2, 4-dimethyl, 3-acetyl-5- carbethoxypyrrole

Glacial acetic acid (1.2 liters) and ethyl acetoacetate (3.09 mol) were taken into a 3 liter three neck flask provided with a stirrer and surrounded by ice bath. To this solution was added a solution of sodium nitrite (3.55 mol) in 400 ml of water with constant stirring such that temperature does not rise above 12⁰C. The mixture was stirred for further 2 hours and then at room temperature for 12 hours without disturbing and then added (3.48 mol) of acetyl acetone at one time. Now 450 gm of zinc dust was added slowly to the reaction mixture in portions of 10 gm. The rate of addition is regulated so that temperature does not rise above 60⁰C. Further the reaction mixture was refluxed for 2 to 3 hours on a hot plate. The hot solution was poured through a fine copper sieve with stirring into 30 liter of ice water. The product was filtered, washed with water and recrystallized from ethanol into get the pure white crystals.

b) Synthesis of 2-chloroquinoline 3-aldehyde ⁽¹³⁾

2-chloroquinoline 3-aldehyde

Dimethylformamide (0.15 mol) was taken in a clean 100 ml round bottom flask, the temperature of which is maintained below -10 ⁰C. Phosphoryl chloride (0.35 mol) was added drop wise to this solution with constant stirring. After complete addition the reaction mixture was stirred for few minutes. Acetanilide (0.05 mol) was added at once to the reaction mixture and stirred until dissolve. The reaction temperature was raised to 75 ⁰C and maintained for 16 hours. The reaction mixture was poured into ice cold water (300 ml) and stirred for 0.5 hours. The precipitate solid was collected by filtration and washed with water, air dried and recrystallized from ethyl acetate to afford the product as faint yellow crystals.

c) Synthesis of Chalcone derivatives: ⁽¹⁴⁾

To a solution of substituted acetophenone (0.01 mol) and substituted aldehyde (0.01 mol) in ethanol at room temperature was added sodium hydroxide (0.01 mol) with constant stirring. The reaction mixture was further stirred until a precipitate was formed. The reaction mixture was diluted with ice water and neutralized by using 0.01 N dilute hydrochloric acid solution. The product was filtered, washed with water and recrystallized from ethanol to get the respective chalcone derivatives.

Table No. 1- Different R and R’ substituents of chalcone derivatives

Compound Code	R	R’
A₁
A₂
A₃
A₄
A₅
A₆

1.2 Synthesis of pyridine urea and 5-bromo phenyl thiourea derivatives.

_a)Synthesis of pyridine urea derivative ⁽¹⁵⁾ B₃

To a solution of ethanolic sodium ethoxide i. e. 0.05 g of sodium metal in absolute ethanol (50 ml) was added 2- amino pyridine (0.01 mol) and urea (0.01 mol). The mixture was heated under reflux for 4 hours then evaporated in vacuo. Resulting solid product was collected by filtration and recrystallized from ethanol.

b) Synthesis of 4-bromo acetanilide (14)

13.5 g (0.1 mol) of finely powdered acetanilide was dissolved in 45 ml f glacial acetic acid in 350 ml conical flask. In another small flask 17 g (5.3 ml, 0.106 mol) of bromine was dissolved in 25 ml of glacial acetic acid, and transferred to separating funnel. The preparation had conducted in fume cupboard. Bromine solution was added slowly and with constant shaking to insure thorough mixing; flask was placed in cold water. When all the bromine had been added, the solution become an orange coloured due to the slight excess of bromine; part of reaction product was crystallized out. The final reaction mixture was kept at room temperature for 30 minutes with occasional shaking. The reaction product was poured into 400 ml of water, rinsed the flask with about 100 ml of water. The reaction mixture was stirred well. The crystalline precipitate was filtered, washed with cold water and recrystallised with dilute ethanol.

Synthesis of 4-bromo aniline ⁽¹⁴⁾

18 g (0.084 mol) of p-bromoacetanilide dissolved in 35 ml of boiling ethanol contained in a 500 ml round bottomed flask equipped with a reflux condenser. With a aid of a pressure equalizing dropping funnel 22 ml of concentrated hydrochloric acid was added down the condensers in small portions to the boiling solution. The reaction mixture was refluxed for 30-40 min. or until a test portion remains clears when diluted with water. Diluted with 150 ml of water, and fitted the flask with a condenser set for downward distillation. The mixture was distilled from an air bath. Approximately 100 ml of distillate was collected; the latter consists of ethyl acetate, ethanol and water. The residual solution of p-bromoaniline hydrochloride was poured into 100 ml of ice water, and with vigorous stirring 5% sodium hydroxide solution was added until just alkaline. The p-bromoaniline was separated as oil, which soon crystallizes. Recrystallised by dilute ethanol. The crystals were filtered, washed with cold water.

c) Synthesis of 4-bromo phenyl thiourea derivative ⁽¹⁶⁾

p-bromoaniline Thiourea B₄

In flask placed 200 g (1.5 mol) of 24 per cent aqueous 4-bromo aniline solution and concentrated hydrochloric acid was added until acidic to methyl red; about 155 cc required. Water was added to bring total weight 500 g, 300 g (5 mol) of thiourea was added and solution was boiled gently under reflux for 2-3 hour. The solution was cooled to room temperature, 100g (1.5 mol) of 95 % sodium nitrite was dissolved in it and whole cooled to 0 ⁰C. The crystals were stirred to a paste with about 50 cc of cold water, sucked as dry as possible and dried in vacuum desiccator to constant weight.

Identification of synthesized compounds:

Table No. 2- IR interpretation of synthesized intermediate compounds

Sr. No.	Compound code	IR cm^-1
1	A₁	1665.29cm^-1(C=O str), 1592.62cm^-1 (CH=CH), 3061.44cm^-1 (CH str), 789cm^-1 (Ar-Cl)
2	A₂	1658.48 cm^-1 (C=O str), 1598.7 cm^-1 (CH=CH), 1516.74 cm-1 (Ar-NO₂)
3	A₃	1665.23cm^-1(C=O), 1588.09 cm^-1 (CH=CH), 723.09 cm^-1 (Ar-Cl), 1015.34cm^-1 (C=O str)
4	A₄	1520.02 cm^-1 ( CH=CH), 1678.73 cm^-1 (C=O str), 3283.21 cm^-1 (NH), 1266.04 cm^-1 (ester)
5	A₅	1685.48 cm^-1 (C=O str), 1577.49 cm^-1 (CH=CH), 3283.21 cm^-1 (NH), 1045.23 cm^-1(Ar-Cl)
6	A₆	1586.16 cm^-1 (C=O str), 1651.73 cm^-1 (CH=CH), 756.92 cm-1 (Ar-Cl), 3335.28 cm^-1 (NH), 835.99 cm^-1 (P-distribution)
7	B₃	3199.33cm^-1 (NH), 1618.95cm^-1 (C=O str), 3292.16cm^-1 (N – pyridine)
8	B₄	503.33 cm^-1 (Ar-Br), 3189.68 cm^-1 (NH), 744.38 cm^-1(C-S), 1394.28 cm^-1 (C-N str)

1.3 Synthesis of oxazine and thiazine mini libraries ⁽¹⁷⁾

a) Oxazine derivatives:

Table No. 3- Different R and R’ substituents of oxazine derivatives

Sr No.	Compound code	R	R’	R’’
1	A1B1			H
2	A2B1			H
3	A3B1			H
4	A4B1			H
5	A5B1			H
6	A6B1			H
7	A1B3
8	A2B3
9	A3B3
10	A4B3
11	A5B3
12	A6B3

b) Thiazine derivatives:

Table No. 4 - Different R and R’ substituents of thiazine derivatives

Sr No.	Compound code	R	R’	R’’
1	A1B2			H
2	A2B2			H
3	A3B2			H
4	A4B2			H
5	A5B2			H
6	A6B2			H
7	A1B4
8	A2B4
9	A3B4
10	A4B4
11	A5B4
12	A6B4

A mixture of 6 different chalcones (0.002mol), thiourea/urea (0.002 mol) were dissolved in ethanolic sodium hydroxide (10ml) was stirred about 2-3 hours with a magnetic stirrer. This was then poured into 400 ml of cold water with continuous stirring for an hour and then kept in refrigerator for 24 hours. The precipitate obtained was filtered, washed and recrystallized. The completion of the reaction was monitored by TLC.

2. Docking study:

2.1. Target protein active site modelling

The 3-D coordinates in the X-ray crystal structure of PDBs complexed with inhibitors, were selected as the receptor model in the virtual screening with docking simulations. After removing the ligand and solvent molecules, hydrogen atoms were added to each protein atom. Then the active sites of receptors were analyzed. The compounds in the docking library were selected with the drug-like filters that select only the compounds with physicochemical properties of potential drug candidates and without reactive functional group(s).

Fig. No. 2 - Active site amino acids of DPP-4 enzyme (PDB ID-2G63)

Fig. No. 3 - Active site amino acids of Glucokinase enzyme (PDB ID-3H1V)

3.2. Docking of synthesized oxazine and thiazine analogues with dpp-4 and glucokinase enzymes

Ligands were designed on Vlife Molecular Design Suite 3.5 by using Biopredicta. All the molecular modeling studies were performed on pentim core2duo workstation using Sybyl. Geometries were optimized by energy minimization using MMFF94 forcefield and Gasteiger-Marsili charges for the atoms, till gradient of 0.001kcal/mol/A⁰ was reached.

Screening of designed molecules was performed with different antidiabetic targets.

After performing Batch GRIP docking the results found are as follows-

Table No. 6 - Docking scores of all synthesized molecules with DPP-4 and glucokinase enzymes

Sr No.	Compound code	DPP-4 binding Energy (Kcal/mol) (PDB ID- 2G63)	Glucokinase binding Energy (Kcal/mol) (PDB ID- 3H1V)
1	A1B1	-50.56	-51.90
2	A2B1	-47.70	-28.45
3	A3B1	-50.53	-45.48
4	A4B1	-46.06	-29.35
5	A5B1	-45.72	-35.20
6	A6B1	-51.90	-55.21
7	A1B2	-47.37	-63.18
8	A2B2	-51.74	-45.04
9	A3B2	-46.50	-52.73
10	A4B2	-52.20	-26.99
11	A5B2	-59.73	-9.41
12	A6B2	-50.52	-46.10
13	A1B3	-55.51	-45.13
14	A2B3	-59.70	-37.48
15	A3B3	-56.88	-50.04
16	A4B3	-56.91	-63.19
17	A5B3	-77.68	-15.36
18	A6B3	-64.12	-68.79
19	A1B4	-50.09	-21.77
20	A2B4	-57.19	-45.00
21	A3B4	-61.59	-53.47
22	A4B4	-48.47	-5.42
23	A5B4	-48.24	-20.33
24	A6B4	-50.27	-54.28
25	Reference Ligand	-57.14	-135.69

Fig. No. 4 – Interaction of reference ligand (blue) with amino acids in the active site of DPP-4 enzyme

Reference ligand showed hydrogenbond interaction (ARG125B, GLU205B, TYR547B), hydrophobic interaction (SER552B, LYS554B, SER630B, TYR631B, VAL656B), VDW interaction (ARG125B, TYR547B, LYS554B, SER630B, TYR666B)

Fig. No. 5 - Interaction of virtually and biologically active molecule A₆B₃(orange) with amino acids in the active site of DPP-4 enzyme

A₆B₃showed PI stacking interaction (PHE357B, TYR547B, TRP629B), VDW interaction (ARG125B, GLU205B, PHE357B, TYR547B, LYS554B, GLN553B, TRP629B)

Fig. No. 6 - Interaction of biologically active molecule A₄B₃ (green) with amino acids in the active site of DPP-4 enzyme

A₄B₃showed hydrogenbond interaction (TRP629B), hydrophobic interaction (TYR547B, SER552B, TRP629B, GLY741B), PI stacking interaction (TYR547B, HIS740B, TRP627B), VDW interaction (VAL546B, TYR547B, LYS554B, TRP627B, CYS551B, TRP629B)

Fig. No. 7 – Orientation of reference ligand (blue) and virtually most active and biologically active molecules A₅B₃ (orange) and A₆B₃ (green) respectively in the active site of DPP-4 enzyme

Fig. No. 8 – Interaction of reference ligand (blue) with amino acids in the active site of Glucokinase enzyme

Reference ligand showed hydrogenbond interaction (ARG63X), hydrophobic interaction (VAL62X, ARG63X, GLY97X, ILE159X), VDW interaction (VAL62X, TYR61X, ARG63X, SER64X, THR65X, GLN98X, GLY97X)

Fig. No. 9 - Interaction of virtually and biologically active molecule A₆B₃ (orange) with amino acids in the active site of Glucokinase enzyme

A₆B₃showed hydrophobic interaction (THR65X, PRO66X), PI stacking interaction (TYR214X), VDW interaction (VAL62X, ARG63X, SER64X, THR65X, PRO66X, GLN98X, ILE211X, TYR214X, MET235X)

Fig. No. 10 - Interaction of biologically active molecule A₄B₃ (green) with amino acids in the active site of Glucokinase enzyme

A₄B₃ showed hydrogenbond interaction (TYR215X), hydrophobic interaction (ILE211X, SER212X, CYS213X, TYR214X, LEU451X), PI stacking interaction (TYR214X), VDW interaction (VAL62X, ARG63X, SER64X, PRO66X, GLN98X, GLU67X, MET210X, ILE211X, CYS213X)

Fig. No. 11 – Orientation of reference ligand (blue) and virtually most active and biologically active molecules A₆B₃ (orange) and A₄B₃ (green) respectively in the active site of Glucokinase enzyme

3.1 Synthesis of lead molecule:

After finding the lead molecule they were synthesized individually. The general procedure for synthesizing the lead molecules as follow-

A mixture of substituted chalcone (0.002mol), pyridine urea (0.002 mol) were dissolved in ethanolic sodium hydroxide (10ml) was stirred about 2-3 hours with a magnetic stirrer. This was then poured into 400 ml of cold water with continuous stirring for an hour and then kept in refrigerator for 24 hours. The precipitate obtained was filtered, washed and recrystallized. The completion of the reaction was monitored by TLC.

IR interpretation of lead molecules:

Table No. 8- IR interpretation of synthesized lead molecules

Comp. No.	IR characteristics cm-1
A₆B₃	3342.03 cm^-1 (NH), 732.10 cm^-1(Ar-Cl), 3212.83 cm^-1 (Aromatic C-H), 1580.38 cm^-1 (C=N), 3212.83 cm^-1(N-pyridine)
A₄B₃	1643.05 cm-1 (C=O str), 3304.43 cm-1 (NH), 2987.27 cm-1 (Ar-CH str), 1513.85 cm-1 (CH=CH), 1140.69 cm-1 (C-O str), 1229.54 cm-1 (C-N str), 1437.67 cm-1 (Ar-OCH3).

NMR Interpretation of compound A₆B₃

Table No. 9- NMR Interpretation of compound A₆B₃

Lead code

δ PPM (CdCl₃)

A₆B₃

7.449-8.130 (m, 5H-Aromatic), 3.337 (s, Aromatic-NH₂), 3.873 (s, Aromatic-NH-Aromatic), 6.676-6.849 (t, 4H-Aromatic), 7.082-7.271 (t, 4H- Aromatic).

INTRODUCTION: