GLP-1 / GIP Dual Agonist, Its Preparation Method and Use

Abstract

The present invention relates to a long-acting GLP-1 / GIP dual agonist compound having a dual agonistic effect on glucagon-like peptide-1 (GLP-1) receptor and human glucose-dependent insulinotropic polypeptide (GIP) receptor.

Description

Technical Field

[0001] The present invention relates to the pharmaceutical field, and in particular, to GLP-1 / GIP dual agonists and their use in the manufacture of medicaments for treating diabetes.

Background Art

[0002] Type II diabetes is a type of chronic metabolic disease clinically characterized by hyperglycemia due to insulin resistance. The increase in blood glucose increases the incidence and mortality of other diseases. Currently, all the methods commonly used to treat this disease, such as doing more exercise, controlling diet, orally administering hypoglycemic agents, injecting therapeutic agents, etc., can only temporarily maintain blood glucose balance and cannot fundamentally treat the various complex complications caused by diabetes and hyperglycemia. Therefore, it is extremely important clinically to widely search for and develop safe medicaments for treating diabetes with a novel hypoglycemic mechanism.

[0003] Glucagon-like peptide-1 (GLP-1) is a type of hormone mainly produced by intestinal L cells and belongs to incretin. GLP-1 is an important incretin hormone secreted from intestinal L cells, and its main physiological functions in the body include promoting the secretion and release of insulin, inhibiting the secretion of glucagon, promoting the proliferation of pancreatic β cells and inhibiting their apoptosis, inhibiting gastric emptying, promoting the production of satiety, etc. The function of glucose-dependent insulinotropic polypeptide (GIP) is to regulate blood glucose by promoting insulin and glucagon, and this has also been widely confirmed and applied.

[0004] Compounds having GLP-1 / GIP dual agonist activity are disclosed in WO2013164483A1, WO2014192284A1 and WO2016111971A1.

[0005] By modifying a polypeptide with a fatty acid and physically binding the fatty acid chain to an albumin subdomain / motif, for example, the half-life can be extended, thereby improving the pharmacokinetics of the peptide. Although the use of fatty acids may improve the half-life of the peptide, specific extension effects and some side effects are technical problems to be solved.

[0006] A series of side effects are caused by GLP-1. For example, the effects of GLP-1 receptor agonists on pancreatic and thyroid tissues are a concern, and animal experiments and analysis of pharmaceutical databases have shown that these drugs are associated with pancreatitis, pancreatic cancer, and thyroid cancer. According to other reported cases, the use of GLP-1 (mainly exenatide) is associated with the occurrence of acute kidney injury caused by hemodynamic disorders mainly due to nausea, vomiting, and diarrhea. The most common symptoms associated with the use of GLP-1 receptor agonist drugs are gastrointestinal symptoms mainly including nausea. Other common adverse reactions include injection site reactions, headache, and nasopharyngitis, etc. (Filippatos TD, Panagiotopoulou TV, Elisaf MS. Adverse Effects of GLP-1 Receptor Agonists. Rev Diabet Stud. 2014 Fall-Winter;11(3-4):202-30. doi: 10.1900 / RDS.2014.11.202. Epub 2015 Feb 10. PMID: 26177483; PMCID: PMC5397288.)

[0007] Side effects of Tirzepatide (Mathiesen DS, Bagger JI, Bergmann NC, Lund A, Christensen MB, Vilsbφll T, Knop FK. The Effects of Dual GLP-1 / GIP Receptor Agonism on Glucagon Secretion-A Review. Int J Mol Sci. 2019 Aug 22;20(17): 4092. doi: 10.3390 / ijms20174092. PMID: 31443356; PMCID: PMC6747202.): At the highest dose, the number of adverse events with tirzepatide exceeded that with dulaglutide, particularly gastrointestinal adverse events (66.0% in subjects taking 15 mg of tirzepatide vs. 42.6% in subjects taking 1.5 mg of dulaglutide), and the total number of hypoglycemic episodes was also higher with tirzepatide than with dulaglutide (7.5% in subjects taking 15 mg of tirzepatide vs. 3.7% in subjects taking dulaglutide). (Frias J.P., Nauck M.A., Van J., Kutner M.E., Cui X., Benson C., Urva S., Gimeno R.E., Milicevic Z., Robins D., et al. Efficacy and safety of LY3298176, a novel dual GIP and GLP-1 receptor agonist, in subjects with type 2 diabetes mellitus: a randomised, placebo-controlled and active comparator-controlled phase 2 trial. Lancet. 2018;392:2180-2193. doi: 10.1016 / S0140-6736(18)32260-8). To avoid this problem, a titration strategy to reduce the adverse events of tirzepatide has been reported.(Frias J.P., Nauck M.A., Van J., Benson C., Bray R., Milicevic Z., Haupt A., Robins D.A. 993-P: A 12-week, randomized, placebo-controlled study assessing the efficacy and safety of three dose-escalation algorithms of Tirzepatide, a novel dual GIP and GLP-1 receptor agonist, in subjects with type 2 diabetes mellitus. Diabetes mellitus. 2019;68:993. doi: 10.2337 / db19-993-P). Furthermore, compared with previously reported studies, the three-step dose-escalation regimen for delivering 15 mg of tirzepatide did not appear to reduce the overall incidence of gastrointestinal side effects. (Frias J.P., Nauck M.A., Van J., Kutner M.E., Cui X., Benson C., Urva S., Gimeno R.E., Milicevic Z., Robins D., et al. Efficacy and safety of LY3298176, a novel dual GIP and GLP-1 receptor agonist, in subjects with type 2 diabetes mellitus: a randomised, placebo-controlled and active comparator-controlled phase 2 trial. Lancet. 2018;392:2180-2193. doi: 10.1016 / S0140-6736(18)32260-8). In subjects receiving 15 mg of tirzepatide, the incidence of hypoglycemic episodes also increased to approximately 16%. Treatment with tirzepatide was still associated with the desired weight loss (approximately 5.6 kg after 12 weeks), but the high incidence of gastrointestinal side effects introduced uncertainty about the clinical potential of tirzepatide.Furthermore, the titration strategy described in the commercial product insert of tirzepatide is long-lasting. The initial dose is 2.5 mg, and the dose is increased to 5 mg after 4 weeks. If the dose needs to be increased to control blood glucose, the current dose must be maintained for 4 weeks and then increased by another 2.5 mg, which is quite inconvenient.

[0008] At present, there is still a need in the medical community to provide a new compound that is a dual agonist of the GIP receptor and the GLP-1 receptor, can be administered once a week, and provides a safer, more effective, and more convenient option.

Summary of the Invention

[0009] An object of the present invention is to provide some long-acting GLP-1 / GIP dual agonist compounds. Compared with the GIP / GLP-1 dual agonist compounds in the art, the compounds according to the present invention have a long half-life and / or a longer duration of action. The compounds according to the present invention have a stronger hypoglycemic effect. The compounds according to the present invention have side effects such as, but not limited to, gastrointestinal reactions and cardiac safety risks, but are relatively few. The compounds according to the present invention can adopt a shorter titration strategy. The compounds according to the present invention have a wider safety margin. Furthermore, the compounds according to the present invention have a protective effect on the liver.

[0010] As part of the present invention, the applicant has found that by selecting the length, composition, and bonding position of the fatty acid chain, as well as the linker between the peptide and the fatty acid chain, the half-life of the polypeptide can be extended, which has an unexpected effect.

[0011] One embodiment of the present invention is formula (AI): Y-X1-E-G-T-X2-T-S-D-Y-A11-A12-A13-L-D-K-A17-A-Q-A20-E-F-V-K-W-L-L-K-A29-G-P-S-S-G-A-P-P-P-S-K Formula (AI) [wherein, X1 is Aib; X2 is αMePhe; A11 is Aib or Ala; A12 is Ala, Ile, Lys, Phe or Pya(4); A13 is Aib, Cha, Leu, αMePhe or αMeTyr; A17 is Gln or Ile; A20 is Ala or Ser; A29 is Gln or Gly; One, two, three or four Ks selected from the group consisting of K at position 16, K at position 24, K at position 28 and K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain, wherein, a is each independently an integer from 0 to 5, b is each independently an integer from 0 to 5, c is each independently an integer from 10 to 24, Z is independently selected from the group consisting of -CH3, carboxylic acid or carboxylic acid bioisostere, phosphonate or sulfonate] and pharmaceutically acceptable salts thereof are provided.

[0012] In a further embodiment, the present invention provides a compound of formula (AI): [wherein, X1 is Aib; X2 is αMePhe; A11 is Aib or Ala; A12 is Ala, Ile, Lys, Phe or Pya(4); A13 is Aib, Cha, Leu, αMePhe or αMeTyr; A17 is Gln or Ile; A20 is Ala or Ser; A29 is Gln or Gly, one, two, three, or four Ks selected from the group consisting of K at position 16, K at position 24, K at position 28, and K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain, wherein, a is independently selected from the integers 1, 2, 3, 4, or 5, b is independently selected from the integers 1, 2, 3, 4, or 5, c is independently selected from the integers 12 to 22; Z is independently selected from the group consisting of -CH3, carboxylic acid or carboxylic acid bioisostere, phosphonate, or sulfonate] and provides a compound or a pharmaceutically acceptable salt thereof. Further, the present invention provides a compound wherein c is independently selected from 14, 16, 18, or 20.

[0013] In the peptide of formula (AI) above or a pharmaceutically acceptable salt thereof, A11 is Aib; and / or In the peptide of formula (AI) above or a pharmaceutically acceptable salt thereof, A12 is Ile; and / or In the peptide of formula (AI) above or a pharmaceutically acceptable salt thereof, A13 is Aib; and / or In the peptide of formula (AI) above or a pharmaceutically acceptable salt thereof, A17 is Gln; and / or In the peptide of formula (AI) above or a pharmaceutically acceptable salt thereof, A20 is Ala; and / or In the peptide of formula (AI) above or a pharmaceutically acceptable salt thereof, A29 is Gly.

[0014] One embodiment of the present invention is formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; One, two, three or four Ks selected from the group consisting of K at position 16, K at position 24, K at position 28 and K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain, here, a is each independently an integer from 0 to 5, b is each independently an integer from 0 to 5, c is each independently an integer from 10 to 24, Z is independently selected from the group consisting of -CH3, carboxylic acid or carboxylic acid bioisostere, phosphonate or sulfonate; and the C-terminal amino acid is amidated as a C-terminal primary amide] and pharmaceutically acceptable salts thereof are provided.

[0015] In some embodiments, the present invention relates to formula (I): [wherein, One K selected from the group consisting of K at position 16, K at position 24, K at position 28 and K at position 40 is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain] and pharmaceutically acceptable salts thereof are provided. For example, the 16-position K is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain. For example, the 24-position K is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain. For example, the 28-position K is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain. For example, the 40-position K is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain.

[0016] In some embodiments, the present invention provides a compound of formula (I): [wherein, Two Ks selected from the group consisting of the 16-position K, 24-position K, 28-position K, and 40-position K are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by two modifying chains through conjugation to the ε-amino of the K side chain] or a pharmaceutically acceptable salt thereof. For example, the 16-position K and 24-position K are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c-Z is chemically modified by two modified chains through conjugation to the ε-amino group of the K side chain. For example, K at position 16 and K at position 28 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by two modified chains through conjugation to the ε-amino group of the K side chain. For example, K at position 16 and K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by two modified chains through conjugation to the ε-amino group of the K side chain. For example, K at position 24 and K at position 28 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by two modified chains through conjugation to the ε-amino group of the K side chain. For example, K at position 24 and K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by two modified chains through conjugation to the ε-amino group of the K side chain. For example, K at position 28 and K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by two modified chains through conjugation to the ε-amino group of the K side chain.

[0017] In some embodiments, the present invention provides a compound of formula (I): [wherein, Three K's selected from the group consisting of 16-bit K, 24-bit K, 28-bit K, and 40-bit K are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by three modified chains through conjugation to the ε-amino of the K side chain] and provides a compound thereof or a pharmaceutically acceptable salt thereof. For example, 16-bit K, 24-bit K, and 28-bit K are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by three modified chains through conjugation to the ε-amino of the K side chain. For example, 16-bit K, 24-bit K, and 40-bit K are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by three modified chains through conjugation to the ε-amino of the K side chain. For example, 16-bit K, 28-bit K, and 40-bit K are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by three modified chains through conjugation to the ε-amino of the K side chain. For example, 24-bit K, 28-bit K, and 40-bit K are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by three modified chains through conjugation to the ε-amino of the K side chain.

[0018] In some embodiments, the present invention is of formula (I): [Wherein, four Ks selected from the group consisting of 16-bit K, 24-bit K, 28-bit K, and 40-bit K are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by four modified chains through conjugation to the ε-amino of the K side chain] or a pharmaceutically acceptable salt thereof. For example, 16-bit K, 24-bit K, 28-bit K, and 40-bit K are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified by four modified chains through conjugation to the ε-amino of the K side chain.

[0019] In some embodiments, the present invention provides a compound of formula (I): [Wherein, Z is independently selected from the group consisting of -CH3, carboxylic acid or carboxylic acid bioisostere, phosphonate or sulfonate. For example, but not limited to, said Z may contain a carboxylic acid (-CO2H) or carboxylic acid bioisostere (e.g.,

Chemical formula

[0020] Regarding carboxylic acid bioisosteres, suitable carboxylic acid bioisosteres are known in the art. Preferably, the bioisostere has a proton with a pK a similar to that of the corresponding carboxylic acid. Examples of suitable bioisosteres include the tetrazoles, acylsulfonamides, acylhydroxylamines, and squaric acid derivatives shown below: [Chemical formula] It can be, but is not limited to, these, and R is Me or CF3.

[0021] In some embodiments, the present invention provides a compound of formula (I): [wherein, a is independently selected from the integers 1, 2, 3, 4 or 5, preferably 1, 2 or 3; b is independently selected from the integers 1, 2, 3, 4 or 5, preferably 1, 2 or 3; c is independently selected from the integers 12 - 22] and a pharmaceutically acceptable salt thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0022] An embodiment of the present invention is formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; One or two Ks selected from the group consisting of K at position 16, K at position 24, K at position 28 and K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain, where Z is independently selected from the group consisting of -CH3, carboxylic acid or carboxylic acid bioisostere, phosphonate or sulfonate, preferably -CO2H. a is, independently of each other, an integer from 0 to 5, b is, independently of each other, an integer from 0 to 5, c is, independently of each other, an integer from 10 to 24, and the C-terminal amino acid is amidated as a C-terminal primary amide and a pharmaceutically acceptable salt thereof.

[0023] In a further embodiment, the present invention provides a compound of formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; one or two Ks selected from the group consisting of K at position 16, K at position 24, K at position 28 and K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z is chemically modified through conjugation to the ε-amino of the K side chain, wherein, Z is independently selected from the group consisting of -CH3, carboxylic acid or carboxylic acid bioisostere, phosphonate or sulfonate, preferably -CO2H, a is independently selected from the integers 1, 2, 3, 4 or 5, b is independently selected from the integers 1, 2, 3, 4 or 5, c is independently selected from the integers 12 to 22 and a pharmaceutically acceptable salt thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0024] An embodiment of the present invention is a compound of formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 24 is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified through conjugation to the ε-amino of the K side chain, where, a is independently selected from the integers 0 to 5, b is independently selected from the integers 0 to 5, c is independently selected from the integers 10 to 24; and The C-terminal amino acid is amidated as a C-terminal primary amide] and pharmaceutically acceptable salts thereof are provided.

[0025] In a further embodiment, the present invention relates to formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 24 is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified through conjugation to the ε-amino of the K side chain, where, a is independently selected from the integers 1, 2, 3, 4 or 5, b is independently selected from the integers 1, 2, 3, 4 or 5, c is independently selected from the integers 12 - 22] and provides a compound or a pharmaceutically acceptable salt thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0026] A further embodiment of the present invention is of formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 28 is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified through conjugation to the ε-amino of the K side chain, wherein, a is independently selected from the integers 0 - 5, b is independently selected from the integers 0 - 5, c is independently selected from the integers 10 - 24; and The C-terminal amino acid is amidated as a C-terminal primary amide] and provides a compound and a pharmaceutically acceptable salt thereof.

[0027] In a further embodiment, the present invention is of formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The 28 - bit K is ([2-(2 - amino - ethoxy)-ethoxy]-acetyl) a -(γ - Glu) b -CO-(CH2) c -CO2H is chemically modified through conjugation to the ε - amino of the K side chain, where a is independently selected from the integers 1, 2, 3, 4, or 5, b is independently selected from the integers 1, 2, 3, 4, or 5, c is independently selected from the integers 12 - 22]] and provides a compound or a pharmaceutically acceptable salt thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18, or 20.

[0028] A further embodiment of the present invention is of formula (I): Y - X1 - E - G - T - X2 - T - S - D - Y - X3 - I - X4 - L - D - K - Q - A - Q - A - E - F - V - K - W - L - L - K - G - G - P - S - S - G - A - P - P - P - S - K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 16 is ([2-(2 - amino - ethoxy)-ethoxy]-acetyl) a -(γ - Glu) b -CO-(CH2) c -CO2H is chemically modified through conjugation to the ε - amino of the K side chain, where a is independently selected from the integers 0 - 5, b is independently selected from the integers 0 - 5, c is independently selected from the integers 10 - 24; and The C - terminal amino acid is amidated as a C - terminal primary amide] and provides a compound and a pharmaceutically acceptable salt thereof.

[0029] In a further embodiment, the present invention provides a compound of formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 16 is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified through conjugation to the ε-amino of the K side chain, where a is independently selected from the integers 1, 2, 3, 4 or 5, b is independently selected from the integers 1, 2, 3, 4 or 5, c is independently selected from the integers 12 to 22] and a pharmaceutically acceptable salt thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0030] A further embodiment of the present invention is a compound of formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 40 is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified through conjugation to the ε-amino of the K side chain, where, a is independently selected from the integers 0 to 5, b is independently selected from the integers 0 to 5, c is independently selected from the integers 10 to 24; and the C-terminal amino acid is amidated as a C-terminal primary amide] and pharmaceutically acceptable salts thereof.

[0031] In a further embodiment, the present invention provides a compound of formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 40 is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified through conjugation to the ε-amino of the K side chain, wherein, a is independently selected from the integers 1, 2, 3, 4 or 5, b is independently selected from the integers 1, 2, 3, 4 or 5, c is independently selected from the integers 12 to 22] and pharmaceutically acceptable salts thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0032] A further embodiment of the present invention is a compound of formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 24 and the K at position 28 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains through conjugation to the ε-amino of the K side chain, where a is independently selected from integers from 0 to 5, b is independently selected from integers from 0 to 5, c is independently selected from integers from 10 to 22; and The C-terminal amino acid is amidated as a C-terminal primary amide] and its pharmaceutically acceptable salts.

[0033] In a further embodiment, the present invention provides a compound of formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 24 and the K at position 28 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains through conjugation to the ε-amino of the K side chain, where a is independently selected from the integers 1, 2, 3, 4 or 5, b is independently selected from the integers 1, 2, 3, 4 or 5, c is independently selected from integers from 12 to 22] and its pharmaceutically acceptable salts. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0034] A further embodiment of the present invention is formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 16 and the K at position 24 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains through conjugation to the ε-amino of the K side chain, wherein, a is independently selected from integers from 0 to 5, b is independently selected from integers from 0 to 5, c is independently selected from integers from 10 to 22; and The C-terminal amino acid is amidated as a C-terminal primary amide] and provides a pharmaceutically acceptable salt thereof.

[0035] In a further embodiment, the present invention is formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 16 and the K at position 24 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains through conjugation to the ε-amino of the K side chain, wherein, a is independently selected from the integers 1, 2, 3, 4 or 5, b is independently selected from the integers 1, 2, 3, 4 or 5, c is independently selected from the integers 12 - 22] and provides a compound or a pharmaceutically acceptable salt thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0036] A further embodiment of the present invention is of formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 16 and the K at position 28 are chemically modified by two modifying chains through conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H to the ε-amino of the K side chain, wherein, a is independently selected from the integers 0 - 5, b is independently selected from the integers 0 - 5, c is independently selected from the integers 10 - 22; and The C-terminal amino acid is amidated as a C-terminal primary amide] and provides a compound and a pharmaceutically acceptable salt thereof.

[0037] In a further embodiment, the present invention is of formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 16 and the K at position 28 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains via conjugation to the ε-amino of the K side chain, where a is independently selected from the integers 1, 2, 3, 4 or 5, b is independently selected from the integers 1, 2, 3, 4 or 5, c is independently selected from the integers 12 to 22] and provides a compound or a pharmaceutically acceptable salt thereof. Further, the present invention provides a compound wherein c is 14, 16, 18 or 20.

[0038] A further embodiment of the present invention is of formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 16 and the K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains via conjugation to the ε-amino of the K side chain, where a is independently selected from the integers 0 to 5, b is independently selected from the integers 0 to 5, c is independently selected from the integers 10 to 22; and The C-terminal amino acid is amidated as a C-terminal primary amide and provides a compound and a pharmaceutically acceptable salt thereof.

[0039] In a further embodiment, the present invention relates to a compound of formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; Lys at position 16 and Lys at position 40 are chemically modified by two modified chains through conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H to the ε-amino of the Lys side chain, wherein, a is independently selected from the integers 1, 2, 3, 4 or 5, b is independently selected from the integers 1, 2, 3, 4 or 5, c is independently selected from the integers 12 to 22] and provides a compound or a pharmaceutically acceptable salt thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0040] A further embodiment of the present invention is a compound of formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; Lys at position 24 and Lys at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a-(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains through conjugation to the ε-amino of the K side chain, wherein, a is independently selected from integers of 0 to 5, b is independently selected from integers of 0 to 5, c is independently selected from integers of 10 to 22; and the C-terminal amino acid is amidated as a C-terminal primary amide] and pharmaceutically acceptable salts thereof.

[0041] In a further embodiment, the present invention provides a compound of formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 and K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains through conjugation to the ε-amino of the K side chain, wherein, a is independently selected from integers of 1, 2, 3, 4 or 5, b is independently selected from integers of 1, 2, 3, 4 or 5, c is independently selected from integers of 12 to 22] and pharmaceutically acceptable salts thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0042] A further embodiment of the present invention is a compound of formula (I): Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K Formula (I) [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 28 and the K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains through conjugation to the ε-amino of the K side chain, wherein, a is independently selected from the integers 0 to 5, b is independently selected from the integers 0 to 5, c is independently selected from the integers 10 to 22; and The C-terminal amino acid is amidated as a C-terminal primary amide] and pharmaceutically acceptable salts thereof are provided.

[0043] In a further embodiment, the present invention relates to formula (I): [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 28 and the K at position 40 are ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -CO2H is chemically modified by two modified chains through conjugation to the ε-amino of the K side chain, wherein, a is independently selected from the integers 1, 2, 3, 4 or 5, b is independently selected from the integers 1, 2, 3, 4 or 5, c is independently selected from the integers 12 - 22] to provide a compound or a pharmaceutically acceptable salt thereof. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20.

[0044] In all of the above embodiments, a is independently an integer from 1 to 3, b is independently an integer from 1 to 3, c is independently an integer from 12 to 22. Further, the present invention further provides a compound wherein c is 14, 16, 18 or 20. In all of the above embodiments, embodiments in which K at position 24 or K at position 28 is modified are preferred.

[0045] In one embodiment, the present invention provides the following formula: Y - X1 - E - G - T - X2 - T - S - D - Y - X3 - I - X4 - L - D - K - Q - A - Q - A - E - F - V - K - W - L - L - K - G - G - P - S - S - G - A - P - P - P - S - K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is chemically modified through conjugation of ([2-(2 - amino - ethoxy)-ethoxy]-acetyl)2-(γ - Glu)1 - CO-(CH2) 16 -CO2H to the ε - amino of the K side chain; and the C - terminal amino acid is amidated as a C - terminal primary amide (SEQ ID NO: 7) to provide a compound or a pharmaceutically acceptable salt thereof.

[0046] In one embodiment, the present invention provides the following formula: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is chemically modified through conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 18 -CO2H to the ε-amino of the K side chain; and the C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 13) to provide a compound of or a pharmaceutically acceptable salt thereof.

[0047] In one embodiment, the present invention provides a compound of the following formula: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is chemically modified through conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H to the ε-amino of the K side chain; and the C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 14) to provide a compound of or a pharmaceutically acceptable salt thereof.

[0048] In one embodiment, the present invention provides a compound of the following formula: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is chemically modified through conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16 -CO2H to the ε-amino of the K side chain; and the C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 15) to provide a compound of or a pharmaceutically acceptable salt thereof.

[0049] In one embodiment, the present invention provides the following formula: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 24 is chemically modified through conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -CO2H to the ε-amino of the K side chain; and the C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 16) to provide a compound of or a pharmaceutically acceptable salt thereof.

[0050] In one embodiment, the present invention provides the following formula: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 28 is chemically modified via conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 16 -CO2H to the ε-amino group of the K side chain; and the C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 8) to provide a compound of or a pharmaceutically acceptable salt thereof.

[0051] In one embodiment, the present invention provides the following formula: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; K at position 28 is chemically modified via conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H to the ε-amino group of the K side chain; and the C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 17) to provide a compound of or a pharmaceutically acceptable salt thereof.

[0052] In one embodiment, the present invention provides the following formula: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; the K at position 28 is chemically modified through the conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16 -CO2H to the ε-amino of the K side chain; and the C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 18) to provide a compound of or a pharmaceutically acceptable salt thereof.

[0053] In one embodiment, the present invention provides a compound of the following formula: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; the K at position 28 is chemically modified through the conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 16 -CO2H to the ε-amino of the K side chain; and the C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 19) to provide a compound of or a pharmaceutically acceptable salt thereof.

[0054] In one embodiment, the present invention provides a compound of the following formula: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [Wherein X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 28 is chemically modified via conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -CO2H to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 20) To provide a compound or a pharmaceutically acceptable salt thereof.

[0055] The present invention further provides a method for increasing the duration of action of a GLP-1 / GIP dual agonist in a subject, characterized by using the acylated GLP-1 / GIP dual agonist provided by the present invention as described above.

[0056] In one embodiment, the present invention provides a pharmaceutical composition comprising a compound according to the present invention and a pharmaceutically acceptable carrier, diluent or excipient.

[0057] One embodiment provides the use of a compound according to any one of the above embodiments in the manufacture of a medicament for treating or preventing a disease selected from hyperglycemia, impaired glucose tolerance, type I diabetes, type II diabetes, obesity, hypertension, metabolic syndrome, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, inflammatory bowel syndrome, indigestion and gastric ulcer.

[0058] One embodiment provides a compound according to any one of the above embodiments in the manufacture of a medicament for delaying or preventing the disease progression of type II diabetes.

[0059] One embodiment provides for the use of a compound according to any one of the above embodiments in the manufacture of a medicament for reducing food intake, reducing β-cell apoptosis, increasing β-cell function and β-cell volume, and / or restoring glucose sensitivity of β-cells.

[0060] In one embodiment, the present invention provides a method of treating type II diabetes, the method comprising administering to a subject in need thereof an effective amount of a compound of the present invention.

[0061] In one embodiment, the present invention provides a method of improving glycemic control in a subject suffering from type II diabetes, the method comprising administering to a subject in need thereof an effective amount of a compound according to the present invention as an adjunct to diet and exercise.

[0062] In one embodiment, the present invention provides a method for long-term weight management in a type 2 diabetic subject with an initial obesity index ≧ 27, the method comprising administering to a subject in need thereof an effective amount of a compound according to the present invention as an adjunct to a low-calorie diet and increased physical activity.

[0063] In one embodiment, the present invention provides a method of treating metabolic syndrome, the method comprising administering to a subject in need thereof an effective amount of a compound according to the present invention. In a further embodiment, the present invention provides a method of treating insulin resistance and lipid abnormalities, obesity and / or fatty liver associated with diabetes, the method comprising administering to a subject in need thereof an effective amount of a compound according to the present invention. Further, the present invention provides a method of treating frailty or increasing bone strength, the method comprising administering to a subject in need thereof an effective amount of a compound according to the present invention.

[0064] In one embodiment, the present invention provides a compound of the present invention as an adjunct to diet and exercise for glycemic control in type II diabetic subjects.In one embodiment, the present invention provides a compound of the present invention for long-term weight management as an adjunct to a reduced calorie diet and increased physical activity in type II diabetic subjects with an initial body mass index of > 27.

[0065] Due to the above-mentioned activity of the compounds according to the invention on the GLP-1 receptor and the GIP receptor, the compounds according to the invention can be used as medicines for treating or preventing various diseases, such as obesity. The compounds according to the present invention are useful for treating symptomatic obesity, obesity due to simple obesity, obesity-related symptoms or diseases, eating disorders, diabetes (e.g., type I diabetes, type II diabetes, gestational diabetes, obesity diabetes mellitus), hyperlipidemia (e.g., hypertriglyceridemia, hypercholesterolemia, hyper LDL cholesterolemia, hypo HDL cholesterolemia, postprandial hyperlipidemia), hypertension, heart failure, diabetic complications (e.g., neuropathy, nephropathy, retinopathy, diabetic cardiomyopathy, cataracts, macroangiopathy, osteopenia, hyperosmolar diabetic coma), infectious diseases (e.g., respiratory tract infections, urinary tract infections, gastrointestinal infections, superficial soft tissue infections, lower limb infections), diabetic gangrene, xerostomia, hearing loss, cerebrovascular disorders, peripheral blood circulation disorders, The present invention can be used as a medicine for preventing or treating diseases such as hypertriglyceridemia (TG), hypo-HDL cholesterol (HDL-C), hypertension, abdominal obesity, and impaired glucose tolerance, and sarcopenia.

[0066] Examples of symptomatic obesity include endocrine obesity (e.g., Cushing's syndrome, hypothyroidism, insulinoma, obese Type II diabetes mellitus, pseudohypoparathyroidism, hypogonadism), central obesity (e.g., hypothalamic obesity, frontal lobe syndrome, Klein-Lewis syndrome), genetic obesity (e.g., Prader-Willi syndrome, Laurence-Moon-Biedl's syndrome), drug-induced obesity (e.g., steroid, phenothiazine, insulin, sulfonylurea (SU) drug, β-blocker-induced obesity), and the like.

[0067] Examples of symptoms or diseases associated with obesity include impaired glucose tolerance, diabetes (especially type II diabetes, obesity diabetes mellitus), lipid metabolism disorder (synonymous with the above hyperlipidemia), hypertension, heart failure, hyperuricemia, fatty liver (including non-alcoholic hepatitis), coronary heart disease (myocardial infarction, angina pectoris), cerebral infarction (cerebral thrombosis, transient ischemic attack), bone and joint diseases (osteoarthritis of the knee, osteoarthritis of the hip, ankylosing spondylitis, low back pain), sleep apnea syndrome / Pickwick syndrome, menstrual disorder (abnormal menstrual cycle, abnormal menstruation and cycle, amenorrhea, abnormal menstrual symptoms), metabolic syndrome, and the like.

[0068] The compounds provided by the present invention have GLP-1 / GIP dual agonist activity, a long half-life, and a long drug efficacy duration.

[0069] The compounds according to the present invention can react with any of various inorganic acids and organic acids to form pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts and the methods generally used for their preparation are well known in the art.

[0070] The amino acid sequences of the present invention include the standard one-letter or three-letter codes of 20 natural amino acids. Also, "Aib" is α-aminoisobutyric acid, "αMePhe" is α-methylphenylalanine, "Pya(4)" is 4-pyridylalanine, "Cha" is cyclohexylalanine, and "αMeTyr" is α-methyltyrosine.

[0071] "AEEA" disclosed in the present invention is an abbreviation for 2-(2-amino-ethoxy)-ethoxy]-acetyl. "Ste" disclosed in the present invention is octadecanediyl-C(O)-C 16 H 32 -C(O)- indicates.

[0072] "Effective amount" or "therapeutically effective amount" as disclosed in the present invention means the amount or dosage of a compound according to the present invention or a pharmaceutically acceptable salt thereof that, upon single or multiple administrations to a subject, brings about a desired effect in the subject being diagnosed or treated.

[0073] "Minimum effective amount" as disclosed in the present invention means the minimum dosage at which a medicinal effect occurs. Specifically, in the medicinal effect experiment by multiple administrations to db / db mice in the present invention, it means the minimum dosage at which the blood glucose AUC of the test compound administration group shows a significant difference (p < 0.05) from that of the vehicle administration group.

[0074] "Effective amount" as disclosed in the present invention means the dosage at which a medicinal effect occurs. Specifically, in the medicinal effect experiment by multiple administrations to db / db mice in the present invention, it means the dosage at which the blood glucose AUC of the test compound administration group shows a significant difference (p < 0.05) from the blood glucose AUC of the vehicle administration group.

[0075] "Vehicle group" as disclosed in this specification means the vehicle control group.

[0076] As used in the present invention, "AUC" refers to the area under the concentration-time curve, that is, the area enclosed by the drug concentration-time curve and the time axis. This parameter is an important indicator for evaluating the degree of drug absorption and reflects the exposure characteristics of the drug in the body.

[0077] As used in the present invention, "MRT" refers to the mean residence time, which is the average value of the residence time of drug molecules in the body and represents the time required for 63.2% of the drug to be excreted outside the body.

[0078] As used in the present invention, "MTD" refers to the maximum tolerated dose, which is the highest dose that does not cause death in test animals.

[0079] As used in the present invention, "internal standard" refers to a predetermined amount of a specific pure compound added to a sample to correct errors caused by signal fluctuations of equipment, human operations, etc.

[0080] As used in the present invention, "inhibition rate of blood glucose AUC" refers to the ratio of the decrease in blood glucose AUC in the group that ingested the test compound compared to the group that ingested the vehicle. Inhibition rate of blood glucose AUC = (blood glucose AUC ビヒクル - blood glucose AUC 試験化合物 ) / blood glucose AUC ビヒクル .

[0081] The "titration strategy" disclosed by the present invention is the method of dose escalation, that is, after the initial dose, the frequency and dose of dose escalation are adjusted to obtain the optimal dose.

[0082] The "subject" disclosed in the present invention includes humans or animals, preferably humans.

[0083] The "modified chain" disclosed in the present invention is ([2-(2-amino-ethoxy)-ethoxy]-acetyl) a -(γ-Glu) b -CO-(CH2) c -Z: [where a is, independently of each other, an integer from 0 to 5, b is, independently, an integer from 0 to 5, c is, independently, an integer from 10 to 22; Preferably, a is, independently, selected from the integers 1, 2, 3, 4 or 5, b is, independently, selected from the integers 1, 2, 3, 4 or 5, c is, independently, selected from the integers 12 to 22] is. Furthermore, the present invention is c is 14, 16, 18 or 20, Z is, independently, selected from the group consisting of -CH3, carboxylic acid or carboxylic acid bioisostere, phosphonate or sulfonate, for example, but not limited to, Z is carboxylic acid (-CO2H) or carboxylic acid bioisostere (for example

Chemical formula

[0084] Regarding carboxylic acid bioisosteres, suitable carboxylic acid bioisosteres are well known in the art. Preferably, the bioisostere has a proton with a pK a similar to that of the corresponding carboxylic acid. Examples of suitable bioisosteres include, but are not limited to, tetrazole, acylsulfonamide, acylhydroxylamine and squaric acid derivatives as shown below:

Chemical formula

[0085] For the peptides described herein, according to the conventional peptide designation, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus). The C-terminus of the peptide is amide (-CONH2), carboxyl (-COOH), carboxylate (-COO- ) can be any one of alkylamide (-CONHR') and ester (-COOR'), and R' is C 1-8 is alkyl. In particular, amide (-CONH2) is preferred.

[0086] In the preparation process of the present invention, "coupling" or "incorporation" means the step of adding a new amino acid to the linked amino acid or peptide.

Brief Description of the Drawings

[0087]

Figure 1

[0088]

Figure 2

[0089]

Figure 3

[0090] The present invention also includes novel intermediates and methods useful for the synthesis of the compounds according to the invention or their pharmaceutically acceptable salts. The intermediates and compounds according to the invention can be prepared by various methods known in the art. In particular, methods using chemical synthesis are exemplified in the following examples. To prepare the compounds according to the invention or their salts, the specific synthesis steps of each route as described can be variously combined. Reagents and starting materials are readily available to those skilled in the art. It should be understood that these examples are not intended to limit the scope of the invention in any way. Mass spectrometry was performed using an Agilent 1260 / 6110 liquid chromatography mass spectrometer, and the scanning range was 100 to 1500.

Example

[0091] Example 1: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 24 is chemically modified via conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16 -CO2H to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 15).

[0092]

Chemical formula

[0093] (1) Synthesis of Peptide Resin Intermediate 1 Take Fmoc-Rink Linker-Nle-MBHA resin (S = 0.49 mmol / g), swell it with an appropriate amount of DCM, wash it 2 - 3 times with DCM, deprotect it with 20% PIP / DMF solution for 30 minutes, filter and wash to obtain NH2-Rink Linker-Nle-MBHA resin with Fmoc removed. Aspirate to remove the solvent and use it for further use.

[0094] Take 4 equivalents of Fmoc-Lys(Boc)-OH and HOBt respectively and dissolve them in an appropriate amount of DMF / DCM. Separately, take 4 equivalents of DIC and dilute it 2-fold with DCM. Slowly add the obtained diluted solution to the DMF / DCM solution and react it with stirring at -5 to 0 °C for 60 minutes or more for activation, and use it for further use.

[0095] Add the activated Fmoc-Lys(Boc)-OH solution to the NH2-Rink linker-Nle-MBHA resin. Control the reaction temperature at 10 - 30 °C. The coupling reaction is carried out for 240 - 480 minutes. After filtration and washing, Fmoc-Lys(Boc)-Rink linker-Nle-MBHA resin is obtained. Deprotect this resin with 20% PIP / DMF solution for 30 minutes. After filtration and washing, Lys(Boc)-Rink linker-Nle-MBHA resin with Fmoc removed is obtained.

[0096] According to the above reaction conditions, each amino acid is sequentially coupled from the second amino acid at the C-terminus to the N-terminus. When the coupling is incomplete (a color reaction occurs), perform a second condensation using HBTU / DIEA to ensure that each amino acid is completely condensed. The coupling is carried out continuously in the following order: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH.H2O, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH.H2O, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Aib-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-α-Me-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Aib-OH and Boc-Tyr(tBu)-OH.

[0097] The following resin is obtained: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Mtt)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA resin.

[0098] Once the above condensation is complete, the Mtt protecting group is removed with a 50% HFIP / DCM solution for 30 minutes. After washing and filtration, the following peptide resin intermediate 1 is obtained: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val- Lys-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA resin.

[0099] (2) Modification step using the modifying chain Take 4 equivalents of Fmoc-AEEA-OH and HOBt and dissolve them in an appropriate amount of DMF / DCM. Separately, take 4 equivalents of DIC and dilute it 2-fold with DCM. Slowly add this diluted solution to the DMF / DCM solution and react with stirring at -5 to 0 °C for 60 minutes or more for activation and use for further reactions.

[0100] Add the activated Fmoc-AEEA-OH solution to the pre-swollen and washed peptide resin intermediate 1. Control the reaction temperature at 10 - 30 °C. The coupling reaction is carried out for 240 - 480 minutes. Filter and wash the resin. Remove the Fmoc protecting group with a 20% PIP / DMF solution for 30 minutes. Filter and wash the resin. According to the above reaction conditions, sequentially couple activated Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, Fmoc-Glu(α-OtBu)-OH, Fmoc-Glu(α-OtBu)-OH and mono-tert-butyl octadecanedioate on the resin, perform Fmoc deprotection, wash with DCM and dry to obtain the following P015 peptide resin: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(tBuO-Ste-γ-Glu(α-OtBu)-γ-Glu(α-OtBu)-γ-Glu(α-OtBu)-AEEA-AEEA)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink-Linker-Nle-MBHA resin.

[0101] Take the P015 peptide resin and add a peptide resin cleavage reagent (TFA: EDT: TIS: H2O, 94:2:2:2 v / v) at 12 - 15 mL / g, and react with stirring at 25 ± 5 °C for 4 hours. Filter the reaction mixture through a sand core funnel. Collect the filtrate. Wash the resin three times with a small amount of TFA, combine the filtrates, and concentrate under reduced pressure. Add methyl tert-butyl ether (MBTE), wash the resulting precipitate three to four times with MBTE, and dry by evaporating MBTE. Dry the crude product under reduced pressure at room temperature to a constant weight to obtain the crude P015 product, and then purify and dry it to obtain the P015 sample (molecular weight determined by MS is 5255.2).

[0102] Example 2: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The 28-position K is chemically modified via conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 16 -CO2H to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 19).

[0103]

Chemical Structure

[0104] The preparation method applies the Fmoc solid-phase polypeptide synthesis strategy and includes the following steps: (1) Synthesis of the peptide resin intermediate 2 Take Fmoc-Rink Linker-Nle-MBHA-MBHA resin (S = 0.49 mmol / g), swell it with an appropriate amount of DCM, wash it 2 - 3 times with DCM, deprotect it with a 20% PIP / DMF solution for 30 minutes, wash it, filter it to obtain NH2-Rink linker-Nle-MBHA resin with Fmoc removed, aspirate to remove the solvent, and use it for further use.

[0105] Take 4 equivalents of Fmoc-Lys(Boc)-OH and HOBt respectively, and dissolve them in an appropriate amount of DMF / DCM. Separately, take 4 equivalents of DIC and dilute it 2-fold with DCM. Slowly add the diluted solution to the DMF / DCM solution, and react and activate it with stirring at -5 to 0 °C for 60 minutes or more for further use.

[0106] An activated Fmoc-Lys(Boc)-OH solution is added to the NH2-Rink Linker-Nle-MBHA resin. The reaction temperature is controlled at 10°C to 30°C. The coupling reaction is carried out for 240 to 480 minutes. After filtration and washing, Fmoc-Lys(Boc)-Rink Linker-Nle-MBHA is obtained. The resin is deprotected with a 20% PIP / DMF solution for 30 minutes, and after filtration and washing, Lys(Boc)-Rink Linker-Nle-MBHA resin with Fmoc removed is obtained.

[0107] According to the above reaction conditions, each amino acid is sequentially coupled from the second amino acid at the C-terminus to the N-terminus. If the coupling is incomplete (a color reaction occurs), a second condensation is performed using HBTU / DIEA to ensure that each amino acid is completely condensed. The coupling is carried out continuously in the following order: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH.H2O, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Lys(Mtt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH.H2O, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH.H2O, Fmoc-Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Aib-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-α-Me-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Glu(OtBu)-Gly-OH, Fmoc-Aib-OH and Boc-Tyr(tBu)-OH.

[0108] The following resin is obtained: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Mtt)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA resin.

[0109] Once the above condensation is complete, the Mtt protecting group is removed with a 50% HFIP / DCM solution for 30 minutes. After washing and filtration, the following peptide resin intermediate 2 is obtained: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA resin.

[0110] (2) Modification step using a modifying chain Take 4 equivalents of Fmoc-AEEA-OH and HOBt and dissolve them in an appropriate amount of DMF / DCM. Separately, take 4 equivalents of DIC and dilute it 2-fold with DCM. Slowly add this dilution to the DMF / DCM solution and react with stirring at -5 to 0 °C for 60 minutes or more to activate it for further use.

[0111] The activated Fmoc-AEEA-OH solution is added to the pre-swollen and washed peptide resin intermediate 2. The reaction temperature is controlled at 10 - 30 °C. The coupling reaction is carried out for 240 - 480 minutes. The resin is filtered and washed. The Fmoc protecting group is removed with 20% PIP / DMF solution for 30 minutes. The resin is filtered and washed. According to the above reaction conditions, activated Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH and mono-tert-butyl octadecanedioate are sequentially coupled on the resin, Fmoc deprotection is performed, washed with DCM, and dried to obtain the following P019 peptide resin: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(tBuO-Ste-γ-Glu(α-OtBu)-AEEA-AEEA-AEEA)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink-Linker-Nle-MBHA resin.

[0112] The P019 peptide resin is taken, and a peptide resin cleavage reagent (TFA: EDT: TIS: H2O, 94:2:2:2 v / v) at 12 - 15 mL / g is added, and the reaction is carried out with stirring at 25 ± 5 °C for 4 hours. The reaction mixture is filtered through a sand core funnel. The filtrate is collected. The resin is washed 3 times with a small amount of TFA, the filtrates are combined, and concentrated under reduced pressure. Methyl tert-butyl ether (MBTE) is added, the obtained precipitate is washed 3 - 4 times with MBTE, and dried by evaporating MBTE. The crude product is dried under reduced pressure at room temperature to a constant weight at room temperature to obtain the crude P019 product, which is then purified and dried to obtain the P019 sample (the molecular weight determined by MS is 5142.0).

[0113] Example 3 Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 24 is chemically modified through conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 16 -CO2H to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide](SEQ ID NO: 7).

[0114]

Chemical formula

[0115] In the same manner as the method described in Example 1 above, a preparation process is carried out to synthesize the peptide of SEQ ID NO: 7 of the present invention (molecular weight determined by MS is 4997.2) using the peptide resin intermediate 1. The difference is that in the modification process using the modification chain, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH and mono-tert-butyl octadecanedioate are sequentially coupled on the resin. The resulting product is deprotected from the Fmoc group and washed with DCM to obtain the P007 peptide resin. The other steps are the same.

[0116] Example 4 Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [In the formula, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 24 is chemically modified through the conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 18 -CO2H to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 13).

[0117]

Chemical formula

[0118] In the same manner as the method described in Example 1 above, a preparation step is carried out to synthesize the peptide of SEQ ID NO: 13 of the present invention (molecular weight determined by MS is 5025.2) using the peptide resin intermediate 1. The difference is that in the modification process using the modification chain, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH and eicosanedioic acid mono-tert-butyl ester are sequentially coupled on the resin. The obtained product is deprotected from the Fmoc group and washed with DCM to obtain the P013 peptide resin. The other steps are the same.

[0119] Example 5: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [In the formula, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 24 is chemically modified through the conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 14).

[0120]

Chemical formula

[0121] In the same manner as the method described in Example 1 above, a preparation step is carried out to synthesize the peptide of SEQ ID NO: 14 of the present invention (molecular weight determined by MS is 5052.4) using the peptide resin intermediate 1. The difference is that in the modification process using the modification chain, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH and docosanedioic acid mono-tert-butyl ester are sequentially coupled on the resin. The obtained product is deprotected from the Fmoc group and washed with DCM to obtain the P014 peptide resin. The other steps are the same.

[0122] Example 6: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [In the formula, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 24 is chemically modified through the conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -CO2H to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 16).

[0123]

Chemical formula

[0124] In the same manner as the method described in Example 1 above, a preparation step is carried out to synthesize the peptide of SEQ ID NO: 16 of the present invention (molecular weight determined by MS is 5198.0) using the peptide resin intermediate 1. The difference is that in the modification process using the modification chain, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH and docosanedioic acid mono-tert-butyl ester are sequentially coupled on the resin. The obtained product is deprotected from the Fmoc group and washed with DCM to obtain the P016 peptide resin. The other steps are the same.

[0125] Example 7: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [In the formula, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 28 is chemically modified through the conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 16 -CO2H to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 8).

[0126] [Chemical formula] The above structure contains the standard one-letter amino acid code except for the residues Aib2, αMePhe6, Aib11, Aib13 and K28 where the structures of the amino acid residues Aib2, αMePhe6, Aib11, Aib13 and K28 are extended.

[0127] In the same manner as the method described in Example 2 above, a preparation process for synthesizing the peptide of SEQ ID NO: 8 of the present invention (molecular weight determined by MS is 4996.8) is carried out using the peptide resin intermediate 2. The difference is that in the modification process using the modification chain, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH and mono-tert-butyl octadecanedioate are sequentially coupled on the resin. The obtained product is deprotected from the Fmoc group and washed with DCM to obtain the P008 peptide resin. The other steps are the same.

[0128] Example 8: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [In the formula, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 28 is chemically modified through the conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)1-CO-(CH2) 20 -CO2H to the ε-amino of the K side chain; The C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 17).

[0129]

Chemical Structure

[0130] In the same manner as the method described in Example 2 above, a preparation process for synthesizing the peptide of SEQ ID NO: 17 of the present invention (molecular weight determined by MS is 5052.8) is carried out using the peptide resin intermediate 2. The difference is that in the modification process using the modification chain, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH and docosanedioic acid mono-tert-butyl ester are sequentially coupled on the resin. The obtained product is deprotected from the Fmoc group and washed with DCM to obtain the P017 peptide resin. The other steps are the same.

[0131] Example 9: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [Wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 28 is chemically modified through conjugation of ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γ-Glu)3-CO-(CH2) 16 -CO2H to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 18).

[0132]

Chemical formula

[0133] In the same manner as the method described in Example 2 above, a preparation step is carried out to synthesize the peptide of SEQ ID NO: 18 of the present invention (molecular weight determined by MS is 5255.2) using the peptide resin intermediate 2. The difference is that in the modification process using the modification chain, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH, Fmoc-Glu(α-OtBu)-OH, Fmoc-Glu(α-OtBu)-OH and mono-tert-butyl octadecanedioate are sequentially coupled on the resin. The obtained product is deprotected from the Fmoc group and washed with DCM to obtain the P018 peptide resin. The other steps are the same.

[0134] Example 10: Y-X1-E-G-T-X2-T-S-D-Y-X3-I-X4-L-D-K-Q-A-Q-A-E-F-V-K-W-L-L-K-G-G-P-S-S-G-A-P-P-P-S-K [Wherein, X1 is Aib; X2 is αMePhe; X3 is Aib; X4 is Aib; The K at position 28 is ([2-(2-amino-ethoxy)-ethoxy]-acetyl)3-(γ-Glu)1-CO-(CH2) 20 -CO2H is chemically modified through conjugation to the ε-amino of the K side chain; and The C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 20).

[0135]

Chemical formula

[0136] In the same manner as the method described in Example 2 above, a preparation step is carried out to synthesize the peptide of SEQ ID NO: 20 of the present invention (molecular weight determined by MS is 5198.4) using the peptide resin intermediate 2. The difference is that in the modification process using the modification chain, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu(α-OtBu)-OH and docosanedioic acid ester mono-tert-butyl are sequentially coupled on the resin. The obtained product is deprotected from the Fmoc group and washed with DCM to obtain the P020 peptide resin. The other steps are the same.

[0137] In some embodiments of the present invention, referring to the method for producing the peptide resin intermediate 1 in Example 1, a peptide resin intermediate 3 (a peptide resin intermediate in which the modified chain is bound to Lys at position 16): Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Boc)-Rink Linker-Nle-MBHA resin, is prepared.

[0138] Referring to the method for producing the peptide resin intermediate 1 in Example 1, a peptide resin intermediate 4 (a peptide resin intermediate in which the modified chain is bound to Lys at position 40): Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-αMePhe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Aib-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Gln(Trt)-Ala-Gln(Trt)-Ala-Glu(OtBu)-Phe-Val-Lys(Boc)-Trp(Boc)-Leu-Leu-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys-Rink Linker-Nle-MBHA resin, is prepared.

[0139] Other dual agonist compounds in the present invention can be prepared according to the above-described method.

[0140] Related assays The conditions and data in several assays related to the examples are provided below.

[0141] I. In Vitro Function (I) In Vitro Binding Activity to Human GlP-1 and GIP Receptors The in vitro binding ability of the compounds according to the present invention to human GIP and GLP-1 receptors is evaluated by measuring the binding affinity Ki using crude cell membranes obtained from a cloned cell line overexpressing either human GLP1R cDNA or human GIP-R cDNA.

[0142] 1) In Vitro Binding Activity to Human GLP-1 Receptor hGLP-1 and the compounds of the present invention are dissolved in DMSO and stored at -80°C. 89 μL of the membrane dissolved in binding buffer (50 mM Hepes, pH 7.4, 5 mM MgCl2, 5 mM EDTA, 0.005% TWEEN, 0.005% HSA) is transferred to a 96-well test plate (5 μg / well). The compounds are serially diluted with DMSO, and then 1 μL of the diluted compound or 100% DMSO is added to the test plate containing the membrane solution. 10 μL of 125 I]GLP-1 is further added (the final concentration in the reaction is 0.15 nM). The test plate is left at room temperature for 90 minutes. The membrane complex is collected into a GF / B plate pre-coated with 0.5% PEI using a Cell Harvester and washed three times with 500 μL of pre-cooled elution buffer (50 mM Hepes, pH 7.4, 500 mM NaCl) at 4°C. After drying at 37°C for 2 hours, 50 μL of scintillation fluid is added to each well. The plate is sealed and allowed to sediment for at least 1 hour, and then read with a MicroBeta2 scintillation counter to determine the membrane-bound radioactive ligand level.

[0143] The absolute IC 50 concentration of the compound is 125 I]derived by non-linear regression of the binding percentage of GLP-1 and the concentration of the added compound. The Cheng-Prusoff equation is used to calculate IC 50Convert the concentration to Ki (Ki is the inhibition constant).

[0144] 2) In vitro binding activity to the human GIP receptor Dissolve hGIP and the compound of the present invention in DMSO and store at -80°C. Transfer 98 μL of the membrane dissolved in the binding buffer (50 mM HEPES pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.1% BSA, 0.005% Tween-20) to a 96-well test plate (15 μg / well). Continuously dilute the compound with DMSO, and then add 2 μL of the diluted compound or 100% DMSO to the test plate containing the membrane solution. Add 100 μL of 125 I]GIP (the final concentration in the reaction is 0.0315 nM). Leave the test plate at room temperature for 90 minutes. Collect this membrane complex into a GF / B plate pre-coated with 0.5% PEI using a Cell Harvester and wash it three times with 500 μL of pre-cooled elution buffer (50 mM Tris-HCl pH 7.4, 125 mM NaCl) at 4°C. After drying at 37°C for 2 hours, add 50 μL of scintillation fluid to each well. Seal the plate, let it settle for at least 1 hour, and then read it with a MicroBeta2 scintillation counter to determine the membrane-bound radioactive ligand level.

[0145] The absolute IC of the compound 50 The concentration is 125 Derived by non-linear regression of the binding percentage of I]GLP-1 and the concentration of the added compound. Use the Cheng-Prusoff equation to convert the IC 50 Convert the concentration to Ki (Ki is the inhibition constant).

[0146]

Table 1

[0147] The binding affinity of all compounds to the receptor is indicated by the Ki ratio, and the greater the Ki ratio of the compound, the stronger the binding affinity. Compared with P001, the binding affinity of the compounds according to the present invention for both human GLP-1R and GIPR decreases to some extent (see Table 1).

[0148] Compared with tildepagliflozin, the binding affinity of the other nine compounds according to the present invention, excluding P015, for human GLP-1R is slightly stronger than that of tildepagliflozin. Furthermore, the binding affinity of five compounds for GIPR, namely P008, P013, P014, P016 and P019, is also slightly stronger than that of tildepagliflozin (see Table 1).

[0149] In conclusion, the binding affinity of the compounds with various modified chains according to the present invention for human GLP-1R and GIPR is somewhat lower than that of P001, but slightly stronger than or equivalent to that of tildepagliflozin.

[0150] (II) Agonist activity against hGLP-1R and hGIPR For the compounds according to the present invention, the in vitro functional activities against human GIP and GLP-1 receptors are measured in HEK-293 cloned cell lines expressing these receptors.

[0151] 1) Agonist activity against human GLP-1 receptor (cAMP reporter gene assay) The agonist activity of the compounds according to the present invention against human GLP-1R is measured by a cAMP reporter gene assay, using P001, tildepagliflozin and the endogenous ligand GLP-1 as references.

[0152] Seed HEK293 / CRE / GLP-1R cells in a 96-well plate at 50,000 cells / well (80 μL / well) and incubate overnight in a 37 °C, 5% CO2 incubator. Add 20 μL / well of test medium (DMEM containing 0.1% casein) containing the compound (the compound of the present invention, P001, tildepazide or GLP-1) to the 96-well plate, continue to incubate in a 37 °C, 5% CO2 incubator for 6 hours, and equilibrate to room temperature. Remove the supernatant. Add 50 μL / well of Bright-Glo reagent, shake at room temperature and dissolve for 10 minutes. Read the luminescence using an Envision microplate reader and measure the luciferase activity.

[0153] Taking the response value to 100 nM GLP-1 as the 100% response value, perform non-linear regression using GraphPad according to the response ratio and the concentration of the added compound to obtain the EC 50 value of each compound.

[0154] 2) Agonist activity against human GIP receptor (LANCE Ultra cAMP assay) The agonist activity of the compound according to the present invention against human GIPR is measured using the LANCE Ultra cAMP Kit, with P001, tildepazide and the endogenous ligand GIP used as references.

[0155] The in vitro agonist activity of the compounds according to the present invention against GIPR is measured in HEK293 cells (HEK293 / GIPR cells) that stably express human GIPR. HEK293 / GIPR cells are formulated in HBSS buffer (0.1% casein, 500 μM IBMX, 5 mM HEPES) and seeded in a 384-well cell culture plate at 1000 cells / well (5 μL / well). 5 μL of HBSS buffer containing 2× compound is added to the above 384-well cell culture plate. After sealing, the plate is placed in a 37 °C, 5% CO2 incubator for about 30 minutes. After the incubation is completed, 5 μL of cAMP-Eu working solution and 5 μL of cAMP-Ulight working solution are sequentially added, and the plate is shaken to mix uniformly. Incubation is carried out at 25 °C for 1 hour. The signal values at 665 nm and 615 nm are read with an Envision microplate reader. The ratio of the signal value at 665 nm to the signal value at 615 nm is calculated and converted to the cAMP concentration using a cAMP standard curve. The response value to 1 μM of GIP is taken as the 100% response value, and non-linear regression is performed using GraphPad with the response ratio and the concentration of the added compound to obtain the EC 50 value of each compound.

[0156]

Table 2

[0157] The agonist activity of all compounds against human GLP-1 and GIP receptors is shown by the EC 50 value, and the greater the EC 50 value, the stronger the activity of the compound. Compared with P001, the compounds of the present invention show a certain degree of decrease in agonist activity against human GLP-1R and GIPR (see Table 2).

[0158] Compared with tirzepatide, all compounds according to the present invention have stronger agonist activity against human GLP-1R. Furthermore, the agonist activity of six compounds against human GIPR, namely P007, P008, P013, P016, P018 and P019, is also slightly stronger than that of tirzepatide (see Table 2).

[0159] In conclusion, the agonist activity of compounds with various modified chains according to the present invention against human GLP-1R and GIPR is somewhat lower than that of P001, but six compounds according to the present invention are stronger than tirzepatide with respect to agonist activity against human GLP-1R and GIPR.

[0160] As shown by in vitro functional assays, since the 10 compounds according to the present invention do not significantly differ in activity, the compounds are further screened using a single-dose hypoglycemia assay in db / db mice.

[0161] II. Pharmacokinetics (I) Pharmacokinetics after single-dose administration in SD rats After subcutaneous or intravenous administration to SD rats, plasma is collected and the drug concentration in the plasma is detected to demonstrate the in vivo pharmacokinetic characteristics of the compounds according to the present invention. The compound is dissolved in phosphate buffer and filtered through a filtration membrane (PTFE, 0.45 μm) to obtain a 25 nmol / mL compound solution. This compound is administered to male SD rats (230 - 268 g, n = 3) at a dose of 50.0 nmol / kg (s.c.) or 25.0 nmol / kg (i.v.). Approximately 150 μL of whole blood is collected from the internal jugular vein into EDTA-K2 anticoagulant tubes at pre-dose, 0.0833 h (intravenous administration only), 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. The blood samples are centrifuged at 1500 g for 10 minutes to obtain plasma, which is stored at -90°C to 60°C and subjected to further analysis and assays.

[0162] Sample treatment (1) Treatment methods for samples of Tilzepide, P007, P008, P013, and P014 Take 30.0 μL of thawed plasma sample. Add 150 μL of acetonitrile solution (containing 5 ng·mL -1 verapamil, 50 ng·mL -1 glyburide, 200 ng·mL -1 tolbutamide, and 200 ng·mL -1 diclofenac) to precipitate proteins. Vortex the mixture for 5 minutes and centrifuge at 3700 rpm for 8 minutes. Take 70.0 μL of the supernatant, add 70.0 μL of 0.5% formic acid aqueous solution thereto. Vortex the mixture for 5 minutes. Sample 15 μL of the mixed solution and inject it into LC-MS / MS to determine the drug concentration in plasma.

[0163] (2) Treatment methods for samples of P015, P016, P017, and P018 Collect 20.0 μL of thawed plasma sample. Add 60 μL of acetonitrile solution (containing 5 ng·mL -1 verapamil, 50 ng·mL -1 glyburide, 200 ng·mL -1 tolbutamide, and 200 ng·mL -1 diclofenac) to precipitate proteins. Vortex the mixture for 1 minute and centrifuge at 13000 rpm for 8 minutes. Take 60.0 μL of the supernatant, add 60.0 μL of 0.5% formic acid aqueous solution thereto. Vortex the mixture for 10 minutes. Sample 10.0 μL of the mixed solution and inject it into LC-MS / MS to determine the drug concentration in plasma.

[0164] (3) Treatment methods for samples of P019 and P020 Collect 30.0 μL of thawed plasma sample. Add 150 μL of acetonitrile solution (containing 5 ng·mL -1 verapamil, 50 ng·mL -1 glyburide, 200 ng·mL -1 tolbutamide, and 200 ng·mL -1(containing diclofenac) is added to precipitate the protein. The mixture is vortexed for 5 minutes and centrifuged at 3700 rpm for 8 minutes. 70.0 μL of the supernatant is taken, and 70.0 μL of an aqueous 0.5% formic acid solution is added thereto. The mixture is vortexed for 5 minutes. 10.0 μL of the mixed solution is sampled and injected into LC-MS / MS to determine the drug concentration in plasma.

[0165] Based on the drug concentration in plasma of the compound in SD rats, the software Winnolin 8.2 was applied to a non-compartmental model to calculate the pharmacokinetic parameters, and the results are shown in Tables 3 and 4 below.

[0166]

Table 3

[0167]

Table 4

[0168] In the pharmacokinetic study in rats by subcutaneous injection of the compound according to the present invention, the following was shown: The half-lives of P007, P008, P013, P014, P015, P016, P017, P018 and P020 are longer than the half-life of tildepazide (an increase of about 23.7% to 64.0%). Compared with tildepazide, a commercially available dual-target product, the compound according to the present invention modified by the modified chain can provide a longer half-life in plasma. Furthermore, compared with P001, the compound according to the present invention has a substantially longer half-life. The half-life is extended by more than 15 times, more than 20 times, and even more than 30 times.

[0169] (II)Pharmacokinetics in C57 mice After subcutaneous or intravenous administration to C57 mice, plasma was collected and the drug concentration in the plasma was measured to illustrate the in vivo pharmacokinetic properties of the compounds according to the present invention. The compound was dissolved in a phosphate buffer containing 0.1% Tween 20 and filtered through a filtration membrane (PTFE, 0.45 μm) to obtain a compound solution of 15 nmol / mL. C57 mice (18 - 22 g) were administered at 30 nmol / kg subcutaneously (s.c.) or 15.0 nmol / kg intravenously (i.v.). Before administration, at 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours and 72 hours, approximately 100 μL of whole blood was collected from the orbital vein into an EDTA-K2 anticoagulant tube. The blood sample was centrifuged at 1500 g for 10 minutes to obtain plasma, which was stored at -90°C to 60°C and subjected to further analysis and assay.

[0170] Sample treatment (1) Treatment methods for samples of tilzepatide, P014, P016 and P020 Collect 20.0 μL of thawed plasma sample. Sequentially add 20 μL of 50% aqueous methanol solution (containing 100 ng / mL of internal standard) and 60 μL of acetonitrile to precipitate proteins. Vortex the mixture for 5 minutes and centrifuge at 3700 rpm for 8 minutes. Take 50.0 μL of the supernatant and add 50.0 μL of 0.5% aqueous formic acid solution thereto. Vortex the mixture for 5 minutes. Sample 50 μL of the mixed solution and inject it into LC-MS / MS to determine the drug concentration in the plasma.

[0171] Based on the plasma drug concentration of the compound in C57 mice, pharmacokinetic parameters were calculated by applying the software Winnolin 8.2 to a non-compartmental model, and the results are shown in Tables 5 and 6 below.

[0172]

Table 5

[0173]

Table 6

[0174] In the pharmacokinetic study in mice by subcutaneous injection of the compounds according to the present invention, the following was shown: The half-lives of P014, P016 and P020 were longer than that of tildepazide (an increase of 116.0% - 150.3%). This means that the half-lives of these compounds are significantly longer than that of tildepazide. Compared with tildepazide, a commercially available dual-target product, the compounds according to the present invention modified by the modified chain can provide a longer half-life in plasma.

[0175] (III) Pharmacokinetics in cynomolgus monkeys After subcutaneous or intravenous administration to cynomolgus monkeys, plasma was collected and the drug concentration in the plasma was measured to exemplify the in vivo pharmacokinetic characteristics of the compounds of the present invention. The compounds were dissolved in 1.4% PG in 8 mM Na2HPO4 buffer (pH 7.39) and filtered through a filtration membrane (PTFE, 0.45 μm) to obtain compound solutions of 25 nmol / mL and 12.5 nmol / mL, respectively. Before administration, at 2 hours, 12 hours, 24 hours, 32 hours, 48 hours, 3 days (72 hours), 4 days (96 hours), 6 days (144 hours), 9 days (216 hours), 12 days (288 hours), 15 days (360 hours), 18 days (432 hours), about 500 μL of whole blood was collected from the radial cutaneous vein or other appropriate vein into an EDTA-K2 anticoagulant tube. The blood sample was centrifuged at 2200 g for 10 minutes to obtain plasma, which was stored at -90°C to -60°C and subjected to further analysis and assay.

[0176] Sample treatment (1) Sample treatment methods for tildepazide, P014, P016, P017 and P020 Collect 70.0 μL of thawed plasma. Add 70 μL of acetonitrile (containing an internal standard of 1 ng / ml) to precipitate the protein. Vortex the mixture for 1 minute and centrifuge at 14,000 rpm for 10 minutes. Take 60.0 μL of the supernatant and add 60.0 μL of 0.5% aqueous formic acid solution thereto. Vortex the mixture for 5 minutes. Sample 10 μL of the mixed solution and inject it into LC-MS / MS to determine the drug concentration in the plasma.

[0177] Based on the plasma drug concentration of the compound in cynomolgus monkeys, the software Winnolin 8.2 was applied to calculate the pharmacokinetic parameters by a non-compartmental model, and the results are shown in Tables 7 and 8 below.

[0178] [Table 7]

[0179] [Table 8]

[0180] In the pharmacokinetic study of the compound according to the present invention by subcutaneous injection in cynomolgus monkeys, the following was shown: The half-lives of P014, P016 and P020 are equivalent to those of tildepazide, and the Tmax of P016 and P017 is delayed compared to tildepazide. Compared with tildepazide, a commercially available dual-target product, the compound according to the present invention modified by a modified chain can provide an equivalent half-life in plasma, and the Tmax of P016 and P017 is twice that of tildepazide. The time to the peak of the drug concentration is significantly delayed compared to tildepazide, and as a result, there may be fewer / lighter gastrointestinal reactions or a shorter titration time.

[0181] III. Pharmacodynamic effects (I) The purpose of the pharmacodynamic effect in db / db mice is to examine the effect of the compound according to the present invention on blood glucose in diabetic model mice (db / db mice).

[0182] In this test, db / db mice were administered a single subcutaneous dose, and changes in blood glucose, food intake, and body weight of the mice were measured to illustrate the blood glucose-lowering effect and the duration of drug efficacy of the compound according to the present invention, and compared with the positive controls, tildepazide and P001. In this test, male db / db mice aged 8 - 9 weeks were used. The db / db mice were placed in an individually ventilated cage (IVC) facility with controlled temperature (20 - 26°C) and humidity (40 - 70%), and a 12-hour:12-hour light-dark cycle. The mice were allowed free access to food and water. Blood was collected from the tip of the tail, and fasting blood glucose was measured using a blood glucose meter from Roche. The mice were randomly divided into groups (n = 6 / group) based on their initial blood glucose and initial body weight, and the body weight and blood glucose values of each group were comparable.

[0183] The compound according to the present invention (10 nmol / kg), or the positive controls, tildepazide (10 nmol / kg) and P001 (10 nmol / kg), were dissolved in a vehicle (PBS containing 0.1% Tween 20, pH 7.2 - 7.4). After a single subcutaneous administration, random blood glucose was recorded at the set time points (0 - 120 hours, and the recording of random blood glucose was stopped when there was no difference in random blood glucose between the compound group and the vehicle group), and the body weight and food intake were recorded daily. The data results were statistically analyzed using GraphPad Prism 8, and the statistical differences between groups were analyzed according to the t-test. A significant difference was confirmed at p < 0.05.

[0184] Duration of blood glucose lowering: The longest time when the random blood glucose value of the compound showed a significant difference (P < 0.05) compared with the vehicle group.

[0185]

Table 9

[0186] Compared with P001, the duration of blood glucose lowering of the compounds according to the present invention is longer than that of P001 by 24 hours or more (P007, P008, P014, P019), further 40 hours or more (P008, P014), and 72 hours or more (P014) (see Table 9, FIGS. 1A and 1B).

[0187] Compared with tildepepide, the duration of blood glucose lowering of the compounds according to the present invention is equal to or longer than that of tildepepide (P007, P008, P013, P014, P016, P017, P018, P019 and P020), further 16 hours or more (P008, P013, P014, P016, P017, P020), more than 24 hours (P013, P014, P016, P017, P020), 48 hours or more (P014, P016, P020), 56 hours or more (P016, P020) (see Table 9).

[0188] When compared with P001, based on the experiments by the above method, during the test period, the blood glucose lowering effects of P008 (p < 0.01), P014 (p < 0.001) and P019 (p < 0.05) are significantly superior to that of P001 (blood glucose AUC, P < 0.05, as shown in FIG. 2A). The inhibitions of blood glucose AUC are 2.3 times, 2.6 times and 2.2 times that of P001 respectively, and the durations of blood glucose lowering effects are 40 hours, 72 hours and 24 hours longer than that of P001 respectively (Table 9). The blood glucose lowering effect of P007 is equivalent to that of P001, but the duration of blood glucose lowering is 24 hours longer than that of P001 (shown in FIG. 2A and Table 9).

[0189] Comparison with tildepepide: The blood glucose lowering effects of P014 (p < 0.05, FIG. 2B), P016 (p < 0.05, FIG. 2C) and P020 (p < 0.05, FIG. 2C) are stronger than that of tildepepide (blood glucose AUC, p < 0.05). The inhibitions of blood glucose AUC are 1.7 times, 2.2 times and 2.0 times that of tildepepide respectively. Furthermore, the durations of drug effects are also significantly longer (48 hours, 56 hours and 56 hours longer than that of tildepepide respectively, Table 9). The blood glucose lowering effects of P013 and P017 are equivalent to that of tildepepide (FIG. 2C), but the durations of drug effects are 24 hours longer than that of tildepepide (Table 9).

[0190] Surprisingly, compared with P001, the acylated GLP-1 / GIP agonist of the present invention significantly improves the hypoglycemic effect while maintaining long-acting properties. Compounds according to the present invention having various forms of modified chains can achieve a hypoglycemic effect that is stronger and more persistent than that of P001. Furthermore, some compounds according to the present invention exhibit a hypoglycemic effect that is stronger and more persistent than that of tirzepatide.

[0191] Furthermore, in relation to the performance of the binding activity and agonist activity of the compounds according to the present invention at the targeted human GLP-1 receptor and GIP receptor, surprisingly, the compounds according to the present invention exhibit significantly reduced binding activity and agonist activity at both the targeted human GLP-1 receptor and the targeted human GIP receptor compared with P001, and the hypoglycemic effect of the compounds according to the present invention was found to be significantly better than that of P001.

[0192] (II) Multiple-dose experiment using db / db mice In this test, db / db mice were administered subcutaneously multiple times, and the changes in blood glucose of the mice were measured to further illustrate the hypoglycemic effect of the compounds according to the present invention and compare with the positive control tirzepatide. In this study, male db / db mice aged 7 - 8 weeks were used. The db / db mice were placed in an individually ventilated cage (IVC) facility controlled at a temperature of (20 - 26°C) and humidity of (40 - 70%) and a 12:12 hour light-dark cycle. The mice were allowed free access to food and water. Blood was collected from the tail tip, and fasting blood glucose was measured using a blood glucose meter manufactured by Roche. The mice were randomly grouped according to their initial blood glucose and initial body weight (n = 6 / group), and the body weight and blood glucose values of each group were comparable.

[0193] Dissolve the compound according to the present invention (3 nmol / kg, 10 nmol / kg, 30 nmol / kg) or the positive control tilsepamate (3 nmol / kg, 10 nmol / kg, 30 nmol / kg) in a vehicle (PBS containing 0.1% Tween 20, pH 7.2 - 7.4). Subcutaneously administer it to mice every three days for four weeks. The subcutaneous administration is performed on the 1st, 4th, 7th, 10th, 13th, 16th, 19th, 22nd, and 25th days. During the test period, record the random blood glucose every three days. Measure the fasting blood glucose on the 28th day. Next, collect blood by orbital blood sampling. After orbital blood sampling, sacrifice the animals and collect and weigh the liver. After 1 - 2 hours, centrifuge the collected blood at 3000 rpm for 10 minutes to separate the serum. Measure biochemical indices such as triglyceride (TG), alanine aminotransferase (ALT), and total bilirubin (TBIL) using a biochemical analyzer. Statistically analyze the data results using GraphPad Prism8, and analyze the statistical differences between groups according to the t - test. Confirm significant differences at p < 0.05.

[0194] In the experiments conducted as described in the above method, all the compounds according to the present invention, such as P014, P016, P017, and P020, showed better blood glucose inhibition than tilsepamate at a dose of 3 nmol / kg. At a dose of 30 nmol / kg, the efficacy of P016 is superior to that of tilsepamate (see Figures 3A - 3D and Table 10). P016, P017, and P020 significantly decreased the liver weight of db / db mice at some doses, while tilsepamate did not decrease the liver weight at the three test doses. Compounds according to the present invention, such as P014, P016, P017, P020, and tilsepamate, can effectively reduce TG in the serum of db / db mice (except for the P017 and tilsepamate groups at 3 nmol / kg), and the P016 and P020 groups at some doses showed better efficacy than tilsepamate (see Table 11). Furthermore, P014 and P016 can also reduce TBIL and / or ALT in the serum.

[0195]

Table 10

[0196]

Table 11

[0197]

Table 12

[0198] In conclusion, under this experimental system, the compound according to the present invention can effectively reduce the blood glucose of db / db mice by subcutaneous injection for 4 weeks. The minimum effective amount (≤3 nmol / kg) of the compound according to the present invention is 1 / 3 of the effective amount (10 nmol / kg) of tildepazide, and the maximum drug efficacy (30 nmol / kg) of some compounds according to the present invention such as P016 is superior to that of tildepazide. Furthermore, some compounds according to the present invention show a decrease in liver weight and a protective effect on the liver in db / db mice, while tildepazide did not show the same effect.

[0199] (III) Repeated administration to male rats for 2 weeks 1. Experimental schedule Select 77 male SD rats (SPF grade), and randomly divide them into 10 groups based on body weight. The first group is the vehicle control group, including 5 rats. The second to fourth groups are control groups administered with commercially available tildepazide, with 8 rats in each group (5 for the main test and 3 for the toxicokinetics test), and the dosing amounts are 1 mg / kg, 3 mg / kg, and 10 mg / kg respectively. The fifth to seventh groups are groups administered with test substance 1, and the eighth to tenth groups are groups administered with test substance 2, with 8 rats in each group (5 for the main test and 3 for the toxicokinetics test), and the dosing amounts are 1 mg / kg, 3 mg / kg, and 10 mg / kg respectively. The above-mentioned each group is administered twice a week for 2 consecutive weeks (administered on the 1st, 5th, 8th, 12th, and 15th days).

[0200] During the experiment, monitoring of the cage state, detailed clinical observations, measurement of food intake, measurement of body weight, measurement of blood biochemical and hematological indices, gross anatomical examination, weighing of organs, and toxicokinetics tests are performed in all animal groups.

[0201] 2. Data Statistics The following table shows the details of the combinations used for statistical comparison. The table is as follows:

Table 13

[0202] Aggregate the original data for each period. For each assay endpoint, calculate the mean value, standard deviation, and / or change between groups based on the group and gender. For each endpoint, compare the treatment group with the control group in the following aspects. Before starting a specific analysis, perform a logarithmic transformation on the endpoint data if necessary.

[0203]

Table 14

[0204] 3. Experimental Results Body weight / weight gain: After the first administration, a decrease in body weight was observed in the following groups: tildepazotide ≥ 3 mg / kg; P014 and P016 ≥ 1 mg / kg. With the continuation of administration, the body weight recovered, and at the end of the administration, the body weight increased in all groups, but the weight gain was lower than that of the control group, indicating an obvious dose-dependent relationship.

[0205] Food intake: Through the test procedure, a dose-dependent decrease in food intake was observed in each dosing group (tilzepide, P014, and P016). Blood chemistry: Amylase and triglyceride levels decreased. Hematology: The reticulocyte level decreased slightly. Gross anatomy: No abnormalities were found in each dosing group (tilzepide, P014, and P016). Organ weights: In each dosing group (tilzepide, P014, and P016), the weights of the heart, liver, spleen, etc. decreased, and no significant difference was observed compared to the vehicle control group. This is considered to be related to the weight loss due to pharmacological action. Toxicokinetics: The C max and AUC of each dosing group (tilzepide, P014, and P016) increased almost proportionally to the dose. In each dosing group (tilzepide, P014, and P016), no substantial accumulation was observed even after twice-weekly administration for 2 consecutive weeks.

[0206] Each dosing group (tilzepide, P014, and P016) had an MTD ≥ 10 mg / kg when administered twice a week for 2 consecutive weeks. The C max , AUC last and the safety margin of the test are as follows:

Table 15

[0207] In conclusion, P016 and P014 have the same toxicity as tilzepide, but have a wide safety margin (based on the exposure amount, the safety margin of P014 is more than 4 times that of tilzepide, and the safety margin of P016 is more than 7 times that of tilzepide).

[0208] Furthermore, the safety margin of the compounds according to the present invention, particularly P014 and P016, is 10 times, or 20 times, or 30 times, or 40 times, or 50 times, or 60 times, or 70 times, or 80 times, or 90 times greater than that of P001.

[0209] On the one hand, under the same exposure, the compound according to the present invention has a lower impact on heart rate (HR) than Tirepatide. The increase in HR of P016 is lower than that of Tirepatide. A sustained increase in HR exceeding 30% is not observed. At the same exposure, the recovery of the heart rate of P016 is faster than that of TZP (recovery time of P016: 72 - 120 hours, recovery time of TZP > 120 hours).

[0210] The peptide compounds provided by the present invention and their uses have been introduced in detail above.

[0211] Specific examples are applied herein to illustrate the principles and embodiments of the present invention. The illustrations of the above examples are only used to assist in the understanding of the methods and central ideas of the present invention. Those skilled in the art should be able to make several improvements and modifications to the present invention without departing from the principles of the present invention, and it should be shown that these improvements and modifications are also included in the protection scope of the claims of the present invention.

[0212]

Table 16

Table 17

Table 18

Table 19

Claims

[Claim 1] The inventions described herein or in the drawings.

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