Exendin-4 derivatives as dual GLP1/GIP or trigonal GLP1/GIP/glucagon agonists

Abstract

The present invention relates to exendin-4 derivatives and their medical use, for example in the treatment of disorders of the metabolic syndrome, including diabetes and obesity, as well as reduction of excess food intake.

Description

[0001] manual field of invention

[0002] The present invention relates to exendin-4 (exendin-4) peptide analogs, which activate glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors, and optionally Activation of glucagon receptors (GCG), and their medicinal use, for example in the treatment of disorders of the metabolic syndrome including diabetes and obesity and in reducing excess food intake. Background of the invention

[0003] Exendin-4 is a 39 amino acid peptide produced by the Gila monster (Heloderma suspectum) (Eng J. et al., J. Biol. Chem. , 267:7402-05, 1992). Exendin-4 is an activator of the glucagon-like peptide-1 (GLP-1) receptor, however it shows only low activation of the GLP receptor and does not activate the glucagon receptor. body (see Table 1).

[0004] Table 1: Potency of exendin-4 at increasing concentrations on human GLP-1, GIP and glucagon receptors (expressed in pM), and cAMP formed measured as described i...

Examples

Embodiment 1

[0597] Synthesis of SEQ ID NO:12

[0598]In Novabiochem Rink-Amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 The solid-phase synthesis was carried out on a mesh with a loading of 0.34 mmol / g. The Fmoc-synthesis strategy was applied with HBTU / DIPEA-activation. Boc-Tyr(tBu)-OH at position 1 and Fmoc-Lys(ivDde)-OH at position 14 were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on the resin with 4% hydrazine hydrate in DMF according to a modified literature procedure (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). Thereafter Palm-Glu(γОSu)-OtBu was coupled to the detethered amino group. Peptides were cleaved from the resin with King's mixture (D.S. King, C.G. Fields, G.B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified by preparative HPLC on a Waters column (Sunfire, Prep C18) with an acetonitrile / water gradient, both buf...

Embodiment 2

[0600] Synthesis of SEQ ID NO:31

[0601] In Novabiochem Rink-Amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 The solid phase synthesis was carried out on the loading amount of 0.34mmol / g. The Fmoc-synthesis strategy was applied with HBTU / DIPEA-activation. Boc-Tyr(tBu)-OH at position 1 and Fmoc-Lys(ivDde)-OH at position 14 were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on the resin with 4% hydrazine hydrate in DMF according to a modified literature procedure (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). Thereafter Stea-Glu(γОSu)-OtBu was coupled to the detethered amino group. Peptides were cleaved from the resin with King's mixture (D.S. King, C.G. Fields, G.B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified by preparative HPLC on a Waters column (Sunfire, Prep C18) with an acetonitrile / water gradient, both buffer...

Embodiment 3

[0603] Synthesis of SEQ ID NO:14

[0604] Solid phase synthesis was performed on Rink-resin from Agilent Technologies with a loading of 0.38 mmol / g, 75-150 μm. The Fmoc-synthesis strategy was applied with HBTU / DIPEA-activation. Boc-Tyr(tBu)-OH at position 1 and Fmoc-Lys(ivDde)-OH at position 14 were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on the resin according to the literature (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). This was followed by coupling of Palm-Glu(γОSu)-OtBu to the detethered amino group using HBTU / DIPEA coupling reagent, followed by Fmoc-deprotection with 20% piperidine in DMF. Peptides were cleaved from the resin using King's mixture (D.S. King, C.G. Fields, G.B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified by preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5 μΜ) with an acetonitrile / water gradient, both buffered with 0.1% TFA. The pur...

[0001] manual field of invention

[0002] The present invention relates to exendin-4 (exendin-4) peptide analogs, which activate glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors, and optionally Activation of glucagon receptors (GCG), and their medicinal use, for example in the treatment of disorders of the metabolic syndrome including diabetes and obesity and in reducing excess food intake. Background of the invention

[0003] Exendin-4 is a 39 amino acid peptide produced by the Gila monster (Heloderma suspectum) (Eng J. et al., J. Biol. Chem. , 267:7402-05, 1992). Exendin-4 is an activator of the glucagon-like peptide-1 (GLP-1) receptor, however it shows only low activation of the GLP receptor and does not activate the glucagon receptor. body (see Table 1).

[0004] Table 1: Potency of exendin-4 at increasing concentrations on human GLP-1, GIP and glucagon receptors (expressed in pM), and cAMP formed measured as described i...

Examples

Embodiment 1

[0597] Synthesis of SEQ ID NO:12

[0598]In Novabiochem Rink-Amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 The solid-phase synthesis was carried out on a mesh with a loading of 0.34 mmol / g. The Fmoc-synthesis strategy was applied with HBTU / DIPEA-activation. Boc-Tyr(tBu)-OH at position 1 and Fmoc-Lys(ivDde)-OH at position 14 were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on the resin with 4% hydrazine hydrate in DMF according to a modified literature procedure (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). Thereafter Palm-Glu(γОSu)-OtBu was coupled to the detethered amino group. Peptides were cleaved from the resin with King's mixture (D.S. King, C.G. Fields, G.B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified by preparative HPLC on a Waters column (Sunfire, Prep C18) with an acetonitrile / water gradient, both buf...

Embodiment 2

[0600] Synthesis of SEQ ID NO:31

[0601] In Novabiochem Rink-Amide resin (4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucylaminomethyl resin), 100-200 The solid phase synthesis was carried out on the loading amount of 0.34mmol / g. The Fmoc-synthesis strategy was applied with HBTU / DIPEA-activation. Boc-Tyr(tBu)-OH at position 1 and Fmoc-Lys(ivDde)-OH at position 14 were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on the resin with 4% hydrazine hydrate in DMF according to a modified literature procedure (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). Thereafter Stea-Glu(γОSu)-OtBu was coupled to the detethered amino group. Peptides were cleaved from the resin with King's mixture (D.S. King, C.G. Fields, G.B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified by preparative HPLC on a Waters column (Sunfire, Prep C18) with an acetonitrile / water gradient, both buffer...

Embodiment 3

[0603] Synthesis of SEQ ID NO:14

[0604] Solid phase synthesis was performed on Rink-resin from Agilent Technologies with a loading of 0.38 mmol / g, 75-150 μm. The Fmoc-synthesis strategy was applied with HBTU / DIPEA-activation. Boc-Tyr(tBu)-OH at position 1 and Fmoc-Lys(ivDde)-OH at position 14 were used in the solid phase synthesis protocol. The ivDde-group was cleaved from the peptide on the resin according to the literature (S.R. Chhabra et al., Tetrahedron Lett. 39, (1998), 1603). This was followed by coupling of Palm-Glu(γОSu)-OtBu to the detethered amino group using HBTU / DIPEA coupling reagent, followed by Fmoc-deprotection with 20% piperidine in DMF. Peptides were cleaved from the resin using King's mixture (D.S. King, C.G. Fields, G.B. Fields, Int. J. Peptide Protein Res. 36, 1990, 255-266). The crude product was purified by preparative HPLC on a Waters column (XBridge, BEH130, Prep C18 5 μΜ) with an acetonitrile / water gradient, both buffered with 0.1% TFA. The pur...

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