALS AND METHODS:

FIG. 2: VIRTUAL SCREENING FLOW OF WORK

Target Selection:

PPAR γ As Antidiabetic Agents: Recent research suggests that PPAR γ has a therapeutic potential to treat diabetes mellitus inflammatory diseases and certain cancers. The metabolic regulatory role of PPAR δ is recently recognised and clinical trials for PPAR δ agonists are key regulators of adipocyte differentiation and used to treat type 2diabetes ⁶. The agonists of PPAR γ is of great interest to the pharmaceutical industry since they regulate the expression of genes associated with diseases like cancer, diabetes, atherosclerosis and obesity⁷.

Selection of X-ray crystal PDB: The protein selection is carried out from the RCSB PDB(Protein Data Bank). Protein data bank is resource for studying biological macromolecules. It contains information about experimentally determined structures of proteins, nucleic acids, and complex assemblies ⁸.

A search in the PDB database retrieved a large number of crystal structure PPAR γ receptors. PDB codes, resolution of crystal structures are shown in tabular column below ⁹.

TABLE 1: LIST OF PPAR CRYSTAL STRUCTURES DEPOSITED IN THE PDB DATABANK AS OF 2013 ¹⁰

Q-Site Software: Using Q-site finder software tool, Some of the recent and efficient PDB file γ receptors with low resolution were selected and further evaluated by its Resolution value, R Free, R value and optimised crystal ligand interaction details. Some of the selected receptors listed below from which the highlighted best PDB targets were selected for present study.

TABLE 2: SELECTION OF PDB USING Q-SITE FINDER SOFTWARE

TABLE 3: ACTIVE SITE OF SELECTED PPAR RECEPTORS WERE IDENTIFIED USING THE SOFTWARE Q-SITE FINDER AND TABULATED AS BELOW

Pharmacophore Identification: When reviewed the efficient journals and research articles, the target-naive screening library was already designed by using compounds from a relatively narrow and low molecular weight range (350-5000D), selected diversity at both the putative “scaffold” core ¹¹ The review of the literature shows that a typical PPAR γ agonist consists of an acidic head attached to an aromatic scaffold, a linker, and a hydrophobic tail.

Common structural features of PPAR agonists: ¹²

FIG. 3: STRUCTURAL COMPONENTS OF PPAR γ AGONISTS ¹³

From the literature survey, the Common heterocycles used in designing PPAR γ agonists resembling the common features of PPARγ agonists have been listed below ¹⁴

TABLE 4: COMMON HETEROCYCLES OF PPAR γ AGONISTS

Based on the above literature facts, some new ligands have been identified, designed for further molecular docking using ARGUS LAB 4.0.

TABLE 5: LIST OF DESIGNED LEADS

Designed leads	IUPAC name	Structure
Lead 1	2-{[2-(4-methoxy phenyl)-1,3-thiazol-4yl]methyl-2-methoxy phenyl}3-(morpholin-4-yl) butanoic acid
Lead 2	6-[2-(4-{[2-(4-chlorophenyl)-1,3-thiazol-4yl]oxy} phenyl)ethyl]pyridine-3-carboxylic acid

Docking Study of Leads: Docking procedures aim to identify correct posses of ligands in the binding pocket of the protein and to identify and to predict the affinity between the ligands and the protein.¹⁵

Snapshots:

3FEJ

LEAD 1                                                                                            LEAD 2

3V9V

LEAD 1                                                                                                       LEAD 2

FIG. 4: INTERACTIONS OF LEADS 1 AND 2 WITH 3FEJ AND 3V9V

Stereo view of LEAD 1 and 2 in the active amino acid binding site of PPARγ receptors, suggested by molecular docking studies. The colors are as

follows: H BONDS: Blue, Steric Interaction: Red, Interaction Overlay: Red and Green dots.

TABLE 6: DOCKINGSCORES OF LEADS USINGARGUS LAB 4.0

From the above obtained docking results and also based on the synthetic feasibility certain   compounds containing thiazoleheterocycles were synthesised and optimised using Lipinski rule of Five. They were also further subjected to molecular docking using ARGUS LAB 4.0.

TABLE 7: BASED ON SYNTHETIC FEASIBILITY, LIST OF COMPOUNDS TO BE SYNTHESISED

Synthesised compounds	IUPAC name	Structure
KPSV 1	4-{[4-(4-chlorophenyl)-1,3-thiazol-2-yl]amino}benzoic acid
KPSV 2	4-{[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]amino}benzoic acid
KPSV 3	4-fluoro-2-{[4-(4-methoxy phenyl)-1,3-thiazol-2-yl]amino}benzoic acid
KPSV 4	4-fluoro-2-{[4-(4-chloro phenyl)-1,3-thiazol-2-yl]amino}benzoic acid

Lead Optimization Using Lipinskis Rule ^16: All the synthesised analogues were checked for Lipinski rule using online version of Mol inspiration software and the results are depicted in table below.

TABLE 8: OPTIMISATION OF COMPUNDS TO BE SYNTHESISED

Synthesis: ^17-19

Procedure:

Step 1: Synthesis of p- (chloro /methoxy) phenacyl bromide: Dissolve 0.25 mol of p- (chloro/ methoxy) acetophenone in 100ml of anhydrous diethyl ether in a 500ml flask. To this add 1gm of anhydrous aluminium chloride followed by the addition of 0.25 mol of bromine in a drop wise manner from a dropping funnel for about 30 minutes. Shake the mixture vigorously during the addition and also maintain the temperature around 20-30⁰C. The final product commences to separate as needles after about half of the bromine has been introduced. When the addition is complete, Cool the mixture in ice water, filter the crude product and recrytallise it using rectified spirit. The purity of product was established by single spot on T.L.C. plate. The percentage of yield was found to be 85%w/w.

Step 2: Synthesis of 2-Amino-4- (p-chloro/ methoxy) phenyl thiazole: Suspend 1mol of p- (chloro/methoxy) phenacyl bromide and 1 mol of thiourea in 100ml of absolute alcohol in a 500ml round bottomed flask fitted with condenser. The reaction mixture was refluxed on water bath for 2 hrs. To this add 2g sodium hydroxide pellets. To this add petroleum ether in a separating flask. Evaporate the ether layer and the resulting solid residue was washed, filtered and recrytallised using rectified spirit. The purity of product was established by single spot on T.L.C. plate. The percentage of yield was found to be 80%w/w.

Step 3: Synthesis of 4-{[4-(4-chlorophenyl)-1,3-thiazol-2-yl]amino}benzoic acid: 2- Amino-4- (p-chloro/methoxy) phenyl thiazole (1.5 gm,0.02mol) and 4 ml of dry benzene were placed in a 100 ml round bottomed flask fitted with water condenser. To this 4-chloro benzoic acid (0.02mol) was added drop wise. The reaction mixture was refluxed on water bath at 80⁰ c for 4 hrs. The resulting solid residue was washed with aqueous solution of sodium bicarbonate (50ml) followed by ice cold water. The resulting product obtained was recrystallised using ethanol. The purity of product was established by single spot on T.L.C. plate. The percentage of yield was found to be 70%w/w.

Scheme:

STEP 1

STEP 2

STEP 3

Where R= Cl / OCH₃                     R1= F, R2= Cl

Physicochemical Properties of Substituted Thiazole:

TABLE 9: PHYSICOCHEMICAL PROPERTIES OF SUBSTITUTED THIAZOLE

Characterisation of Synthesised Compounds:

Comp. code. KPSV 1: (C₁₆H₁₁ClN₂O₂S);4-{[4-(4-chlorophenyl) - 1, 3-thiazol-2-yl] amino}benzoic acid; Mol. wt: 330.788; C(58.09%), H(3.35%), Cl (10.72%), N(8.47%), O(9.67%), S(9.69%); IR Values: 3448.47cm^-1 (O-H str); 3070.45cm^-1(C=H str); 1689.52cm^-1(C=O str); 1596.94cm^-1(C=N str); 678.89cm^-1 (C-Clstr)

Comp. code: KPSV 2: (C₁₇H₁₄N₂O₃S);4-{[4-(4-methoxyphenyl)-1,3-thiazol-2-yl] amino} benzoic acid; Mol. wt : 326.369; C(62.56%), H(4.32%), N(8.58%), O(14.71%), S(9.83%) IR Values: 3456.18cm^-1 (O-H str); 3070.45cm^-1 (C=H str); 1596.94cm^-1 (C=N str); 678.89cm^-1 (C-Clstr)

Comp. code: KPSV 3: C₁₇H₁₃FN₂O₃S;4-fluoro-2-{[4-(4-methoxy phenyl)-1,3-thiazol-2-yl] amino} benzoicacid:Mol.Wt:344.360;C(59.29%),H(3.81%,N(8.13%), O(13.94%), S(9.83%), F(5.52%); IR Values:1311.50cm^-1(C-C str); 2923.87cm^-1 (C-H str); 1689.52cm^-1(C-Ostr); 1596.94cm^-1(C-N str); 678.89cm^-1(C-Cl str)

Comp.code:4 KPSV;C₁₆H₁₀ClFN₂O₂S;4-fluoro-2-{[4-(4-chloro phenyl)-1,3-thiazol-2-yl] amino} benzoic acid; Mol. wt: 348.77; C(55.10%), H(2.89%), N(8.03%), O(9.17%), S(9.19%), F(5.45%) 1311.50cm^-1 (C-C str); 3085.88cm^-1 (C-H str); 1689.52cm^-1 (C-O str); 1596.94cm^-1 (C-Nstr); 678.89cm^-1; (C-Cl str); 3425.33cm^-1 (O-H str);

Docking Studies of Synthesised Compounds:

Snap Shots: ARGUS LAB 4.0 docking results between Peroxisome Proliferator Activated Receptor (PPAR γ) along with four synthesised ligands PPAR γ:

3FEJ

                   KPSV 1                                                                   KPSV 2

       
KPSV 3                                                                  KPSV 4

FIG. 5: INTERACTIONS OF KPSV 1, 2, 3, 4 WITH 3FEJ

Stereo view of KPSV 1, 2, 3, 4, in the active amino acid binding site of PPARγ receptor 3 FEJ, suggested by molecular docking studies. The colors

are as follows: H Bonds: Blue, Steric Interaction: Red, Interaction Overlay: Red and Green dots.

3V9V

KPSV 1                                                                                  KPSV 2

KPSV 3                                                                                  KPSV 4

FIG. 6: INTERACTIONS OF KPSV 1, 2, 3, 4 WITH 3V9V

Stereo view of KPSV 1, 2, 3, 4 in the active amino acid binding site of PPARγ receptors 3V9V, suggested by molecular docking studies. The

colors are as follows: H Bonds: Blue, Steric Interaction: Red, Interaction Overlay: Red and Green dots.

Docking View with 3FEJ

KPSV 1                                                                                 KPSV 2

KPSV 3                                                                                  KPSV 4

FIG.7: DOCKING VIEW OF KPSV 1, 2, 3, 4 WITH 3FEJ

Docking View with 3V9V

KPSV 1                                                                                  KPSV 2

KPSV 3                                                                                  KPSV 4

FIG. 8: DOCKING VIEW OF KPSV 1, 2, 3, 4 WITH 3V9V

TABLE 10: RESULTS OF MOLECULAR DOCKING STUDIES USING ARGUS LAB 4.0

Evaluation of Anti-Diabetic Activity:

α Amylase Inhibitory Method: ^{20, 21} α-amylase was dissolved in phosphate buffer saline (PBS, 0.02 mol/L, pH 6.8) at a concentration of 0.1 mg/mL. Various concentrations of sample solutions (0.25 mL) were mixed with α-amylase solution (0.25 mL) and incubated at 37°C for 5 min. Then the reaction was initiated by adding 0.5 mL 1.0% (w/v) starch substrate solution to the incubation medium. After incubation at 37°C for 3 min, the reaction was stopped by adding 0.5mL DNS reagent (1% Dinitrosalicylic acid, 0.05% Na₂SO₃ and 1% NaOH solution) to the reaction mixture and boiling at 100 °C for 5 min. After cooling to room temperature, the absorbance (Abs) at 540 nm was recorded by a spectrophotometer. The inhibition percentage was calculated by the following equation:

Inhibition (%) = [(Abs1 − Abs2)/Abs1] × 100

where, Abs1=sample and Abs2 = control.

TABLE 11: α AMYLASE INHIBITORY ACTIVITY

FIG. 9: PERCENTAGE GROWTH INHIBITION

GRAPH 1: α AMYLASE INHIBITORY ACTIVITY OF SYNTHESISED SUBSTITUTED THIAZOLES AND ACARBOSE

α-Glucosidase Inhibitory Activity: ^{20, 21} The α glucosidase reaction mixture contains 2.9 mM p-nitrophenyl-α D-glucopyranoside (p NPG) (Sigma-Aldrich),0.25 ml of extract(varying concentrations) in DMSO and 0.6 U/ml bakers  yeast α-glucosidase in sodium phosphate buffer, pH 6.9.Control tubes contain only DMSO, enzyme and substrate, while in positive control acarbose replaced the synthesised compounds. Mixture without enzyme, synthesised compounds and acarbose served as blanks. The reaction mixture were incubated at 25 C for 5 min, after which the reaction was stopped by boiling for 2 min.

Absorbance of the resulting p-Nitrophenol (pNP) was determined at 405 nm and was considered directly to the activity of the enzyme. Glucosidase activity inhibition was determined as percentage of control as follows:

% Glucosidase inhibition (%) = [(Abs1 − Abs2)/Abs1] × 100

where, Abs1=sample and Abs2 = control.

TABLE 11: α - GLUCOSIDASE INHIBITORY ACTIVITY

FIG. 10: PERCENTAGE GROWTH INHIBITION

GRAPH 2: α GLUCOSIDASE INHIBITORY ACTIVITY OF SYNTHESISED SUBSTITUTED THIAZOLES AND ACARBOSE

RESULTS AND DISCUSSIONS:

PPAR (Peroxisome Proliferator Activated Receptor) agonist play a critical role in treating metabolic diseases, especially the type-2 diabetes mellitus (T2DM). In a search for more effective anti-diabetic treatment, we first aimed to work on a series of compounds that selectively modulate the activities of Peroxisome Proliferator-activated receptor γ. By reviewing the literature many scientific medical journals of existing PPAR γ agonists, it was found that a typical PPAR γ agonist consists of an acidic head attached to an aromatic scaffold, a linker, and hydrophobic tail. Based on these facts, new ligands containing thiazole heterocyclic nucleus have been designed and made ready for docking studies.

The PPAR activities of the designed NCEs were predicted by the developed models and then they were subjected to docking studies using ARGUS LAB 4.0 PPARγ (PDB code 3FEJ, 3V9V) to predict the binding affinities an interactions of the ligands at the active sites of the receptors. The hydrogen bonding interactions of the selected molecules at the active sites of PPARγ was also determined to identify which lead interacts better with the target proteins and results were tabulated. Based on the synthetic feasibility, certain compounds containing thiazoleheterocycles were synthesised and optimised using Lipinski rule of five. The synthesised compounds were characterised by IR, NMR, and Spectraldata’s.

All the four synthesised compounds revealed a significant inhibitory action on α amylase and α glucosidase enzyme when evaluated for their antidiabetic activity. The percentage inhibition at 10μg to 1000μg/ml concentrations of all the four synthesised compounds showed a concentration dependent increase in percentage inhibition in both the α- amylase and α –glucosidase inhibitory methods. Compound KPSV 4 exhibited a percentage inhibition varying from 61.92±0.65 to 21.6±0.35 for highest concentration1000μg/ml to lowest concentration 100μg/ml against α amylase enzyme and    80.46±0.46   to 15.47±0.32 for highest concentration 1000μg/ml to lowest concentration 100μg/ml against α glucosidase enzyme.

For Acarbose, 10 μg of sample exhibits 21.52 % growth of inhibition. 50μg of sample exhibits 34.73 % growth of inhibition. 100μg of sample exhibits 60.75 % growth of inhibition. 500μg of sample exhibits 70.83 % growth of inhibition. 1000μg of sample exhibits 82.23 % growth of inhibition.

SUMMARY AND CONCLUSIONS: Based on the literature facts, all the existing partial agonists found to posses mainly three parts of the pharmacophore acidic head, a linker ,and a hydrophobic tail. Four different compounds have been synthesised and subjected to docking studies. Then the synthesised compounds were subjected to optimization using Lipinksi’s rule of five.

Docking is performed by using ARGUS LAB 4.0 docking studies showed the important interactions of lead molecules posses with some of the common active site residues like ARG 288, CYS 28, HIS 323, HIS 449, LEU 353, LYS 367, MET 364, MET 348, PHE 363 of different PPARγ receptors like 3FEJ, 3V9V.

The synthesised compounds were evaluated for their antidiabetic activity by α amylase and α glucosidase inhibitory methods and all were found to exhibit an effective inhibition against both the enzymes. Out of the four synthesised compounds, KPSV 4 was found to be most effective with percentage growth inhibition of 61.92% and 80. 46% at 1000μg when compared with other three compounds.

ACKNOWLEDGEMENT: The Authors are thankful to IIT, Chennai for providing us analytical and spectral datas and also to Dr. Suresh M. Pharm Ph.D for helping us to carry out in vitro antidiabetic studies.

CONFLICT OF INTEREST: The authors have no conflict of interest.

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    How to cite this article:

    Priyadarsini R, Durga V and Ahmed S: Virtual screening, synthesis of newer heterocycles as PPAR γ agonists with antidiabetic activity. Int J Pharm Sci Res 2017; 8(2): 631-45.doi: 10.13040/IJPSR.0975-8232.8(2).631-45.

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