GLP-1/GIP DUAL AGONISTS

Abstract

The present invention relates to long acting glucagon-like peptide-1 and human glucose-dependent insulinotropic polypeptide (GIP) dual agonist polypeptide which may be useful for treating type 2 diabetes mellitus (T2D), diabetes with obesity, obesity and hyperlipidemia.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof comprising an amino acid sequence: TABLE-US-00020 (SEQIDNO:3) Y-X1-E-G-T-F-T-S-D-Y-S-I-X2-L-D-K-I-A-Q-X3-A-F-V- Q-W-L-X4-A-G-G-P-S-S-G-A-P-P-P-S, wherein: X1 is Aib or (L)-norvaline; X2 is Leu or (L)-norvaline; X3 is Lys, wherein the side chain amino (-amino) group of the Lys is acylated with a moiety having the formula:
{-U-W-Y-Z wherein: U is C(O)CH.sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2NH}, wherein } is point of attachment to W; W is C(O)C (CH.sub.3).sub.2NH], wherein ] is point of attachment to Y; Y is C(O)(CH.sub.2).sub.2CH(COOH)NH, wherein is point of attachment to Z; and Z is C(O)(CH.sub.2).sub.nCOOH or C(O)(CH.sub.2).sub.nCH.sub.3, wherein n is an integer from 14 to 20; X4 is Ile; and the acid group of the C-terminal amino acid is a free carboxylic acid group or is amidated as a C-terminal primary amide.

2. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein X1 is Aib.

3. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein X1 is (L)-norvaline.

4. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein X2 is Leu.

5. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein X2 is (L)-norvaline.

6. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein X1 is Aib and X2 is Leu.

7. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein X1 is Aib and X2 is (L)-norvaline.

8. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein X1 is (L)-norvaline and X2 is Leu.

9. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein X1 is (L)-norvaline and X2 is (L)-norvaline.

10. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein Z is C(O)(CH.sub.2).sub.nCOOH and n is 16 or 18.

11. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 2, wherein Z is C(O)(CH.sub.2).sub.nCOOH and n is 16 or 18.

12. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 3, wherein Z is C(O)(CH.sub.2).sub.nCOOH and n is 16 or 18.

13. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 4, wherein Z is C(O)(CH.sub.2).sub.nCOOH and n is 16 or 18.

14. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 5, wherein Z is C(O)(CH.sub.2).sub.nCOOH and n is 16 or 18.

15. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 6, wherein Z is C(O)(CH.sub.2).sub.nCOOH and n is 16 or 18.

16. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 7, wherein Z is C(O)(CH.sub.2).sub.nCOOH and n is 16 or 18.

17. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 8, wherein Z is C(O)(CH.sub.2).sub.nCOOH and n is 16 or 18.

18. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 9, wherein Z is C(O)(CH.sub.2).sub.nCOOH and n is 16 or 18.

19. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, the polypeptide comprising an amino acid sequence selected from the group consisting of: TABLE-US-00021 (SEQIDNO:4) i.)TyrAibGluGlyThrPheThrSerAspTyrSer IleL-norvalineLeuAspLysIleAlaGlnLys AlaPheValGlnTrpLeuIleAlaGlyGlyPro SerSerGlyAlaProProProSer-NH.sub.2; (SEQIDNO:6) ii.)TyrAibGluGlyThrPheThrSerAspTyrSer IleLeuLeuAspLysIleAlaGlnLysAlaPhe ValGlnTrpLeuIleAlaGlyGlyProSerSer GlyAlaProProProSer-NH.sub.2; and (SEQIDNO:7) iii.)TyrL-norvalineGluGlyThrPheThrSerAsp TyrSerIleL-norvalineLeuAspLysIleAla GlnLysAlaPheValGlnTrpLeuIleAlaGly GlyProSerSerGlyAlaProProProSer-NH.sub.2.

20. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein {-U-W-Y-Z represents Moiety A or Moiety B, and wherein Moiety A and Moiety B have the following structures, respectively: TABLE-US-00022 Moiety A Moiety B

21. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein the C-terminal amino acid is amidated as a C-terminal primary amide.

22. The polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, wherein the C-terminal amino acid is a free carboxylic acid.

23. A polypeptide or a pharmaceutically acceptable salt thereof selected from the group consisting of: ##STR00052## wherein: Moiety A is ##STR00053## and Moiety B is ##STR00054##

24. A pharmaceutical composition comprising the polypeptide or the pharmaceutically acceptable salt thereof according to claim 1, and one or more of pharmaceutically acceptable excipient.

Description

EXAMPLES

[0209] Instruments and analytical methods: Instruments used for characterization and analysis of the compounds of the present invention are HPLC (Waters e2695 Alliance; Detector Waters (2489 UV/Visible)).

[0210] Mass instrument: HPLC: Waters e2695 Alliance; Detector: Acquity-QDa.

[0211] The final compounds of the present disclosure were purified by preparative HPLC procedure as outlined below:

[0212] Preparative HPLC: WATERS 2555 Quaternary gradient module (Max Total Flow: 300 mL/min, Max Pressure: 3000 psi) or Shimadzu LC-8A (Max Total Flow: 150 mL, Max Pressure: 30 Mpa), Column: Phenyl, 10 Flow: 75 mL/min

Mobile Phase:

TABLE-US-00008 For first For second For third purification purification purification Mobile pH 8.0 Phosphate 1% Acetic acid in pH 8.2 Ammonium Phase A buffer water formate buffer Mobile Acetonitrile 1% Acetic acid in Acetonitrile Phase B Acetonitrile:n- Propanol (50:50) Gradient 15 to 45% Mobile 20 to 50% Mobile 20 to 50% Mobile Phase-B in Phase-B in Phase-B in 300 min 250 min 250 min

[0213] The purity of the compounds of the present disclosure were analyzed by one of the RP-HPLC methods as outlined below:

HPLC Method A:

[0214] Column: Xbridge Peptide BEH C18 (4.6 mm250 mm, 3.5)

[0215] Eluent: Mobile Phase A: Buffer: Acetonitrile (900:100)

[0216] Mobile phase B: Buffer: Acetonitrile (300:700)

[0217] Buffer: Potassium dihydrogen orthophosphate in water, pH adjusted to 3.00.1 with orthophosphoric acid

[0218] Flow rate: 0.8 mL/min

[0219] Detection: UV detection at 210 nm

[0220] Column Temperature: 65 C.

[0221] Sample Tray temperature: 5 C.

[0222] Run Time: 90 min.

TABLE-US-00009 Time (min) Mobile Phase A % Mobile Phase B % 0 55 45 3 55 45 5 40 60 60 39 61 65 0 100 75 0 100 75.01 55 45 90 55 45

HPLC Method B:

[0223] Column: XSelect CSH C18 (4.6 mm150 mm, 2.5)

[0224] Eluent: Mobile Phase A: Buffer: Acetonitrile (900:100)

[0225] Mobile phase B: Buffer: Acetonitrile (300:700)

[0226] Buffer: Potassium dihydrogen orthophosphate in water, pH adjusted to 3.00.1 with orthophosphoric acid

[0227] Flow rate: 0.8 mL/min

[0228] Detection: UV detection at 210 nm

[0229] Column Temperature: 65 C.

[0230] Sample Tray temperature: 5 C.

[0231] Run Time: 90 min.

TABLE-US-00010 Time (min) Mobile Phase A % Mobile Phase B % 0 55 45 3 55 45 5 40 60 60 39 61 65 0 100 75 0 100 75.01 55 45 90 55 45

HPLC Method C:

[0232] Column: Xbridge Peptide BEH C18 (4.6 mm250 mm, 3.5) Eluent: Mobile Phase A: Buffer: Acetonitrile (900:100)

[0233] Mobile phase B: Buffer: Acetonitrile (300:700)

[0234] Buffer: Potassium dihydrogen orthophosphate in water, pH adjusted to 3.00.1 with orthophosphoric acid

[0235] Flow rate: 1.0 mL/min

[0236] Detection: UV detection at 210 nm

[0237] Column Temperature: 65 C.

[0238] Sample Tray temperature: 5 C.

[0239] Run Time: 60 min.

TABLE-US-00011 Time (min) Mobile Phase A % Mobile Phase B % 0 55 45 2 41 59 50 40 60 51 55 45 60 55 45

HPLC Method D:

[0240] Column: XSelect CSH C18 (4.6 mm150 mm, 2.5)

[0241] Eluent: Mobile Phase A: Buffer: Acetonitrile (900:100)

[0242] Mobile phase B: Buffer: Acetonitrile (300:700)

[0243] Buffer: Potassium dihydrogen orthophosphate in water, Added Triethylamine and pH adjusted to 2.50.1 with orthophosphoric acid

[0244] Flow rate: 0.5 mL/min

[0245] Detection: UV detection at 214 nm

[0246] Column Temperature: 60 C.

[0247] Sample Tray temperature: 10 C.

[0248] Run Time: 90 min.

TABLE-US-00012 Time (min) Mobile Phase A % Mobile Phase B % 0 55 45 6 55 45 10 40 60 80 39 61 80.1 0 100 85 0 100 85.1 55 45 90 55 45

METHOD OF PREPARATION

Example 1

Preparation of 2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl) amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid (Moiety A-di-tert-butyl ester)

##STR00046##

[0249] Moiety A-di-tert-butyl ester was prepared using solid phase synthesis using 2-chlorotrityl chloride resin. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to 2-chlorotrityl chloride resin in presence of DIPEA to yield 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc protecting group was removed by selective de-blocking of amino group using piperidine followed by coupling with Fmoc-Aib-OH in THF using DIPC and HOBt which yielded 2-[2-[2-[(2-Fmoc-amino-2-methyl-propanoyl)amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group was removed by selective de-blocking using piperidine and the free amino group was then coupled with Fmoc-Glu-OtBu using HOBt and DIPC to yield 2-[2-[2-[[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group of the resultant compound was selectively de-blocked using piperidine and the free amino group was then coupled with 20-(tert-butoxy)-20-oxo-icosanoic acid to give 2-[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. This intermediate was then cleaved from 2-Cl-Trt-Resin using trifluoroethanol: DCM (1:1) to obtain 2-[2-[2-[[2-[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl) amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid (Moiety A-di-tert-butyl ester). (LCMS=m/z: 814.10 (M+H.sup.+)).

Example 2

Preparation of 2-[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid

##STR00047##

[0250] Moiety B-di-tert-butyl ester was prepared using solid phase synthesis using 2-chlorotrityl chloride resin. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to 2-chlorotrityl chloride resin in presence of DIPEA to yield 2-[2-(2-Fmoc-aminoethoxy) ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc protecting group was removed by selective de-blocking of amino group using piperidine followed by coupling with Fmoc-Aib-OH in THF using DIPC and HOBt which yielded 2-[2-[2-[(2-Fmoc-amino-2-methyl-propanoyl)amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group was removed by selective de-blocking using piperidine and the free amino group was coupled with Fmoc-Glu-OtBu using HOBt and DIPC to yield 2-[2-[2-[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc group of the resultant compound was selectively de-blocked using piperidine and the free amino group was then coupled with octadecanedioic acid mono tert butyl ester to give 2-[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]-amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The intermediate was then cleaved from 2-Cl-Trt-Resin using trifluoroethanol: DCM (1:1) to obtain 2-[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl) amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid (Moiety B-di-tert-butyl ester). (LCMS=m/z: 786.39 (M+H.sup.+)).

Example 3

Preparation of 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (Moiety C-di-tert-butyl ester)

##STR00048##

[0251] Moiety C-di-tert-butyl ester was prepared using solid phase synthesis using 2-chlorotrityl chloride resin. 2-[2-(2-Fmoc-aminoethoxy) ethoxy]acetic acid was attached to 2-chlorotrityl chloride resin in presence of DIPEA to yield 2-[2-(2-Fmoc-aminoethoxy) ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc protecting group was removed by selective de-blocking of amino group using piperidine followed by coupling with 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid in THE using DIPC and HOBt which yielded {(Fmoc-amino-ethoxy)-ethoxy}-acetyl-{(-amino-ethoxy)-ethoxy}-acetic acid-2-Cl-Trt-Resin. The

[0252] Fmoc group was removed by selective de-blocking using piperidine and the free amino group was coupled with Fmoc-Glu-OtBu using HOBt and DIPC to yield Fmoc-Glu({(amino-ethoxy)-ethoxy}-acetyl-{(-amino-ethoxy)-ethoxy}-acetic acid-2-Cl-Trt-Resin)-OtBu. The Fmoc group of the resultant compound was selectively de-blocked using piperidine and the free amino group was then coupled with octadecanedioic acid mono tert butyl ester to give 2-[2-[2-[[2-[2-[2-[5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The intermediate was then cleaved from 2-Cl-Trt-Resin using trifluoroethanol: DCM (1:1) to obtain 2-[2-[2-[[2-[2-[2[[5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (Moiety C-di-tert-butyl ester) (LCMS=m/z: 846.10 (M+H.sup.30 )).

Example 4

Preparation of 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid.

(Moiety D-di-tert-butyl Ester)

##STR00049##

[0253] Moiety D-di-tert-butyl ester was prepared using solid phase synthesis using 2-chlorotrityl chloride resin as schematically represented below. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to 2-chlorotrityl chloride resin in presence of DIPEA to yield 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-Resin. The Fmoc protecting group was removed by selective de-blocking of amino group using piperidine followed by coupling with 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid in THF using DIPC and HOBt which yielded {(Fmoc-amino-ethoxy)-ethoxy}-acetyl-{(-amino-ethoxy)-ethoxy}-acetic acid-2-Cl-Trt-Resin The Fmoc group was removed by selective de-blocking using piperidine and the free amino group was coupled with Fmoc-Glu-OtBu using HOBt and DIPC to yield Fmoc-Glu ({(amino-ethoxy)-ethoxy}-acetyl-{(-amino-ethoxy)-ethoxy}-acetic acid-2-Cl-Trt-Resin)-OtBu The Fmoc group of the resultant compound was selectively de-blocked using piperidine and the free amino group was then coupled with 20-(tert-Butoxy)-20-oxoicosanoic acid to give 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl) amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-Resin. The intermediate was then cleaved from 2-Cl-Trt-Resin using trifluoroethanol: DCM (1:1) to obtain 2-[2-[2-[[2-[2-[2-[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl) amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (Moiety D-di-tert-butyl ester) (LCMS=m/z: 874.15 (M+H.sup.+)).

Example 5

Preparation of Compound 1

[0254] The parent peptide was synthesized by solid-phase method. The starting resin used for synthesis was Fmoc-Rink amide resin. Selective de-blocking of Fmoc protected amino group of rink amide resin was carried out using piperidine to yield Rink amide resin which was then coupled with Fmoc-Ser(tBu)-OH to yield Fmoc-Ser(tBu)-Rink amide Resin. This coupling reaction was performed by using diisopropylcarbodiimide. N-hydroxy benzotriazole (DIPC-HOBt) as coupling reagent. This completed one cycle. Acetic anhydride and diisopropylethyl amine/pyridine was used to terminate/cap the uncoupled amino groups at every amino acid coupling. Selective de-blocking of the amino group of Fmoc-Ser (tBu)-Rink amide Resin was done using piperidine. Then coupling with Fmoc-Pro-OH using HOBt and DIPC yielded Fmoc-Pro-Ser(tBu)-rink amide Resin. This completed the second cycle. Acetic anhydride and diisopropylethyl amine/pyridine were used to terminate the uncoupled amino groups at every amino acid coupling.

[0255] The above three steps, i.e., selective Capping. deblocking of Fmoc-protection of amino acid attached to the resin and coupling of next amino acid residue in sequence with Fmoc-protected amino group, were repeated for the remaining 37 amino acid residues. The selective deblocking, i.e., capping of uncoupled amino group done by using acetic anhydride and diisopropylethylamine/pyridine. deprotection of Fmoc group was done using piperidine and coupling with next Fmoc protected amino acid was done using HOBt/DIPC. The side chain of the Fmoc-protected amino acids were protected orthogonally, e.g., hydroxyl group of Serine. Tyrosine or Threonine were protected with tert-butyl(-tBu) group, amino group of Lysine was protected with tert-butyloxycarbonyl(-Boc) and (4.4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl(IVDde) group respectively and carboxylic acid groups of aspartic acid or glutamic acid were protected with-tBu group and amide group of glutamine was protected with trityl (-Trt) group. The above mentioned three steps. i.e., selective capping, deblocking and then coupling with next Fmoc protected amino acid, were performed to get Fmoc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-[L-norvaline]-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin.

[0256] De-blocking of Fmoc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-[L-norvaline]-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln Trt)-Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin was carried out using piperidine followed by Boc protection of peptide resin using Boc anhydride to yield Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-[L-norvaline]-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin. De-protection of the IVDde group of the peptide resin was carried out using hydrazine hydrate and then it was coupled with Moiety A-di-tert-butyl ester using diisopropylcarbodiimide, N-hydroxy benzotriazole (DIPC-HOBt) as coupling reagent to yield an intermediate compound resin, Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-[L-norvaline]-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(NH-Moiety A di-tert-butyl ester)-Ala-Phe-Val-Gln (Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin, which on cleavage and de-protection using trifluoroacetic acid with ethane-1,2-dithiol and triisopropylsilane followed by purification through preparative HPLC resulted in Compound 1.

[0257] Mass (LCMS): m/z=1192.7 (MH.sub.4.sup.4+), Calculated Mass=4766.77: HPLC Purity (Method B): 98.60%, RT=33.8 min.

Example 6

Synthesis of Compound 2

[0258] Compound 2 was prepared by solid phase method as per the analogous process given for Example 5, wherein, Fmoc-[L-norvaline]-OH was used at position 2 instead of Fmoc-Aib-OH and Fmoc-Aib-OH was used at position 13th instead of Fmoc-[L-norvaline]-OH to get Boc-Tyr(tBu)-[L-norvaline]-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu) -Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe -Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin.

[0259] Then coupling with Moiety A-di-tert-butyl ester followed by cleavage, de-protection and preparative HPLC purification as per Example 5 resulted in Compound 2. Mass (LCMS): m/z=1192.6 (MH.sub.4.sup.4+), Calculated Mass=4766.4; HPLC Purity (Method B): 96.09%, RT=25.6 min.

Example 7

Synthesis of Compound 3

[0260] Compound 3 was prepared by solid phase method as per the analogous process given for Example 5, wherein Fmoc-Leu-OH was used at position 13 instead of Fmoc-[L-norvaline]-OH to get Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu) -Ser(tBu)-Ile-Leu-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe -Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin.

[0261] Then coupling with Moiety A-di-tert-butyl ester followed by cleavage, de-protection and preparative HPLC purification as per Example 5 resulted in Compound 3.

[0262] Mass (LCMS): m/z=1196.1 (MH.sub.4.sup.4+), Calculated Mass=4780.4: HPLC Purity (Method A): 95.27%, RT=39.1 min.

Example 8

Synthesis of Compound 4

[0263] Compound 4 was prepared by solid phase method as per the analogous process given for Example 5, wherein Fmoc-[L-norvaline]-OH was used at position 2 instead of Fmoc-Aib-OH to get Boc-Tyr (tBu)-[L-norvaline]-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu) -Tyr(tBu)-Ser(tBu)-Ile-[L-norvaline]-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt) -Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu) -Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin.

[0264] Then coupling with Moiety A-di-tert-butyl ester followed by cleavage, de-protection and preparative HPLC purification as per Example 5 resulted in compound 4.

[0265] Mass (LCMS): m/z=1196.32 (MH.sub.4.sup.4+), Calculated Mass=4781.25; HPLC Purity (Method A): 94.21%, RT=29.8 min.

Example 9

Synthesis of Compound 5

[0266] De-protection of IVDde group of peptide resin: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu) -Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-[L-norvaline]-Leu-Asp(OtBu) -Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala -Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin (prepared as per Example 5) was carried out using hydrazine hydrate and then it was coupled with Moiety C-di-tert-butyl ester using diisopropylcarbodiimide, N-hydroxy benzotriazole (DIPC-HOBt) as coupling reagent to yield an intermediate compound resin, Boc-Tyr(tBu)-Aib-Glu(OtBu) -Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-[L-norvaline]-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(NH-Moiety C di-tert-butyl ester)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro -Pro-Pro-Ser(tBu)-resin, which on cleavage and de-protection using trifluoroacetic acid with ethane-1,2-dithiol and triisopropylsilane followed by purification through preparative HPLC resulted in Compound 5.

[0267] Mass (LCMS): m/z=1600.80 (MH.sub.3.sup.3+), Calculated Mass=4799.376; HPLC Purity (Method A): 98.64%, RT=15.9 min.

Example 10

Synthesis of Compound 6

[0268] Compound 6 was prepared by solid phase method as per the analogous process given for Example 9, wherein coupling with Moiety D-di-tert-butyl ester was carried out, followed by cleavage, de protection and preparative HPLC purification as per Example 9 resulted in compound 6.

[0269] Mass (LCMS): m/z=1609.98 (MH.sub.3.sup.3+), Calculated Mass=4826.916:

[0270] HPLC Purity (Method A): 96.31%, RT=26.7 min.

Example 11

Synthesis of Compound 7

[0271] Compound 7 was prepared by solid phase method as per the analogous process given for Example 9, wherein coupling with Moiety B-di-tert-butyl ester carried out, followed by cleavage, de protection and preparative HPLC purification as per Example 9 resulted in compound 7.

[0272] Mass (LCMS): m/z=1580.64 (MH.sub.3.sup.3+), Calculated Mass=4738.896;

[0273] HPLC Purity (Method A): 98.43%, RT=18.5 min.

Example 12

Synthesis of Compound 8

[0274] The parent peptide was synthesized by solid-phase method. The starting resin used for the synthesis was Fmoc-Rink amide resin. Selectively de-blocking of Fmoc protected amino group of rink amide resin using piperidine followed by coupling with Fmoc-Lys (IVDde)-OH with the Rink amide resin. The coupling was performed by using DIPC-HOBt to yield Fmoc-Lys (IVDde)-Rink amide Resin, this completed one cycle. Acetic anhydride and diisopropylethyl amine/pyridine was used to terminate/cap the uncoupled amino groups at the end of every amino acid coupling. Selective de-blocking Fmoc of amino group of Fmoc-Lys(IVDde)-Rink amide Resin using piperidine, Then coupling with second amino acid i.e., Fmoc-Ser (tBu)-OH using HOBt and DIPC yielded Fmoc-Ser (tBu)-Lys (IVDde)-rink amide Resin. This completed the second cycle. As stated earlier acetic anhydride and diisopropylethyl amine/pyridine was used to terminate the uncoupled amino groups [Capping] after each amino acid coupling.

[0275] The above three steps, i.e., deblocking of Fmoc-protection of amino acid attached to the resin, coupling of next amino acid residue in sequence with Fmoc-protected amino group and selective Capping, were repeated for the remaining 38 amino acid residues. The side chain of the Fmoc-protected amino acids used were protected orthogonally, e.g., hydroxyl group of Serine. Tyrosine or Threonine were protected with tert-butyl (-tBu) group. amino group of Lysine was protected with tert-butyloxycarbonyl (-Boc) and (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (IVDde) group respectively and carboxylic acid groups of aspartic acid or glutamic acid were protected with-tBu group, amide group of glutamine and asparagine was protected with trityl (-Trt) group. The above mentioned three steps, i.e., selective capping, deblocking and then coupling with next Fmoc protected amino acid were performed to get Fmoc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu) -Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-L-Norvaline-Leu-Glu (OtBu)-Lys(Boc)-Ile-Ala -Ala-Gln(Trt)-Glu(OtBu)-Phe-Val-Asn(Trt)-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu) -Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(IVDde)-Rink amide resin.

[0276] De-blocking of Fmoc group from Fmoc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu) -Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-L-Norvaline-Leu-Glu(OtBu)-Lys(Boc) -Ile-Ala-Ala-Gln(Trt)-Glu(OtBu)-Phe-Val-Asn(Trt)-Trp-Leu-Leu-Ala-Gly-Gly-Pro -Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(IVDde)-Rink amide resin was done using piperidine followed by Boc protection of Peptide resin using Boc anhydride to yield Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu) -Ser(tBu)-Ile-L-Norvaline-Leu-Glu(OtBu)-Lys(Boc)-Ile-Ala-Ala-Gln(Trt)-Glu(OtBu) -Phe-Val-Asn(Trt)-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro -Pro-Pro-Ser(tBu)-Lys(IVDde)-Rink amide resin.

[0277] De-protection of the IVDde group of peptide resin using Hydrazine hydrate followed by coupling of moiety-A-di-tert butyl ester was performed using diisopropylcarbodiimide, N-hydroxy benzotriazole (DIPC-HOBt) as the coupling reagent to yield protected compound 8-resin.

[0278] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu) -Ile-L-Norvaline-Leu-Glu(OtBu)-Lys(Boc)-Ile-Ala-Ala-Gln(Trt)-Glu(OtBu)-Phe-Val -Asn(Trt)-Trp-Leu-Leu-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu) -Lys(NH moiety A-di-tert butyl ester)-Rink amide resin cleavage and de-protection using trifluoroacetic acid with ethane-1,2-dithiol and triisopropylsilane followed by purification through preparative HPLC resulted in Compound 8. The HPLC purity of Compound 8 was assessed by Method given below Mass (LCMS): m/z=980.42 (MH.sub.5.sup.5+), Calculated Mass=4897.06 HPLC Purity (Method D): 94.55%. RT=44.9 min.

Example 13

Synthesis of Compound 9

[0279] The parent peptide was synthesized by solid-phase method. The starting resin used for synthesis was Wang resin. Fmoc protected Arg(Pbf) was used for coupling with the Wang resin. The coupling was performed by using diisopropylcarbodiimide, N-hydroxybenzotriazole (DIC-HOBt) as coupling reagent in presence of 4-dimethylaminopyridine (DMAP) which yielded Fmoc-Arg(Pbf)-Wang Resin. Selective de-blocking of amino group of Fmoc-Arg(Pbf)-Wang Resin using piperidine followed by coupling with Fmoc-Ser(tBu)-OH using HOBt/DIPC yielded Fmoc-Ser(tBu)-Arg(Pbf)-Wang Resin. This completed one cycle. Acetic anhydride and diisopropylethyl amine/pyridine were used to terminate the uncoupled amino groups at every amino acid coupling.

[0280] The above two steps, i.e., selective deblocking of Fmoc-protection of amino acid attached to the resin and coupling of next amino acid residue in sequence with Fmoc-protected amino group were repeated for the remaining 38 amino acid residues. The side chain of the Fmoc-protected amino acids were protected orthogonally, e.g., hydroxyl group of Serine, Tyrosine or Threonine were protected with tert-butyl(-tBu) group, amino group of Lysine was protected with tert-butyloxycarbonyl(-Boc) and (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (IVDde) group respectively and carboxylic acid groups of aspartic acid or glutamic acid were protected with-tBu group. The above mentioned three steps, i.e., selective capping, deblocking and then coupling with next Fmoc protected amino acid were performed to get Fmoc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu) -Tyr(tBu)-Ser(tBu)-Ile-L-Norvaline-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt) -Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu) -Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Arg(Pbf)-Wang resin.

[0281] De-blocking of Fmoc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu) -Tyr(tBu)-Ser(tBu)-Ile-L-Norvaline-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt) -Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu) -Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Arg(Pbf)-Wang resin, using piperidine followed by Boc protection of Peptide resin using Boc anhydride to yield Boc-Tyr(tBu)-Aib-Glu(OtBu) -Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-L-Norvaline -Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe-Val-Gln(Trt) -Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Arg(Pbf) -Wang resin. De-protection of IVDde group of peptide resin using Hydrazine hydrate followed by coupling of moiety A-di-tert butyl ester was performed by using diisopropylcarbodiimide, N-hydroxy benzotriazole (DIPC-HOBt) as coupling reagent to yield Compound 9-Wang resin.

[0282] Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu) -Ile-L-Norvaline-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(NH moiety A-di-tert butyl ester)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly -Ala-Pro-Pro-Pro-Ser(tBu)-Arg(Pbf)-Wang resin.

[0283] Cleavage and de-protection from resin using trifluoroacetic acid with ethane-1,2-dithiol, triisopropylsilane followed by purification through preparative HPLC resulted in Compound 9. Mass (LCMS): m/z=985.88 (MH.sub.5.sup.5+), Calculated Mass=4924.36 HPLC Purity (Method C): 93.52%, RT=27.8 min.

Example 14

Synthesis of Compound 10

[0284] Compound 10 was prepared by solid phase method as per the analogous process given for Example 5, wherein, Fmoc-[2-amino butyric acid] was used at position 13 instead of Fmoc-[L-norvaline]-OH to get Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu) -Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-2-amino butyric acid-Leu-Asp(OtBu)-Lys(Boc) -Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro -Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin.

[0285] Then coupling with Moiety A-di-tert-butyl ester followed by cleavage, de-protection and preparative HPLC purification as per Example 5 resulted in Compound 10.

[0286] Mass (LCMS): m/z=1189.57 (MH.sub.4.sup.4+), Calculated Mass=4754.248; HPLC Purity (Method D): 96.79%, RT=42.6 min.

Example 15

Synthesis of Compound 11

[0287] Compound 11 was prepared by solid phase method as per the analogous process given for Example 5, wherein, Fmoc-norleucine was used at position 13 instead of Fmoc-[L-norvaline]-OH to get Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu) -Tyr(tBu)-Ser(tBu)-Ile-Norleucine-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde) -Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala -Pro-Pro-Pro-Ser(tBu)-resin.

[0288] Then coupling with Moiety A-di-tert-butyl ester followed by cleavage, de-protection and preparative HPLC purification as per Example 5 resulted in Compound 11. Mass (LCMS): m/z=1196.66 (MH.sub.4.sup.4+), Calculated Mass=4782.608: HPLC Purity (Method D): 95.43%, RT=58.7 min.

Example 16

Synthesis of Compound 12

[0289] Compound 12 was prepared by solid phase method as per the analogous process given for Example 5, wherein, Fmoc-Ile-OH was used at position 13 instead of Fmoc-[L-norvaline]-OH to get Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu) -Tyr(tBu)-Ser(tBu)-Ile-Ile-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde) -Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin.

[0290] Then coupling with Moiety A-di-tert-butyl ester followed by cleavage, de-protection and preparative HPLC purification as per Example 5 resulted in Compound 12. Mass (LCMS): m/z=1196.32 (MH.sub.4.sup.4+), Calculated Mass=4781.25: HPLC Purity (Method D): 94.38%, RT=47.7 min.

BIOLOGICAL STUDIES

Example 12

Efficacy Study in db/db Mice at 10 nM/kg Dose

[0291] The effect of compounds of present invention on blood glucose, food intake and body weight was studied in mice. This study was performed in a type 2 diabetic mouse (db/db) model. The animals were divided into 4 treatment groups (n=6)a diabetic control group,

[0292] Compound 1 (10 nM/kg), Compound 2 (10 nM/kg) and Tirzepatide (10 nM/kg). Baseline blood glucose was measured from all the animals. All the animals were administered with test items subcutaneously. Blood glucose was measured at 4 hr, 8 hr, 12 hr, 24 hr, 48 hr, 72 hr and 96 hr post treatment. Delta blood glucose (mM) was calculated. The results are provided in Table 3. Similarly, body weight changes and cumulative food consumption was measured at 48 hr and 96 hr post treatment. The results of body weight changes are provided in Table 4 and cumulative food consumption are provided in Table 5. Similarly, the efficacy of Compounds 3, 4, 5, 6 and 7 in db/db mice at 10 nM/kg dose was carried out in separate study. The animals were divided into 7 treatment groups (n=6)a diabetic control group, Compound 1, Compound 3, Compound 4, Compound 5, Compound 6 and Compound 7.

[0293] Baseline blood glucose was measured from all the animals. All the animals were administered with test items subcutaneously. Blood glucose was measured at 4 hr, 8 hr, 12 hr, 24 hr, 48 hr, 72 hr and 96 hr post treatment. Delta blood glucose (mM) was calculated. The results are also provided in Table 3. Similarly, body weight changes and cumulative food consumption was measured at 48 hr and 96 hr post treatment. The results of body weight changes are also provided in Table 4 and cumulative food consumption are also provided in Table 5. Similarly, the efficacy of Compound 8 and 9 in db/db mice at 10 nM/kg dose were carried out in a separate study. The animals were divided into 3 treatment groups (n=5)a diabetic control group, Compound 8 and Compound 9. Baseline blood glucose was measured from all the animals. All the animals were administered with test items subcutaneously. Blood glucose was measured at 4 hr, 8 hr, 12 hr, 24 hr, 48 hr, 72 hr and 96 hr post treatment. Delta blood glucose (mM) was calculated. The results are provided in Table 3. Similarly, body weight changes and cumulative food consumption was measured at 48 hr and 96 hr post treatment. The results of body weight changes are provided in Table 4 and cumulative food consumption are provided in Table 5. Another separate efficacy study for

[0294] Compound 10, 11 and 12 in db/db mice at 10 nM/kg dose were carried out. The animals were divided into 4 treatment groups (n=5)a diabetic control group, Compound 10, Compound 11 and Compound 12. Baseline blood glucose was measured from all the animals. All the animals were administered with test items subcutaneously. Blood glucose was measured at 4 hr, 8 hr, 12 hr, 24 hr, 48 hr, 72 hr and 96 hr post treatment. Delta blood glucose (mM) was calculated. The results are provided in Table 3. Similarly, body weight changes and cumulative food consumption was measured at 48 hr and 96 hr post treatment. The results of body weight changes are provided in Table 4 and cumulative food consumption are provided in Table 5.

TABLE-US-00013 TABLE 3 Effect on blood glucose Delta Blood Glucose (mM) Mean (SD) Study 1 Treatment Groups (n = 6) 0 hr 4 hr 8 hr 12 hr 24 hr 48 hr 72 hr 96 hr Diabetic 0 1.0 1.4 4.6 2.8 5.3 4.5 5.6 Control (3.3) (3.3) (3.7) (2.0) (3.6) (5.4) (3.5) Compound 1, 0 12.7*** 12.0*** 10.5*** 15.5*** 6.7*** 4.3*** 2.2*** 10 nM/kg/s.c (3.2) (3.9) (3.1) (4.0) (2.0) (3.0) (3.0) Compound 2, 0 7.4*** 7.6*** 9.7*** 9.5*** 3.6*** 2.1** 1.1** 10 nM/kg/s.c (4.0) (1.8) (4.3) (4.0) (2.2) (2.0) (2.6) Tirzepatide, 0 9.9*** 8.7*** 8.6*** 11.4*** 5.3*** 3.7*** 3.7 10 nM/kg/s.c (5.1) (1.2) (3.2) (3.9) (2.2) (1.4) (1.8) Study 2 Treatment Groups (n = 6) 0 hr 4 hr 8 hr 12 hr 24 hr 48 hr 72 hr 96 hr Diabetic 0 1.1 0.8 1.2 0.8 2.2 2.7 2.7 Control (3.0) (2.5) (1.8) (2.3) (2.0) (0.8) (2.3) Compound 3, 0 10.3*** 13.9*** 15.8*** 10.9*** 6.6*** 0.1 0.7 10 nM/kg/s.c/ (3.2) (3.5) (3.7) (5.7) (8.0) (2.1) (2.9) single dose Compound 4, 0 10.4*** 13.3*** 13.8*** 4.9* 3.7* 0.2 1.9 10 nM/kg/s.c/ (6.0) (4.5) (6.4) (3.7) (4.7) (2.5) (5.4) single dose Compound 5, 0 10.7*** 11.9*** 11.6*** 7.2** 2.8 0.8 0.9 10 nM/kg/s.c/ (4.8) (6.5) (7.2) (7.4) (4.8) (2.0) (1.8) single dose Compound 6, 0 15.6*** 15.9*** 16.3*** 11.8*** 9.5*** 2.6* 1.6 10 nM/kg/s.c/ (4.7) (5.6) (4.4) (4.4) (6.6) (2.9) (2.3) single dose Compound 7, 0 8.5*** 8.0*** 4.1 1.8 0.7 1.0 0.7 10 nM/kg/s.c/ (2.4) (5.7) (3.7) (3.6) (1.3) (1.5) (0.9) single dose Compound 1, 0 10.0*** 13.5*** 12.3*** 11.5*** 7.9*** 4.3** 1.4 10 nM/kg/s.c/ (3.0) (1.4) (1.8) (5.0) (3.8) (1.4) (3.1) single dose Study 3 Treatment Groups (n = 5) 0 hr 4 hr 8 hr 12 hr 24 hr 48 hr 72 hr 96 hr Diabetic 0 0.1 0.1 2.1 0.7 1.2 1.0 1.1 Control (1.2) (2.8) (1.7) (4.1) (1.8) (2.2) (2.3) Compound 8, 0 13.6*** 13.6*** 13.6*** 8.5*** 5.6* 1.8 0.6 10 nM/kg/s.c/ (2.5) (3) (3.5) (2.7) (2.1) (2.4) (2.3) single dose Compound 9, 0 13.1*** 12.7*** 12.1*** 8.7*** 7.1*** 5.6* 0 10 nM/kg/s.c/ (3.6) (1.8) (2.5) (2.2) (2) (2.6) (2.9) single dose Study 4 Treatment Groups (n = 5) 0 hr 4 hr 8 hr 12 hr 24 hr 48 hr 72 hr 96 hr Diabetic 0 0.2 1.2 2.2 1.7 2.5 3.6 7 Control (2.7) (2.4) (3.3) (3.1) (2.4) (4) (1.7) Compound 10, 0 12.1*** 13.6*** 14.7*** 15.6*** 4.0** 2.3 1.7 10 nM/kg/s.c/ (3.1) (3.2) (2.9) (3.5) (3.8) (1.8) (2.7) single dose Compound 11, 0 13.3*** 13.3*** 13.6*** 15.4*** 6.1*** 3.6 6 10 nM/kg/s.c/ (4.4) (3.8) (3.2) (3.8) (4.5) (3.6) (2.7) single dose Compound 12, 0 14.6*** 14.8*** 14.9*** 4.8** 2.5 3.9 6.8 10 nM/kg/s.c/ (0.7) (1.3) (1.9) (2.3) (3.6) (3.2) (1.8) single dose *p < 0.05, **p < 0.01, ***p < 0.001 vs Diabetic Control; Two way ANOVA followed by Bonferroni's post-test.

TABLE-US-00014 TABLE 4 Effect on body weight Body Wt. Body Wt. Change (%) Change (%) 48 hr. vs. Baseline 96 hr. vs. Baseline Mean SD Mean SD Study 1 Treatment Groups (n = 6) Diabetic Control 1.0 0.5 1.4 0.5 Compound 1, 10 nM/kg/s.c 5.1*** 0.3 4.2*** 0.9 Compound 2, 10 nM/kg/s.c 4.3*** 0.7 3.3*** 1.3 Tirzepatide, 10 nM/kg/s.c 4.2*** 0.8 2.8*** 0.9 Study 2 Diabetic Control 1.8 1.6 4.1 3.5 Compound 3, 5.6*** 0.8 4.9*** 1.4 10 nM/kg/s.c/single dose Compound 4, 3.8*** 1.0 1.9** 1.3 10 nM/kg/s.c/single dose Compound 5, 4.6*** 4.0 3.3*** 6.0 10 nM/kg/s.c/single dose Compound 6, 5.5*** 2.0 2.2** 1.3 10 nM/kg/s.c/single dose Compound 7, 4.8*** 0.5 2.9*** 1.1 10 nM/kg/s.c/single dose Compound 1, 5.1*** 1.2 3.5*** 0.7 10 nM/kg/s.c/single dose Study 3 Treatment Groups (n = 5) Diabetic Control 1.1 0.7 1.8 1.0 Compound 8, 3.1*** 1.0 2.8*** 1.3 10 nM/kg/s.c/single dose Compound 9, 3.4*** 0.6 3.8*** 0.5 10 nM/kg/s.c/single dose Study 4 Diabetic Control 0.9 1.7 0.9 1.0 Compound 10, 4.5*** 0.6 3.1*** 0.9 10 nM/kg/s.c/single dose Compound 11, 4.7*** 1.8 2.0*** 1.1 10 nM/kg/s.c/single dose Compound 12, 3.0*** 1.7 0.4 2.5 10 nM/kg/s.c/single dose *p < 0.05, **p < 0.01, ***p < 0.001 vs. Diabetic Control; One way ANOVA followed by Bonferroni's post test.

TABLE-US-00015 TABLE 5 Effect on food consumption Cumulative food Cumulative food Intake (g) Intake (g) 0-48 hr. vs. Baseline 0-96 hr. vs. Baseline Mean SD Mean SD Study 1 Treatment Groups (n = 6) Diabetic Control 11.7 1.6 24.6 4.0 Compound 1, 10 nM/kg/s.c 7.5*** 2.0 13.9*** 2.4 Compound 2, 10 nM/kg/s.c 6.5*** 0.1 21.6 1.1 Tirzepatide, 10 nM/kg/s.c 4.9*** 1.4 13.0*** 1.3 Study 2 Diabetic Control 12.0 0.1 25.8 4.9 Compound 3, 6.0*** 4.8 16.0*** 3.8 10 nM/kg/s.c/single dose Compound 4, 4.6*** 2.4 14.4*** 4.3 10 nM/kg/s.c/single dose Compound 5, 6.2*** 1.6 18.4** 4.5 10 nM/kg/s.c/single dose Compound 6, 4.2*** 1.5 16.0*** 3.6 10 nM/kg/s.c/single dose Compound 7, 5.6*** 0.7 19.6* 0.3 10 nM/kg/s.c/single dose Compound 1, 3.8*** 0.7 12.5*** 2.5 10 nM/kg/s.c/single dose Study 3 Treatment Groups (n = 5) Diabetic Control 14.9 0.5 29.8 1.2 Compound 8, 4.0*** 1.0 13.9*** 1.6 10 nM/kg/s.c/single dose Compound 9, 4.2*** 0.9 16.7*** 3.4 10 nM/kg/s.c/single dose Study 4 Diabetic Control 14.4 0.8 25.7 0.4 Compound 10, 8.2*** 2.2 20.5** 3.9 10 nM/kg/s.c/single dose Compound 11, 8.2*** 1.1 21.7* 1.0 10 nM/kg/s.c/single dose Compound 12, 10.2* 0.7 26.0 0.5 10 nM/kg/s.c/single dose *p < 0.05, **p < 0.01, ***p < 0.001 vs. Diabetic Control; One way ANOVA followed by Bonferroni's post test.

[0295] The results demonstrate that Compound 1 and Compound 2 showed statistically significant blood glucose reduction upto 96 hr post treatment. The effect on blood glucose reduction for the compounds was superior to tirzepatide when tested at the same concentration.

[0296] Compound 1 and Compound 2 also showed statistically significant body weight reduction which was comparable to tirzepatide. Compound 1 showed a significant reduction in food consumption which was comparable to tirzepatide. No significant reduction in food consumption was observed for Compound 2 as compared to diabetic control.

[0297] Similarly the results demonstrate that Compounds 3, 4, 5, 6 and 7 of present invention showed statistically significant blood glucose reduction upto 96 hr post treatment. Also statistically significant reduction in food intake and body weight was observed for these compounds compared to diabetic control.

Example 13

Efficacy Study in db/db Mice at 3 and 20 nM/kg Dose

[0298] The effect of compounds of present invention on blood glucose, food intake and body weight was studied on mice. This study was performed in type 2 diabetic mouse (db/db) model. The animals were divided into 5 treatment groups (n=8)a diabetic control group, Compound 1 (3 nM/kg and 20 nM/kg) and Tirzepatide (3 nM/kg and 20 nM/kg). Baseline blood glucose was measured from all the animals. All animals were administered with test items subcutaneously. Blood glucose was measured at 4 hr, 24 hr, 48 hr, 72 hr post treatment. Delta blood glucose (mM) was calculated. Body weight changes and cumulative food consumption was measured at 72 hr post treatment. The results of delta blood glucose are provided in Table 6. Similarly the results of body weight changes are provided in Table 7 and food consumption are provided in Table 8.

TABLE-US-00016 TABLE 6 Effect of Compound 1 on blood glucose Treatment Delta Blood Glucose (mM), Mean (SD) Groups (n = 8) 0 hr 4 hr 24 hr 48 hr 72 hr Diabetic 0.0 0.1 0.9 0.1 0.3 Control (1.9) (1.6) (1.2) (1.8) Compound 1, 0.0 8.9*** 9.9*** 5.8** 3.2 3 nM/kg/s.c (3.1) (4.7) (3.4) (2.3) Compound 1, 0.0 13.7*** 13.6*** 14.0*** 7.9*** 20 nM/kg/s.c (3.1) (5.9) (6.6) (4.9) Tirzepatide, 0.0 4.4* 1.6 0.7 1.4 3 nM/kg/s.c (3.2) (2.9) (1.8) (1.8) Tirzepatide, 0.0 8.2*** 11.1*** 7.6*** 1.3 20 nM/kg/s.c (3.6) (4.0) (4.7) (2.1) *p < 0.05, **p < 0.01, ***p < 0.001 vs. Diabetic Control; Two way ANOVA followed by Bonferroni's post-test.

TABLE-US-00017 TABLE 7 Effect of Compound 1 on body weight Treatment Groups Body weight change (%) 72 hr vs. Baseline (n = 8) Mean SD Diabetic Control 1.3 1.1 Compound 1, 3 nM/kg/s.c 4.0*** 0.7 Compound 1, 20 nM/kg/s.c 6.2*** 1.4 Tirzeatide, 3 nM/kg/s.c 3.4*** 2.2 Tirzepatide, 20 nM/kg/s.c 4.9*** 0.8 *p < 0.05, **p < 0.01, ***p < 0.001 vs. Diabetic Control; One way ANOVA followed by Bonferroni's post test

TABLE-US-00018 TABLE 8 Effect of Compound 1 on food consumption Treatment Groups Cumulative Food Intake (g), 0-72 hr (n = 8) Mean SD Diabetic Control 16.4 1.40 Compound 1, 3 nM/kg/s.c 10.2*** 1.66 Compound 1, 20 nM/kg/s.c 6.3*** 1.03 Tirzepatide, 3 nM/kg/s.c 9.6*** 1.71 Tirzepatide, 20 nM/kg/s.c 8.4*** 2.16 *p < 0.05, **p < 0.01, ***p < 0.001 vs. Diabetic Control; one way ANOVA followed by Bonferroni's post test

[0299] The results demonstrate that Compound 1 at 3 nM/kg and 20 nM/kg showed dose dependent improvement in glucose lowering effect upto 72 hours. The effect was superior to tirzepatide at similar dosage.

[0300] The effect compound 1 on food intake and body weight at 3 and 20 nM/kg dose was significant, dose dependent and comparable to tirzepatide.

[0301] The results presented above demonstrate that the compounds of present invention are potent inhibitors of GLP-1 and GIP receptors and can be effective in treatment of type 2 diabetes, diabetes with obesity, obesity and hyperlipidemia.

Example 16

Cellular CAMP Assay

[0302] In-vitro potency determination was performed using a cAMP assay. G protein coupled receptor (GPCR) activation following ligand binding initiates a series of second messenger cascades that results in a cellular response. Signaling by the GLP-1R and GIP-R involves activation of adenylate cyclase and cAMP production. Cellular cAMP production was determined using the CAMP Hunter eXpress GPCR Assay (Eurofins DiscoveRx).

[0303] Cellular CAMP Assay of tirzepatide, Compound 1, Compound 3, Compound 6, Compound 10 and Compound 11 was performed and the half effective concentrations on GLP-1R-expressing cells and GIPR-expressing cells was as mentioned in below Table 9:

TABLE-US-00019 TABLE 9 Half-effective concentrations on GLP-1R-expressing cells and GIPR-expressing cells Half effective Half effective Concentration (EC50) Concentration (EC50) GLP-1R GIPR Compound GLP-1R GIPR (Exenatide) (GIP) Tirzepatide 6.8 nM 1.9 nM 78.9 pM 306.1 pM Compound 1 12.4 pM 27.6 pM 35.9 pM 179.2 pM Compound 3 32.7 pM 12.8 pM 87.3 pM 41.1 pM Compound 6 19.6 pM 17.7 pM Compound 10 3.6 pM 7.3 pM 15 pM 50.4 pM Compound 11 5.9 pM 6.4 pM