WO2020110152A1 - Agonistes non peptidiques du récepteur du peptide-1 de type glucagon et leur procédé de préparation - Google Patents

Agonistes non peptidiques du récepteur du peptide-1 de type glucagon et leur procédé de préparation Download PDF

Info

Publication number
WO2020110152A1
WO2020110152A1 PCT/IN2019/050874 IN2019050874W WO2020110152A1 WO 2020110152 A1 WO2020110152 A1 WO 2020110152A1 IN 2019050874 W IN2019050874 W IN 2019050874W WO 2020110152 A1 WO2020110152 A1 WO 2020110152A1
Authority
WO
WIPO (PCT)
Prior art keywords
unsubstituted
substituted
compound
mice
cells
Prior art date
Application number
PCT/IN2019/050874
Other languages
English (en)
Inventor
Khyati GIRDHAR
Pankaj GAUR
Prosenjit MONDAL
Original Assignee
Girdhar Khyati
Gaur Pankaj
Mondal Prosenjit
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Girdhar Khyati, Gaur Pankaj, Mondal Prosenjit filed Critical Girdhar Khyati
Publication of WO2020110152A1 publication Critical patent/WO2020110152A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism

Definitions

  • the present invention relates to small molecule Glucagon-like Peptide- 1 Receptor (GLP-1R) agonists and their method of preparation thereof.
  • GLP-1R Glucagon-like Peptide- 1 Receptor
  • 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.
  • Type 2 Diabetes T2DM
  • T2DM Type 2 Diabetes
  • GLP-1 Glucagon-Like Peptide- 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.
  • 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 .
  • DPP-4 ubiquitously expressed enzyme diaminopeptidyl peptidase IV
  • Exenatide synthetic analog of exendin-4, a hormone extracted from a lizard Gila Monster
  • Liraglutide a derivative of human GLP-1 derivatized at position- 26 with palmitic acid with a glutamic acid as spacer
  • These two peptidic-GLP-lR agonists have the distinct disadvantage of being administrated via subcutaneous route.
  • GLP-1 R ligands 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.
  • GLP-1 R agonist many synthetic small molecules have been reported as GLP-1 R agonist, but they have not been pharmacologically approved.
  • GLP-1R Glucagon-like Peptide- 1 Receptor
  • 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.
  • GLP-1R Glucagon-like Peptide- 1 Receptor
  • 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.
  • 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.
  • 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.
  • FIG. 10 shows PK2 protects mice from Streptozotocin (STZ).
  • STZ Streptozotocin
  • 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.
  • mice Streptozodocin-65mg/kg (STZ), and STZ+ PK2 (pre and post-treatment) mice groups on day of sacrifice.
  • STZ Streptozodocin-65mg/kg
  • 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.
  • FIG. 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.
  • 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.
  • FIG. 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
  • 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.
  • ChREBP Carbohydrate-response element-binding protein
  • 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.
  • FIG. 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.
  • 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.
  • GLP-1R Glucagon-like Peptide-1 Receptor
  • 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 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.
  • PK2 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.
  • the present invention provides compound of Formula I:
  • 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.
  • 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.
  • the compound of Formula I of the present invention is selected from:
  • the present invention provides a pharmaceutical composition comprising the compound of Formula I along with one or more pharmaceutically acceptable carriers or excipients.
  • the process for preparing the compound of formula I comprises the steps of:
  • step ( ii ) filtering the precipitate, triturating the product obtained from step (i) with a solvent to get the purified compound of formula 3;
  • 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).
  • THF Tetrahydrofuran
  • 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.
  • the present invention provides a kit comprising the non-peptidic compound of Formula I.
  • 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.
  • the compound of present invention has the following formula:
  • Example 1 Screening of small molecule using autodock
  • 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.
  • 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.
  • DMEM high Glucose; Invitrogen Dulbecco's modified Eagle's medium
  • fetal bovine serum Invitrogen, US origin
  • HepG2 and INS-1 cells were kindly provided by Dr. Debabrata Ghosh, CSIR- IIT Lucknow, India.
  • 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).
  • FBS fetal bovine serum
  • penicillin 100 U/ml
  • streptomycin 100 g/ml streptomycin
  • lmM sodium pyruvate 100 g/ml
  • lOmM HEPES 11 mM glucose
  • b-mercapto ethanol under standard cell culture conditions (humidified atmosphere, 5% CCh and 31° C).
  • HepG2 and INS-1 cells were seeded in a 96- well plate at a density of 6 x 10 3 /well and 4 x 10 4 /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).
  • 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.
  • 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.
  • 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.
  • sulpho-NHS-biotin Thermo Fisher Scientific#24510
  • 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).
  • HepG2 cells were grown at an initial density of 10 4 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.
  • DMEM Dulbecco's modified Eagle's medium
  • NaHCCb Sodium bicarbonate
  • penicillin 100 g/ml bovine serum
  • streptomycin 100 g/ml streptomycin
  • 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 10 th day using tri-reagent (#Sigma), RNA was extracted, and cDNA was prepared using iscript cDNA synthesis kit (BIO-RAD).
  • 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.
  • BIO-RAD primary and secondary antibodies
  • 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.
  • mice were housed and maintained under a 12 hrs light/dark cycle at a temperature of 23 ⁇ 2 °C.
  • mice 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 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.
  • CMC carboxymethyl cellulose sodium salt
  • Glucose was injected 2mg/Kg body weight, blood was collected from tail vein at 0, 10, 20, and 30 mins from each mouse.
  • mice 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).
  • 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.
  • 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.
  • mice 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.
  • 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).
  • FLD fluorescence detector
  • 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 (V d ), systemic plasma clearance (C L ), were determined by using standard methods.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PK2 binds and activate the GLP-1R and thereby activate PKA, which in turn induced CREB phosphorylation at Seri 33.
  • PK2 Oral administration of PK2 resulted in fast absorption from gastrointestinal tract, detected 12.6 ng/mL in plasma (Co) and rapidly reached T max in 1 hr.
  • PK2 shows a higher volume of distribution (V D ) at steady state which was found to be 38.88 L which shows that it has greater binding with the tissue and plasma protein.
  • V D volume of distribution
  • PK2 upon oral administration, PK2 exhibited a plasma half-life of 4.8 hrs (T1 / 2) and 7.79 L/hr. of plasma clearance (C L ), 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 protects against STZ induced pancreatic b-cell apoptosis and dysfunction
  • TUNEL assay was performed to determine the apoptotic b-cell in the pancreatic sections from mice received 18 days of treatment.
  • Proliferating b-cells were identified by nuclear co-localization of proliferation marker Ki67, in insulin-positive islet cells (Figure 12C).
  • PK2 showed more prominent effect in the enhancement of b-cell proliferation.
  • 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
  • mice were divided in three groups. NCD group mice were fed with normal chow diet (NCD), HFD and HFD-PK2 were fed with HFD.
  • NCD normal chow diet
  • HFD normal chow diet
  • HFD HFD
  • HFD-PK2 HFD-PK2
  • 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.
  • 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.
  • FIG. 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.
  • 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).
  • 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
  • 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.
  • H&E Hematoxylin and Eosin
  • PK2 reduce initiation of HFD-induced fatty liver pathologies.
  • 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).
  • 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
  • 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.
  • FIG. 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.
  • 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.
  • PK2 treatment inhibit TXNIP expression and thereby protects b-cell apoptosis.
  • PK2 treatment activates AMPK in liver and thereby inhibits lipid accumulation or NAFLD in mice.
  • PK2 orally active, non-peptidic GLP-1 R agonist
  • the PK2 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.
  • PK2 treatment ameliorates liver steatosis, hepatotoxicity, and confers protection against diet-induced obesity and improves glucose tolerance.
  • 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 reveals PK2 can also be tested for in vivo as an anti-obesity drug.
  • 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.

Abstract

La présente invention concerne de nouveaux composés qui se lient spécifiquement au GLP-1R et qui ont des effets bénéfiques en tant qu'agents thérapeutiques contre le diabète et d'autres maladies métaboliques. La présente invention concerne également le procédé de synthèse dudit nouveau composé et de sa capacité à se lier au GLP-1R.
PCT/IN2019/050874 2018-11-29 2019-11-29 Agonistes non peptidiques du récepteur du peptide-1 de type glucagon et leur procédé de préparation WO2020110152A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN201811045023 2018-11-29
IN201811045023 2018-11-29

Publications (1)

Publication Number Publication Date
WO2020110152A1 true WO2020110152A1 (fr) 2020-06-04

Family

ID=70853888

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2019/050874 WO2020110152A1 (fr) 2018-11-29 2019-11-29 Agonistes non peptidiques du récepteur du peptide-1 de type glucagon et leur procédé de préparation

Country Status (1)

Country Link
WO (1) WO2020110152A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115536638A (zh) * 2022-08-15 2022-12-30 上海交通大学 一种三氮唑类化合物及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011122815A2 (fr) * 2010-03-29 2011-10-06 Dong-A Pharm.Co.,Ltd. Nouveaux dérivés de quinoxaline

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011122815A2 (fr) * 2010-03-29 2011-10-06 Dong-A Pharm.Co.,Ltd. Nouveaux dérivés de quinoxaline

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 291, no. 20, 2016 *
ANA B. BUENO ET AL., POSITIVE ALLOSTERIC *
CHRIS DE GRAAF ET AL.: "Glucagon-Like Peptide-1 and Its Class B G Protein-Coupled Receptors: A Long March to Therapeutic Successes", PHARMACOLOGICAL REVIEWS, vol. 68, 2016, pages 954 - 1013, XP055712755 *
SREENIVAS AVULA ET AL.: "Synthesis and cytotoxicity of novel 6H-indolo[2,3-b]quinoxaline derivatives", MEDICINAL CHEMISTRY RESEARCH, vol. 22, 2013, pages 3712 - 3718, XP028370964 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115536638A (zh) * 2022-08-15 2022-12-30 上海交通大学 一种三氮唑类化合物及其应用
CN115536638B (zh) * 2022-08-15 2023-10-13 上海交通大学 一种三氮唑类化合物及其应用

Similar Documents

Publication Publication Date Title
US20210228599A1 (en) Farnesoid x receptor modulators
ES2874682T3 (es) Derivados de 3-desoxi y composiciones farmacéuticas de los mismos
ES2929829T3 (es) Alcoxipiridinil indolsulfonamidas sustituidas
KR20220151616A (ko) 에스트로겐 수용체-연관 질환의 치료 방법
US8008346B2 (en) Chemotherapeutic agents for inhibition of protein translation
Chen et al. Discovery of new LXRβ agonists as glioblastoma inhibitors
CN112656795A (zh) 防己诺林碱在抗结膜黑色素瘤中的作用机制及应用
WO2020110152A1 (fr) Agonistes non peptidiques du récepteur du peptide-1 de type glucagon et leur procédé de préparation
Wang et al. Gomisin D alleviates liver fibrosis through targeting PDGFRβ in hepatic stellate cells
WO2012126280A1 (fr) Composé rutaecarpine, son procédé de préparation et son utilisation
Xin et al. Structure-guided identification of novel dual-targeting estrogen receptor α degraders with aromatase inhibitory activity for the treatment of endocrine-resistant breast cancer
Gao et al. Design, synthesis and biological evaluation of novel bouchardatine analogs as potential inhibitors of adipogenesis/lipogenesis in 3T3-L1 adipocytes
CN106265620A (zh) 阿特匹灵c及其类似物在制备防治代谢性疾病的药物中的应用
AU2019366819A1 (en) Biomarkers of MetAP2 inhibitors and applications thereof
Chen et al. Design, synthesis and biological evaluation of novel 9-N-substituted-13-alkylberberine derivatives from Chinese medicine as anti-hepatocellular carcinoma agents
Shang et al. Schisandrin B, a potential GLP-1R agonist, exerts anti-diabetic effects by stimulating insulin secretion
Huo et al. Discovery of pyrimidine-bridged CA-4 CBSIs for the treatment of cervical cancer in combination with cisplatin with significantly reduced nephrotoxicity
EP3955922A1 (fr) Inhibiteurs à petites molécules de rcpg gpr68 et récepteurs associés pour le traitement du cancer, du glioblastome et d'autres indications
Zhu et al. Design, synthesis and triglyceride-lowering activity of tricyclic matrine derivatives for the intervention of non-alcoholic fatty liver disease
Obianom Novel WNT/β-Catenin Signaling Pathway Inhibitors for the Treatment of Metabolic Disorders
GB2599528A (en) Potassium salt crystal form B of phosphodiesterase type 5 inhibitor, and preparation method and use therefor
NZ714082B2 (en) 11-hydroxyl-derivatives of bile acids and amino acid conjugates thereof as farnesoid x receptor modulators

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19889229

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19889229

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 19889229

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 03.11.2022)

122 Ep: pct application non-entry in european phase

Ref document number: 19889229

Country of ref document: EP

Kind code of ref document: A1