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1. WO2020110152 - NON-PEPTIDIC GLUCAGON-LIKE PEPTIDE-1 RECEPTOR AGONISTS AND METHOD OF PREPARATION THEREOF

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[ EN ]
“NON-PEPTIDIC GLUCAGON-LIKE PEPTIDE- 1 RECEPTOR AGONISTS

AND METHOD OF PREPARATION THEREOF”

FIELD OF THE INVENTION

The present invention relates to small molecule Glucagon-like Peptide- 1 Receptor (GLP-1R) agonists and their method of preparation thereof.

BACKGROUND OF INVENTION

The incidences of diabetes, metabolic syndrome, and obesity have reached epidemic status in India; and as per current reports in The Lancet, India is the top third country having a high number of obese population in the world. Obesity develops from an imbalance between energy intake and energy expenditure. Obesity leads to disproportionate accumulation of visceral fat as well as ectopic lipid accumulation in peripheral organs and liver in particular. This ectopic fat accumulation in the liver is termed as Non-Alcoholic Fatty Liver Disease (NAFLD), a condition in which excess fat accumulation happens in the liver with no history of alcohol use. Recruitment of the brown-like phenotype in white adipocytes (browning) and activation of existing brown adipocytes are currently being investigated as a means to combat obesity.

Insulin resistance is the common link between metabolic diseases like obesity, diabetes and NAFLD. The pathogenesis of diabetes is dysfunction of pancreatic b-cells to secrete insulin in response to nutrients. Insulin plays an important role in maintaining the blood glucose and fatty acids level. One of the hallmark features of Type 2 Diabetes (T2DM) is a deficit in the functional beta-cell mass, which results from a futile compensatory response to insufficient insulin secretion, to counter metabolic demands of insulin resistance and to maintain euglycemia. There are a number of organs involved in complicated pathogenesis of diabetes, therefore, a range of drug classes are available to target those organs. In all these, one of the major targets for diabetes therapy is Glucagon-Like Peptide- 1 (GLP-1); an incretin hormone which gives a number of beneficial effects that make GLP-1 one of the popular drugs for multi drug approach to T2DM.

GLP-1 is a posttranslational proteolytic product of the proglucagon gene released from intestinal L-cells within a minute in response to nutrient injection. GLP-1 has beneficial effects on many other organs. These include increasing insulin gene expression, delaying gastric emptying, increasing b-cell mass, increasing cardio protection. Other than that, it has roles in inhibition of glucagon release and in augmenting glycogen synthase activity in adipose, muscle, and hepatic cells. In addition, GLP-1 also reduces steatosis and enhances peripheral insulin sensitivity. GLP-1 also acts on nervous system and reduces apoptosis, augments proliferation and neogenesis of neural cells. GLP-1R agonists are also known to stimulate AMPK activity. AMPK activation has favorable effects on nutrients (carbohydrate and lipid) metabolism in the skeletal muscle and liver cells, and there has been keen interest in developing AMPK-activating drugs for therapeutic use in the treatment of metabolic diseases, such as T2DM and obesity. All beneficial roles of GLP-1 make it more attractive to be one of the most important therapeutics for diabetes. GLP-1 is active only as GLP-1 (7-36) amide, once GLP-1 (7-36) amide gets in circulation; it has half-life of less than 2 minutes because of rapid cleavage by ubiquitously expressed enzyme diaminopeptidyl peptidase IV (DPP-4), which gives rise to GLP-1 (9-36) amide, an inactive form of GLP-1 .

Therefore, there is very high demand for the development of therapeutics, which can mimic the GLP-1 activity and are more stable analogues of GLP-1 for the management of metabolic disorders, obesity and diabetes. Exenatide (synthetic analog of exendin-4, a hormone extracted from a lizard Gila Monster) and Liraglutide (a derivative of human GLP-1 derivatized at position-26 with palmitic acid with a glutamic acid as spacer) are the GLP-1 mimetic, which are resistant to DPP-4 cleavage and have an extended half-life. These two peptidic-GLP-lR agonists have the distinct disadvantage of being administrated via subcutaneous route. Some non-peptide GLP-1 R ligands have been identified and include Quinoxalines, Thiophenes, Pyrimidines, (S)-8, Boc-5/ S4P, Azoanthracenes, Pyrazole-Carboxamides, Phenylalanines, Imidazopyridines and Flavonoids. However, many synthetic small molecules have been reported as GLP-1 R agonist, but they have not been pharmacologically approved.

Therefore, there is a need for small molecules which can mimic GLP-1 R agonists with increased efficacy and lesser side effects to overcome the pitfall of present day GLP-1 R peptidic agonists and significantly show their effect in treatment of diabetes and other metabolic disorders like obesity. The present invention thus aims to identify and develop small molecule Glucagon-like Peptide- 1 Receptor (GLP-1R) agonists which overcome the above discussed problems of the prior art.

OBJECTIVES OF THE INVENTION

An important objective of the present invention is to provide novel small molecule Glucagon-like Peptide- 1 Receptor (GLP-1R) agonist compounds, having beneficial effect in various diseases like diabetes, non-alcoholic fatty liver disease and obesity.

Another important objective of the present invention is to design, synthesize and screen a novel compound library of said compounds.

Yet another objective of the present invention is to perform in vitro and in vivo studies to determine the significant binding and beneficial effect of said compounds in different cell lines and mouse models.

Still another objective of the present invention is to perform in vivo studies to study the efficacy of compound in different organs like liver and pancreas for treatment of non-alcoholic fatty liver disease, obesity and diabetes.

BRIEF DESCRIPTION OF FIGURES

Figure 1 shows Molecular docking of small molecule library. (A) Structure of ECD of GLP-1R PDB: [3iol] (B) Chemical structure of selected small molecule library (C) Confine representation of the binding site of selected small molecule library.

Figure 2 shows synthesis strategy of small molecule library of GLP-1R agonists and route for analogs synthesis.

Figure 3 shows (A) 1H and (B) 13C NMR and (C) HR-MS spectra of compound PK2.

Figure 4 shows (A) 1H and (B) 13C NMR and (C) HR-MS spectra of compound PK3.

Figure 5 shows (A) 1H and (B) 13C NMR and (C) HR-MS spectra of compound PK4.

Figure 6 shows (A) 1H and (B) 13C NMR and (C) HR-MS spectra of compound PK5.

Figure 7 shows cell viability studies in HepG2 and INS-1 cells using MTT assay for 24hr (A) HepG2 (B) INS-1 cells treated with different concentration of PK2.

Figure 8 shows validation of binding of small molecule with GLP-1R. (A) HepG2 cells were treated each compound PK2-PK5 (50 mM) for 1 hr, Ex-4 (20 nM) as positive control and DMSO as a vehicle. (B) HepG2 cells were treated with 300 nM of Ex-9 for 15 min and stimulated with PK2-50 mM for 1 hr, and the cells were analyzed under confocal microscope. (C) 293 A cells were overexpressed with hGLP-lR stimulated with PK2-50 mM and DMSO as vehicle, cells analyzed for PKA activity. Data expressed mean ± SEM of triplicates, ***p<0.05 of control (DMSO). (D) HepG2 cells were serum-starved for 12 hrs treated with PK2-50 mM for another 1 hr and analyzed for pCREB protein expression. (E) The phosphorylation of CREB was quantified using ImageJ software. Data expressed mean ± SEM of triplicates, ***p<0.05 of control (DMSO).

Figure 9 shows Pharmacokinetics and tissue distribution study of PK2. (A) PK2 concentration was measured in blood plasma after oral administration at doses of 25 mg/kg body weight at different time-points. (B) PK2 Concentrations were measured in various organs (Pancreas, Heart, Brain, Lung, Spleen, Kidney and Liver) after 2 hrs of oral administration. (C) Glucose levels of vehicle (Black) and PK2 administered (Green) group after glucose infusion at different time points. (D) Insulin levels of vehicle (Black) and PK2 administered (Green) group after glucose infusion at different time points.

Figure 10 shows PK2 protects mice from Streptozotocin (STZ). (A) Schematic diagram of the treatment strategy used in this study. (B) Bodyweight in Control, STZ, Pre-PK2, and Post-PK2 treatment groups. (C) Plasma glucose levels after 5 days STZ treatment mice groups, *** *p<0.001 of STZ Pre-PK2, ****p<0.001 of STZ, Post-PK2 (D) Serum insulin level of all mice groups. Data expressed mean ± SEM of triplicates, *p<0.05. (E) Immunofluorescence images of the pancreatic tissue sections are raised with insulin (insulin staining in red, nuclei staining in blue). Scale bars, 20mhi. control, Streptozodocin-65mg/kg (STZ), and STZ+ PK2 (pre and post-treatment) mice groups on day of sacrifice. (F-G) b-cell morphometric analysis. Graphical representation of indicated parameters of n >5 in each group, mean ± SEM; (F-G) ***p < .0001.

Figure 11 shows PK2 protect islets anatomy. (A) Pancreas weight in Control, STZ, Pre-PK2 and Post-PK2 mice groups on the day of sacrifice n >3 in each group, mean ± SEM; **P< 0.05, (B) Representative images of the pancreatic tissue sections are stained with H & E.

Figure 12 shows PK2 effect in b-cell protection via preventing apoptosis and augmenting proliferation. (A) Immunofluorescence images of the pancreatic tissue sections are raised with

insulin followed by TUNEL staining (insulin staining in red, nuclei staining in blue, TUNEL staining -green). (B) Quantitative Analysis of TUNEL (+) cells. Data expressed mean ± SEM, ***p<0.0001 of Control to STZ, **p<0.05 of STZ to PK2. (C) Images of the pancreatic tissue sections are stained by immunofluorescence raised with insulin and Ki67 (Ki67 in green). Scale bars-20pm. (D) Quantitative Analysis of Ki67 (+) cells. Data expressed mean ± SEM, **p<0.05 of STZ. (E) INS-1 cells transfected with TXNIP promoter-luciferase construct. Luminescence was measured after 45 mins PK2 treatment. Data expressed mean ± SEM, **p<0.05 of Control (F) INS-1 cells were treated with PK2 for 30 mins and DMSO in control as vehicle followed by western blotting using TXNIP antibody.

Figure 13 shows PK2 effect on diet-induced-obese mice. (A) Schematic outline of the treatment regimens used in this study. (B) Food intake of NCD, HFD, and HFD-PK2 mice groups after treatment regimens. (C) Bodyweight in NCD, HFD, and HFD-PK2 mice groups after treatment regimens. (D) Representative epidermal fat morphology of NCD, HFD, and HFD-PK2 mice groups after completion of regimens. (E) Weight of epididymal white adipose tissue (eWAT) in NCD, HFD, and HFD-PK2 mice groups after treatment regimens. (F) Fasting blood glucose levels in NCD, HFD, and HFD-PK2 mice groups after 8 weeks of treatment regimens. (G) Circulating serum insulin levels in NCD, HFD, and HFD-PK2 mice groups after 8 weeks of treatment regimens. (H) Plasma glucose levels during ipITT in NCD, HFD, and HFD-PK2 mice groups. (mean ± SEM, ** P < 0.05; n >3).

Figure 14 shows PK2 improves hepatic insulin resistance. (A) Western blot results of PK2 induced phosphorylation of pAkt and pGSK3p. HepG2 cells were treated with Insulin (lOOnM) along with vehicle (DMSO) and PK2 (25 & 50 mM) for 24 hrs. After completion of incubation, cells were given an induction of 100 nM insulin for 10 mins and analyzed for protein expression. (B & C) The phosphorylation of Akt and GSK3P was quantified using Image J software. Data expressed as mean ± SEM.

Figure 15 shows PK2 improves HFD induced liver toxicity. (A) Triglyceride, (B) Total cholesterol, (C) AST and (D) ALT, in serum were detected. (E) Representative images for H & E staining using the Bouins’ fixed liver tissues at 40/ and 100/ magnification; blue arrow-head represents microvascular steatosis, Red arrow-head represents multiple lipid droplets inside hepatocyte, Green arrow-head represents macrovascular steatosis, a signet-ring appearance. (F) Western blot results of PK2 induced phosphorylation of AMPK and ACC in liver tissues samples. (G) PK2 reduces transcript-level expression of ACC and Carbohydrate-response element-binding protein (ChREBP) in liver tissue.

Figure 16 shows PK2 enhance AMPK and ACC phosphorylation in HepG2 cells. (A) Western blot results of PK2 induced phosphorylation of AMPK. HepG2 cells were treated with vehicle (DMSO) and PK2 (50 mM) different time-points 8 hrs, 10 hrs, and 12 hrs. (B) The phosphorylation of AMPK was quantified using ImageJ software. Data expressed as mean ± SEM. (C) Western blot results of PK2 induced phosphorylation of ACC. HepG2 cells were treated with vehicle (DMSO) and PK2 (50 mM) for 2 hrs. (D) The phosphorylation of ACC was quantified using ImageJ software. Data expressed as mean ± SEM.

Figure 17 shows PK2 restricts ChREBP to the cytoplasm. (A) Photomicrograph of immunostained HepG2 cells represents PK2 inhibited the translocation of ChREBP from cytoplasm to nucleus. HepG2 cells treated with 2.5 mM + vehicle (DMSO), 30 mM + vehicle (DMSO), and 30 mM Glucose + PK2 (50 mM) for 24 hrs followed by ICC as described in methods. The image scale bar is 20 m. (B) Western blot results of nuclear fractions of HepG2 cells represents PK2 restrict ChREBP translocation to cytoplasm. HepG2 cells treated with 2.5 mM and 30 mM glucose with or without PK2 for 24 hrs.

Figure 18 shows transcription level studies of browning inducing genes on treatment of 50mM of PK2 in maintenance media for 6 days.

SUMMARY

The present invention relates to synthesis and development of small molecule Glucagon-like Peptide- 1 Receptor (GLP-1R) agonists which have important application in metabolic disorders like Impaired Glucose Tolerance (IGT), Type 1 Diabetes (T1DM), Type 2 Diabetes (T2DM), non alcoholic fatty liver disease, cardiac dysfunction and obesity. Method of preparation of these Glucagon-like Peptide-1 Receptor (GLP-1R) agonists is also provided. The present invention also provides in vitro and in vivo experimental studies for determining the beneficial effect of said compounds in the treatment of various diseases like diabetes, non-alcoholic fatty liver disease, cardiac dysfunction and obesity.

OFT ATT FT) DESCRIPTION

The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention. The embodiments of the invention which are apparent to one skilled in the art after reading the present disclosure and on applying the common general knowledge of the technical field are within the scope of this invention.

Definitions:

The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”,“includes”, and“including” are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

The present invention provides novel non-peptidic compounds that specifically bind to GLP-1R and enhance cAMP level (second messenger) comparable to a GLP agonist, Exendin-4 in vitro in different cell lines. The present invention also provides compositions comprising these novel compounds and their method of preparation thereof. The non-peptidic compounds of the present invention are also able of doing GLP-1R internalization. Treatment of said compounds to HepG2 cells also rescue the palmitate induced lipid accumulation via inhibition of promoter activity of fatty acid synthase by suppressing the AMPK phosphorylation. Treatment of PK2 also exalts insulin sensitivity in hyperinsulinemia induced insulin resistant HepG2 cells comparable to control. Most importantly, in an in vivo mice model, PK2 could protect against pancreatic b-cell apoptosis and dysfunction caused by STZ and therefore PK2 may be a potential pharmacological agent for preventing pancreatic b-cell damage caused by oxidative stress associated with diabetes.

In an embodiment, the present invention provides compound of Formula I:

R 2


Formula I

or a stereoisomer, salt, hydrate, solvate, or crystalline form thereof;

Wherein

Ri is independently selected from hydrogen, halogen, unsubstituted or substituted hydroxyl, unsubstituted or substituted sulphonate, unsubstituted or substituted nitro, unsubstituted or substituted isatin, unsubstituted or substituted alkyl;

R2 is independently selected from hydrogen, -COPh, unsubstituted or substituted sulphonate, unsubstituted or substituted alkyl, unsubstituted or substituted halide, unsubstituted or substituted dihalide; and

R3 is independently selected from hydrogen, halogen, unsubstituted or substituted hydroxyl, unsubstituted or substituted sulphonate, unsubstituted or substituted nitro, unsubstituted or substituted isatin, unsubstituted or substituted alkyl, unsubstituted or substituted alkoxy.

In another embodiment, the compound of Formula I of the present invention has Rl as Hydrogen, R2 as Hydrogen or - COPh and R3 is -N02 or -CH3 or -OCH3.

In yet another embodiment, the compound of Formula I of the present invention is selected from:


In still another embodiment, the present invention provides a pharmaceutical composition comprising the compound of Formula I along with one or more pharmaceutically acceptable carriers or excipients.

In another embodiment, the process for preparing the compound of formula I, comprises the steps of:

(i) adding a mixture of isatin (1) and ortho-phenylenediamine (2) in acetic acid;


(ii) filtering the precipitate, triturating the product obtained from step (i) with a solvent to get the purified compound of formula 3;

(1i1n1) adding anhydrous metal carbonate in dry acetone to the mixture of compound of formula 3 of step (ii) and then adding propargyl bromide in the reaction mixture and refluxing the said mixture;


(IV) removing the solvent, extracting the residue and drying over anhydrous Na2SC>4, concentrating the same and triturating with hexane to get the purified product;

(V) dissolving aryl bromide in 1 : 1 : acetone: water mixture and sodium azide, reacting the same followed by removal of acetone under reduced pressure and extracting the product using a solvent;


(vi) dehydrating and evaporating the solvent to obtain the azide; and adding freshly prepared solution of CUSO4.5H2O and sodium ascorbate in water to a mixture of 9- substituted-6-propargyl-indolo [2,3-b]quinoxalines and 1 -azido-arylbenzene, in

Tetrahydrofuran (THF) to get the final product

In yet another embodiment, the reaction mixture in step (i) is irradiated for around 10 min at about 120°C at 25W and the concentration of CUSO4.5H2O is around 5 mole% and that of sodium ascorbate is around 15 mole% in water and the ratio of 9-substituted-6- propargyl-indolo [2,3-b]quinoxalines and 1 -azido-arylbenzene is around 1: 1.5, in dry Tetrahydrofuran (THF).

In still another embodiment, the present invention provides a method for treating diabetes and/or other metabolic diseases in a subject in need of such treatment, the method comprising the step of administering to a subject a therapeutically active amount of compound of the Formula I.

In another embodiment, the present invention provides a kit comprising the non-peptidic compound of Formula I.

In yet another embodiment, the compound of Formula I is used in the treatment of diabetes and other metabolic diseases including but not limited to non-alcoholic fatty liver disease, cardiac dysfunction and obesity.

In another embodiment, the compound of present invention has the following formula:


Material and Methods

All solvents were procured from, Alfa Aesar, Merck, SD Fine, Fischer Scientific, and Sigma-Aldrich and used without further purification. 1H and 13C NMR spectra were recorded on 500 MHz Jeol ECS-400 (or 125 MHz for 13C) spectrometer in CDCh and DMSO-d6. Mass spectra

were recorded on a Bruker Impact-HD spectrometer. High-Pressure Liquid Chromatography (HPLC) was performed using Agilent 1200 series. Cary Eclipse spectrophotometers and Shimadzu UV-245 were used for examining the fluorescence spectra and absorption respectively. All spectral data were detailed at room temperature.

Example 1: Screening of small molecule using autodock

Virtual screening of number of small molecules was performed using Autodock 4.2 software on crystal structure of glucagon-like peptide-1 receptor extracellular domain (PDB file (3C59)). Kollman charge and Polar hydrogen were assigned to the protein structure using Autodock tools. The grid map consisted of 104* 126 c 102 with a grid spacing of 0.375 A. 100 run were performed during docking experiment per molecule and the remaining parameter were set as default. For each docking, a root mean square deviation (RMSD) was set as 2.0. At the end of each docking, all conformation were clustered and were ranked according to Binding energy and inhibitory constant. The interaction of each docking conformation was studied using Discovery Studio Visualizer (DS) tool.

Example 2: Synthesis of Compounds

Preparation of 6,10b-dihydro-5aH-indolo[2,3-b]quinoxaline (Compound 1)

A mixture of isatin (250 mg, 1.7 mmol) and ortho-phenylenediamine (183.5 mg, 1.7 mmol) in 500 L glacial acetic acid was irradiated in a sealed tube for 10 min at 120°C and 25 W. The reaction mixture was dispensed in crushed ice; the precipitates were filtered and washed with ice-cold water thrice. Further, the dry yellow solid was triturated with a mixture of dichloromethane (DCM): Methanol to get the pure product.

Preparation of (6H-indolo[2,3-b]quinoxalin-2-yl)(phenyl) methanone (Compound 3)

A mixture of isatin (lOOmg, 6mmol) and 3,4-diaminobenzophenone (144.28mg, 6mmol) in 250 L DMSO was irradiated in a sealed tube for 5 min at 90°C and 20 W. The reaction mixture was poured in crushed ice; the precipitates were filtered and washed with ice-cold water thrice. Further, the dry yellow solid was triturated with ether to get the pure product.

Preparation of 6-(prop-2-yn-l-yl)-6H-indolo[2,3-b]quinoxaline (Compound 2)

To the mixture of compound 1/3 (leq), anhydrous potassium carbonate (2 eq) in dry acetone (10 ml), propargyl bromide (1.5 eq) was added, and the reaction mixture was refluxed for four hrs. The completion of the reaction was checked by using thin-layer chromatography (TLC). After the completion of the reaction, solvent was removed under reduced pressure, and the crude product was extracted using ethyl acetate. The organic layer was washed with water, dehydrated over anhydrous Na2SC>4, and concentrated on getting the orange-yellowish solid. The crude product was triturated with hexane to get the pure product.

Preparation of azido-benzene


Benzyl bromide (1 eq) was dissolved in a 1 : 1 acetone: water mixture and sodium azide (2 eq) was added. The reaction was placed in a microwave oven for 20 min at 10 W and 65°C in an open vessel. After completion of the 20 min of the reaction, acetone was removed under reduced pressure, and the product was extracted using ethyl acetate. The ethyl acetate layer was dehydrated using sodium sulfate and evaporated. The azide formation was analyzed using infrared spectroscopy. The product was directly used in the next reaction.

Preparation of PK2-PK5

To a mixture of 9-substituted-6-propargyl-indolo [2,3-b] quinoxalines (1 eq) and 5’ (1.5 eq) in dry THF, freshly prepared solution of CUSO4.5H2O (5 moles%) and sodium ascorbate (15 moles%) in water was added. The reaction was carried at room temperature overnight. After the end of the reaction, the reaction mixture was filtered using Whatman filter paper. Compound 6-((l-(4-nitrobenzyl)-lH-l,2,3- triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline (PK2), 6-((l-(4-methylbenzyl)-lH-l,2,3-triazol-4- yl)methyl)-6H-indolo[2,3-b]quinoxaline (PK3), 6-((l-(4-methoxybenzyl)-lH-l,2,3-triazol-4-yl)methyl)- 6H-indolo[2,3-b]quinoxaline (PK4) and (6-((l-(4-nitrobenzyl)-lH-l,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxalin-2

yl)(phenyl)methanone (PK5) were further purified using column chromatography. Characterization of synthesized compounds was performed using lH-NMR, 13C-NMR and UR-MS (Figure 3-6).

Example 3: Cell Culture

The HepG2 (human hepatocellular carcinoma) /HEK 293A cells were grown in Dulbecco's modified Eagle's medium (DMEM high Glucose; Invitrogen) containing 3.7g/L NaHCCb, supplemented with 10% fetal bovine serum (Invitrogen, US origin), 100 U/ml penicillin and 100 g/ml streptomycin. HepG2 and INS-1 cells were kindly provided by Dr. Debabrata Ghosh, CSIR-IIT Lucknow, India. In addition to that, 293A cells were supplemented with MEM non-essential amino acid. INS-1 cells were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 g/ml streptomycin, lmM sodium pyruvate, lOmM HEPES, 11 mM glucose and 50 mM b-mercapto ethanol under standard cell culture conditions (humidified atmosphere, 5% CCh and 31° C).

Example 4: Cell Viability Assay

HepG2 and INS-1 cells were seeded in a 96- well plate at a density of 6 x 103/well and 4 x 104/well respectively. Cells were treated with different concentrations (0-100 M) of PK2 for 24 hrs. 4 hrs prior to the incubation 5 mg/mL of MTT dye was added. After completion of incubation, formazan crystals were solubilized in solubilization solution, and absorbance was measured at 570 nm and normalized with absorbance at 630 nm (Figure 7A and 7B).

Example 5: GLP-1R Internalization

HepG2 cells were seeded in 35mm plate in the density of 60-70% confluency and incubated overnight. Next day, cells were transfected with GLP-1R-GFP construct and after 4hrs transfection; reaction media was changed to complete media and incubated for 48hrs. GLP-1R transfected cells were trypsinized and seeded on cover slips. After that, cells were given a treatment of lOnM of Ex-4 (positive control) and PK2 (50mM) for lhr followed by fixation using formalin and subsequently cover slips were mounted on glass slide using DAPI containing mounding media. Internalization of GLP-1R was observed using Confocal Microscope by exciting laser at 488nm.

In another experiment, HepG2 cells were transfected with hGLP-lR-spark-GFP construct (Sino biologies)) using lipofectamine-3000 (Thermo Fisher Scientific). Transfected cells were treated with 20 nM of Ex-4 (positive control), PK2 (50 M), PK4 (50 M), PK5 (50 M), PK6 (50 M) and DMSO as vehicle for 1 hr. After completion of incubation, cells were washed with PBS and fixed using formalin. For antagonism of GLP-1R receptor 300 nM of Ex-9 was pre-treated for 15 min followed by compound (PK2-50 M) treatment as described above.

For cell surface labeling, cells were incubated with 1 mg/mL sulpho-NHS-biotin (Thermo Fisher Scientific#24510) in PBS for 30 mins at 4°C, washed with PBS containing 100 mM glycine and then fixed in 4% paraformaldehyde for 15 mins. Cells were blocked in 1% BSA in PBS for another 30 mins and then incubated in 5 g/mL of Texas-Red-avidin (Thermo Fisher Scientific) for 30 mins. After extensive washing with PBS, the coverslips were mounted, and internalization of hGLP-lR was observed using Nikon Confocal Microscope.

Example 6: PKA activity Assay

293A cells were transfected using lipofectamine-3000 with hGLP-lR-spark-GFP construct. After 48 hrs of transfection; cells were seeded at a density of 8.0 x 103 cells/well in 96-well plate. Next day, cells were treated with PK2 (50 M) and DMSO as a vehicle for 45 mins in serum-free media. PKA activity was measured as per manufacturer’s protocol (Thermo Fisher Scientific#EIAPKA).

Example 7: Oil Red O (ORO) Staining

HepG2 cells were grown at an initial density of 104 cells/well on coverslips and treated with 0.5 mM palmitate along with the vehicle as DMSO or with 25 M PK2 for 24 hrs. Control cells were treated with BSA. Cells were then washed with PBS and fixed with 4% paraformaldehyde for 15 mins. After fixation, cells were washed with PBS three times and stained with Oil Red O (ORO) solution 29 for 5-7 mins. Cells were washed with 40% isopropanol to remove unbound staining, and slides were mounted using DAPI mounting media (#vectashield). Images were captured using Nikon confocal microscopy using excitation wavelength of 630nm.

Example 8: Cell culture and differentiation of 3T3-L1

3T3-L1 fibroblasts were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 3.7g/L Sodium bicarbonate (NaHCCb), supplemented with 10% fetal bovine serum (Invitrogen, US origin), lOOU/ml penicillin, 100 g/ml streptomycin and lOmM HEPES at 37°C in a humidified atmosphere of 5% CO2.

3T3-L1 cells were seeded in 6- well plate and incubated for 48hrs to achieve high confluence. Differentiation was induced in confluent cells by replacing complete media with differentiation media consisting of 10pg/ml of insulin (Sigma), I mM dexamethasone (Sigma), and 0.5 mM 3-isobutyl-l-methylxanthine (IBMX, Sigma) in DMEM. After 2 days, cells were switched to Insulin media for another 2 days followed by switching to maintenance media with 50mM of PK2 for 6 days with media replacement every 24 h along with PK2. Cells treated with DMSO (0.5%) during maintenance were used as a control. Cells were harvested on 10th day using tri-reagent (#Sigma), RNA was extracted, and cDNA was prepared using iscript cDNA synthesis kit (BIO-RAD).

Example 9: Western Blot

Liver tissue samples/ treated cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors, and the lysate was clarified by centrifugation at 15,000 rpm for 20 mins at 4°C. The protein concentration of the lysate was measured using BCA protein assay (Thermo Fisher Scientific#23225). Sample buffer containing 2-mercaptoethanol was added to the total protein and heated at 95 °C for 5 mins. The whole-cell lysate protein was resolved via (4-10%) SDS-PAGE unit and blotted onto polyvinylidene difluoride membranes (PVDF# BIO-RAD). Membranes were blocked in 5% bovine serum albumin or skimmed milk, and specific proteins were detected by incubation with appropriate primary and secondary (BIO-RAD) (horseradish peroxidase-conjugated) antibodies in TBST containing 5% BSA. pCREB/CREB, p-MAPK/MAPK, pAMPK/AMPK, pACC/ACC, pAKT/AKT, pGSK3p/GSK3p, ChREBP, LaminA/C, a-tubulin and b-actin were purchased from Cell Signaling Technology and TXNIP from Novus Biologicals. Next day, blots were probed with appropriate secondary (horseradish peroxidase conjugated) antibodies for 2 hrs at room temperature and analyzed using Amersham Imager 680 blot and gel imager (#GE Healthcare Life Sciences).

Example 10: Pharmacokinetics and tissue distribution of PK2

Animal experiments were conducted in accordance with animal care and use committee-approved protocol at the CSIR-IITR (Indian Institute of Toxicology and Research) and Visva Bharati Santiniketan University, India. Mice were housed and maintained under a 12 hrs light/dark cycle at a temperature of 23 ± 2 °C.

Six to seven weeks old age BALB/c male mice were acclimated for 1 week before starting the experimental procedure. All animals were maintained on a control chow diet with free access to food and water. For GSIS, mice were starved for 6 hrs, PK2 were administered at the dose of 25 mg/Kg body weight in treated group and control group was ad-ministered with 0.25% carboxymethyl cellulose sodium salt (CMC) as vehicle. After lhr of treatment, Glucose was injected 2mg/Kg body weight, blood was collected from tail vein at 0, 10, 20, and 30 mins from each mouse.

Streptozotocin model: Six to seven weeks old age BALB/c male mice were acclimated for 1 week before starting the experimental procedure. All animals were maintained on a control chow diet with free access to food and water. Mice were divided into 4 groups wherein mice were subjected to different conditions, 1. Vehicle group receiving 0.25% CMC and citrate buffer (Control), 2. The group receiving STZ alongside 0.25% CMC (STZ), 3. Mice were infused with PK2 three days before STZ injection (Pre-PK2) and 4. Mice were injected with STZ and PK2 was administered one day after STZ injection (Post-PK2). Low-dose (65 mg/kg) STZ in citrate buffer pH-4.0 injected IP for 5 days after overnight fasting, and PK2 (25 mg/Kg) was administered orally over the course of 18 days, and then mice were sacrificed. The complete scheme is described schematically in Figure 10.

HFD (High fat diet) Model: Male Swiss-Albino outbred mice were randomized into three groups (5 animals/group). Group 1, NCD mice were fed with regular chow diet and administered 0.25% CMC-Na as a vehicle, Group 2, HFD mice, were fed with a HFD and administered with 0.25% CMC-Na. Group-3, HFD-PK2 mice were fed with a HFD and administered with PK2 orally (25 mg/Kg B.W.). Mice were treated with vehicle (0.25% CMC-Na) and PK2 (25 mg/Kg B.W.) on every alternate day for 60 days. During food intake study, mice were provided with a measured amount of food with free access to water. The amount of food taken per group was measured. To determine the food intake, the amount of food consumed was normalized with the body weight. For insulin tolerance test (ipITT) mice were fasted for 12 hrs and each mouse have injected insulin (0.50 U/kg B.W.) through i.p. and blood glucose level was measured at the indicated time points. After the completion of the study, animals were sacrificed, and tissue samples were collected which were snap-freezed and fixed in Bouin’s fixative overnight.

Balb/c male mice (5-6 weeks old, bodyweight 25 ± 2 g) were administered PK2 orally at an equivalent dose of 25 mg/kg at different time-points (0-24 hrs). Mice were sacrificed at different time points (5 min, 30 min, 1 hr, 2 hrs, 4 hrs, 8 hrs, 16 hrs, and 24 hrs) after PK2 administration. Plasma, liver, heart, kidneys, lungs, spleen, and pancreas samples were harvested and stored at -80°C until analyzed. Concentrations of PK2 in plasma and tissue were examined using reverse-phase HPLC. Briefly, acetonitrile: methanol (1 : 1) is added into 100 L of plasma and homogenized tissue samples, sample was sonicated, and vortexed for 2 min and debris were removed by centrifugation at 5000 rpm for 10 min. The organic layer was transferred to the fresh tubes and evaporated to dryness. The remaining residue was resuspended in 100 L of mobile phase and 20 L resuspended aliquot injection was boosted into the HPLC column. Chromatographic separation was achieved using an Agilent reverse-phase Cl 8-column (2.1 x 50 mm) at 28°C. The mobile phase comprised of 0.1% trifluoroacetic acid in water/acetonitrile (40:60) was eluted at a flow rate of 1 ml/min, and discharge was monitored at excitation 350 nm and emission of 497 nm using a fluorescence detector (FLD). The amount of PK2 in the samples was quantified by measurement of the peak area ratios of the PK2 using standard curve. The pharmacokinetic parameters including the area under the plasma concentration (AUC), half-life (T1/2), the apparent volume of distribution (Vd), systemic plasma clearance (CL), were determined by using standard methods.

Example 11: Immunohistochemistry

The tissues from each mouse were fixed in 4% paraformaldehyde and implanted in paraffin for sectioning. Paraffin sections were deparaffinized using xylene, washed with ethanol followed by rehydration with decreasing concentration of ethanol. The tissue slides were subjected to antigen retrieval using citrate buffer of pH-6.0 after then sections were incubated at 4°C for another 1 hr.

Sections were washed using PBS and then permeabilized using 0.1% TritonX-100. The sections were blocked in 5% horse serum in PBS at room temperature for 1 hr, followed by primary antibody overnight incubation with anti-insulin (1 :200) and anti-Ki67 (1 : 100) (Abeam) in 5% horse serum at 4°C. Sections were then washed with TBST for 3 x 5 mins and were incubated with the Alexa f uor secondary antibody (Jackson Immuno Research) at dilution of 1:500 at room temperature for 3 hrs. DAPI was used for staining the nuclei. Microscopic images were then captured using a Zeiss microscope (40□ objectives). The quantification was carried out with the ImageJ software.

Example 12: TUNEL assay

The immunofluorescent double staining of insulin and TUNEL were performed according to the standard procedures. In brief, the sections were incubated with primary antibody anti-insulin (1 :200). Sections were then incubated with Alexa fluor-conjugated secondary antibody (Jackson Immuno research) at 1 : 500 diluted in PBST at room temperature for 2 hrs in the dark. The sections were fixed using paraformaldehyde for 15 mins, and TUNEL signal was stained according to the manufacturer’s instructions (Promega). Microscopic images were then captured in Zeiss microscope using a 40 x objective.

Example 13: TXNIP Promoter activity assay

INS-1 cells were seeded in 35 mm dish at a confluence of 60-70%. Next day, cells were transfected with TXNIP promoter-luciferase construct (Addgene) for 48 hrs. After the transfection, cells were trypsinized and seeded in 96-well white plate. Cells were given a treatment of 50 M-PK2 and DMSO as a vehicle in control for 45 mins. After completion of incubation, steady glo reagent (Promega) was added to each well and incubated for another 10 mins. Luminescence was determined by using iTECAN multi-plate reader.

Example 14: Immunocytochemistry

HepG2 cells were seeded on coverslips and treated with 2.5 mM and 30 mM glucose for 18 hrs with or without PK2. Cells were washed with PBS, followed by fixation with 4% paraformaldehyde and permeabilized using PBST with 0.1% Tween-20. Cells were blocked in 2%

Fetal Bovine Serum (FBS) in PBST at room temperature (RT.) for 2 hrs, followed by primary antibody overnight incubation with ChREBP (1 :200#Novus). Cells were then washed with PBST for 3 x 10 min and were incubated with the Alexa fluor secondary antibody at room temperature for another 2 hrs. DAPI was used for the staining of nuclei. Microscopic images were then captured using a Zeiss microscope.

Quantification and Statistical Analysis

Data are presented as mean ± SEM. The analysis of significant differences between means using GraphPad Prism was determined using student’s t-test. Statistics are mentioned in the figure legends or brief description of figures, and a p-value of < 0.05 was considered statistically significant.

Results

Molecular docking for small molecule agonist of GLP-1R

Crystal structure of the ECD of GLP-1R (Figure 1A) and its interaction with its allosteric agonists have revealed an exciting opportunity for structure-based drug design. To discover non-peptidyl agonist of GLP-1R, structurally modified small molecules were screened with an in silico technique utilizing, AutoDock. The ligand-protein complexes of selected docked small molecule resulting from the docking were ranked according to their binding affinity (Table 1). The structure of selected five molecules having a high binding affinity is shown in Figure IB. The binding modes of the lowest binding conformer of best-ranked molecules were analyzed using the DSV, as shown in (Figure 1C).

Data revealed that PK2, PK3, and PK4 exhibit almost comparable binding modes and interactions while PK5; having a lowest binding affinity, array different interactions with the ECD of GLP-IR. All these three compounds, PK2, PK4, and PK5 develop p-p stacking with the residue Trp42, TRP87, Trp39, and Trp69. PK2 and PK4 show some conventional H-bond with the Glu45 and Arg40, Arg43 respectively while PK3 displays no H-bond formations.

Table 1: Comparison of binding energies of set of PK compounds with existing agonists



Synthesis

Preparation of 6,10b-dihydro-5aH-indolo[2,3-b]quinoxaline (1).

Yellow solid, M. P. = 264°C, 1H NMR (1) (DMSO-de, 500 MHz, d): 12.04 (br s, -NH, 1H), 8.33-8.35 (d, J = 7.55 Hz,IH), 8.24-8.25 (d, J = 8.25 Hz, 1H), 8.05-8.07 (d, J = 8.25 Hz, 1H), 7.77-7.805 (m, 1H), 7.68-7.72 (m, 2H), 7.57-7.59 (d, J = 8.25 Hz, 1H), 7.34-7.37 (t, J = 7.55 Hz, 1H) ppm.

13C NMR (125 MHz, DMSO-de, d): 145.83, 143.99, 140.13, 139.78, 138.58, 131.28, 129.04, 128.71, 127.47, 125.92, 122.24, 120.68, 118.97, 111.98ppm.

HRMS (ESI): Calc for C14H9N3 [M-H]-: 219.0796; Found: 218.0739.

Preparation of (6H-indolo[2,3-b]quinoxalin-2-yl)(phenyl) methanone (Compound 3):

Brick red solid, M.P: 190°C, HRMS (ESI): Calc for C21H13N3O [M-H] : 323.1059; Found: 322.0974.

Preparation of 6-(prop-2-yn-l-yl)-6H-indolo[2,3-b]quinoxaline (2):

Yellow solid, M. P. = Lit. 205-206°C; 1H NMR (2) (DMSO-de, 500 MHz, d): 8.47-8.49 (d, J=7.55Hz, 1H), 8.29-8.30 (dd, J= 8.25 Hz, 1H), 8.13-8.15 (dd, J= 8.25 Hz, 1H), 7.65-7.78 (m, 3H),

7.44-7.64 (d, J= 8.25Hz, 1H), 7.2-7.4 (t, J= 7.55 Hz, 1H), 5.29-5.30 (s, 2H), 2.30-2.31 (s,lH) ppm.

13C NMR (125 MHz, DMSO-de, d): 144.80, 143.48, 140.36, 140.11, 139.56, 131.06, 129.33, 128.95, 127.77, 126.32, 122.72, 121.56, 119.84, 110.12, 77.34, 72.64, 30.55ppm.

HRMS (ESI): Calc for C17H11N3 [M-H] : 257.0953; Found: 256.2342.

Yellow solid, M. P. = 180°C; 1H NMR (4) (CDC13, 500 MHz, d): 8.69 (s, 1H), 8.45-8.46 (d, J=7.55Hz, 1H), 8.30-8.32 (dd, J= 8.925 Hz, 1H), 8.23-8.24 (d, J= 8.95 Hz, 1H), 7.91-7.92 (m, 2H), 7.76-7.79 (m, 1H), 7.68-7.70 (d, J= 7.55, 1H), 7.62-7.65 (m, 1H), 7.53-7.56 (t, J= 7.55Hz, 2H),

7.45-7.48 (t, J= 7.55 Hz, 1H), 5.33 (s, 2H), 2.30-2.34 (s,lH) ppm.

13C NMR (125 MHz, CDCh, d): 196.11, 145.57, 143.67, 142.79, 141.33, 138.27, 137.72, 134.89, 133.22, 132.43, 131.65, 130.08, 129.01, 128.44, 128.27, 122.93, 122.11, 119.62, 110.44, 76.87, 72.96, 30.72 ppm.

HRMS (ESI): Calc for C24H15N3O [M+H]+: 361.1215; Found: 362.1288

Characterisation data of (PK2-PK5):

Yellow solid, M. P. = 232°C; 1H NMR (PK2) (DMSO-de, 500 MHz,d): 8.38-8.40 (d, J= 7.55Hz, 1H), 8.28-8.30 (dd, J = 8.25 Hz, 1H), 8.23 (s, 1H), 8.13-8.18 (m, 3H), 7.74-7.86 (m, 4H), 7.41-7.45 (m, 3H), 5.68 (s, 2H), 5.81 (s, 2H) ppm.

13C NMR (125 MHz, DMSO-de, d): 147.17, 144.80, 143.89, 143.36, 142.94, 139.91, 139.60, 138.86, 131.43, 129.14, 128.96, 127.59, 126.32, 124.07, 123.86, 122.24, 121.35, 118.79, 110.89,

51.85, 36.40ppm.

HRMS (ESI): Calc for C24H17N7O2 [M+H]+: 435.1444; Found: 436.1545

Yellow solid, M. P. = 216°C; 1H NMR (PK3) (DMSO-de, 500 MHz,d): 8.38-8.39 (d, J= 7.55Hz, 1H), 8.28-8.29 (d, J = 8.25 Hz, 1H), 8.13-8.14 (d, J=7.55, 2H), 7.83-7.86 (m, 1H), 7.73-7.79 (m,3H), 7.40-7.43 (m, 1H), 7.09-7.13 (m, 4H), 5.78 (s, 2H), 5.43 (s, 2H), 2.23 (s,3H) ppm.

13C NMR (125 MHz, DMSO-de, d): 144.81, 143.91, 139.91, 139.60, 138.85, 137.43, 132.95, 131.39, 129.28, 129.21, 129.13, 128.54, 127.90, 127.59, 126.29, 124.93, 123.46, 122.22, 121.31, 118.78, 110.90, 52.55, 20.67ppm.

HRMS (ESI): Calc for C25H20N6 [M+H]+: 404.1749; Found: 405.1819

Yellow solid, M. P. = 192.4-195°C; 1H NMR (PK4) (DMSO-de, 500 MHz, d): 8.38-8.39 (d, J= 7.55Hz, 1H), 8.28-8.29 (d, J = 8.25 Hz, 1H), 8.17 (s, 1H), 8.13-8.15 (m, 1H), 7.93-7.86 (m, 1H), 7.73-7.80 (m, 3H), 7.40-7.43 (t, J= 7.55Hz, 1H), 7.19-7.23 (t, J= 8.25Hz, 1H), 6.77-6.85 (m, 1H), 6.76-6.77 (m, 2H), 5.79 (s, 2H), 5.46 (s, 2H), 3.64 (s, 3H) ppm.

13C NMR (125 MHz, DMSO-de, d): 159.34, 144.82, 143.90, 142.74, 139.92, 139.60, 138.85, 137.42, 131.37, 129.83, 129.13, 127.58, 126.29, 123.69, 122.22, 121.31, 119.86, 118.79, 113.45, 110.88, 54.99, 52.65, 36.40 ppm.

HRMS (ESI): Calc for C25H20N6O [M+H]+: 420.1699; Found: 421.1798.

Yellow solid, M. P.-225°C; 1H NMR (PK5) (DMSO-de, 500 MHz,d): 8.52 (s,lH), 5.69 (s, 2H), 8.38-8.39 (d, J= 7.55Hz, 1H), 8.25-8.28 (m, 2H), 8.16-8.21 (m, 3H), 7.73-7.88 (m, 5H), 7.62-7.65 (t, J= 7.55 Hz, 2H), 7.44 -7.45 (d, J= 8.25Hz, 3H), 5.84 (s, 2H), 5.69 (s, 2H) ppm.

13C NMR (125 MHz, DMSO-de, d): 195.05, 147.17, 145.66, 144.18, 143.33, 142.64, 142.29, 141.07, 137.51, 137.19, 134.00, 132.76, 132.38, 132.01, 129.71, 128.96, 128.70, 128.53, 128.17, 124.19, 123.87, 122.54, 121.88, 118.59, 111.21, 51.88, 36.55 ppm.

HRMS (ESI): Calc for C31H21N7O [M+H]+: 539.1706; Found: 540.1774.

Internalization of GLP-1R: A ligand-binding assay

GLP-1R involved in conserved signaling events including receptor phosphorylation by GRKs (G protein-coupled receptor kinases), b-arrestin recruitment and receptor internalization. The GLP-1R is a GPCR B receptor, have property to internalize after activation by its cognate agonist. For determining the efficacy of synthesized compound, spark-GFP tagged human GFP-1R plasmid (hGFP-lR) was transfected in HepG2 cells. Cells were stimulated with 50 M concentration of each compound (PK2-PK5), alongside positive control group was receiving 20 nM concentration of Ex-4 and control group was treated with DMSO as vehicle for 1 hr. It can be observed from confocal microscopy images, compound PK2, PK3, and PK4 rapidly internalized GFP-1R receptor (Figure 8A). Nevertheless, PK5 appears unable to internalize GFP-IR.

These results suggest that GFP-IR internalization triggered by the compound PK2, PK3 and PK4 are due to the binding of these compound with the receptor domain which leads to activation of some signaling cascade and thereby receptor internalization. However, the inability of the PK5 to internalize the GFP-IR, may be because of either failure of PK5 to form p-p stacking or due to formation of unfavourable acceptor-acceptor interaction. By taking these results into

consideration, it can be concluded that the variation of any group at position-9 does not affect the binding position.

For further examination, considering the solubility criteria; PK2 having highest solubility of 1 Omg/ml in DMSO and have zero violation as drug-likeness (follow Lipinski, Veber, Ghose, Egan, Muegge rules), was chosen for in-vitro and in-vivo studies. Additionally, agonistic activity of PK2 for GLP-1R were further proved by utilizing GLP-1R antagonist, Ex-9 where, Ex-9 (300 nM) significantly (p = 0.04, *p < 0.05 ) block PK2 mediated GLP-1R internalization (Figure 8B).

Moreover, PK2 agonism was also validated by examining the downstream signaling. For verification full agonist activity of PK2, PKA activity in the presence of PK2 (50 mM) treatment was determined in hGLP-lR transfected cells. The outcome recommended (Figure 8C). a noteworthy increment (p = 0.0128, *p < 0.05) in PKA activity on treatment with PK2. Furthermore, PK2 (50 mM) treatment on HepG2 cells for 30 mins observed to have the ability to enhance (p= 0.0015, *p < 0.05) CREB phosphorylation at Seri 33 (Figure 8D-E). That suggests, PK2 binds and activate the GLP-1R and thereby activate PKA, which in turn induced CREB phosphorylation at Seri 33. These findings likewise demonstrate that PK2 has full agonistic activity on hGLP-lR.

In vivo pharmacokinetic and tissue distribution study in mice

In-silico and in-vitro observation indicates PK2 as a stable GLP-1R agonist, to further analyse the PK2 activity in mice model of diabetes, it is necessary to study PK2 distribution and pharmacokinetics in-vivo. Safe and the efficient dose of PK2 (25 mg/Kg) was selected based on dose dependent study (0.1, 1, 10, 25, 50 mg/kg body weight) in BALB/c mice. Furthermore, no abnormal behaviour or acute toxicity was observed in animals in the selected dose in 30 days of treatment. In-vivo pharmacokinetics studies demonstrated the mean plasma concentration versus time profiles of PK2 after a single oral administration of 25 mg/kg body weight (Figure 9A). Oral administration of PK2 resulted in fast absorption from gastrointestinal tract, detected 12.6 ng/mL in plasma (Co) and rapidly reached Tmax in 1 hr. PK2 shows a higher volume of distribution (VD) at steady state which was found to be 38.88 L which shows that it has greater binding with the tissue and plasma protein. However, upon oral administration, PK2 exhibited a plasma half-life of 4.8 hrs (T1/2) and 7.79 L/hr. of plasma clearance (CL), indicating rapid absorption with normal rate of clearance. Tissue distribution results of PK2 among the various tissues of mice after 2 hrs of oral administration are listed in Figure 9B. PK2 was distributed highly among the liver, kidney and pancreas with no traces in heart, lungs and spleen. A small amount of PK2 was detectable in brain which indicates that the PK2 may be able to cross the blood brain barrier.

PK2 administration increase GSIS

Mice were starved for 6 hrs, and Glucose stimulated insulin secretion levels were measured in Control and PK2 administered mouse groups, Figure 9B revels that mice administered with PK2 showed less increase in glucose on infusion of glucose through intraperitoneal route as compared to control mice. Moreover, PK2 administered mice showed prolonged insulin secretion as compared to control mice (Figure 9C). This data clearly indicates that PK2 significantly increases insulin on administration to glucose.

PK2 protects against STZ induced pancreatic b-cell apoptosis and dysfunction

To analyze the protective as well as reversal effect of PK2 on pancreatic b-cells in-vivo, mice were divided into four groups as mentioned in method sections. Blood glucose was measured on every alternate day and body weight was measured on random days till mice were sacrificed (18 days) (Figure 10). Improved plasma glucose levels were observed (p = 0.004, **p <0.05) in both the groups of mice receiving PK2 dosing (Figure IOC). To further explore the recovery of the blood glucose levels, serum insulin levels were detected from all experimental groups at the end of the study. It was observed from Figure 10D, that there was a significant reduction (p = 0.046, *p <0.05) in the serum insulin levels in STZ group nevertheless PK2 treated group indicating significantly (p = 0.02, *p <0.05) higher serum insulin levels. This indicates PK2 may either protect b-cell mass or enhance stored insulin secretion.

Weight of pancreas at the termination of study (Figure 11 A) revealed protection in pancreas weight by PK2. To examine effect of PK2 on islets mass H & E staining of pancreatic sections were carried out, a significant bigger islets area in mice receiving STZ-PK2 in contrast with STZ mice group, PK2 appears to protect mice from STZ induced damage to islets mass (Figure 1 IB). This data clearly implies, there is conservation of islets area in mice treated with PK2. Most importantly PK2 mediated enhanced islets mass are restricted to the endocrine pancreas, the exocrine part of pancreas was unaltered in PK2 treated mice group (Figure 11B). However, further prospective

studies are required to determine if any potential risk of developing pancreatic cancer is associated with long-term treatment with PK2.

In order to find out whether PK2 is protecting b-cells from STZ-mediated apoptosis or by enhancing b-cell proliferation or both (attenuating b-cell apoptosis and inducing b-cell proliferation simultaneously). TUNEL and Ki67/ insulin co-staining of pancreas section were performed.

TUNEL assay was performed to determine the apoptotic b-cell in the pancreatic sections from mice received 18 days of treatment. Figure 12A-B represents the number of TUNEL-positive cells, which is significantly higher (p = 0.01., *p < 0.05) in the pancreatic islets from streptozotocin-treated mice than those of the PK2 -treated mice.

Proliferating b-cells were identified by nuclear co-localization of proliferation marker Ki67, in insulin-positive islet cells (Figure 12C). The results (Figure 12C-D) showed that there was significant proliferation (p = 0.04, *p <0.05) in the pancreas of STZ treated mice. Moreover, PK2 showed more prominent effect in the enhancement of b-cell proliferation. Overall, these results confirm that, PK2 is capable of protecting pancreatic b-cell apoptosis and dysfunction induced by STZ in-vivo. It is the rationale to replenish lost b-cells in T1DM patients as obvious therapeutics. PK2 is mainly conferring its b-cell protecting effect by attenuating b-cell apoptosis and inducing b-cell proliferation this in turn increases b-cell mass and thereby conferring improves glycemic control.

PK2 treatment in diet induced obese mice reduces fat accumulation, improves fasting blood glucose and insulin sensitivity

To study the effect of PK2 on HFD-induced in vivo model, mice were divided in three groups. NCD group mice were fed with normal chow diet (NCD), HFD and HFD-PK2 were fed with HFD. PK2 at a dose of 25 mg/Kg body weight was administered orally from the first day of high-fat feeding in HFD-PK2 group for 60 days (Figure 13 A). The amount of food intake was measured and normalizes according to body weight which shows decrease in food intake of PK2 administered mice in comparison to HFD control (Figure 13B). It can be observed from the average body weight trend of each animal that there is a significant increase in the body weight of HFD

animals while mice receiving PK2 showed protection in HFD induced body weight gain (Figure 13C). Figure 13D clearly shows the increase in the adipose content in HFD mice as compared to HFD-PK2 mice. For quantitative analysis, the weight of adipose tissue was measured, which advises the exact fat gain in adipose of NC, HFD, and HFD-PK2 mice. Data signify the reduced-gain of fat amount in adipose of HFD-PK2 mice in comparison to HFD mice (Figure 13E). This result clearly showed that HFD-PK2 mice were resistant to HFD-induced obesity.

To investigate whether PK2 possesses its effect in glycemic control, on the 8th week of the treatment regimen, fasting blood glucose was measured. Figure 13F suggests there is a significant increase in fasting blood glucose levels of HFD mice as compared to NCD and interestingly there is reduction in fasting blood glucose levels of HFD-PK2 mice group as compared to HFD. This indicates that PK2 rescues high blood glucose levels in HFD induced mice models without causing hypoglycemia. Moreover, PK2 treatment significantly found to increase whole body insulin sensitivity and decreases the circulating concentrations of insulin that was found high in HFD mice group (Figure 13G). Decreased circulating insulin concentrations indicate improved insulin sensitivity that was also represented by insulin tolerance of HFD-PK2 mice (Figure 13H). Moreover, PK2 was also found to rescue hyperinsulinemia induced insulin resistance by activating Akt signaling (Figure 14). Taken together, the results of in vivo PK2 treatment indicate that PK2 have effects in decreasing food intake, fat accumulation and improvement of fasting glucose level, insulin sensitivity and resistant to diet-induced weight gain.

PK2 ameliorates HFD induced liver toxicity

To examine the effect of PK2 on liver toxicity and fat accumulation, circulating levels of triglyceride and cholesterol were analyzed; the results portrayed a significant increase in cholesterol and triglyceride levels in mice receiving HFD while PK2 treated mice shows reduced cholesterol and triglyceride contents (Figure 15A-B). Further, serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) were estimated, as ALT and AST are the most sensitive hepatic biomarkers which directly explain the extent of hepatic damage and toxicity. HFD-saline group had a significantly higher level of AST (Figure 15C) and ALT (Figure 15D) indicating impaired liver function. PK2 treatment significantly reduced HFD induced

pathologically elevated liver enzyme levels (ALT and AST). Furthermore, the histological analysis of liver using Hematoxylin and Eosin (H&E) staining revealed marked vacuolar degeneration in the 40x/100x HFD fed mice as compared with the NCD mice. This infers extreme fat accumulation within the livers of these mice. The white coloring and lipid droplets in hepatocytes were dramatically improved in HFD-induced mice after PK2 treatment compared with the HFD group (Figure 15E). These findings also seem consistent with the serum analytes for liver function assay. Taken together, these observations thereby indicate and present a protective role of PK2 by rescuing the HFD fed mice models from hepatotoxicity and fatty liver pathologies.

PK2 rescues HFD induced fatty liver pathologies through AMPK signaling axis

From all of the above experiments, it is clear that PK2 reduce initiation of HFD-induced fatty liver pathologies. To investigate the signaling pathway of PK2 in reducing the condition of NAFLD, impact of PK2 on hepatic AMPK axis was determined. Long acting peptidic GLP-1R agonists are well known to inhibit hepatic steatosis by activating AMPK. AMPK is an abg heterotrimer has a role in the inhibition of hepatic lipogenesis either by inhibiting acetyl-CoA carboxylase 1 and 2 (ACC1 and ACC2) by phosphorylating at residues Ser79/212 respectively. Western blot results of liver tissue of HFD mice suggests decrease in phosphorylation of AMPK while PK2 can significantly (<0.0001) stimulate AMPK activity by inducing phosphorylation at Thrl72 in HFD fed mice (Figure 15F).

To further analyze the downstream signaling, which of the axis of AMPK is activated upon PK2 treatment, ACC phosphorylation was determined. Figure 15F reveals that PK2 significantly ( 0.05) enhance ACC phosphorylation at Ser79 residue that is downstream to the AMPK pathway, thereby cause inhibition of DNL. This result confirms that PK2 shown its effect via activating AMPK signaling axis and inhibition of ACC by AMPK inhibits lipid accumulation in liver. Above data clearly indicates PK2 affects posttranslational modification to alter hepatic lipid flux towards oxidation, further the effect of PK2 treatment on hepatic lipogenic gene expression was examined. qPCR analysis of liver tissue samples revels PK2 treatment downregulates the transcription of lipogenic genes like, ACC and ChREBP (Figure 15G). Additionally, PK2 also ameliorate palmitate induced lipid accumulation in HepG2 cells as depicted by the ORO staining (Figure 16). These results taken together presents PK2 can activates AMPK, which phosphorylates ACC and

thereby lowering DNL and mitigating the enhanced hepatic lipid accumulation induced by HFD feeding.

PK2 treatment restricts ChREBP shuttling to cytoplasm

To further investigate the effect of PK2 in carbohydrate metabolism and how PK2 affects transcription level expression of lipogenic genes, translocation of ChREBP was examined. ChREBP, a glucose-responsive transcription factor is well known to enhance many lipogenic genes expression once translocated to the nucleus. PKA and AMPK are both known to inhibit ChREBP translocation to the nucleus and thereby inhibit its metabolic regulation. PK2, a GLP-1 agonist activates both PKA and AMPK, to determine the effect of PK2 on ChREBP translocation, HepG2 cells were induced with low (2.5 mM), high (30 mM) glucose and high glucose with PK2 (50 M) for 24 hrs. To determine the endogenous ChREBP localization, ICC was performed with ChREBP antibody. Figure 17 indicates that in the presence of cells induced with high glucose, translocate ChREBP to the nucleus while PK2 restricts the shuttling of ChREBP to nucleus. This data was supplementary authorized by Western blot after the same treatment profile; cells were harvested at the end of the study; cytosolic and nuclear fractions were extracted. Western blot results of the nuclear fraction of ChREBP indicates that on the induction of high glucose to HepG2 cells led to ChREBP translocation to the nucleus while PK2 treatment reverses the effect of high glucose by inhibiting shuttling of ChREBP to the nucleus (Figure 17B). All the above experiments indicate PK2 ability to reverse the lipid accumulation by inhibiting ChREBP shuttling to the nucleus.

In vivo results on two different models of diabetes represents, PK2 treatment protects mice from STZ-induced b-cell dysfunction mainly via protecting b-cells from apoptosis and inducing b-cell proliferation and as well protect NAFLD by restricting ChREBP to cytoplasm. Mechanistically, PK2 treatment inhibit TXNIP expression and thereby protects b-cell apoptosis. Moreover, PK2 treatment activates AMPK in liver and thereby inhibits lipid accumulation or NAFLD in mice. It is anticipated that the PK2 (orally active, non-peptidic GLP-1 R agonist) can be useful to human health and in diabetes treatment, where, maintaining a b-cell mass either by inducing b-cell replication and attenuate b-cell apoptosis could be a potential therapeutic in preventing the development of T1DM and T2DM. In addition, in an in vivo model (HFD obese mice), PK2 treatment ameliorates liver steatosis, hepatotoxicity, and confers protection against diet-induced obesity and improves glucose tolerance.

PK2 induce browning in 3T3-L1 adipocyte

In-vitro, 3T3- LI cells were differentiated in presence of PK2 (IOmM) and in presence of vehicle as DMSO. The cells were harvested and RNA were extracted from the control as well as PK2 treated group. qPCR data (Figure 18) from both control and treatment groups indicate that PK2 induce browning in 3T3-L1 adipocytes. This data revels PK2 can also be tested for in vivo as an anti-obesity drug.

ADVANTAGES

The present invention provides compounds and the preparations having significant applications in treatment of various diseases like diabetes, non-alcoholic fatty liver disease, cardiac dysfunction and obesity.