PRODRUGS AND USES THEREOF

Abstract

Prodrug compounds of GLP-1/GIP receptor co-agonists are provided wherein the GLP-1/GIP receptor co-agonists have been modified by the linkage of a dipeptide to the GLP-1/GIP receptor co-agonist through an amide bond. The prodrugs disclosed herein have extended half-lives and are converted to the active GLP-1/GIP receptor co-agonist at physiological conditions through a non-enzymatic reaction driven by chemical instability.

Claims

1. A compound
B-Z  (Formula I) or a pharmaceutical acceptable salt, ester or amide thereof; wherein B is a dipeptide or a derivative thereof; wherein Z is a glucagon-like peptide 1/glucose-dependent insulinotropic polypeptide (GLP-1/GIP) receptor co-agonist or a derivative thereof; wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LX.sub.1X.sub.1X.sub.17AX.sub.1X.sub.2X.sub.21FX.sub.23X.sub.24WLX.sub.27X.sub.2GX.sub.30X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39 (SEQ ID NO.: 1); wherein X.sub.2 is Aib or A, X.sub.6 is F or V, X.sub.12 is I or Y, X.sub.13 is Y, A, L, I or Aib, X.sub.15 is D or E, X.sub.16 is K or E, X.sub.17 is Q or I, X.sub.19 is A or Q, X.sub.20 is Q, R, E, H, or K, X.sub.21 is A or E, X.sub.23 is I or V, X.sub.24 is E, Q or N, X.sub.27 is L or I, X.sub.28 is A or R, X.sub.30 is G or absent, X.sub.31 is P or absent, X.sub.32 is E, S or absent, X.sub.33 is S, K or absent, X.sub.34 is G or absent, X.sub.35 is A or absent, X.sub.36 is P or absent, X.sub.37 is P or absent, X.sub.38 is P or absent, X.sub.39 is S or absent; and wherein the N-terminal amino group of the GLP-1/GIP receptor co-agonist is linked to B via a peptide bond.

2. The compound according to claim 1, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is Y-Aib-EGTFTSDYSILLEX.sub.16QAAREFIEWLLAGGPSX33GAPPPS (SEQ ID NO.: 3), wherein X.sub.16 is K or E, and wherein X.sub.33 is S or K.

3. The compound according to claim 2, wherein X.sub.16 is E and X.sub.33 is K.

4. The compound according to claim 2, wherein X.sub.16 is K and X.sub.33 is S.

5. The compound according to claim 1, further comprising a substituent z attached to the GLP-1/GIP receptor co-agonist (SEQ ID NO.: 1) via a lysine (K) in position 16 or 33.

6. The compound according to claim 5, wherein the substituent z is selected from the group consisting of ##STR00100##

7. The compound according to claim 1, wherein the dipeptide B is:
X-Y  (Formula II), wherein X is an alpha-amino acid and Y is an N-alkylated alpha-amino acid, wherein X is linked to Y via an amide bond formed between the alpha-carboxylic acid group of X and the alpha-amino group of Y, wherein Y is linked to Z via a peptide bond formed between the alpha-carboxylic acid group of Y and the N-terminal amino group of the GLP-1/GIP receptor co-agonist.

8. The compound according to claim 7, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamate, N-methylnorleucine, N-methylhomoalanine, N-methylalanine, N-methyllysine, N-(2-aminoethyl)glycine, N-hexylhomoalanine, N-propylalanine, homoproline, N-propylglycine, N-ethylglycine, and N-methylphenylalanine.

9. The compound according to claim 7, wherein X is selected from the group consisting of lysine, 4-aminophenylalanine, D-lysine, alanine, glycine, proline, D-valine, homoproline, D-proline, D-homoproline, D-alanine, and azetidine-2-carboxylic acid.

10. The compound according to claim 7, wherein X is selected from the group consisting of lysine, D-lysine, and glycine, and wherein Y is sarcosine or N-(2-aminoethyl)glycine.

11. The compound according to claim 1, wherein the dipeptide comprises substituent b selected from a group consisting of ##STR00101##

12. The compound according to claim 1, wherein the compound is selected from the group consisting of: ##STR00102## ##STR00103## ##STR00104## ##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110## ##STR00111## ##STR00112##

13. The compound according to claim 12, wherein the compound is ##STR00113##

14. The compound according to claim 12, wherein the compound is ##STR00114##

15. The compound according to claim 12, wherein the compound is ##STR00115##

16. The compound according to claim 12, wherein the compound is ##STR00116##

17. The compound according to claim 12, wherein the compound is ##STR00117##

18. The compound according to claim 12, wherein the compound is ##STR00118##

19. The compound according to claim 12, wherein the compound is ##STR00119##

20. The compound according to claim 12, wherein the compound is ##STR00120##

21. The compound according to claim 12, wherein the compound is ##STR00121##

22. The compound according to claim 12, wherein the compound is ##STR00122##

23. The compound according to claim 12, wherein the compound is ##STR00123##

24. The compound according to claim 12, wherein the compound is ##STR00124##

25. The compound according to claim 12, wherein the compound is ##STR00125##

26. The compound according to claim 12, wherein the compound is ##STR00126##

27. The compound according to claim 12, wherein the compound is ##STR00127##

28. The compound according to claim 12, wherein the compound is ##STR00128##

29. The compound according to claim 12, wherein the compound is ##STR00129##

30. The compound according to claim 12, wherein the compound is ##STR00130##

Description

EMBODIMENTS

[0108] The invention is further described by the following non-limiting embodiments:

[0109] 1. A compound of Formula I:

B-Z  (Formula I)

or a pharmaceutical acceptable salt, ester or amide thereof,
wherein B is a dipeptide, said dipeptide optionally comprising a substituent b;
wherein Z is a GLP-1/GIP receptor co-agonist, said GLP-1/GIP receptor co-agonist optionally comprising a substituent z; and
wherein the N-terminal amino group of the GLP-1/GIP receptor co-agonist is linked to B via a peptide bond.

[0110] 2. The compound according to embodiment 1, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is

YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LX.sub.15X.sub.16X.sub.17AX.sub.19X.sub.20X.sub.21FX.sub.23X.sub.24WLX.sub.27X.sub.28GX.sub.3X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39 (SEQ ID NO.: 1), wherein [0111] X.sub.2 is Aib or A [0112] X.sub.6 is F or V [0113] X.sub.12 is I or Y [0114] X.sub.13 is Y, A, L, I or Aib [0115] X.sub.15 is D or E [0116] X.sub.16 is K or E [0117] X.sub.17 is Q or I [0118] X.sub.19 is A or Q [0119] X.sub.20 is Q, R, E, H, or K [0120] X.sub.21 is A or E [0121] X.sub.23 is I or V [0122] X.sub.24 is E, Q or N [0123] X.sub.27 is L or I [0124] X.sub.28 is A or R [0125] X.sub.30 is G or absent [0126] X.sub.31 is P or absent [0127] X.sub.32 is E, S or absent [0128] X.sub.33 is S, K or absent [0129] X.sub.34 is G or absent [0130] X.sub.35 is A or absent [0131] X.sub.36 is P or absent [0132] X.sub.37 is P or absent [0133] X.sub.38 is P or absent [0134] X.sub.39 is S or absent.

[0135] 3. The compound according to embodiment 1, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is

Y-Aib-EGTFTSDYSIX.sub.13LX.sub.15X.sub.16X.sub.17AX.sub.19X.sub.20X.sub.21FX.sub.23X.sub.24WLX.sub.27AGGPSX.sub.33GAPPPS (SEQ ID NO.: 2), wherein

X.SUB.13 .is L or Aib,

X.SUB.15 .is D or E,

X.SUB.16 .is K or E,

X.SUB.17 .is Q or I,

X.SUB.19 .is A or Q,

X.SUB.20 .is R or K

X.SUB.21 .is A or E

X.SUB.23 .is I or V

X.SUB.24 .is E or Q

X.SUB.27 .is L or I;

X.SUB.33 .is S or K.

[0136] 4. The compound according to embodiment 1, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is

Y-Aib-EGTFTSDYSILLEX.sub.16QAAREFIEWLLAGGPSX33GAPPPS (SEQ ID NO.: 3), wherein

X.SUB.16 .is K or E,

X.SUB.33 .is S or K.

[0137] 5. The compound according to any one of embodiments 2 to 4, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X.sub.16 is E and X.sub.33 is K.

[0138] 6. The compound according to any one of embodiments 2 to 4, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X.sub.16 is K and X.sub.33 is S.

[0139] 7. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS (SEQ ID NO.: 4).

[0140] 8. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is selected from the group consisting of Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS (SEQ ID NO.: 4), Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO.: 5), and Y-Aib-EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO.: 6).

[0141] 9. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO.: 5) or Y-Aib-EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO.: 6).

[0142] 10. The compound according to any one of embodiments 1 to 9, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the GLP-1/GIP receptor co-agonist comprises a substituent z, and wherein the substituent z is attached to the GLP-1/GIP receptor co-agonist via a lysine (K).

[0143] 11. The compound according to any one of embodiments 2 to 10, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X.sub.16 and/or X.sub.20 and/or X.sub.33 is lysine.

[0144] 12. The compound according to any one of embodiments 2, 3, 11, or 6, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X.sub.16 is lysine.

[0145] 13. The compound according to any one of embodiments 2, 11, or 7, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X.sub.20 is lysine.

[0146] 14. The compound according to any one of embodiments 2, 3, 11, or 5, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X.sub.33 is lysine.

[0147] 15. The compound according to any one of embodiments 1 to 14, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent z is attached to the GLP-1/GIP receptor co-agonist via a lysine (K) at position 16, 20 or 33.

[0148] 16. The compound according to any one of embodiments 1 to 15, or a pharmaceutically acceptable salt, ester or amide thereof, wherein GLP-1/GIP receptor co-agonist has the amide modification of the C-terminus.

[0149] 17. The compound according to any one of embodiments 5, 8 or 9, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the lysine at position 33 is chemically modified through conjugation to the epsilon-amino group of the lysine side-chain with Chem. 8, Chem. 7 or Chem. 10.

[0150] 18. The compound according to any one of embodiments 6, 8 or 9, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the lysine at position 16 is chemically modified through conjugation to the epsilon-amino group of the lysine side-chain with Chem. 7.

[0151] 19. The compound according to embodiment 7 or embodiment 8, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the lysine at position 20 is chemically modified through conjugation to the epsilon-amino group of the lysine side-chain with Chem. 11.

[0152] 20. The compound according to any one of embodiments 1 to 19, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide B is of Formula II:

X-Y  (Formula II),

wherein X is any alpha-amino acid linked to Y via an amide bond formed between the alpha-carboxylic acid group of X and the alpha-amino group of Y,
wherein Y is an N-alkylated alpha-amino acid linked to Z via a peptide bond formed between the alpha-carboxylic acid group of Y and the N-terminal amino group of the GLP-1/GIP receptor co-agonist.

[0153] 21. The compound according to any one of embodiments 1 to 16, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide B is of Formula II:

X-Y  (Formula II),

wherein X is any alpha-amino acid linked to Y via an amide bond formed between the alpha-carboxylic acid group of X and the alpha-amino group of Y,
wherein Y is an N-alkylated alpha-amino acid linked to Z via a peptide bond formed between the alpha carboxylic acid group of Y and an N-terminal amino group of Z.

[0154] 22. The compound according to embodiment 20 or embodiment 21, or a pharmaceutically acceptable salt, ester or amide thereof, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamate, N-methylnorleucine, N-methylhomoalanine, N-methylalanine, N-methyllysine, N-(2-aminoethyl)glycine, N-hexylhomoalanine, N-propylalanine, homoproline, N-propylglycine, N-ethylglycine, and N-methylphenylalanine.

[0155] 23. The compound according to any one of embodiments 20 to 22, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from the group consisting of lysine, 4-aminophenylalanine, D-lysine, alanine, glycine, proline, D-valine, homoproline, D-proline, D-homoproline, D-alanine, and azetidine-2-carboxylic acid.

[0156] 24. The compound according to any one of embodiments 20 to 23, or a pharmaceutically acceptable salt, ester or amide thereof, wherein, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamate, N-methylnorleucine, N-methylhomoalanine, N-methylalanine, N-methyllysine, N-hexylhomoalanine, N-propylalanine, homoproline, N-propylglycine, N-ethylglycine, and N-methylphenylalanine.

[0157] 25. The compound according to any one of embodiments 20 to 24, or a pharmaceutically acceptable salt, ester or amide thereof, wherein Y is sarcosine or N-(2-aminoethyl)glycine.

[0158] 26. The compound according to any one of embodiments 20 to 25, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from the group consisting of lysine, D-lysine, alanine, leucine, glycine, proline, and aspartic acid.

[0159] 27. The compound according to any one of embodiments 20 to 26, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from the group consisting of lysine, D-lysine, and glycine.

[0160] 28. The compound according to any one of embodiments 1 to 27, or a pharmaceutically acceptable salt, ester or amide thereof, wherein dipeptide is capable of undergoing an intramolecular cyclisation to form a 2,5-diketopiperazine (DKP) such that the amide bond between A and Z is cleaved.

[0161] 29. The compound according to any one of embodiments 1 to 27, or a pharmaceutically acceptable salt, ester or amide thereof, wherein dipeptide is capable of undergoing an intramolecular cyclization to form a 2,5-diketopiperazine (DKP) such that the peptide bond between A and Z is cleaved.

[0162] 30. The compound according to any one of embodiments 1 to 29, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide comprises a substituent b.

[0163] 31. The compound according to any one of embodiments 1 to 29, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide carries a substituent b.

[0164] 32. The compound according to any one of embodiments 1 to 29, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide has a substituent b.

[0165] 33. The compound according to any one of embodiments 21 to 30, or a pharmaceutically acceptable salt, ester or amide thereof, wherein a substituent b is covalently attached to X optionally via an amide bond.

[0166] 34. The compound according to any one of embodiments 21 to 30, or a pharmaceutically acceptable salt, ester or amide thereof, wherein a substituent b is covalently attached to Y optionally via an amide bond.

[0167] 35. The compound according to any one of embodiments 1 to 34, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent b is an albumin binding moiety.

[0168] 36. The compound according to any one of embodiments 1 to 35, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent b comprises or consists of a protractor and optionally a linker.

[0169] 37. The compound according to embodiment 36, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the protractor is a fatty acid such as a C.sub.16-C.sub.22 carboxylic acid.

[0170] 38. The compound according to embodiment 36 or embodiment 37, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the protractor is Chem. 1.

[0171] 39. The compound according to any one of embodiments 1 to 38, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent comprises a linker and optionally wherein the linker comprises or consists of linker elements.

[0172] 40. The compound according to any one of embodiments 38 to 39, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the linker is of formula IV

A.sub.1-A.sub.2-A.sub.3-A.sub.4-A.sub.5  (Formula IV),

[0173] wherein A.sub.1 is covalently bound to an amino acid of the dipeptide via an amide bond and optionally also covalently bound to the protractor via an amide bond and is selected from a group consisting of Chem. 2, Chem. 3, Chem. 4, and Chem. 5; wherein A.sub.5 is covalently bound to Chem. 1 and is Chem. 2 or absent; wherein each of A.sub.2, A.sub.3, and A.sub.4, are individually selected from the group consisting of Chem. 2, Chem. 3, Chem. 4, and Chem. 5, or is absent.

[0174] 41. The compound according to any one of embodiments 1 to 40, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent b is selected from the group consisting of Chem. 16, Chem. 17, Chem. 18, Chem 19, Chem. 20, Chem 21, and Chem. 22.

[0175] 42. The compound according to any one of embodiments 1 to 41, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is selected from the group consisting of:

##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067## ##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072##

[0176] 43. The compound according to any one of embodiments 1 to 42, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is selected from the group consisting of compound no. 1, 2, 3, 9, and 10.

[0177] 44. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is compound no. 1.

[0178] 45. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is compound no. 2.

[0179] 46. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is compound no. 3.

[0180] 47. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is compound no. 4.

[0181] 48. The compound according to any one of embodiments 1 to 43, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is compound no. 5.

[0182] 49. The compound according to any one of embodiments 1 to 48, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is a prodrug and does not exert any significant potency in vitro.

[0183] 50. The compound according to any one of embodiments 1 to 49, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is a prodrug and has a conversion half-life.

[0184] 51. The compound according to embodiment 50, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life is measured in vitro at pH 7.4 at 37° C.

[0185] 52. The compound according to embodiments 50 or 51, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life is measured as described herein in General Methods for Measuring Conversion Half-life.

[0186] 53. The compound according to any one of embodiments 50 to 52 or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life is suitable for once daily administration.

[0187] 54. The compound according to any one of embodiments 50 to 52 or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life is suitable for weekly daily administration.

[0188] 55. The compound according to any one of embodiments 50 to 52 or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life measured in vitro is 90-4300 hours.

[0189] 56. The compound according to any one of embodiments 50 to 52 or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life measured in vitro is 90-4300 hours.

[0190] 57. The compound according to any one of embodiments 50 to 52 or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life measured in vitro is 300-1100 hours.

[0191] 58. The compound according to any one of embodiments 50 to 52 or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life measured in vitro is 450-650 hours.

[0192] 59. The compound according to any one of embodiments 50 to 52 or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life measured in vitro is at least 100 hours.

[0193] 60. The compound according to any one of embodiments 50 to 52 or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life measured in vitro is at least 200 hours.

[0194] 61. The compound according to any one of embodiments 50 to 52 or a pharmaceutically acceptable salt, ester or amide thereof, wherein the conversion half-life measured in vitro is at least 300 hours.

[0195] 62. The compound according to any one of embodiments 1 to 61, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound has a terminal half-life.

[0196] 63. The compound according to any one of embodiments 1 to 61, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound has a terminal half-life and the terminal half-life is suitable for once daily administration.

[0197] 64. The compound according to any one of embodiments 1 to 61, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound has a terminal half-life and the terminal half-life is suitable for once weekly administration.

[0198] 65. The compound according to any one of embodiments 62 to 64, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is determined in minipigs and measured as described herein in General Methods for Measuring Terminal Half-Life in Minipigs.

[0199] 66. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is >90 hours when determined in minipigs.

[0200] 67. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is >110 hours when determined in minipigs.

[0201] 68. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is >250 hours when determined in minipigs.

[0202] 69. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is >180 hours when determined in minipigs.

[0203] 70. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is 80-240 hours when determined in minipigs.

[0204] 71. The compound according to any one of embodiments 62 to 65, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the terminal half-life is 110-191 hours when determined in minipigs.

[0205] 72. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 71 and at least one pharmaceutically acceptable excipient.

[0206] 73. The pharmaceutical composition according to embodiment 72, wherein the pharmaceutical composition is a liquid formulation.

[0207] 74. The pharmaceutical composition according to embodiment 72, wherein the pharmaceutical composition is a solid formulation.

[0208] 75. The pharmaceutical composition according to embodiment 72, wherein the pharmaceutical composition is for oral administration.

[0209] 76. The pharmaceutical composition according to any one of embodiments 74 to 76, wherein the composition if in form of a tablet.

[0210] 77. The pharmaceutical composition according to any one of embodiments 74 to 77, wherein at least one pharmaceutical acceptable excipient is a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid such as N-(8-(2 hydroxybenzoyl)amino)caprylic acid is sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC).

[0211] 78. The pharmaceutical composition according to any one of embodiments 74 to 78, further comprising a lubricant such as magnesium stearate.

[0212] 79. A tablet comprising a compound according to any one of embodiments 1-71, a salt of N-(8-(2-hydroxybenzoyl)amino)caprylic acid, a lubricant, and optionally one or more pharmaceutically acceptable excipients.

[0213] 80. The tablet according to embodiment 79, wherein the salt of N-(8-(2 hydroxybenzoyl)amino)caprylic acid is sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC).

[0214] 81. The tablet according to embodiment 79 or embodiment 80, wherein the lubricant is magnesium stearate.

[0215] 82. A compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 for use as a medicament.

[0216] 83. A compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 for use in the treatment of type 2 diabetes.

[0217] 84. A compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 for use in the treatment of obesity.

[0218] 85. A compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 for use in the treatment of liver diseases. such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation, and/or fatty liver.

[0219] 86. Use of a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 in the manufacture of a medicament for [0220] a. prevention and/or treatment of liver diseases. such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation, and/or fatty liver; [0221] b. prevention and/or treatment of obesity; and/or [0222] c. prevention and/or treatment of type 2 diabetes.

[0223] 87. Use of a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 in the manufacture of a medicament for the treatment of type 2 diabetes.

[0224] 88. Use of a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81 in the manufacture of a medicament for obesity.

[0225] 89. A method for prevention and/or treatment of type 2 diabetes administering a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81, to a subject in need thereof.

[0226] 90. A method for prevention and/or treatment of obesity administering a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81, to a subject in need thereof.

[0227] 91. A method for prevention and/or treatment of liver diseases. such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation, and/or fatty liver administering a compound according to any one of embodiments 1 to 71 or a pharmaceutical composition according to any one of embodiments 72 to 78 or a tablet according to any one of embodiments 79 to 81, to a subject in need thereof.

[0228] The invention is further described by the following further non-limiting embodiments: [0229] 1. A compound of Formula I:

B-Z  (Formula I)

or a pharmaceutical acceptable salt, ester or amide thereof, [0230] wherein Z is a GLP-1/GIP receptor co-agonist or derivative thereof; [0231] wherein B is a dipeptide of formula II:

X-Y  (Formula II), [0232] wherein X is any alpha-amino acid linked to Y via an amide bond formed between the alpha-carboxylic acid group of X and the alpha-amino group of Y, [0233] wherein Y is an N-alkylated alpha-amino acid linked to Z via an amide bond formed between the alpha-carboxylic acid group of Y and an amine of Z. [0234] 2. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamate, N-methylnorleucine, N-methylhomoalanine, N-methylalanine, N-methyllysine, N-(2-aminoethyl)glycine, N-hexylhomoalanine, N-propylalanine, homoproline, N-propylglycine, N-ethylglycine, and N-methylphenylalanine. [0235] 3. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from the group consisting of lysine, 4-aminophenylalanine, D-lysine, alanine, glycine, proline, D-valine, homoproline, D-proline, D-homoproline, D-alanine, and azetidine-2-carboxylic acid. [0236] 4. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein Y is selected from the group consisting of sarcosine, N-sec-butylglycine, proline, trans-4-hydroxyproline, N-methylglutamate, N-methylnorleucine, N-methylhomoalanine, N-methylalanine, N-methyllysine, N-hexylhomoalanine, N-propylalanine, homoproline, N-propylglycine, N-ethylglycine, and N-methylphenylalanine. [0237] 5. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein Y is sarcosine or N-(2-aminoethyl)glycine. [0238] 6. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from the group consisting of lysine, D-lysine, alanine, leucine, glycine, proline, and aspartic acid. [0239] 7. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X is selected from the group consisting of lysine, D-lysine, and glycine. [0240] 8. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein dipeptide is capable of undergoing an intramolecular cyclisation to form a 2,5-diketopiperazine (DKP) such that the amide bond between B and Z is cleaved. [0241] 9. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide comprises a substituent b. [0242] 10. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the dipeptide has a substituent b. [0243] 11. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein a substituent b is covalently attached to X optionally via an amide bond. [0244] 12. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent b comprises or consists of a protractor and optionally a linker. [0245] 13. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the protractor is Chem. 1. [0246] 14. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent b is selected from the group consisting of Chem. 16, Chem. 17, Chem. 18, Chem. 19, Chem. 20, Chem. 21, and Chem. 22. [0247] 15. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is [0248] YX.sub.2EGTX.sub.6TSDYSX.sub.12X.sub.13LX.sub.15X.sub.16X.sub.17AX.sub.19X.sub.20X.sub.21FX.sub.23X.sub.24WLX.sub.27X.sub.28GX.sub.3X.sub.31X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39 (SEQ ID NO.: 1), wherein [0249] X.sub.2 is Aib or A [0250] X.sub.6 is F or V [0251] X.sub.12 is I or Y [0252] X.sub.13 is Y, A, L, I or Aib [0253] X.sub.15 is D or E [0254] X.sub.16 is K or E [0255] X.sub.17 is Q or 1 [0256] X.sub.19 is A or Q [0257] X.sub.20 is Q, R, E, H, or K [0258] X.sub.21 is A or E [0259] X.sub.23 is I or V [0260] X.sub.24 is E, Q or N [0261] X.sub.27 is L or I [0262] X.sub.28 is A or R [0263] X.sub.30 is G or absent [0264] X.sub.31 is P or absent [0265] X.sub.32 is E, S or absent [0266] X.sub.33 is S, K or absent [0267] X.sub.34 is G or absent [0268] X.sub.35 is A or absent [0269] X.sub.36 is P or absent [0270] X.sub.37 is P or absent [0271] X.sub.38 is P or absent [0272] X.sub.39 is S or absent. [0273] 16. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is [0274] Y-Aib-EGTFTSDYSIX.sub.13LX.sub.15X.sub.16X.sub.17AX.sub.19X.sub.20X.sub.21FX.sub.23X.sub.24WLX.sub.27AGGPSX.sub.33GAPPPS (SEQ ID NO.: 2), wherein [0275] X.sub.13 is L or Aib, [0276] X.sub.15 is D or E, [0277] X.sub.16 is K or E, [0278] X.sub.17 is Q or I, [0279] X.sub.19 is A or Q, [0280] X.sub.20 is R or K [0281] X.sub.21 is A or E [0282] X.sub.23 is I or V [0283] X.sub.24 is E or Q [0284] X.sub.27 is L or I; [0285] X.sub.33 is S or K. [0286] 17. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, the amino acid sequence of the GLP-1/GIP receptor co-agonist is [0287] Y-Aib-EGTFTSDYSILLEX.sub.16QAAREFIEWLLAGGPSX33GAPPPS (SEQ ID NO.: 3), [0288] wherein [0289] X.sub.16 is K or E, [0290] X.sub.33 is S or K. [0291] 18. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X.sub.16 is E and X.sub.33 is K. [0292] 19. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein X.sub.16 is K and X.sub.33 is S. [0293] 20. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the amino acid sequence of the GLP-1/GIP receptor co-agonist is selected from the group consisting of Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS (SEQ ID NO.: 4), Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPSKGAPPPS (SEQ ID NO.: 5), and Y-Aib-EGTFTSDYSILLEKQAAREFIEWLLAGGPSSGAPPPS (SEQ ID NO.: 6). [0294] 21. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the GLP-1/GIP receptor co-agonist comprises a substituent z, and wherein the substituent z is attached to the GLP-1/GIP receptor co-agonist via a lysine (K). [0295] 22. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the substituent z is attached to the GLP-1/GIP receptor co-agonist via a lysine (K) at position 16, 20 or 33. [0296] 23. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is selected from the group consisting of compound no. 1, compound no. 2, compound no. 3, compound no. 4, compound no. 5, compound no. 6, compound no. 7, compound no. 8, compound no. 9, compound no. 10, compound no. 11, compound no. 12, compound no. 13, compound no. 14, compound no. 15, compound no. 16, compound no. 17, and compound no. 18. [0297] 24. The compound according to any one of the preceding embodiments, or a pharmaceutically acceptable salt, ester or amide thereof, wherein the compound is selected from the group consisting of compound no. 1, 2, 3, 9, and 10. [0298] 25. A pharmaceutical composition comprising a compound any one of the preceding embodiments and at least one pharmaceutically acceptable excipient. [0299] 26. The pharmaceutical composition according to any one of the preceding embodiments, wherein the pharmaceutical composition is a liquid formulation. [0300] 27. The pharmaceutical composition according to any one of the preceding embodiments, wherein the pharmaceutical composition is a solid formulation. [0301] 28. The pharmaceutical composition according to any one of the preceding embodiments, wherein the pharmaceutical composition is for oral administration. [0302] 29. The pharmaceutical composition according to any one of the preceding embodiments, wherein the pharmaceutical composition is for parenteral administration. [0303] 30. The pharmaceutical composition according to any one of the preceding embodiments, wherein the pharmaceutical composition is in form of a tablet. [0304] 31. A compound according to any one of the preceding embodiments for use as a medicament. [0305] 32. A compound according to any one of the preceding embodiments for use in the prevention and/or treatment of type 2 diabetes. [0306] 33. A compound according to any one of the preceding embodiments for use in the prevention and/or treatment of obesity. [0307] 34. A compound according to any one of the preceding embodiments for use in the prevention and/or treatment of liver diseases. such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver inflammation, and/or fatty liver.

EXAMPLES

[0308] This experimental part starts with a list of abbreviations and is followed by a section on the general methods for compound preparation and a section on the methods for measuring properties relevant for the exposure profile. A number of specific examples have been included in each of the sections to illustrate the invention. All example compounds were prepared according to the general methods described herein. Where appropriate, chemical names of substituents were generated using Accelrys Draw version 4.1 SP1 software and IUPAC nomenclature.

Abbreviations

[0309] The following abbreviations are used in the following, in alphabetical order:

Ado: 8-amino-3,6-dioxaoctanoic acid
Aeg: N-(2-aminoethyl)glycine
Aib: α-aminoisobutyric acid
Alloc: allyloxycarbonxyl
API: atmospheric pressure ionization
AUC: area under the curve
BHK: baby hamster kidney
Boc: t-butyloxycarbonyl
Cl-HOBt: 6-chloro-1-hydroxybenzotriazole
DCM: dichloromethane
DIC: diisopropylcarbodiimide

DIPEA: N,N-diisopropylethylamine

[0310] DKP: 2,5-diketopiperazine

DMEM: Dulbecco's Modified Eagle's Medium

[0311] DPBS: Dulbecco's phosphate buffered saline
EDTA: ethylenediaminetetraacetic acid
ELISA: enzyme linked immunosorbent assay
equiv: molar equivalent
FBS: fetal bovine serum
Fmoc: 9-fluorenylmethyloxycarbonyl
GIP: glucose-dependent insulinotropic polypeptide
GIPR: glucose-dependent insulinotropic polypeptide receptor
GLP-1: glucagon-like peptide 1
GLP-1R: glucagon-like peptide 1 receptor
h: hours
HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HFIP: 1,1,1,3,3,3-hexafluoro-2-propanol or hexafluoroisopropanol
HPLC: high performance liquid chromatography
HSA: human serum albumin
i.v. intravenously
LCMS: liquid chromatography mass spectroscopy
MeCN: acetonitrile
MeOH: methanol
mM: millimolar
mmol: millimoles
min: minutes
Mtt: 4-methyltrityl
NMP: 1-methyl-pyrrolidin-2-one
OtBu: tert-butyl ester
Oxyma Pure®: cyano-hydroxyimino-acetic acid ethyl ester
Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
PBS: phosphate buffered saline
PK: pharmacokinetic
pM: picomolar
p.o.: oral
rpm: rounds per minute
Rt: retention time
Sar: sarcosine
s.c.: subcutaneous
SNAC: sodium N-[8-(2-hydroxybenzoyl)amino]caprylate
SPPS: solid phase peptide synthesis
tBu: tert-butyl
T2D: type 2 diabetes mellitus
TFA: trifluoroacetic acid
TIS: triisopropylsilane
Trt: triphenylmethyl or trityl
UPLC: ultra-performance liquid chromatography

General Methods for Preparation of the Compounds of the Invention

[0312] Methods for solid phase peptide synthesis (SPPS methods, including methods for de-protection of amino acids, methods for cleaving the peptide from the resin, and for its purification), as well as methods for detecting and characterising the resulting peptide (LCMS methods) are described here below.

[0313] Resins employed for the preparation of C-terminal peptide amides were H-Rink Amide-ChemMatrix resin (loading e.g. 0.5 mmol/g). Resins employed for the preparation of C-terminal peptide acids were Wang-polystyrene resin pre-loaded with the suitably protected C-terminal amino acid derivative (loading e.g. 0.5 mmol/g). All operations stated below were performed within a 0.1-1.0 mmol synthesis scale range. The Fmoc-protected amino acid derivatives used, unless specifically stated otherwise, were the standard recommended: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Aib-OH, etc. supplied from e.g. AAPPTEC, Anaspec, Bachem, ChemImpex, Iris Biotech, Midwest Biotech, Gyros Protein Technologies or Novabiochem.

[0314] Where nothing else is specified, the proteinogenic L-form of the amino acids are used. For coupling of the N-terminal amino acid of each compound, a reagent was used bearing Boc protected at the alpha-amino group.

[0315] In case of dipeptide attachment using SPPS, the following suitably protected building blocks such as but not limited to Alloc-Aeg(Fmoc)-OH, Boc-Ala-OH, Boc-Asp(OtBu)-OH, Boc-Gly-OH, Boc-Leu-OH, Boc-Lys(Fmoc)-OH, Boc-D-Lys(Fmoc)-OH, Boc-Pro-OH, Fmoc-Aeg(N)—OH, and Fmoc-Sar-OH were used. In case of substituent attachment using SPPS, the following suitably protected building blocks such as but not limited to Fmoc-8-amino-3,6-dioxaoctanoic acid (Fmoc-Ado-OH), Boc-Lys(Fmoc)-OH, Fmoc-Glu-OtBu, Fmoc-Gly-OH, hexadecanedioic acid mono-tert-butyl ester, octadecanedioic acid mono-tert-butyl ester, or eicosanedioic acid mono-tert-butyl ester were used.

1. Synthesis of Resin-Bound Protected Peptide Backbone:

Method: SPPS_A

[0316] SPPS was performed using Fmoc based chemistry on a Protein Technologies SymphonyX solid-phase peptide synthesizer, using the manufacturer supplied protocols with minor modifications. Mixing was accomplished by occasional bubbling with nitrogen. The step-wise assembly was performed using the following steps: 1) pre-swelling of resin in DMF; 2) Fmoc-deprotection by the use of 20% (v/v) piperidine in DMF with or without 1% (v/v) TFA for two treatments of 10 min each; 3) washes with DMF to remove piperidine; 4) coupling of Fmoc-amino acid by the addition of 3-12 equiv each of Fmoc-amino acid, Oxyma Pure®, and DIC as a solution in DMF with or without 2,4,6-collidine, then mixing for at least 30 min; 4) washes with DMF to remove excess reagents; 5) final washes with DCM at the completion of the assembly. Some amino acids such as, but not limited to, those following a sterically hindered amino acid (e.g. Aib) were coupled with an extended reaction time (e.g. 4 h or overnight) to ensure reaction completion.

Method: SPPS_B

[0317] SPPS was performed using Fmoc based chemistry on an Applied Biosystems 431A solid-phase peptide synthesizer, using the manufacturer supplied general Fmoc protocols. Mixing was accomplished by vortexing and occasional bubbling with nitrogen. The step-wise assembly was done using the following steps: 1) activation of Fmoc-amino acid by dissolution of 10 equiv each of solid Fmoc-acid acid in a 1 M solution of Cl-HOBt in NMP, then addition of 10 equiv of DIC as a 1 M solution in NMP, then mixing simultaneously to steps 2-3; 2) Fmoc-deprotection by the use of 20% (v/v) piperidine in NMP for one treatment of 3 min then a second treatment of 15 min; 3) washes with NMP to remove piperidine; 4) addition of activated Fmoc-amino acid solution to resin, then mixing for at least 45 min; 4) washes with NMP to remove excess reagents; 5) final washes with DCM at the completion of the assembly. Some amino acids such as, but not limited to, those following a sterically hindered amino acid (e.g. Aib) were coupled with an extended reach time (eg 4 h) and/or repeatedly treated with fresh coupling reagents to ensure reaction completion.

2. Attachment of Dipeptide and Substituents to Resin-Bound Protected Peptide Backbone

Method: DS_A

[0318] For compounds containing a substituent-carrying N-terminal Lys or D-Lys, SPPS was continued using the same protocols as in SPPS_A to attach the amino acids of dipeptide B and the elements of substituent b.

Method: DS_B

[0319] For compounds containing a substituent-carrying Aeg within dipeptide B, SPPS was continued using the same protocols as in SPPS_A to attach Fmoc-Aeg(N.sub.3)—OH and the Na-Boc-protected N-terminal amino acid. The azido protecting group was reduced to the amine by treating the resin-bound peptide with 5-10 equiv of tris(2-carboxyethyl) phosphine as a solution in 9:1 DMF/water for 2-3 h. The resin was drained and washed with 9:1 DMF/water and DMF, followed by attachment of the elements of substituent b using the same protocols as in SPPS_A.

Method: DS_C

[0320] As an alternative to DS_B for compounds containing substituent-carrying Aeg within dipeptide B, SPPS was continued using the same protocols as in SPPS_A to attach Alloc-Aeg(Fmoc)-OH and the elements of substituent b. The Alloc protecting group was removed by treating the resin-bound peptide with 10 equiv of borane dimethylamine complex and 20 equiv of morpholine as a solution in DMF for 5 min under an argon atmosphere, then adding 0.1 equiv of palladium-tetrakis(triphenylphosphine) as a solution in DMF and treating for an additional 30 min. The resin was drained and washed with DCM, DMF, MeOH, water, and DMF. The Na-Boc-protected N-terminal amino acid was then attached using the same protocols as in SPPS_A.

Method: DS_D

[0321] To attach substituent z, the NE-Mtt protection of the substituent-carrying Lys was removed by washing the resin with 30% HFIP in DCM for two treatments of 45 min each or 80% HFIP in DCM for treatments of 5 min, 5 min, 10 min, 10 min, 15 min, 20 min, and 30 min. The resin was drained and washed with DCM, DMF, 10% DIPEA/DCM, DCM, and DMF. SPPS was continued using the same protocols as in SPPS_A to attach the elements of substituent z.

3. Cleavage of Resin-Bound Peptide and Purification:

Method: CP_A

[0322] Following completion of the sidechain synthesis, the peptidyl resin was washed with DCM and dried, then treated with 95:2.5:2.5 (v/v/v) TFA/water/TIS or 92.5:5:2.5 (v/v/v) TFA/water/TIS for 2-3 h, followed by precipitation with diethyl ether. The precipitate was isolated (e.g. by filtration or centrifugation), washed with diethyl ether, dissolved in a suitable solvent (e.g. 2:1 water/MeCN), and let stand until all labile adducts decomposed. Purification was performed by reversed-phase preparative HPLC on a Phenomenex Luna C8(2) column (10 μm particle size, 100 Å pore size, 250×21.2 mm dimensions) or a Phenomenex Gemini-NX C18 column (5 μm particle size, 110 Å pore size, 250×50 mm dimensions). Separation of impurities and product elution was accomplished using an increasing gradient of MeCN in water containing 0.1% TFA. Relevant fractions were checked for identity and purity by analytical LCMS. Fractions containing the pure desired product were pooled and freeze-dried to afford the peptide TFA salt as a white solid.

4. Salt Exchange from TFA to Sodium Salt:

Method: SX_A

[0323] The freeze-dried peptide isolated from method CP_A was dissolved to 5-20 mg/mL in an appropriate aqueous buffer (e.g. 4:1 water/MeCN, 0.2 M sodium acetate) and adjusted to pH 7-8 with 1 M NaOH if necessary to achieve full solubility. The buffered solutions containing the peptide were salt-exchanged using a Sep-Pak C18 cartridge (0.5-2 g): The cartridge was first equilibrated with 4 column volumes of isopropanol, then 4 column volumes of MeCN, then 8 column volumes of water. The peptide solution was applied to the cartridge, and the flow through was reapplied to ensure complete retention of peptide. The cartridge was washed with 4 column volumes of water, then 10 column volumes of a buffer solution (e.g. pH 7.5) containing such as, but not limited to, NaHCO.sub.3, NaOAc, or Na.sub.2HPO.sub.4. The column was washed with 4 column volumes of water, and the peptide was eluted with 5-10 column volumes of 50-80% MeCN in water. The peptide-containing eluent was freeze-dried to afford the peptide sodium salt as a white solid, which was used as such.

General Methods of Detection and Characterisation

LCMS Methods:

Method: LCMS_A

[0324] The analysis was performed on Agilent 1260 Infinity series HPLC/MS system by injecting an appropriate volume of sample onto a Phenomenex Kinetex C8 column (2.6 μm particle size, 100 Δ pore size, 4.6×75 mm dimensions) equilibrated at 37 C. Eluent A was 0.05% TFA in water; eluent B was 0.05% TFA in 9:1 MeCN/water. Elution was achieved with a linear gradient of 20-100% eluent B over 10 min at a flow rate of 1.0 mL/min. UV detection was set to 214 nm. MS ionization was run in API-ES mode and positive polarity with a scan mass range of 500-2000 amu. The most abundant isotope of each m/z is reported.

Method: LCMS_B

[0325] The analysis was performed on a Waters ACQUITY UPLC/MS system by injecting an appropriate volume of sample onto a ACQUITY UPLC BEH130 column (1.7 μm particle size, 130 Δ pore size, 2.1×150 mm dimensions) equilibrated at 40 C. Eluent A was 0.05% TFA in water; eluent B was 0.05% TFA in MeCN. Elution was achieved with a linear gradient of 5-95% eluent B over 16 min at a flow rate of 0.4 mL/min for UV detection and a linear gradient of 5-60% eluent B over 4 min at a flow rate of 0.45 mL/min for MS detection. UV detection was set to 214 nm. MS ionization was run in API-ES mode and positive polarity with a scan mass range of 100-2000 amu. The most abundant isotope of each m/z is reported.

Example 1: Synthesis of Compounds

[0326] The compounds are in the following described using single letter amino acid codes, except for Aeg, Aib, D-Lys, and Sar. Each substituent is included in brackets after the residue to which it is attached.

Parent Compound No. 1

[0327] Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00073##

SEQ ID NO.: 5; substituent: Chem. 8 attached to Lys33
Synthesis methods: SPPS_A; DS_D; CP_A
Molecular weight (average) calculated: 4901.4 Da
LCMS_A: Rt=6.3 min; found [M+3H].sup.3+ 1634.6, [M+4H].sup.4+ 1226.1

Parent Compound No. 2

[0328] Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPS-OH

##STR00074##

SEQ ID NO: 5; Substituent: Chem. 10 attached to Lys33
Synthesis methods: SPPS_A; DS_D; CP_A
Molecular weight (average) calculated: 4867.5 Da
LCMS_A: Rt=6.1 min; found [M+3H].sup.3+ 1623.1, [M+4H].sup.4+ 1217.6

Parent Compound No. 3

[0329] Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPS-OH

##STR00075##

SEQ ID NO: 5; Substituent: Chem. 7 attached to Lys33
Synthesis methods: SPPS_A; DS_D; CP_A
Molecular weight (average) calculated: 4895.5 Da
LCMS_A: Rt=6.3 min; found [M+3H].sup.3+ 1632.4, [M+4H].sup.4+ 1224.6

Parent Compound No. 4

[0330] Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-QAAREFIEWLLAGGPSSGAPPPS-OH

##STR00076##

SEQ ID NO: 6; Substituent: Chem. 7 attached to Lys16
Synthesis methods: SPPS_A; DS_D; CP_A
Molecular weight (average) calculated: 4853.5 Da
LCMS_A: Rt=5.9 min; found [M+3H].sup.3+ 1618.5, [M+4H].sup.4+ 1214.2

Parent Compound No. 5

[0331] Y-Aib-EGTFTSDYSI-Aib-LDKIAQK[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-AFVQWLIAGGPSSGAPPPS-NH.sub.2

##STR00077##

SEQ ID NO.: 4 with C-terminal amide modification; substituent: Chem. 11 attached to Lys20
Synthesis methods: SPPS_A; DS_D; CP_A
Molecular weight (average) calculated: 4813.5 Da
LCMS_A: Rt=6.2 min; found [M+3H].sup.3+ 1605.2, [M+4H].sup.4+ 1204.3

Compound No. 1

[0332] (D-Lys)[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00078##

Z: Parent compound no. 1; X=D-Lys; Y=Sar; substituent b: Chem. 16 attached to X; substituent z: Chem. 8 attached to Lys33 of Z.
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5498.2 Da
LCMS_A: Rt=6.8 min; found [M+3H].sup.3+ 1833.4, [M+4H].sup.4+ 1375.3

Compound No. 2

[0333] G-Aeg[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00079##

Z: Parent compound no. 1; X=Gly; Y=Aeg; substituent b: Chem. 16 attached to Y; substituent
z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_B; DS_B; DS_D; CP_A
Molecular weight (average) calculated: 5456.1 Da
LCMS_A: Rt=7.0 min; found [M+3H].sup.3+ 1819.4, [M+4H].sup.4+ 1364.8

Compound No. 3

[0334] K[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00080##

Z: Parent compound no. 1; X=Lys; Y=Sar; substituent b: Chem. 16 attached to X; substituent
z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5498.2 Da
LCMS_A: Rt=6.9 min; found [M+3H].sup.3+ 1833.7, [M+4H].sup.4+ 1375.6

Compound No. 4

[0335] A-Aeg[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00081##

Z: Parent compound no. 1; X=Ala; Y=Aeg; substituent b: Chem. 16 attached to Y; substituent
z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_B; DS_B; DS_D; CP_A
Molecular weight (average) calculated: 5470.1 Da
LCMS_A: Rt=7.0 min; found [M+3H].sup.3+ 1823.8, [M+4H].sup.4+ 1368.2

Compound No. 5

[0336] L-Aeg[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00082##

Z: Parent compound no. 1; X=Leu; Y=Aeg; substituent b: Chem. 16 attached to Y; substituent
z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_B; DS_B; DS_D; CP_A
Molecular weight (average) calculated: 5512.2 Da
LCMS_A: Rt=7.1 min; found [M+3H].sup.3+ 1838.1, [M+4H].sup.4+ 1378.9

Compound No. 6

[0337] P-Aeg[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00083##

Z: Parent compound no. 1; X=Pro; Y=Aeg; substituent b: Chem. 16 attached to Y; substituent
z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_B; DS_B; DS_D; CP_A
Molecular weight (average) calculated: 5496.2 Da
LCMS_A: Rt=7.0 min; found [M+3H].sup.3+ 1832.4, [M+4H].sup.4+ 1374.6

Compound No. 7

[0338] D-Aeg[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00084##

Z: Parent compound no. 1; X=Asp; Y=Aeg; substituent b: Chem. 16 attached to Y; substituent
z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_A; DS_C; DS_D; CP_A
Molecular weight (average) calculated: 5514.1 Da
LCMS_B: Rt=10.2 min; found [M+3H].sup.3+ 1838.8, [M+4H].sup.4+ 1379.3.

Compound No. 8

[0339] K[(4S)-4-carboxy-4-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]butanoyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00085##

Z: Parent compound no. 1; X=Lys; Y=Sar; substituent b: Chem. 18 attached to X; substituent
z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_B; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5627.3 Da
LCMS_A: Rt=6.9 min; found [M+3H].sup.3+ 1876.3, [M+4H].sup.4+ 1407.6

Compound No. 9

[0340] K[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]acetyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00086##

Z: Parent compound no. 1; X=Lys; Y=Sar; substituent b: Chem. 19 attached to X; substituent
z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_B; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5555.2 Da
LCMS_A: Rt=6.9 min; found [M+3H].sup.3+ 1852.3, [M+4H].sup.4+ 1389.7

Compound No. 10

[0341] (D-Lys)[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00087##

Z: Parent compound no. 1; X=D-Lys; Y=Sar; substituent b: Chem. 21 attached to X;
substituent z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_B; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5788.5 Da
LCMS_A: Rt=6.9 min; found [M+3H]3.sup.+ 1930.3, [M+4H]4.sup.+ 1447.8

Compound No. 11

[0342] (D-Lys)[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00088##

Z: Parent compound no. 1; X=D-Lys; Y=Sar; substituent b: Chem. 20 attached to X;
substituent z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_B; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5626.4 Da
LCMS_A: Rt=6.7 min; found [M+3H].sup.3+ 1876.2, [M+4H].sup.4+ 1407.2

Compound No. 12

[0343] K[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPS-OH

##STR00089##

Z: Parent compound no. 2; X=Lys; Y=Sar; substituent b: Chem. 16 attached to X; substituent
z: Chem. 10 attached to Lys33 of Z
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5464.2 Da
LCMS_A: Rt=6.6 min; found [M+3H].sup.3+ 1822.2, [M+4H].sup.4+ 1366.7

Compound No. 13

[0344] K[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPS-OH

##STR00090##

Z: Parent compound no. 2; X=Lys; Y=Sar; substituent b: Chem. 17 attached to X; substituent
z: Chem. 10 attached to Lys33 of Z
Synthesis methods: SPPS_B; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5492.3 Da
LCMS_A: Rt=6.8 min; found [M+3H].sup.3+ 1831.4, [M+4H].sup.4+ 1373.7

Compound No. 14

[0345] K[(2S)-2,6-bis[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPS-OH

##STR00091##

Z: Parent compound no. 2; X=Lys; Y=Sar; substituent b: Chem. 22 attached to X; substituent
z: Chem. 10 attached to Lys33 of Z
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5989.9 Da
LCMS_A: Rt=7.0 min; found [M+3H].sup.3+ 1997.1, [M+4H].sup.4+ 1498.0

Compound No. 15

[0346] K[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]-Sar-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPS-OH

##STR00092##

Z: Parent compound no. 3; X=Lys; Y=Sar; substituent b: Chem. 17 attached to X; substituent
z: Chem. 7 attached to Lys33 of Z
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5520.3 Da
LCMS_A: Rt=7.0 min; found [M+3H].sup.3+ 1840.9, [M+4H].sup.4+ 1380.9

Compound No. 16

[0347] K[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Sar-Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-QAAREFIEWLLAGGPSSGAPPPS-OH

##STR00093##

Z: Parent compound no. 4; X=Lys; Y=Sar; substituent b: Chem. 16 attached to X; substituent
z: Chem. 7 attached to Lys16 of Z
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5450.2 Da
LCMS_A: Rt=6.3 min; found [M+3H].sup.3+ 1817.3, [M+4H].sup.4+ 1363.1

Compound No. 17

[0348] G-Aeg[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-QAAREFIEWLLAGGPSSGAPPPS-OH

##STR00094##

Z: Parent compound no. 4; X=Gly; Y=Aeg; substituent b: Chem. 16 attached to Y; substituent
z: Chem. 7 attached to Lys16 of Z
Synthesis methods: SPPS_A; DS_C; DS_D; CP_A
Molecular weight (average) calculated: 5408.2 Da
LCMS_B: Rt=9.5 min; found [M+3H].sup.3+ 1803.6, [M+4H].sup.4+ 1353.0

Compound No. 18

[0349] K[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Sar-Y-Aib-EGTFTSDYSI-Aib-LDKIAQK[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-AFVQWLIAGGPSSGAPPPS-NH.sub.2

##STR00095##

Z: Parent compound no. 5; X=Lys; Y=Sar; substituent b: Chem. 16 attached to X; substituent
z: Chem. 11 attached to Lys20 of Z
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5410.2 Da
LCMS_A: Rt=6.6 min; found [M+3H].sup.3+ 1804.0, [M+4H].sup.4+ 1353.5

Non-Converting Compound No. 1

[0350] K[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Val-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-GAPPPS-OH

##STR00096##

Z: Parent compound no. 1; X=Lys; Y=Val; substituent b: Chem. 16 attached to X; substituent
z: Chem. 8 attached to Lys33 of Z
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5526.2 Da
LCMS_A: Rt=6.9 min; found [M+3H].sup.3+ 1842.8, [M+4H].sup.4+ 1382.2

Non-Converting Compound No. 2

[0351] K[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Val-Y-Aib-EGTFTSDYSILLEEQAAREFIEWLLAGGPS-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-GAPPPS-OH

##STR00097##

Z: Parent compound no. 2; X=Lys; Y=Val; substituent b: Chem. 16 attached to X; substituent
z: Chem. 10 attached to Lys33 of Z
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5492.3 Da
LCMS_A: Rt=6.6 min; found [M+3H].sup.3+ 1831.4, [M+4H].sup.4+ 1373.9

Non-Converting Compound No. 3

[0352] K[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Val-Y-Aib-EGTFTSDYSILLE-K[(2S)-2-amino-6-[[(2S)-2-amino-6-[[(4S)-4-carboxy-4-(17-carboxyheptadecanoylamino)butanoyl]amino]hexanoyl]amino]hexanoyl]-QAAREFIEWLLAGGPSSGAPPPS-OH

##STR00098##

Z: Parent compound no. 4; X=Lys; Y=Val; substituent b: Chem. 16 attached to X; substituent
z: Chem. 7 attached to Lys16 of Z
Synthesis methods: SPPS_A; DS_A; DS_D; CP_A
Molecular weight (average) calculated: 5478.3 Da
LCMS_A: Rt=6.1 min; found [M+3H].sup.3+ 1826.7, [M+4H].sup.4+ 1370.3
Non-converting compound No. 4
K[(4S)-4-carboxy-4-(15-carboxypentadecanoylamino)butanoyl]-Val-Y-Aib-EGTFTSDYSI-Aib-LDKIAQK[2-[2-[2-[[2-[2-[2-[[(4S)-4-carboxy-4-(19-carboxynonadecanoylamino)butanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]-AFVQWLIAGGPSSGAPPPS-NH.sub.2

##STR00099##

Z: Parent compound no. 5; X=Lys; Y=Val; substituent b: Chem. 16 attached to X; substituent
z: Chem. 11 attached to Lys20 of Z
Synthesis methods: SPPS_A: DS_A: DS_D; CP_A
Molecular weight (average) calculated: 5438.3 Da
LCMS_A: Rt=6.6 min; found [M+3H].sup.3+ 1813.4, [M+4H].sup.4+ 1360.2

General Methods for Measuring Conversion Half-Life

[0353] The assay was performed to investigate the conversion half-life of prodrug to drug of the prodrugs of the invention. The conversion half-life was investigated in vitro at pH 7.4 upon incubation at 37° C.

Preparation of Formulation and Sampling

[0354] Test compounds were dissolved to a concentration of 100 μM in phosphate-buffered saline (140 mM NaCl, 2.07 mM KCl, 8.05 mM Na.sub.2HPO.sub.4, 1.96 mM KH.sub.2PO.sub.4, pH 7.4). Solution pH was measured after dissolution and adjusted to pH 7.4 with aqueous NaOH if necessary. The solution was filtered through a 0.22 μm syringe filter, then incubated in a water bath at 37° C. Aliquots were withdrawn at defined timepoints, e.g. every 24-72 hours, for LCMS analysis.

Analysis and Calculations

[0355] LCMS analysis was performed using the procedure defined in LCMS_A above. The AUC of the prodrug, active drug, and DKP were determined using UV detection at 214 nm, and the ratio of prodrug AUC over total AUC of prodrug plus active drug plus DKP was calculated. The negative natural logarithm of this ratio was plotted over time, and a linear regression of this relationship was then performed. The conversion half-life was calculated from this linear regression as the time at which the AUC ratio=0.5.

Example 2

[0356] The prodrug to drug conversion half-life of the compounds of the invention was measured as described in General Methods for Measuring Conversion Half-life. The results are presented in Table 6. The compounds of the invention are associated with surprisingly long conversion half-lives, which can be tuned through subtle modifications of the chemical structure of the DKP moiety.

TABLE-US-00007 TABLE 6 Prodrug conversion half-life in PBS buffer at pH 7.4 and 37° C. Compound No. Conversion half-life [h] 1 244 2 363 3 111 4 97 5 74 6 440 7 38 8 125 9 132 10 195 11 182 12 129 13 137 14 166 15 127 16 117 18 101

General Methods for Measuring Terminal Half-Life in Minipigs

[0357] The purpose of this method is to determine the half-life in vivo of the derivatives of the present invention after i.v. administration to minipigs, i.e. the prolongation of their time in the body and thereby their time of action. This is done in a pharmacokinetic (PK) study, where the terminal half-life of the derivative in question is determined. By terminal half-life is generally meant the period of time it takes to halve a certain plasma concentration, measured after the initial distribution phase.

Study

[0358] Female Göttingen minipigs were obtained from Ellegaard Göttingen Minipigs (Dalmose, Denmark) approximately 7-14 months of age and weighing from approximately 16-35 kg were used in the studies. The minipigs were housed individually and fed restrictedly once daily with SDS minipig diet (Special Diets Services, Essex, UK).

[0359] After at 3 weeks of acclimatisation two permanent central venous catheters were implanted in vena cava caudalis in each animal. The animals were allowed 1 week recovery after the surgery, and were then used for repeated pharmacokinetic studies with a suitable wash-out period between successive derivative dosing.

[0360] The animals were fasted for approximately 18 hours before dosing and from 0 to 4 hours after dosing but had adlibitum access to water during the whole period.

[0361] The sodium salts of compounds of Examples 1 were prepared using method SX_A under General Methods for preparation of the compounds of the invention. The resulting sodium salts were dissolved to a concentration of 50-300 nmol/mL in a buffer containing 0.007% polysorbate 20, 50 mM sodium phosphate, 70 mM sodium chloride, pH 7.4. Intravenous injections (the volume corresponding to usually 1-20 nmol/kg, for example 0.02-0.05 mL/kg) of the compounds were given through one catheter, and blood was sampled at predefined time points for up to 21 days post dosing (preferably through the other catheter). Blood samples (for example 0.8 mL) were collected in 8 mM EDTA buffer and then centrifuged at 4° C. and 1942 g for 10 minutes.

Sampling and Analysis

[0362] Plasma was pipetted into Micronic tubes on dry ice and kept at −20° C. until analysed for plasma concentration of the compounds using ELISA, or a similar antibody-based assay, or LCMS. Individual plasma concentration-time profiles were analysed by a non-compartmental model in Phoenix WinNonLin ver. 6.4. (Pharsight Inc., Mountain View, Calif., USA), and the resulting terminal half-lives (harmonic mean) determined.

Example 3

[0363] The terminal half-lives and/or the observed terminal half-lives, measured as described herein in General Methods for Measuring Terminal Half-Life in Minipigs are shown in Table 7. Parent compound Nos. 1, 2, and 4, administered in their free form, are associated with surprisingly high observed terminal half-lives, which is extended further by addition of a non-converting dipeptide and substituent (eg Non-converting compound No. 1). The use of a converting prodrug (eg. compound Nos. 1, 3, 9, and 10) results in a shorter half-life than the non-converting counterpart (eg Non-converting compound No. 1), since conversion contributes to the elimination of the converting prodrug but not to the non-converting compound, and conversion cannot be differentiated from other mechanisms of elimination. This provides evidence that conversion of prodrug to parent compound is occurring in vivo. The terminal half-life of the prodrug may be faster or slower than the corresponding parent compound due to the contribution of conversion on the terminal half-life of the prodrug.

TABLE-US-00008 TABLE 7 Terminal half-life as measured after i.v. administration to minipigs Dose of test compound Compound No. (nmol/kg) Terminal t.sub.1/2 (h) Parent compound 2 121 1 Parent compound 1 104 2 Parent compound 1 106 4 Non-converting 2 170 compound 1 Compound 1 20 118 Compound 3 2 119 Compound 9 20 102 Compound 10 20 118

General Methods for Measuring Oral Bioavailability in Beagle Dogs

[0364] The purpose of this method is to determine the terminal half-life and plasma exposure in vivo of the compounds of the present invention after p.o. administration to beagle dogs, i.e. the terminal half-life and concentration of test substance that reaches circulation with time. This is done in a pharmacokinetic (PK) study, where these parameters of the compound in question are determined. By terminal half-life is generally meant the period of time it takes to halve a certain plasma concentration, measured after the initial distribution phase.

Preparation of Tablet Compositions

[0365] Tablet compositions comprising the test compounds obtained from Example 1 and SNAC (sodium N-(8-(2-hydroxybenzoyl)amino)caprylate) were prepared according to methods known to the person skilled in the art by mixing test substance with roller compacted SNAC and magnesium stearate as e.g. described in WO 2019/149880. Each tablet was comprised of 7.7 mg magnesium stearate, 2-4 mg of each compound tested, and 300 mg SNAC.

Animals, Dosing, and Sampling

[0366] Male beagle dogs, 1-7 years of age and weighing 9-17 kg during the study period, were included in the study. The dogs were dosed in a fasting state. The dogs were group housed in pens (12 hours light: 12 hours dark), and fed individually and restrictedly once daily with Royal Canin Medium Adult dog food (Royal Canin Products, China Branch, or Brogaarden A/S, Denmark). The dogs were used for repeated PK studies with a suitable wash-out period between successive dosing. An appropriate acclimatisation period was given prior to initiation of the first PK study. All handling, dosing, and blood sampling of the animals were performed by trained and skilled staff. Before the studies, the dogs were fasted overnight and from 0 to 4 hours after dosing. The dogs were restricted to water 1 hour before dosing until 4 hours after dosing, but otherwise had adlibitum access to water during the whole period.

[0367] The compositions were administered by a single oral dosing to the dogs in groups of 6-8 dogs. The tablets were administered in the following manner: 10 min prior to tablet administration, the dogs may be dosed subcutaneously with approximately 3 nmol/kg of SEQ ID NO.: 7 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNT), then tablets were placed in the back of the mouth of the dog to prevent chewing. The mouth was then closed, and 10 mL of tap water was given by syringe or gavage to facilitate swallowing of the tablet.

[0368] One blood sample was drawn before dosing, and additional samples were drawn at predefined time points after dosing, such as for up to 600 hours, to adequately cover the full plasma concentration-time absorption profile of the test substance. For each blood sample time point, approximately 0.8 mL of whole blood was collected in a 1.5 mL EDTA-coated tube, which was gently turned to mix the sample with EDTA. Blood samples were collected in EDTA buffer (8 mM) and then centrifuged at 4° C. and 2000 g for 10 minutes. Plasma was pipetted into Micronic tubes on dry ice, and kept at −20° C. or lower until analysis. Blood samples were taken as appropriate, for example from a venflon in the cephalic vein in the front leg for the first 2 hours and then with syringe from the jugular vein for the rest of the time points. The first few drops were allowed to drain from the venflon to avoid heparin saline from the venflon in the sample. All blood samples were collected into test tubes containing EDTA for stabilisation and kept on ice until centrifugation. Plasma was separated from whole blood by centrifugation and the plasma was stored at −20° C. or lower until analysis.

Analysis and Calculations

[0369] The plasma was analysed for test substance using LC-MS (Liquid Chromatography-Mass Spectrometry) as known to the person skilled in the art. The system consisted of either: a Thermo Fisher QExactive mass spectrometer equipped with a 10-valve interface module TurboFlow system, CTC HTS PAL autosampler, Accela 1250 pumps, and Hot Pocket column oven; or a Thermo Fisher QExactive Plus mass spectrometer equipped with a valve interface module TurboFlow system, TriPlus RSI autosampler, Dionex UltiMate 3000 pumps, and Hot Pocket column oven. Reversed-phase HPLC separation was achieved using a linear gradient of 1:1 acetonitrile/methanol in 1% aqueous formic acid using either: a Phenomenex Onyx Monolithic C18 column (50×2.0 mm) and a flow rate of 0.8 mL/min at 30° C.; or an Agilent Poroshell 120 SB-C18 column (50×2.1 mm, 2.7 μm) at a flow rate of 0.4 mL/min at 60° C. The mass spectrometer was operated in either positive ionization SIM mode or positive ionization PRM mode.

[0370] For each individual animal, a plasma concentration-time profile was analysed by a non-compartmental model in Pharsight Phoenix WinNonLin ver. 6.4 software or other relevant software for PK analysis, and the resulting terminal half-life (t.sub.1/2), maximum plasma concentration per dose (C.sub.max/D), time for maximum plasma concentration (t.sub.max), and area under the curve to infinity per dose (AUC/D) were determined. Summary statistics of pharmacokinetic results were presented as median (for t.sub.max), hormonic mean (t.sub.1/2), or arithmetic mean (C.sub.max, AUC).

Example 4

[0371] The pharmacokinetic properties, measured as described herein in General Methods for Measuring Oral Bioavailability in Beagle Dogs, are shown in Table 8. All tested compounds of the present invention demonstrate oral bioavailability in this model, as concentrations of the compounds in plasma were detected (C.sub.max/D>0 and AUC/D>0) following oral administration. All tested compounds are also associated with surprisingly high observed terminal half-lives as observed in Example 3.

TABLE-US-00009 TABLE 8 Pharmacokinetic parameters as measured after p.o. administration to Beagle dogs Dose of test C.sub.max/ AUC/ compound Terminal Dose Dose Compound No. (mg/tablet) T.sub.max (h) t.sub.1/2 (h) (kg/L) (kg*h/L) Parent compound 1 3 1.5 104 0.67 60.7 Parent compound 3 2.9 1.5 131 0.22 21.4 Parent compound 4 2.9* 1.3* 56* 0.35* 17.9* Parent compound 5 3 1.0 134 0.22 13.0 Non-converting 2.8 1.5 137 0.21 20.1 compound 1 Non-converting 3.5 1.3 130 0.13 10.8 compound 2 Non-converting 3.0 4.0 115 0.27 31.4 compound 3 Non-converting 3.0 7.0 136 0.16 18.9 compound 4 ( Compound 1 3.1 4.0 146 0.66 41.2 Compound 2 2.9 4.0 142 0.18 22.9 Compound 3 3.0 7.0 139 0.32 38.5 Compound 5 2.8 7.0 106 0.25 23.4 Compound 12 2.7 1.3 143 0.46 50.8 Compound 13 2.8 4.3 121 0.36 41.3 Compound 14 3.2 5.5 119 0.38 43.6 Compound 15 2.0 1.5 124 0.20 21.7 Compound 16 2.9 4.0 96 0.73 67.6 Compound 17 3.2 1.5 105 0.30 30.4 Compound 18 2.9 4.0 88 0.15 13.3 * = averaged data from three experiments

General Methods for Measuring In Vitro Functional Potency

[0372] The purpose of this example is to test the functional activity, or potency, of the compounds in vitro at the human GLP-1 and GIP receptors. The in vitro functional potency is the measure of target receptor activation in a whole cell assay. The potencies of parent compound Nos. 1-5 of Example 1 were determined as described below. Human GLP-1(7-37) (HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G; SEQ ID NO.: 8) and human GIP (YAEGT FISDY SIAMD KIHQQ DFVNW LLAQK GKKND WKHNI TO; SEQ ID NO.: 9) were included as reference compounds in appropriate assays for comparison.

Principle

[0373] In vitro functional potency was determined by measuring the response of the target receptor in a reporter gene assay in individual cell lines. The assay was performed in stably transfected BHK cell lines that expresses one of the following G-protein coupled receptors: human GLP-1 receptor or human GIP receptor; and where each cell line contains the DNA for the cAMP response element (CRE) coupled to a promoter and the gene for firefly luciferase (CRE luciferase). When the respective receptor is activated, it results in the production of cAMP, which in turn results in expression of the luciferase protein. When assay incubation is completed, luciferase substrate (luciferin) is added resulting in the enzymatic conversion of luciferin to oxyluciferin and producing bioluminescence. The luminescence is measured as the readout for the assay.

Cell Culture and Preparation

[0374] The cells lines used in these assays were BHK cells with BHKTS13 as a parent cell line. The cell lines were derived from a clone containing the CRE luciferase element and were established by further transfection with the respective human receptor to obtain the relevant cell line: BHK CRE luc2P hGLP-1R or BHK CRE luc2P hGIPR. The cells were cultured at 37° C. with 5% CO.sub.2 in Cell Culture Medium. They were aliquoted and stored in liquid nitrogen. The cells were kept in continuous culture and were seeded out the day before each assay.

Materials

[0375] The following chemicals were used in the assay: Pluronic F-68 10% (Gibco 2404), human serum albumin (HSA; Sigma A9511), 10% fetal bovine serum (FBS; Invitrogen 16140-071), chicken egg white ovalbumin (Sigma A5503), DMEM w/o phenol red (Gibco 21063-029), DMEM (Gibco 12430-054), 1 M Hepes (Gibco 15630), Glutamax 100x (Gibco 35050), G418 (Invitrogen 10131-027), hygromycin (Invitrogen 10687-010), and steadylite plus (PerkinElmer 6016757).

Buffers

[0376] GLP-1R Cell Culture Medium consisted of DMEM medium with 10% FBS, 500 μg/mL G418, and 300 μg/mL hygromycin. GIPR Cell Culture Medium consisted of DMEM medium with 10% FBS, 400 μg/mL G418, and 300 μg/mL hygromycin. Assay Buffer consisted of DMEM w/o phenol red, 10 mM Hepes, 1× Glutamax. 1% ovalbumin, and 0.1% Pluronic F-68 with the addition of HSA at twice the final assay concentration. The Assay Buffer was mixed 1:1 with an equal volume of the test compound in Assay Buffer to give the final assay concentration of HSA.

Procedure

[0377] 1) Cells were plated at 5000 cells/well and incubated overnight in the assay plate.
2) Cells were washed once in DPBS.
3) Stocks of the test compounds and reference compounds in concentrations ranging from 100-300 μM were diluted 1:150 in Assay Buffer. Compounds were then diluted 1:10 in column 1 of a 96 deep well dilution plate and then carried across the row creating a 3.5 fold, 12 point dilution curve.
4) Assay Buffer (50 μl aliquot) with or without HSA was added to each well in the assay plate.
5) A 50 μl aliquot of compound or blank was transferred from the dilution plate to the assay plate containing the Assay Buffer with or without HSA.
6) The assay plate was incubated for 3 h in a 5% CO.sub.2 incubator at 37° C.
7) The cells were washed once with DPBS.
8) A 100 μl aliquot of DPBS was added to each well of the assay plate.
9) A 100 μl aliquot of steadylite plus reagent (light sensitive) was added to each well of the assay plate.
10) Each assay plate was covered with aluminum foil to protect it from light and shaken at 250 rpm for 30 min at room temperature.
11) Each assay plate was read in a microtiter plate reader.

Calculations and Results

[0378] The data from the microtiter plate reader was first regressed in an Excel in order to calculate the x-axis, log scale concentrations based on the individual test compound's stock concentration and the dilutions of the assay. This data was then transferred to GraphPad Prism software for graphing and statistical analysis. The software performs a non-linear regression (log(agonist) vs response). EC.sub.50 values which were calculated by the software and reported in pM are shown in Table 9 below. A minimum of two replicates was measured for each sample. The reported values are averages of the replicates. The compounds of the present invention display potent functional activation of the human GLP-1R and human GIPR receptors under the given conditions.

Example 5

[0379] The GLP-1 and GIP receptor functional potencies, measured as described herein in General methods for measuring in vitro functional potency, are shown in Table 9. Parent compounds 1-5, administered in their free form, display potent functional activation of the human GLP-1 receptor and human GIP receptor under the given conditions.

TABLE-US-00010 TABLE 9 Functional potencies at human GLP-1R and GIPR in the presence of 0% and 1% HSA. hGLP-1R, hGLP-1R, hGIPR, hGIPR, CRE CRE CRE CRE Compound Luc 0% HSA Luc 1% HSA Luc 0% HSA Luc 1% HSA No. EC.sub.50 (pM) EC.sub.50 (pM) EC.sub.50 (pM) EC.sub.50 (pM) hGLP- 8.4 6.7 nd nd 1(7-37) hGIP nd nd 11.3 6.4 Parent 2.1 154.5 3.1 155.7 compound 1 Parent 1.5 110.8 1.8 97.0 compound 2 Parent 4.0 359.5 5.6 444.5 compound 3 Parent 4.2 202.0 4.7 188.5 compound 4 Parent 9.7 530.9 4.2 136.8 compound 5 nd = not determined.

[0380] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.