TWI428139B - A novel glucagon-like peptide analogue, a composition and use thereof - Google Patents

Description

Novel glucagon-like peptide analog, composition and use thereof

The present invention relates to a novel glucagon-like peptide analog and a composition thereof for use in modulating insulin expression and treating diabetes in a mammal. In particular, these polypeptide derivatives are useful for the long-term treatment of diseases such as diabetes and other diseases associated with insulin secreting polypeptides, gastrointestinal function and activity associated with glucagon levels. The invention also relates to the use of said glucagon-like peptide analogs and compositions thereof for use in the above conditions.

The islet endocrine process is regulated by a complex set of control mechanisms. This mechanism is not only affected by blood-derived metabolites such as glucose, amino acids and catecholamines, but also by local paracrine. The major islet hormones, glucagon, insulin, and somatostatin interact with specific islet cells (A, B, and D cells) to regulate the secretory response. Although insulin secretion is mainly determined by blood glucose levels, auxin can also inhibit the secretion of glucose-regulated glycemic insulin. In addition to the endo-colonial regulation of insulin secretion, there is evidence of the presence of intestinal insulinotropic factors. The concept of insulinotropics stems from the observation that the same amount of intravenous infusion of energy (glucose), energy from food intake or intestinal glucose absorption can provoke a greater stimulus to promote insulin release ( Elrick, H., et al., J. Clin. Endocrinol. Metab. 1964, 24: 1076-1082; McIntyre, N., et al., J. Clin. Endocrinol. Metab., 1965, 25: 1317- 1324). Thus, assuming that when energy is taken up through the gastrointestinal tract rather than the parenteral route, the intestinal-derived signal stimulated by oral nutrient uptake represents an effective insulin secretagogue that promotes increased insulin release (Dupre, J., et al.,. Diabetes. 1966, 15: 555-559). Although some neurotransmitters and gastrointestinal hormones have similar incretin-like activities, a large body of evidence from studies of immunization, antagonists, and knockout suggests that Glucose-dependent insulinotropic polypeptide (GIP) and Glucagons-like peptide-1 (GLP-1) represents a polypeptide that plays a leading role in insulin secretion caused by most nutrient stimulation. Studies have shown that patients with type 2 diabetes show a significant reduction in insulin-stimulated release stimulated by meals, and this observation has raised interest in the following studies: determining whether defective incretin release or incretin resistance is resistant It is related to the pathophysiology of β-cell dysfunction in diabetic patients.

In 1987, human glucagon-like peptide-1 (GLP-1) was first discovered as an endocrine hormone, a polypeptide secreted by the intestine after food intake. GLP-1 is secreted by intestinal L cells after proteolytic process by a 160 amino acid precursor protein (ie, proglucagon). After cleavage of proglucagon, a peptide GLP-1 consisting of 37 amino acids, GLP-1(1-37)OH, is first formed, and the activity of the peptide is very weak. Subsequently, it is cleaved at the 7-position to produce bioactive GLP-1(7-37)OH. After the glycine residue at the end of the L-cell was removed, approximately 80% of the synthesized GLP-1 (7-37) OH was amylated on the C-terminus. The biological effects and metabolic turnover of the free acid GLP-1 (7-37) OH and the guanamine compound GLP-1 (7-37) NH ₂ cannot be distinguished.

GLP-1 is known to stimulate insulin secretion that causes glucose uptake by cells, thereby lowering blood glucose levels (Mojsov, S., et al., J. Clin. Invest., 1987, 79: 616-619; Kreymann, B., et Al., Lancet ii, 1987, 1300-1304; Orskov, C., et al., Endocrinology, 1988, 123: 2009-2013). Acute intraventricular injection of GLP-1 or GLP-1 receptor agonists produces a transient reduction in food intake (Turton MD, et al., Nature, 1996, 379: 60-72). However, some studies have shown that administration of more long-term intraventricular or parenteral GLP-1 receptor agonists is associated with weight loss (Meeran, K., et al., Endocrinology, 1999, 140: 244-250; Davies , HR Jr., Obes. Res., 1998, 6: 147-156; Szayna, M., et al., Endocrinology, 2000, 141: 1936-1941; Larsen, PJ, et al., Diabetes, 2001, 50: 2530-2539). A large number of GLP-1 analogs with insulinotropic effects are known in the art. For example, these analogs include GLP-1 (7-36), Gln ⁹ -GLP-1 (7-37), D-Gln9-GLP-1 (7-37), acetyl-Lys9-GLP-1 ( 7-37), Thr16-Lys18-GLP-1 (7-37) and Lys18-GLP-1 (7-37). For example, GLP-1 derivatives include acid addition salts, carboxylates, lower alkyl esters, and guanamine compounds (WO 91/11457, EP 0733644, and US 5,512,549).

Most of the effects of GLP-1 described in preclinical experiments have also been demonstrated in human studies. In the glucose-loaded or food-ingested state, injection of GLP-1(7-36)NH ₂ into normal humans stimulates insulin secretion and significantly lowers blood glucose levels (Orskov, C., et al., Diabetes, 1993, 42: 658-661; Qualmann, C., et al., Acta. Diabetol., 1995, 32: 13-16).

For diabetic patients who have failed sulfonylurea therapy, the use of GLP-1-like peptides for insulin therapy will certainly have a good effect (Nauck, M.A., et al., Diabetes Care, 1998, 21:1925-1931). GLP-1 stimulates insulin secretion only in the case of hyperglycemia. This property of GLP-1 makes it safer than insulin, and it has been observed that the amount of insulin secreted is proportional to the degree of hyperglycemia. In addition, GLP-1 treatment will result in the release of insulin from the pancreas and the first action of insulin on the liver. These results can result in a low level of circulating insulin compared to subcutaneous insulin injections. GLP-1 slows the gastrointestinal emptying process, which is more conducive to allowing nutrients to be absorbed over a longer period of time, thereby reducing postprandial glucose peaks. Some reports indicate that GLP-1 can increase the sensitivity of insulin in peripheral tissues such as muscle, liver and fat. GLP-1 also acts as a potential appetite regulator.

If considered for use in patients with type 1 diabetes, the potential for treatment of GLP-1 and its analogs will be more prominent. Numerous studies have demonstrated the effectiveness of native GLP-1 in the treatment of insulin-dependent diabetes mellitus (IDDM). Similar to patients with non-insulin-dependent diabetes mellitus (NIDDM), GLP-1 is effective in alleviating pre-prandial hyperglycemia by stabilizing glucagon properties. Other studies have also shown that GLP-1 can also reduce postprandial glucose excursion in IDDM patients by prolonging gastric emptying. These observations indicate that GLP-1 can be used to treat IDDM and NIDDM.

However, the biological half-life of native GLP-1 molecules is affected by dipeptidase IV (DPPIV) activity, which has a very short biological half-life. For example, the biological half-life of GLP-1(7-37)OH is only 3-5 minutes (US5118666). Only by continuous instillation of GLP-1 can the blood glucose concentration be continuously reduced, as demonstrated by the study, which can control blood glucose levels by intravenous infusion over 24 hours (Larsen, J., et al., Diabetes Care, 2001, 24: 1416-1421). The enzyme DPP IV is a serine protease which preferentially hydrolyzes the NH ₂ -terminal peptide of the proline (Xaa-Pro-) or alanine (Xaa-Ala-) in the penultimate position (Mentlein, R. , Regul. Pept., 1999, 85: 9-25), DPP IV enzymes have demonstrated the ability to rapidly metabolize GLP-1 in vitro. Therefore, long-acting GLP-1-like peptides that are resistant to DPP IV have enormous therapeutic potential in the treatment of diabetes.

It is an object of the present invention to provide a novel GLP-1 analog which is more durable and which is completely resistant to the hydrolysis of the enzyme DPP IV compared to native GLP-1.

The present invention includes at least one compound having the following formula (I) or a pharmaceutically acceptable salt thereof,

Xaa ₇ -Q-Gly-Thr-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Tyr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ -Ala-Ala-Xaa ₂₆ -Xaa ₂₇ -Phe- Ile-Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -B ------------------(I)

Wherein, Xaa ₇ is natural or non-natural, selected from the group consisting of L-histidine, D-histidine, dea-histidine, 2-amino-histidine, β-hydroxy-histidine, and high An amino acid of histidine, α-fluoromethyl-histidine or α-methyl-histidine;

Q is selected from the following linkers (II), (III), (IV):

Wherein R _{1 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ -C ₆ )alkoxy; R _{2 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ - C ₆ ) alkoxy; R ₃ is H, (C ₁ -C ₆ )alkyl, or form a 5-8 membered ring with R ₁ or R ₂ ; X is selected from H, F, OH, CF ₃ or O; Y is selected from H, OH, F or (C ₁ -C ₆ )alkyl; Z is selected from N, C, O or S; wherein, when Z is selected from N, O or S, W is absent; when Z is When C, W is selected from H or F; Xaa ₁₄ is a natural or non-natural amino acid selected from serine or histidine, wherein one or more carbon atoms of the amino acid may also be arbitrarily one or more Alkyl substitution; Xaa ₁₆ is a natural or non-natural amino acid selected from the group consisting of valine, lysine or leucine, wherein one or more carbon atoms of the amino acid may also be arbitrarily one or more Alkyl substitution; or Xaa ₁₆ is a lysine linked to TU; Xaa ₁₈ is a natural or non-natural amino acid selected from the group consisting of serine, arginine or lysine, wherein one or more carbons of said amino acid atoms may be optionally substituted with one or more alkyl groups; Xaa ₂₂ and Xaa ₂₃ are natural or unnatural, selected Amino acid, amino isobutyric acid or glutamic acid, wherein one or more carbon atoms of said amino acids may also be optionally substituted by one or more alkyl _{groups; Xaa 26, Xaa 27, Xaa} 34, Xaa 35 And Xaa ₃₆ are natural or non-natural, an amino acid selected from the group consisting of glycine, lysine, arginine, leucine, aspartame or aminoisobutyric acid, wherein one or more carbon atoms of the amino acid It may also be optionally substituted by one or more alkyl groups; or Xaa ₂₆ is a lysine linked to TU; T is selected from γ-glutamic acid, β-alanine, γ-aminobutyric acid or HOOC (CH _{2 ) n} COOH, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27; or T is selected from Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; U is only an amino acid selected from γ-glutamic acid, β-alanine or γ-aminobutyric acid at T, or T is selected from T When present, wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; U is selected from C ₈ -C ₂₀ fatty acids, and B is selected from glycine, glycinamine or a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and having one amino acid being cysteine.

The present invention further provides a preferred technical solution of the above technical solution: in the formula (I), Xaa ₂₆ is natural or non-natural, selected from glycine, lysine, arginine, leucine and aspartame or not A-aminoisobutyric acid linked to TU, B is a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and one amino acid is cysteine.

The above peptide fragment is cysteine-serine-glycine, cysteine-alanine or methoxypolyethylene glycol maleimide linked to cysteine.

The present invention also provides a GLP-1 analogue represented by the formula (V), a composition thereof, or a pharmaceutically acceptable salt thereof,

In formula (V), R _{1 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ -C ₆ )alkoxy; R _{2 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ -C ₆ ) alkoxy; R 3 is selected from H, (C ₁ -C ₆ )alkyl, or forms a 5-8 membered ring with R ₁ or R ₂ ; X is selected from H, F, OH, CF ₃ or O; D is Gly-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Thr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ -Ala-Ala-Xaa ₂₆ -Xaa ₂₇ -Phe-Ile -Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -B; Xaa ₇ is natural or non-natural, selected from the group consisting of L-histidine, D-histidine, dea-histidine , amino acid of 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl-histidine; Xaa ₁₄ is natural or unnatural An amino acid selected from the group consisting of serine or histidine, wherein one or more carbon atoms of the amino acid may also be optionally substituted by one or more alkyl groups; Xaa ₁₆ is natural or non-natural, selected from the group consisting of hydrazine ammonia An amino acid of acid, lysine or leucine, wherein one or more carbon atoms of said amino acid may also be optionally substituted by one or more alkyl groups; or Xaa ₁₆ is a lysine linked to TU; Xaa ₁₈ Natural or unnatural, selected from serine, arginine or lysine amino acid, wherein one or more carbon atoms of said amino acids may also be optionally substituted by one or more alkyl groups; Xaa ₂₂ and Xaa ₂₃ Is a natural or non-natural amino acid selected from the group consisting of glycine, aminoisobutyric acid or glutamic acid, wherein one or more carbon atoms of the amino acid may also be optionally substituted by one or more alkyl groups; Xaa ₂₆ , Xaa ₂₇ , Xaa ₃₄ , Xaa ₃₅ and Xaa ₃₆ are natural or non-natural amino acids selected from the group consisting of glycine, lysine, arginine, leucine, aspartame or aminoisobutyric acid, wherein One or more carbon atoms of the amino acid may also be optionally substituted by one or more alkyl groups; or Xaa ₂₆ is a lysine linked to TU; T is selected from γ-glutamic acid, β-alanine, γ- Aminobutyric acid or HOOC(CH ₂ ) _n COOH, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ₁₆ , 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27; or T is selected from Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; U is only an amino acid selected from γ-glutamic acid, β-alanine or γ-aminobutyric acid at T, or T is selected from T When present, wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; U is selected from C ₈ -C ₂₀ fatty acids; B is selected from glycine, glycosamine or a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and having one amino acid being cysteine.

The present invention also provides a preferred technical solution of the above technical solution: in the above formula (V), the Xaa ₂₆ contained in D is natural or non-natural, and is selected from the group consisting of glycine, lysine, arginine, leucine and day. Winter oxime or α-aminoisobutyric acid without TU, B is a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and one amino acid is cysteine.

The above peptide fragment is cysteine-serine-glycine, cysteine-alanine or methoxypolyethylene glycol maleimide linked to cysteine.

In the above formula (V), R ₁ , R ₂ and R ₃ are each selected from H or CH ₃ , or R ₁ is CH ₃ , R ₂ and R ₃ are both H; or both R ₁ and R ₃ are H, R ₂ is CH ₃ ; or R ₃ and R ₁ form a 5-8-membered ring, R ₂ is H; or R ₃ and R ₂ form a 5-8-membered ring, and R ₁ is H.

The present invention still further provides a GLP-1 analogue represented by the formula (VI), a composition thereof, or a pharmaceutically acceptable salt thereof,

R _{1 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ -C ₆ )alkoxy; R _{2 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ -C ₆ ) Alkoxy; R _{3 is} selected from H, (C ₁ -C ₆ )alkyl, or forms a 5-8 membered ring with R ₁ or R ₂ ; Y is selected from H, OH, F or (C ₁ -C ₆ ) Alkyl; D is Gly-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Thr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ -Ala-Ala-Xaa ₂₆ -Xaa ₂₇ -Phe-Ile- Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -B; Xaa ₇ is natural or non-natural, selected from the group consisting of L-histidine, D-histidine, dea-histidine, Amino acid of 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl-histidine; Xaa ₁₄ is natural or unnatural An amino acid selected from the group consisting of serine or histidine, wherein one or more carbon atoms of the amino acid may also be optionally substituted by one or more alkyl groups; Xaa ₁₆ is natural or non-natural, selected from the group consisting of valine An amino acid of lysine or leucine, wherein one or more carbon atoms of said amino acid may also be optionally substituted by one or more alkyl groups; or Xaa ₁₆ is a lysine linked to TU; Xaa ₁₈ Is natural Or non-natural, an amino acid selected from the group consisting of serine, arginine or lysine, wherein one or more carbon atoms of said amino acid may also be optionally substituted by one or more alkyl groups; Xaa ₂₂ and Xaa ₂₃ are A natural or non-natural amino acid selected from the group consisting of glycine, aminoisobutyric acid or glutamic acid, wherein one or more carbon atoms of the amino acid may also be optionally substituted by one or more alkyl groups; Xaa ₂₆ , Xaa ₂₇ , Xaa ₃₄ , Xaa ₃₅ and Xaa ₃₆ are natural or non-natural amino acids selected from the group consisting of glycine, lysine, arginine, leucine, aspartame or aminoisobutyric acid, wherein the amino acid One or more carbon atoms may also be optionally substituted by one or more alkyl groups; or Xaa ₂₆ is a lysine linked to TU; T is selected from γ-glutamic acid, β-alanine, γ-amino Butyric acid or HOOC(CH ₂ ) _n COOH, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ₁₆ , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27; or T is selected from Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; U is only an amino acid selected from γ-glutamic acid, β-alanine or γ-aminobutyric acid at T, or T is selected from T When present, wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; U is selected from C ₈ -C ₂₀ fatty acids; B is selected from glycine, glycosamine or a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and having one amino acid being cysteine.

The present invention also provides a preferred technical solution of the above technical solution: in the above formula (VI), X contains Xaa _{26 which} is natural or non-natural, and is selected from the group consisting of glycine, lysine, arginine, leucine and day. Winter oxime or α-aminoisobutyric acid without TU, B is a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and one amino acid is cysteine.

The above peptide fragment is cysteine-serine-glycine, cysteine-alanine or methoxypolyethylene glycol maleimide linked to cysteine.

In the above formula (VI), R _{1 is} selected from H or (C ₁ -C ₆ )alkyl; R _{2 is} selected from H or (C ₁ -C ₆ )alkyl; R ₃ is H; Y is selected from H or ( C ₁ -C ₆ )alkyl.

The present invention still further provides a GLP-1 analogue represented by the formula (VII), a composition thereof, or a pharmaceutically acceptable salt thereof,

In formula (VII), R _{1 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ -C ₆ )alkoxy; R _{2 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ -C ₆ )alkoxy; R _{3 is} selected from H, (C ₁ -C ₆ )alkyl, or forms a 5-8 membered ring with R ₁ or R ₂ ; Y is selected from H, OH, F or (C ₁ -C ₆ )alkyl; W is selected from H or F; D is Gly-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Thr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ -Ala-Ala-Xaa ₂₆ -Xaa ₂₇ -Phe-Ile-Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -B; Xaa ₇ is natural or non-natural, selected from L-histamine Acid, D-histidine, dea-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl An amino acid of histidine; Xaa ₁₄ is a natural or non-natural amino acid selected from the group consisting of serine or histidine, wherein one or more carbon atoms of the amino acid may also be optionally substituted by one or more alkyl groups. Xaa ₁₆ is a natural or non-natural amino acid selected from the group consisting of valine, lysine or leucine, wherein one or more carbon atoms of the amino acid may also be optionally substituted by one or more alkyl groups. ; or Xaa ₁₆ is connected TU lysine; Xaa ₁₈ is natural or unnatural, selected from serine, arginine or lysine amino acid, wherein said one or more amino acids of carbon atoms can optionally be substituted with one or more Alkyl substitution; Xaa ₂₂ and Xaa ₂₃ are natural or non-natural, an amino acid selected from the group consisting of glycine, aminoisobutyric acid or glutamic acid, wherein one or more carbon atoms of the amino acid may also be arbitrarily one or Multiple alkyl substitutions; Xaa ₂₆ , Xaa ₂₇ , Xaa ₃₄ , Xaa ₃₅ and Xaa ₃₆ are natural or non-natural, selected from glycine, lysine, arginine, leucine, aspartame or amino An amino acid of butyric acid, wherein one or more carbon atoms of the amino acid may also be optionally substituted by one or more alkyl groups; or Xaa ₂₆ is a lysine linked to TU; T is selected from γ-glutamic acid , β-alanine, γ-aminobutyric acid or HOOC(CH ₂ ) _n COOH, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27; or T is selected from .

Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Only T is an amino acid selected from γ-glutamic acid, β-alanine, γ-aminobutyric acid, or T is selected from T In the formula, k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. Or 10; U is selected from C ₈ -C ₂₀ fatty acids; B is selected from glycine, glycosamine or a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and having one amino acid being cysteine.

The present invention also provides a preferred technical solution of the above technical solution: in the above formula (VII), the Xaa ₂₆ contained in D is natural or non-natural, and is selected from the group consisting of glycine, lysine, arginine, leucine and day. Winter oxime or α-aminoisobutyric acid without TU, B is a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and one amino acid is cysteine.

The above peptide fragment is cysteine-serine-glycine, cysteine-alanine or methoxypolyethylene glycol maleimide linked to cysteine.

In the above formula (VII), R ₃ is H or forms a 5-8 membered ring with R ₁ or R ₂ ; Y is H or F.

The present invention also provides a GLP-1 analogue represented by the formula (VIII), a composition thereof, or a pharmaceutically acceptable salt thereof,

Wherein, Xaa ₇ is natural or non-natural, selected from the group consisting of L-histidine, D-histidine, dea-histidine, 2-amino-histidine, β-hydroxy-histidine, and high An amino acid of histidine, α-fluoromethyl-histidine or α-methyl-histidine; Q is selected from the following linkers (II), (III), (IV):

Wherein R _{1 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ -C ₆ )alkoxy; R _{2 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ - C ₆ ) alkoxy; R _{3 is} selected from H, (C ₁ -C ₆ )alkyl, or forms a 5-8 membered ring with R ₁ or R ₂ ; X is selected from H, F, OH, CF ₃ or O Y is selected from H, OH, F or (C ₁ -C ₆ )alkyl; Z is selected from N, C, O or S; wherein, when Z is N, O or S, W is absent; when Z is When C, W is selected from H or F; Xaa ₁₄ is a natural or non-natural amino acid selected from serine or histidine, wherein one or more carbon atoms of the amino acid may also be arbitrarily one or more Alkyl substitution; Xaa ₁₆ is a natural or non-natural amino acid selected from the group consisting of valine, lysine or leucine, wherein one or more carbon atoms of the amino acid may also be arbitrarily one or more Alkyl substituted; Xaa ₁₈ is a natural or non-natural amino acid selected from the group consisting of serine, arginine or lysine, wherein one or more carbon atoms of the amino acid may also be optionally substituted by one or more alkyl groups. substituted; Xaa ₂₂ and Xaa ₂₃ are natural or unnatural, selected from glycine, aminoisobutyric acid or glutamic acid Acid, wherein one or more carbon atoms of said amino acids may also be optionally substituted by one or more alkyl _{groups; Xaa 27, Xaa 34, Xaa} 35 and Xaa ₃₆ are natural or unnatural, selected from glycine, An amino acid of lysine, arginine, leucine, aspartame or aminoisobutyric acid, wherein one or more carbon atoms of said amino acid may also be optionally substituted by one or more alkyl groups; Selected from γ-glutamic acid, β-alanine, γ-aminobutyric acid or HOOC(CH ₂ ) _n COOH, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27; or T is selected from Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; U is only an amino acid selected from γ-glutamic acid, β-alanine, γ-aminobutyric acid at T, or T is selected from T When present, wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; U is selected from C ₈ -C ₂₀ fatty acids, and B is selected from glycine or glycine.

The present invention also provides a GLP-1 analogue represented by the formula (IX), a composition thereof, or a pharmaceutically acceptable salt thereof,

Xaa ₇ -Q-Gly-Thr-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Tyr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ -Ala-Ala-Xaa ₂₆ -Xaa ₂₇ -Phe- Ile-Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -Gly-Xaa ⁿ -Xaa ⁿ⁺¹ -Xaa ⁿ⁺² -Xaa ⁿ⁺³ -Xaa ⁿ⁺⁴ -Cys ^(PEG) -Xaa ^m -Xaa ^m+1 -Xaa ^m+2 -Xaa ^m+3 -Xaa ^m+4 IX

Wherein Xaa ₇ is natural or non-natural, selected from the group consisting of L-histidine, D-histidine, dea-histidine, 2-amino-histidine, β-hydroxy-histidine, An amino acid of homohistidine, α-fluoromethyl-histidine or α-methyl-histidine; Q is selected from the following linkers (II), (III), (IV):

Wherein R _{1 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ -C ₆ )alkoxy; R _{2 is} selected from H, (C ₁ -C ₆ )alkyl or (C ₁ - C ₆ ) alkoxy; R ₃ is H, (C ₁ -C ₆ )alkyl, or form a 5-8 membered ring with R ₁ or R ₂ ; X is selected from H, F, OH, CF ₃ or O; Y is selected from H, OH, F or (C ₁ -C ₆ )alkyl; Z is selected from N, C, O or S; wherein, when Z is N, O or S, W is absent; when Z is C When W is selected from H or F; Xaa ₁₄ is a natural or non-natural amino acid selected from serine or histidine, wherein one or more carbon atoms of the amino acid may also be optionally substituted with one or more alkane Substituent; Xaa ₁₆ is a natural or non-natural amino acid selected from the group consisting of valine, lysine or leucine, wherein one or more carbon atoms of the amino acid may also be optionally substituted with one or more alkane Substituent; or Xaa ₁₆ is a lysine linked to TU; Xaa ₁₈ is a natural or non-natural amino acid selected from the group consisting of serine, arginine or lysine, wherein one or more carbon atoms of the amino acid may optionally be substituted with one or more alkyl groups; Xaa ₂₂ and Xaa ₂₃ are natural or unnatural, selected Gan Amino acids, amino isobutyric acid or glutamic acid, wherein one or more carbon atoms of the amino acid may optionally be substituted with one or more alkyl _{groups; Xaa 26, Xaa 27, Xaa} 34, Xaa 35 , and Xaa ₃₆ is a natural or non-natural amino acid selected from the group consisting of glycine, lysine, arginine, leucine, aspartame or aminoisobutyric acid, wherein one or more carbon atoms of the amino acid are also It may be optionally substituted by one or more alkyl groups; or Xaa ₂₆ is a lysine linked to TU; Xaa ⁿ , Xaa ⁿ⁺¹ , Xaa ⁿ⁺² , Xaa ⁿ⁺³ , Xaa ⁿ⁺⁴ , Xaa ^m , Xaa ^m+1 , Xaa ^m+2 , Xaa ^m+3 and Xaa ^m+4 together constitute 1, 2, 3 or 4 natural or non-natural amino acids. That is, Xaa ⁿ , Xaa ⁿ⁺¹ , Xaa ⁿ⁺² , Xaa ⁿ⁺³ , Xaa ⁿ⁺⁴ , Xaa ^m , Xaa ^m+1 , Xaa ^m+2 , Xaa ^m+3 and Xaa ^m+4 The cysteine attached to the methoxypolyethylene glycol maleimide forms a fragment of 2 to 5 amino acids.

To assist the reader in understanding the compounds of the present invention, the present invention also provides a compound selected from the group consisting of:

‧[Q-linker-d8, glutamic acid 22] GLP-1-(7-37)-peptide;

‧[Q-linker-a8-9, glutamic acid 22] GLP-1-(7-37)-peptide;

‧[Q-linker-b8-9, glutamic acid 22] GLP-1-(7-37)-peptide;

‧[Q-linker-c8, glutamic acid 22] GLP-1-(7-37)-peptide;

‧[Q-linker-e8-9, glutamic acid 22] GLP-1-(7-37)-peptide;

‧[Q-linker-f8-9, arginine 34] GLP-1-(7-37)-peptide;

‧N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-c8, arginine 34]GLP-1-(7-37)- Peptide

‧N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-d8, arginine 34]GLP-1-(7-37)- Peptide

‧N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker- _e 8,arginine 34]GLP-1-(7-37) -peptide;

‧N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-f8, arginine 34]GLP-1-(7-37)- Peptide

‧N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-a8-9,arginine 34]GLP-1-(7-37 )-peptide;

‧N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-b8-9,arginine 34]GLP-1-(7-37 )-peptide;

‧N-ε ²⁶ -[(N ^ε -ω-carboxyheptadecanyl)]-[Q-linker-c8, arginine 34]GLP-1-(7-37)-peptide;

‧N-ε ²⁶ -[(N ^ε -ω-carboxynonanoyl)]-[Q-linker-c8, arginine 34] GLP-1-(7-37)-peptide;

‧ [Q-linker-d8] GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ ;

‧ [Q-linker-c8] GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ ;

‧ [Q-linker-a8-9] GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ ;

‧ [Q-linker-b8-9] GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ ;

‧ [Q-linker-e8-9] GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ ;

‧[Q-linker-f8-9]GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ .

The invention also provides a pharmaceutical composition comprising any of the compounds described above and a pharmaceutically acceptable adjuvant.

The present invention also provides a preferred technical solution of the above pharmaceutical composition: the pharmaceutical composition is suitable for gastrointestinal administration.

The invention also provides the use of any of the above compounds or pharmaceutical compositions for the preparation of a medicament.

A preferred technical solution for the preparation of a medicament by any of the compounds or pharmaceutical compositions provided by the present invention: in the preparation of a treatment or prevention of hyperglycemia, type II diabetes, impaired glucose tolerance, type I diabetes, obesity, hypertension, X synthesis Applications in signs, dyslipidemia, cognitive impairment, arteriosclerosis, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcer medications.

The use in the preparation of a medicament for treating the delayed or preventable deterioration of the type 2 diabetes.

Use in the preparation of a medicament for reducing food intake, reducing beta cell apoptosis, increasing islet beta cell function, increasing beta-cell mass and/or restoring glucose sensitivity to beta-cells.

The core of the present invention is to replace the amino bond of Ala ⁸ at the amino terminus of GLP-1 with a peptide bond mimicking linker, which is a DPP-IV enzyme recognition site. The peptide bond mimic linker is a classical approach in drug discovery discovery by mimicking native peptide bonds and retaining the ability to interact with biological targets and produce the same biological effects (Curr. Chem. Bio. 2008) , 12: 292-296). Based on the same principle, the peptide-linked mock-modified GLP-1 analog may retain the same biological activity as native GLP-1 and have a sustained action like long-acting insulinotropic action.

The compounds provided herein may contain one or more asymmetric centers, such as the A-linker of formula (I). The compound may have one or more stereoisomers. These compounds may be in any active form, such as a racemate, a mixture of stereoisomers enriched in enantiomers. The single enantiomers required therein, such as any of the active forms, can be synthesized by known means (e.g., asymmetric synthesis) using optically active starting materials, or by racemic resolution. The racemic resolution method can be carried out by a conventional method. For example, crystallization in a resolving reagent, derivatization with an enantiomerically or enantiomerically resolving reagent after separation of the target isomer; or chromatography, such as a chiral HPLC column.

The terms "polypeptide" and "peptide" as used herein mean a compound containing at least 5 amino acid components joined by peptide bonds. The amino acid component may be selected from the group consisting of an amino acid encoded by a gene code, a natural amino acid not encoded by a gene code, and a synthetic amino acid. The natural amino acid not encoded by the genetic code may be, for example, v-carboxyglutamate, ornithine, phosphoserine, D-alanine, and D-glutamic acid. Synthetic amino acids include chemically synthesized amino acids, such as D-isomers encoded by the gene code, such as D-alanine and D-leucine, α-aminoisobutyric acid (Aib), α-amino --aminobutyric acid (Abu), tetrabutylglycine (Tie), β-alanine, 3-aminomethylphenyl methionine, anthranilic acid.

The 22 natural amino acids are: alanine, arginine, aspartame, aspartic acid, cysteine, cystine, glutamine, glutamic acid, glycine, histidine , hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, valine, serine, threonine, tryptophan, tyrosine, valine.

Therefore, an unnatural amino acid as a component and bound to a peptide by a peptide bond can be incorporated into the peptide category instead of the natural amino acid. Specific examples are γ-carboxyglutamic acid, ornithine, phosphoserine, D-amino acids such as D-alanine and D-glutamic acid. Synthetic non-natural amino acids include chemically synthesized amino acids, such as D-isomers of amino acids encoded by the gene code, such as D-alanine and D-leucine, alpha-aminoisobutyric acid (α-aminoisobutyric acid, Aib), α-aminobutyric acid (Abu), tetrabutylglycine (Tie), 3-aminomethylbenzoic acid, aminobenzoic acid, deaminohistidine, amino acid β Such as β-alanine and the like. D-histidine, deaminohistidine, 2-amino-histidine, β-hydroxy-valine, homohistidine, N ^α -acetyl-histidine, α-fluoromethyl- Histidine, α-methyl-histidine, 3-pyridyl alanine, 2-pyridyl alanine or 4-pyridyl alanine, (1-amino-cyclopropyl)carboxylic acid, ( 1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxyl, (1-aminocycloheptyl)carboxylic acid or (1-aminocyclooctyl)carboxylate Acid; has been reported for the amino acid sequence of GLP (Lopez, LC et al., Proc. Nat'l, Acad. Sci., USA 80 , 5485-5489, 1983; Bell, GI, et al., Nature 302 : 716- 718 (1983); Heinrich, G., et al, Endocrinol, 115 : 2176-2181 (1984). The structure of the proglucagonogen mRNA and its corresponding amino acid sequence are known to the public. The precursor gene product and proglucagon hydrolyzed into glucagon and insulinotropic peptides were characterized. The GLP-1 (1-37) label described herein refers to all from 1 position (N -end) a GLP-1 polypeptide to amino acid at position 37 (C-terminal). Similarly, the labeling of GLP-1 (7-37) described herein refers to all from the 7 position (N- a GLP-1 polypeptide to amino acid at position 37 (C-terminal). Similarly, the labeling of GLP-1 (7-36) as used herein refers to all from 7 (N-terminal) to 36 (C) - a terminal amino acid GLP-1 polypeptide.

Another object of the invention is to provide a pharmaceutical composition comprising at least one compound of the invention and one or more pharmaceutically acceptable carriers, diluents or adjuvants.

The principle of solid phase synthesis of polypeptides is well known in the art and can also be found in general publications in the art, for example: Dugas, H. and Penney, C., Bioorganic Chemistry (1981) Springer-Verlag, New York, page 54-92; Merrifield, JM, Chem. Soc., 85 , 2149, 1962, and Stewart and Young, Solid Phase Peptide Synthesis, page 24-66, Freeman (San Francisco, 1969).

For example, the peptide fragment provided by the present invention can be used as a 430 peptide synthesizer (Applied Biosystems, Inc., 850 Lincoln Centre Drive, Foster City, CA 94404 (Applied Biosystems Co., Ltd., Lincoln Center Area 850, Foster City, Canada) Lieutenant State, 94404)) and solid phase synthesis of synthetic devices supplied by Applied Biosystems. The Boc protected amino acids and reagents are commercially available from Applied Biosystems, Inc. or other chemical suppliers. A continuous Boc chemistry using a duplex scheme can be applied to the p-methylbenzhydrylamine resin to produce a C-terminal carboguanamine. In order to obtain the C-terminal acid, the corresponding PAM resin can be used. The aspartic acid, glutamine and arginine can be coupled using a pretreated hydroxybenzotriazole ester.

A further object of the present invention is to provide a pharmaceutical preparation comprising at least one compound of the invention comprising at least one compound of the invention at a concentration of from 0.1 mg/ml to 25 mg/ml, wherein the pH of the pharmaceutical preparation is from 3.0 to 9.0. The formulation further includes a buffer solution system, a preservative, a penetrant, a chelating agent, a stabilizer, and a surfactant. In a particular embodiment of the invention, the pharmaceutical preparation chamber is an aqueous preparation, such as an aqueous preparation. The formulation is preferably a solution or suspension. In another embodiment of the invention, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" as used herein refers to a formulation containing at least 50% w/w water. Similarly, the term "aqueous solution" as used herein refers to a solution containing at least 50% w/w water, and the term "aqueous suspension" refers to a suspension containing at least 50% w/w water.

In another embodiment of the invention, the pharmaceutical formulation is a lyophilized formulation, and the physician or patient needs to add a solvent and/or diluent prior to use.

In another embodiment of the invention, the pharmaceutical formulation is a dry formulation such as a lyophilized or spray dried formulation which does not require dissolution prior to use.

In another embodiment of the invention, the invention relates to a pharmaceutical preparation comprising at least one aqueous preparation of a compound of the invention and a buffer comprising at least one compound of the invention at a concentration of 0.1 mg/ml or more, having a pH of 3.0 ~9.0. In another embodiment of the invention, the pH of the formulation is preferably from 7.0 to 9.5. In another embodiment of the invention, the formulation has a pH of from 3.0 to 7.0. In another embodiment of the invention, the formulation has a pH of from 5.0 to 7.5. In another embodiment of the invention, the formulation has a pH of from 7.5 to 9.0. In another embodiment of the invention, the formulation has a pH of from 7.5 to 8.5. In another embodiment of the invention, the formulation has a pH of from 6.0 to 7.5.

In another embodiment of the invention, the pH of the formulation is more preferably from 6.0 to 7.0. In another embodiment, the pharmaceutical formulation has a pH of from 8.0 to 8.5.

In a preferred embodiment of the invention, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, diglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, phosphoric acid Disodium hydrogen, sodium phosphate and tris(hydroxymethyl)-aminomethane, diglycine, triglycine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or a mixture thereof. Each of the above specific buffers may constitute an optional embodiment of the invention.

In a further preferred embodiment of the invention, the formulation further comprises a pharmaceutically acceptable preservative. In a preferred embodiment of the invention, the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2 -phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, thimerosal, bronopol, benzoic acid, imidazolidine, chlorhexidine, sodium dehydroacetate, Chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride hydrochloride, chlorophenylglycol (3 p-chlorophenoxypropane-1,2-diol). In one embodiment, the preservative is selected from the group consisting of phenol or m-cresol. In a preferred embodiment of the invention, the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In a preferred embodiment of the invention, the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In a preferred embodiment of the invention, the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In a preferred embodiment of the invention, the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. Each of the specific preservatives described above may constitute an optional embodiment of the invention. Preservatives for use in the pharmaceutical compositions of the invention are well known to those skilled in the art. For convenience, reference is Remington: The Science and Practice of Pharmacy , 19 th edition, 1995.

In another preferred embodiment of the invention, the formulation further comprises at least one penetrant. In a preferred embodiment of the invention, the penetrant is selected from the group consisting of a salt (such as sodium chloride), a sugar or a sugar alcohol, an amino acid (such as L-glycine, L-histidine, arginine, lysine, Isoleucine, aspartic acid, tryptophan, threonine), aldol (such as glycerol (glycerol), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1 , 3-butanediol), polyethylene glycol (such as PEG400) or a mixture thereof. In one embodiment, the penetrant is propylene glycol, any sugar, such as a monosaccharide, disaccharide or polysaccharide, or a water soluble glucan, including if sugar, glucose, mannose, sorbose, xylose, maltose, lactose , sucrose, trehalose, dextran, amylopectin, dextrin, cyclodextrin water-soluble starch, hydroxyethyl starch and carbonylmethyl-cellulose sodium. In one embodiment, the sugar additive is sucrose. The sugar alcohol refers to a C ₄ -C ₈ hydrocarbon containing at least one hydroxyl group, including, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol . In one embodiment, the sugar alcohol is mannitol. The above sugars or sugar alcohols may be used singly or in combination. As long as the sugar or sugar alcohol is soluble in the liquid preparation, the amount of the sugar or sugar alcohol is not fixedly limited without adversely affecting the stability effect of the method used in the present invention. In one embodiment, the concentration of the sugar or sugar alcohol is from about 1 mg/ml to about 150 mg/ml. In a preferred embodiment of the invention, the concentration of the penetrant is from 1 mg/ml to 50 mg/ml. In a preferred embodiment of the invention, the penetrant is present in a concentration from 1 mg/ml to 7 mg/ml. In a preferred embodiment of the invention, the concentration of the penetrant is from 5 mg/ml to 7 mg/ml. In a preferred embodiment of the invention, the concentration of the penetrant is from 8 mg/ml to 24 mg/ml. In a preferred embodiment of the invention, the penetrant is present in a concentration from 25 mg/ml to 50 mg/ml. Each of the above specific penetrants may constitute an optional embodiment of the invention. Penetrants for use in the pharmaceutical compositions of the invention are well known to those skilled in the art. For convenience, reference is Remington: The Science and Practice of Pharmacy , 19 th edition, 1995.

In another preferred embodiment of the invention, the formulation further comprises a chelating agent. In a preferred embodiment of the invention, the chelating agent is selected from the group consisting of salts of ethylenediaminetetraacetic acid (EDTA), citric acid, aspartic acid or mixtures thereof. In another preferred embodiment, the concentration of the chelating agent is from 0.1 mg/ml to 5 mg/ml. In a preferred embodiment of the invention, the concentration of the chelating agent is from 0.1 mg/ml to 2 mg/ml. In a preferred embodiment of the invention, the concentration of the chelating agent is from 2 mg/ml to 5 mg/ml. Each of the above specific chelating agents may constitute an optional embodiment of the invention. Chelating agents for use in the pharmaceutical compositions of the invention are well known to those skilled in the art. For convenience, reference is Remington: The Science and Practice of Pharmacy , 19 th edition, 1995.

In another preferred embodiment of the invention, the formulation further comprises a stabilizer. Stabilizers for use in the pharmaceutical compositions of the invention are well known to those skilled in the art. For convenience, reference is Remington: The Science and Practice of Pharmacy , 19 th edition, 1995.

More specifically, the compositions of the present invention are stable liquid pharmaceutical compositions. The therapeutically active ingredient of the liquid pharmaceutical composition comprises a polypeptide which is capable of exhibiting a polymeric form in the storage of a liquid pharmaceutical preparation. The term "forming aggregates" hereinafter refers to the physical interaction between the molecules of the polypeptide that result in the formation of the oligomer, the oligomers are still soluble, or the macroscopic aggregates are precipitated from the solution. The term "in storage" hereinafter refers to a liquid pharmaceutical composition or a one-time preparation that is not immediately administered to a subject. Quite, the formulations described below may be packaged for storage in liquid form, in frozen form or in dry form for later processing into a liquid form or other form suitable for administration of the article. The term "dry form" means that the liquid pharmaceutical composition or formulation is subjected to lyophilization (for example, lyophilization, see Williams and PoIIi (1984) J. Parenteral Sci. Technol. 38: 48-59), spray drying (see Masters). (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, UK), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1 169-1206; Mumenthaler et al. (1994) Pharm. Res. 11: 12-20) or air drying (Carpenter and Crowe (1988) Cryobiology 25: 459-470; and Roser (1991) Biopharm. 4: 47-53) dry. The aggregate formed by the polypeptide of the liquid formulation pharmaceutical composition in storage can adversely affect the biological activity of the polypeptide, resulting in a loss of therapeutic effect of the pharmaceutical composition. Further, aggregate formation may also cause other problems, such as when the pharmaceutical composition containing the polypeptide is administered using an infusion system, the aggregate morphology can clog the needle, filter or infusion pump.

The pharmaceutical compositions of the present invention may further comprise a dose of an amino acid oxime sufficient to reduce aggregate formation by the polypeptide storage process of the composition. The term "amino acid oxime" refers to an amino acid or a combination of amino acids, wherein any amino acid used may be either in its free oxime form or in its salt form. When a combination of amino acids is used, all of the amino acids may be in the form of their free oxime or salt, or when other amino acids are in the form of a salt, the amino acid free oxime may also be used in part. In one embodiment of the invention, the amino acid used to prepare the compositions of the invention is a charged side chain, for example, arginine, lysine, aspartic acid, glutamic acid. The pharmaceutical composition of the present invention may use certain specific amino acids (such as methionine, histidine, imidazole, arginine, isoleucine, aspartic acid, tryptophan, threonine or a mixture thereof). Any stereoisomer (such as the L configuration, the D configuration or a mixture thereof) or a mixture of these isomers, provided that the particular amino acid is in the form of its free oxime or its salt. In one embodiment of the invention, the amino acid is an L-stereoisomer. Compositions of the invention may also be prepared using these amino acid analogs. The term "amino acid analog" refers to derivatives of naturally occurring amino acids which are capable of achieving the desired effect of reducing the formation of aggregates of polypeptides in storage by the liquid pharmaceutical compositions of the present invention. Suitable arginine analogs include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable methionine analogs include ethylthiobutyric acid and butyl methionine, suitable cysteines Analogs include S-methyl-L cysteine. Similar to other amino acids, the compositions of the present invention also contain the free oxime or salt form of the above amino acid analogs. In a preferred embodiment of the invention, the concentration of the above amino acid or amino acid derivative is sufficient to prevent or delay the aggregation process of the protein. In a preferred embodiment of the invention, when the polypeptide therapeutically active ingredient is a fragment comprising at least one oxidizable methionine, methionine (or other sulfur-containing amino acid or amino acid analog) may be added to inhibit oxidation of the methionine fragment to methionine. . The term "inhibiting" means minimizing the cumulative amount of methionine oxide throughout the process. Inhibition of methionine oxidation allows the polypeptide to be held in the proper molecular form to the maximum extent possible. Any methionine stereoisomer (L or D configuration) or a mixture thereof can be used. A certain amount of a methionine stereoisomer or mixture may be added to inhibit the oxidation of the methionine residue, such that the content of methionine hydrazide is acceptable for the conditioning reagent. Typically, this means that the pharmaceutical compositions of the present invention contain no more than about 10% to about 30% methionine hydrazide. In general, the above requirements can be accomplished by the addition of methionine, whereby the amount of methionine to methionine residue added ranges from about 1:1 to about 1000:1, such as from about 10:1 to about 100:1.

In a preferred embodiment of the invention, the formulation further comprises a stabilizer selected from the group consisting of high molecular weight polymers or low molecular weight molecular compounds. In a preferred embodiment of the invention, the stabilizer is selected from the group consisting of polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carbonyl cellulose or hydroxy cellulose or derivatives thereof ( Such as HPC, HPC-SL, HPC-L, HPMC), cyclodextrin, sulfur-containing substances (such as thioglycerol, thioglycolic acid, 2-methylthioethanol) or different salts (such as sodium chloride) . Each of the above specific stabilizers may constitute an optional embodiment of the invention.

The pharmaceutical compositions may also contain other stabilizers to further enhance the stability of the therapeutically active ingredient polypeptide. Stabilizers that are particularly useful for the present invention include, but are not limited to, methionine and EDTA for blocking the oxidation of the polypeptide methionine; a nonionic surfactant for preventing the concomitant aggregation associated with freeze-thaw or mechanical shearing of the polypeptide.

In another preferred embodiment of the invention, the formulation further comprises a surfactant. In another embodiment of the invention, the pharmaceutical composition contains two different surfactants. The term "surfactant" as used herein refers to any molecule or ion that contains a water soluble (hydrophilic) moiety, a head group, and a fat soluble (lipophilic) moiety. The surfactant preferably aggregates at the interface, the hydrophilic portion of which is directed to the aqueous phase (hydrophilic phase) and the lipophilic portion to the aqueous phase of the oil phase (eg, glass, air, oil, etc.). The concentration at which the surfactant begins to form ionic micelles is defined as the critical micelle concentration (CMC). In addition, the surface tension of the surfactant is lower than the surface tension of the liquid. Surfactants are well known amphoteric compounds. In general, the term "cleanser" can be used synonymously with surfactants.

The anionic surfactant is selected from the group consisting of: chenodeoxycholic acid, sodium chenodeoxycholate, cholic acid, dehydrocholic acid, deoxycholic acid, methyl deoxycholate, digitonin, digitoxin, N, N - dimethyldodecyl-N-oxide, sodium docusate, sodium sulphate deoxycholate, glycocholic acid hydrate, glycodeoxycholic acid monohydrate, sodium glycodeoxycholate, Glycine cholic acid-3-sulfate disodium salt, glycine bile acid ethyl ester, N-dodecyl sarcosinate sodium, N-dodecyl sarcosine, lithium lauryl sulfate, Lu Lugol, sodium 1-octylsulfonate, sodium 1-butylsulfonate, sodium 1-decanesulfonate, sodium 1-dodecanesulfonate, sodium 1-heptanesulfonate, 1- Sodium decane sulfonate, sodium 1-propane sulfonate monohydrate, sodium 2-bromoethane sulfonate, sodium cholate hydrate, bovine bile or sheep bile, sodium bile hydrate, sodium cholesteric acid, deoxycholic acid Sodium, sodium lauryl sulfate, sodium hexanesulfonate, sodium octyl sulfonate, sodium butane sulfonate, sodium taurocholate, sodium tauroche-deoxycholate, sodium tauro-sodeoxycholate Hydrate, taurochenodeoxycholic acid-3-sulfate disodium salt, taurours sodium deoxycholate , Dodecyl sulfonic acid, docusate sodium (DSS, CAS registration number [577-11-7]), docusate calcium (CAS registration number [128-49-4]), potassium docusate (CAS registration number [7491-09-0]), sodium dodecyl sulfate (sodium lauryl sulfate, SDS), dodecylphosphocholine (FOS-Choline-12), 癸Decylphosphocholine (FOS-Choline-10), Nonylphosphocholine (FOS-Choline-9), dipalmitoside phosphatidic acid, sodium octanoate and/or ursodeoxycholic acid.

The cationic surfactant is selected from the group consisting of alkyltrimethylammonium bromide, benzylmethane chloride, benzyldimethylhexadecyl ammonium chloride, benzyldimethyltetradecyl ammonium chloride, benzyl Dimethyltetradecyl ammonium iodide, dimethyl dioctadecyl ammonium bromide, dodecyl ethyl dimethyl ammonium bromide, dodecyl trimethyl ammonium bromide, B Cetyldimethylammonium bromide, cetyltrimethylammonium bromide, polyoxyethylene (10)-N-tallow-1,3-diaminopropane, hexadecyl/brominated Ammonium and/or trimethyltetradecyl ammonium bromide.

The nonionic surfactant is selected from the group consisting of: BigCHAP, bis(polyethylene glycol dimercaptocarbonyl), block copolymers such as polyethylene oxide, or polyoxypropylene block copolymers such as poloxamer, poloxa M-188 and Polosham-407, 35. 56, 72, 76. 92V, 97. 58P, EL, lauryl polyoxyethylene ether, N-mercapto-N-methyl-glucosamine, n-dodedecyl-N-methylglucamine, alkyl-polyglucoside, ethoxylation Castor oil, heptaethylene glycol monoterpene ether, heptaethylene glycol monododecyl ether, heptaethylene glycol monotetradecyl ether, hexaethylene glycol monododecyl ether, hexaethylene glycol monohexadecyl ether, six ethyl Glycol octadecyl ether, hexaethylene glycol tetradecyl ether, lgepal CA-630, lgepal CA-630, methyl-6-0-(N-heptylaminocarboxamidine)-β-D-glucopyranose Ethylene glycol monododecyl ether, N-mercapto-N-methylglucamine, N-mercapto-N-methylglucamine, octaethylene glycol monoterpene ether, octaethylene glycol Monododecyl ether, octaethylene glycol monohexadecyl ether, octaethylene glycol monostearyl ether, octaethylene glycol monotetradecyl ether, octa-β-D-glucopyranose, pentaethylene glycol monoterpene Ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monohexadecyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monostearyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol II Glycidyl ether, polyethylene glycol ether W-1, polyoxyethylene tridecyl ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 olefinene , polycyanoethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyethylene oxide bis(imidazolylcarbonyl), polyethylene oxide 25 propylene glycol stearate, soap hull Tree (Quillaja bark) extract saponin, division 20( 20), Division 40, Division 60, Division 65, Division 80, Division 85, Tergitol Type 15-S-12, Tergitol Type 15-S-30, Tergitol Type 15-S-5, Tergitol Type 15-S-7, Tergitol Type 15-S-9, Tergitol Type NP-10, Tergitol Type NP-4, Tergitol Type NP-40, Tergitol Type NP-7, Tergitol Type NP-9, tetradecyl-β-D-maltoside, tetraethylene glycol monoterpene ether, tetraethylene glycol monododecyl ether , tetraethylene glycol monotetradecyl ether, triethylene glycol monoterpene ether, triethylene glycol monododecyl ether, triethylene glycol monohexadecyl ether, triethylene glycol monooctyl ether, triethylene glycol mono Tetraether, Triton CF-21, Triton CF-32, Triton DF-12, Triton DF-16, Triton GR-5M, Triton QS-15, Triton QS-44, Triton X-100, Triton X-102, Triton X -15, Triton X-151, Triton X-200, Triton X-207, X-100, X-114, X-165 solution, X-305 solution, X-405, X-45, X-705-70, spit 20( 20), spit 40, spit 60, spit 6, spit 65, spit 80, spit 81, spit 85. Teresa, sphingomyelin, sphingomyelin, sphingoglycolipids, ceramides, gangliosides, phospholipids, and/or n-undecyl-β-D-glucopyridine Unsweetened.

The zwitterionic surfactant is selected from the group consisting of CHAPS, CHAPSO, 3-(decyldimethylamino)propanesulfonic acid inner salt, 3-(dodecyldimethylamino)propanesulfonic acid inner salt, 3-(12 Alkyl dimethylamino)propane sulfonate, 3-(N,N-dimethylmyristylamino)propane sulfonate, 3-(N,N-dimethyloctadecylamino)- Propane sulfonate, 3-(N,N-dimethyloctyl)propane sulfonate, 3-(N,N-dimethylpalmitoylamino)propane sulfonate, N-alkyl-N , N-dimethylamino-1-propane sulfonate, 3-cholestyramine-1-propyldimethylamino-1-propane sulfonate, dodecylphosphocholine, myristyl hemolysis Lecithin choline, Zwittergent 3-12 (N-dodecyl-N,N-dimethyl-3-amino-1-propane sulfonate), Zwittergent 3-10 (3-(mercaptodimethyl dimethyl) Base amino)-propanesulfonic acid inner salt), Zwittergent 3-08 (3-(octyldimethylamino)propane sulfonate), glycerophospholipid (lecithin, cephalin, phospholipid thiol serine), glyceroglycolipid (galactopyranoside), lysophosphatidylcholine and phospholipid choline alkyl, alkoxy (alkyl ester), alkoxy (alkyl ether) derivatives, such as lysophosphatidylcholine Clams and nutmeg Derivatives, dipalmitoside phosphocholine choline and polar head group of choline, ethanolamine, phosphatidic acid, serine, threonine, glycerol, cellulose, lysophosphatidylcholine and hemolytic phosphothionine, a modified product of mercaptocarnitine or a derivative thereof, an N ^beta -thiolated derivative of lysine, arginine or histidine, or a side chain thiolated derivative of lysine or arginine, An N ^beta -thiolated derivative of a dipeptide comprising lysine, arginine or histidine and a neutral or acidic amino acid, containing a neutral amino acid and a tripeptide having an arbitrary composition of two charged amino acids N ^beta -thiolated derivative; or the surfactant is selected from the group consisting of imidazoline derivatives, C ₆ -C ₁₂ long chain fatty acids or salts thereof (such as oleic acid and lanolin), N-hexadecyl -N,N-dimethyl-3-amino-1-propane sulfonate, anionic (alkyl-aromatic hydrocarbon-sulfonate) monovalent surfactant, palmitoyl lysophosphatidyl thiol-L-color ammonia Acid, lysophospholipids (such as 1-ethanolamine, choline, serine, threonyl-sn-glycerol-3-phosphate of threonine) or mixtures thereof.

The term "alkyl-polyglycoside" as used herein refers to a branched or branched C _5-20 alkyl group, C _5-20 alkenyl group or C substituted by one or more sugar ligands such as maltosides, glycosides and the like. _5-20 alkynyl chain. The above alkyl-polyglycosides include C _6-I8 alkyl-alkyl-polyglycosides. Said alkyl - polyglycoside particular embodiment comprises an even number of carbon chains, e.g. C ₆ alkyl chain, C ₈ alkyl chains, C ₁₀ alkyl chains, C ₁₂ alkyl chains, C ₁₄ alkyl chains, C ₁₆ An alkyl chain, a C ₁₈ alkyl chain, and a C ₂₀ alkyl chain. Specific embodiments of the sugar ligands include pyranosides, glucopyranosides, maltosides, glucosides, and sucrose. In an embodiment of the invention, the number of sugar ligands attached to the alkyl group is less than 6. In an embodiment of the invention, the number of sugar ligands attached to the alkyl group is less than 5. In an embodiment of the invention, the number of glycosyl groups attached to the alkyl group is less than four. In an embodiment of the invention, the number of sugar ligands attached to the alkyl group is less than three. In an embodiment of the invention, the number of glycosyl groups attached to the alkyl group is less than two. A specific embodiment of the alkyl-polyglycoside is an alkyl glucoside such as n-fluorenyl-β-D-glucopyranoside, thiol-β-D-maltopyranoside, dodecyl-β -D-glucopyranoside, n-dodecyl-β-D-maltoside, n-dodecyl-β-D-maltoside, n-dodecyl-β-D-maltoside, Tetradecyl-β-D-glucopyranoside, thiol-β-D-maltoside, cetyl-β-D-maltoside, thiol-β-D-maltoside, dodecyl -β-D-maltotrioside, tetradecyl-β-D-maltotrioside, cetyl-β-D-maltotrioside, n-dodecyl-sucrose, n-癸Base-sucrose, sucrose monodecanoate, sucrose monolaurate, cane monostearate and sucrose palmitate. Surfactants for use in the pharmaceutical compositions of the invention are well known to those skilled in the art. For convenience, reference is Remington: The Science and Practice of Pharmacy , 19 th edition, 1995.

In a preferred embodiment of the invention, the formulation further comprises a protease inhibitor, such as ethylenediamine tetraacetic acid (EDTA) and benzamidine hydrochloride, as well as other commercially available protease inhibitors. In pharmaceutical compositions containing proproteinase, the use of protease inhibitors has particular utility and can be used to inhibit autocatalytic reactions.

The peptide pharmaceutical preparation of the present invention may further contain other components. The above additional components may include wetting agents, emulsifiers, antioxidants, bulking agents, permeability improvers, chelating agents, metal ions, oily carriers, proteins such as human serum albumin, gelatin and proteins, and zwitterions (eg Amino acids such as betaine, taurine, arginine, glycine, lysine and histidine). The above-mentioned added components should not adversely affect the overall stability of the pharmaceutical preparation of the present invention.

Depending on the needs of the treatment, the pharmaceutical composition containing the compound of the present invention can be administered to the patient from several sites, for example, typical sites such as skin, mucous membranes, bypass absorption such as arteries, blood vessels, heart, and areas having absorption such as skin, Subcutaneous, muscular or abdominal cavity.

Depending on the therapeutic needs of the patient, the pharmaceutical compositions of the invention may be administered by several routes of administration, such as the tongue, sublingual, buccal, buccal, oral, intragastric, nasal, pulmonary (eg, via the bronchioles, alveoli, or Combination), epidermis, dermis, percutaneous, vaginal, rectal, eye (eg through the conjunctiva), ureter and parenteral.

The composition of the present invention can be administered in several dosage forms, such as solutions, suspensions, emulsions, microemulsions, multiple emulsions, foams, ointments, pastes, ointments, ointments, tablets, coated tablets, lotions. Capsules (such as hard gelatin capsules and soft gelatin capsules), suppositories, rectal capsules, drops, gels, sprays, powders, aerosols, inhalants, eye drops, eye ointments, ophthalmic cleansers, Uterine support, vaginal ring, vaginal ointment, injection, in situ transfer of liquid (such as in situ gel, in situ placement, in situ precipitation, in situ crystallization, infusion and implant). The composition of the present invention may further further incorporate or link a drug carrier, a drug dispersion system and a high-grade drug dispersion system by covalent action, hydrophobic action and electrostatic action, thereby further enhancing stability, improving biological activity, and increasing solubility. The side effects are reduced, the time therapy well known to those skilled in the art is achieved, the effect of increasing patient compliance, or a combination of the above effects is achieved. Specific examples of the carrier, the drug dispersion system or the advanced drug dispersion system are selected from, but not limited to, a polymer such as cellulose or a derivative thereof, a polysaccharide such as dextran or a derivative thereof, starch or a derivative thereof, polyvinyl alcohol, acrylate Polymer and methacrylate polymers, polylactic acid and polyglycolic acid and their block polymers, polyvinylidene alcohol, carrier proteins such as serum, gels such as thermal gelation systems, as is well known to those skilled in the art Copolymerization systems, micelles, liposomes, microspheres, nanoparticles, liquid crystals and their dispersions, L2 phases and their dispersions, those skilled in the art are familiar with the phase behavior of oil-water systems, polymeric micelles, Multiple emulsions, self-emulsifying, in microemulsification, cyclodextrin or its derivatives, and dendrimers.

The compositions of the present invention are suitable for use in solution formulations for solid, semi-solid, powder and pulmonary administration, such as metered dose inhalers, dry powder inhalers and nebulizers, all of which are well known to those skilled in the art. The compositions of the present invention are particularly useful in controlled, sustained, extended, delayed recoil and slow release drug delivery systems. The compositions of the present invention are further suitable for, but not limited to, parenteral controlled release and sustained release systems well known to those skilled in the art (both systems use multiple fold reductions). Further preferred is a controlled release and sustained release system for subcutaneous administration. The examples provided below do not limit the scope of the invention, and examples of suitable retardation systems and compositions are selected from the group consisting of hydrogels, oily gels, liquid crystals, polymer micelles, microspheres, nanoparticles, and are suitable for use in production. A method of controlling the release system of the composition of the present invention, including but not limited to, crystallization, condensation, crystallization, precipitation, coprecipitation, emulsification, dispersion, high pressure homogenization, embedding, spray drying Method, microcapsule method, coacervation method, phase separation method, solvent evaporation method for preparing microspheres, extrusion method and supercritical fluid method.

Reference is generally made to: Handbook of Pharmaceutical Controlled Release (Wise, DL, ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, EJ, ed. Marcel Dekker, New York , 2000). For parenteral administration, a syringe can be used, and any pen-type syringe is administered subcutaneously, intramuscularly, intraperitoneally or intravenously. Preferably, parenteral administration can be accomplished in the form of an infusion pump. More preferably, the dosage form of the solution or suspension or powder composition for administration of the compound of the present invention may be a nasal liquid, a liquid for the lung or a powder spray. More preferably, the pharmaceutical compositions containing the compounds of the invention are also suitable for transdermal administration, for example by needle-free injection or patch, any iontophoresis, or transmucosal administration such as buccal. The compounds of the present invention can be administered by the pulmonary route in a carrier such as a solution, suspension or dry powder using any known type of device suitable for pulmonary drug administration. These specific examples include, but are not limited to, three general models of aerosol generators for pulmonary administration, and may include nozzles or ultrasonic nebulizers, metered dose inhalers or dry powder inhalers (Cf. Yu J, Chien YW. Pulmonary drug delivery: Physiologic and mechanistic aspects. Crit Rev Ther Drug Carr Sys 14(4) (1997) 395-453).

Based on the standard test methodology, the aerodynamic diameter (d _a ) of _a particle refers to the geometric equivalent diameter of a spherical particle of reference standard per unit density (1 g/cm ³ ). In the simplest case, for spherical particles, d _a is the reference root diameter (d) as a function of the square root of the density ratio, which is described as follows: For clustering particles, this relationship needs to be corrected (cf. Edwards DA, Ben- Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385). The terms "MMAD" and "MMEAD" have been reported in detail and are known to the skilled person (cf. Edwards DA, Ben-Jebria A, Langer R and represents a measure of the median value of an aerodynamic particle size distribution. Recent advances in pulmonary) Drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385). Mass median aerodynamic diameter (MMAD) and mass median effective aerodynamic diameter (MMEAD) are used interchangeably. They are two statistical parameters for empirical description. The size factor of the relationship between the deposition potential of aerosol particles in the lungs, ie the actual actual shape, the actual size or the actual density (cf. Edwards DA, Ben-Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles J Appl Physiol 84 (2) (1998) 379-385). The MMAD is typically calculated from a collision sampler that measures the inertial motion of the particles in the air. In a preferred embodiment, the formulation can be atomized by any known spray technique, such as atomization, to achieve an aerosol particle having an MMAD of less than 10 μm, particularly preferably 1-5 μm, more preferably 1-3 μm. The preferred particle size is based on the most effective size for deep lung administration for optimal protein absorption (cf. Edwards DA, Ben-Jebria A, Langer R. Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84 (2) (1998) 379-385).

Deep lung deposition of a pulmonary administration formulation containing a compound of the invention can optionally be further optimized using inhalation techniques such as, but not limited to, slow inspiratory flow rates (e.g., 30 L/min), breath holding breath, and stimulation timing.

The term "stabilized formulation" refers to a formulation that has high physical stability, high chemical stability, or high physical stability.

The term "physical stability" of a protein preparation herein means: a tendency for the protein to form a biologically inert; and/or the protein is exposed to thermo-mechanical pressure; and/or due to an unstable interface and surface interaction such as hydrophobicity, The tendency to form insoluble aggregates. The physical stability of the aqueous protein preparation can be evaluated by using a container filled with the preparation (such as a cassette or a kit) at different temperatures for a different period of time under mechanical/physical pressure (such as vibration). Visual and/or turbidity measurements are evaluated. The visual inspection of the formulation was done in highly focused light with a dark background. The turbidity of the formulation can be characterized by the degree of turbidity of a visual score, as in the 0-3 grade (the formulation is not turbid relative to the visual score of 0, and the visual turbidity of the formulation in white light is consistent with the visual score of 3) . When the formulation showed visual turbidity in white light, the formulation was classified as physically unstable for protein aggregation. Alternatively, the turbidity of the formulation can be assessed using simple turbidity measurements well known to the skilled artisan. The physical stability of aqueous protein preparations can also be assessed using protein spectroscopy reagents or protein conformational morphological probes. The above probes are preferably small molecules which are capable of preferentially binding to the non-native conformer of the protein. An example of a protein structure small molecule spectroscopy probe is Thioflavin T, a fluorescent dye widely used for amyloid-like fiber detection. When small fibers and possibly other protein structures are present, thioflavin T binds to the fibrin form, increasing the new excitation maximum at about 450 nm, enhancing the emission peak at about 482 nm. The unbound thioflavin T is substantially free of fluorescent excitation or emission at the above wavelengths.

Other small molecules can also be used as probes to transform the protein structure from a native conformation to a non-native conformation. For example, a "hydrophobic region" fluorescent probe preferentially binds to an exposed hydrophobic region of the protein. The hydrophobic region of a protein is usually embedded in the tertiary structure of its native conformation, but when the protein begins to open or denature, the hydrophobic region of the protein is exposed. Examples of such small molecules, spectral probes are aromatic hydrophobic dyes such as hydrazine, acridine, phenanthroline or their analogs. Other spectroscopy probes are metal-amino acid complexes such as cobalt metal complexes such as phenylalanine, leucine, isoleucine, methionine, valine and the like hydrophobic amino acids.

The term "chemical stability" of a protein preparation herein refers to a change in a covalent chemical bond in a protein structure that produces a chemical degradation product that has a potentially low biological activity and/or potentially produces an immune system that is greater than the native protein structure. The formation of various chemical degradation products depends on the type and nature of the native protein and the environment in which the protein is exposed. Elimination of chemical degradation products is not completely avoidable, and an increase in the amount of chemical degradation products is common during storage and use of protein preparations well known to those skilled in the art. Most proteins tend to undergo a deamination process, i.e., the glutamine azide residue or the side chain amino group of the aspartamide residue is hydrolyzed to form a free carboxylic acid. Other degradation pathways also include the formation of high molecular weight conversion products, ie, covalent bonding of two or more protein molecules to each other during conversion, and/or disulfide resulting in covalently bound dimers, oligomers And formation of polymeric degradation products (Stability of Protein Pharmaceuticals, Ahem. TJ & Manning M. C, Plenum Press, New York 1992). In addition, oxidation (such as oxidation of methionine residues) is also mentioned as another chemical degradation pathway. The chemical stability of protein preparations is evaluated by exposing the protein preparation to different environmental conditions (the formation of degradation products is usually proportional to environmental conditions such as elevated temperatures) and measuring the chemical degradation product content at different time points. The amount of each individual degradation product typically depends on the separation method based on molecular size and/or charge degradation products using different chromatographic equipment (eg, SEC-HPLC and/or RP-HPLC).

Thus, as outlined above, a "stable formulation" refers to a formulation that has high physical stability and high chemical stability. In general, the formulation must be stable in use and storage (according to recommended consumer and storage conditions) until the expiration date.

In one embodiment of the invention, the pharmaceutical composition comprising a compound of the invention is stable for more than 6 weeks and for 3 years of storage.

In another embodiment of the invention, the pharmaceutical composition comprising a compound of the invention is stable for more than 4 weeks and for 3 years of storage. In still another embodiment of the invention, the pharmaceutical composition comprising a compound of the invention is stable for more than 4 weeks and for 2 years of storage.

In still another embodiment of the present invention, the pharmaceutical composition containing the compound of the present invention is stable for 2 weeks or more and for 2 years of storage.

Another aspect of the invention relates to the use of a compound prepared according to the medicament of the invention.

The invention also relates to salts of GLP-1 analogs. The GLP-1 analogs of the invention may be sufficiently acidic or sufficiently basic to react with any equivalent of an inorganic or inorganic acid to form a salt. Commonly used acids which form acid addition salts are: inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid and similar inorganic acids; organic acids such as m-benzenesulfonic acid, methanesulfonic acid, oxalic acid , m-bromophenyl-sulfonic acid, carbonic acid, citric acid, benzoic acid, acetic acid and similar organic acids. Examples of the above salts include sulfates, hydrogen sulfates, heavy sulfates, sulfites, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, hydrochlorides, bromates, and iodine. Acid salt, propionate, citrate octanoate, acrylate, formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate, suberic acid Salt, sebacate, fumarate, maleate, butyne-1,4-diate, hexyne-1,6-diate, present formate, chlorobenzoate, methyl Formate, dinitrobenzoate, hydroxybenzoate, methoxy carboxate, phthalate, sulfonate, xylene sulfonate, phenylpropionate, phenyl Butyrate, citrate, lactate, γ-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propyl sulfonate, naphthyl-1-sulfonate, naphthyl-2 a sulfonate, a mandelate and a similar acid addition salt. Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid, hydrobromic acid or hydrofluoric acid, especially hydrochloric acid.

Base addition salts include those derived from inorganic bases such as ammonium or alkali metal hydroxides or alkaline earth metal hydroxides, carbonates, hydrogencarbonates and similar inorganic bases. Accordingly, these bases for the preparation of the salts of the present invention include sodium hydroxide, potassium hydroxide, aqueous ammonia, potassium carbonate and similar bases thereof. Salt forms of the GLP-1 analog are particularly preferred. Of course, when the compounds of the invention are used for therapeutic purposes, these compounds may also be in the form of a salt, but these salts must be pharmaceutically acceptable.

The inventors have found that the modified GLP-1 analogs of the present invention have versatile uses, including use as a method of treating diabetes, a sedative, as a treatment for essential diseases of the nervous system, for inducing an anxiolytic effect on the central nervous system (CNS). For the stimulation of the central nervous system, for postoperative treatment and as a treatment for insulin resistance.

A. Diabetes treatment

The modified GLP-1 analogs of the invention generally normalize hyperglycemia by a glucose-dependent mechanism. Similarly, the modified GLP-1 analog can be used as a primary agent for the treatment of type 2 diabetes and as an adjunct to the treatment of type 1 diabetes.

An advantage of using an effective amount of a modified GLP-1 analog for treating diabetes is that it is more effective than an unmodified GLP-1 analog. Since the modified GLP-1 analog is more stable in vivo, a smaller dose of the molecule can achieve an effective therapeutic effect. The invention is particularly useful for treating diabetic patients, including type I and type II diabetes. In the treatment of diabetic patients, the effect of the GLP-1 analog polypeptide is dependent on the concentration of blood glucose in the blood, thus greatly reducing the risk of side effects of hypoglycemia using existing therapies.

The invention also provides a method of treating diabetes in a subject, wherein the method comprises providing at least one dose of a modified GLP-1 analogue sufficient to treat diabetes; the composition comprising at least one modified GLP-1 similar Things.

B. Treatment of nervous system disorders

The inventors have found that the modified GLP-1 analogs of the invention are useful as sedatives. In one aspect of the invention, the invention further provides a method of sedating a mammalian article: for a mammal having increased activity of the central or peripheral nervous system due to an abnormality, a sufficient dose of modified GLP-1 is used Analogs, which may have produced an induced or anxiolytic effect in these mammals, thereby calming them. These modified GLP-1 analogs can be administered intracerebroventricularly, orally, subcutaneously, intramuscularly, or intravenously. The method can be used to treat or ameliorate nervous system symptoms such as anxiety, movement disorders, aggressive behavior, confusion, epilepsy, panic attacks, hysteria and sleep disorders.

In another aspect, the invention comprises a method of increasing the activity of a mammalian subject comprising administering to the subject a dose of a modified GLP-1 analogue to produce and activate the subject in a mammal. Preferably, the subject has a central nervous system or peripheral nervous system activity reduction profile. The inventors have found that modified GLP-1 analogs are particularly useful for treating or ameliorating a number of symptoms such as depression, schizoaffective disorder, sleep apnea, attention distraction, amnesia, amnesia, narcolepsy and the like. Among these conditions, awakening the central nervous system may be advantageous.

The modified GLP-1 analogs of the invention can also be used to induce challenge for the treatment or alleviation of depression, schizoaffective disorder, sleep apnea, attention distraction, amnesia, amnesia, narcolepsy. The therapeutic effects of modified GLP-1 analog therapies can be assessed by patient consultations, assessed by psychological or neurological experiments, or by improvements in symptoms associated with these conditions. For example, the therapeutic effect on narcolepsy can be evaluated from the degree of control over the onset of narcolepsy. As another example, any of a variety of different diagnostic tests well known to those skilled in the art can be used to assess the effect of the modified GLP-1 analog upon focusing on the patient or enhancing memory.

C. Postoperative treatment

The modified GLP-1 analogs of the invention are useful for post-operative treatment. The patient is in need of the modified GLP-1 analog of the invention, either 1-16 hours before surgery, or during surgery, or within 5 days of surgery.

The modified GLP-1 analog was administered to the patient from 16 hours to 1 hour prior to the start of the procedure. The compounds of the invention are administered to a patient to reduce catabolism and insulin resistance, and the length of administration depends on a number of factors. These factors are routinely known to physicians, including, during the most important pre-operative preparation phase, whether the patient is fasting or has undergone glucose infusion or cites a drink or other form of food. Other important factors include the patient's gender, weight, age, severity of blood glucose, any potential cause of regulating blood sugar incompetence, severity of surgically anticipated trauma, route of administration, and bioavailability, individual tolerance, prescription, and efficacy. The preferred time to start administering the modified GLP-1 analog used in the present invention is from about 1 hour to about 10 hours before surgery. A further preferred start administration time is 2 hours to 8 hours before surgery.

After a particular type of surgery, for example, insulin resistance after selective abdominal surgery is most severe on the first day after surgery, lasting at least 5 days, and may last for three weeks to return to normal. Therefore, after undergoing surgical trauma, the patient needs to administer the modified GLP-1 analogue of the present invention for a period of time after surgery, depending on whether the patient is fasted or has undergone glucose infusion or drinking a drink or Other forms of food. At the same time, not limited to, the patient's gender, weight and age, the severity of blood glucose disorders, potential causes of loss of blood glucose regulation, the actual severity of surgical trauma, route of administration, bioavailability, individual tolerance, prescription And efficacy is also a factor. The preferred duration of administration of the compounds used in the present invention is no more than five days after surgery.

D. Insulin resistance treatment

The modified GLP-1 analogs of the invention are useful for treating insulin resistance, whether or not they are used in post-operative treatment. The cause of insulin resistance may be a decrease in the amount of insulin bound by cell surface receptors, or a change in intracellular metabolism. The first cause of insulin resistance is characterized by a decrease in insulin sensitivity, which can be overcome by increasing the insulin concentration. The second cause of insulin resistance is characterized by a decrease in insulin reactivity, which is ineffective by high doses of insulin. The trauma secondary to insulin resistance can be overcome by increasing the insulin dose in proportion to insulin resistance, so this trauma is clearly caused by a decrease in insulin sensitivity.

The dose of the modified GLP-1 analogue of the invention used to normalize the blood glucose level of a patient is affected by factors including, but not limited to, the patient's gender, weight and age, the severity of loss of glycemic regulation, and the ability to cause glycemic regulation. The underlying cause of loss of function, whether glucose or other sugars are administered simultaneously, the route of administration and bioavailability, patient tolerance, prescription, and efficacy.

To detect the ability of GLP-1 analogs to stimulate insulin secretion, GLP-1 analogs can be injected into artificially cultured animal cells, such as RIN-38 fractional islet beta cell tumor cells, and then monitored for release into the artificial ligand base. The number of immunoreactive insulin (IRI). Another method is to inject the GLP-1 analogue into the animal and then monitor the concentration of immunoreactive insulin (IRI) in the plasma.

The presence of IRI is detected by a radioimmunoassay for insulin. Any radioimmunoassay method capable of detecting IRI can be used, one of which is an improved method invented by Albano, JDM, etc. (Albano, JDM et al., Acta Endocrinol. 1972, 70: 487-509). In this modified method, a phosphate/albumin buffer of pH 7.4 is used. 500 μL of phosphate buffer, 50 μL of perfusate sample or a perfusion solution containing rat insulin standard amount, 100 μL of anti-insulin antiserum (Wellcome Laboratories: 1:40,000 dilution) and 100 μL of [ ¹²⁵ I] insulin were added to the culture solution in turn, and the total volume was added. 750 μL was placed in a 10 x 75 mm disposable glass test tube. Incubate at 4 ° C for 2-3 days to separate free insulin and antibody-bound insulin with activated carbon. The sensitivity of this assay is 1-2 uU/ml. To determine the amount of cell growth Kidide IRI released into tissue culture, the proinsulin can be radiolabeled. Although any radiolabel that can be labeled can be used, it is preferred to use ³ H leucine to obtain the labeled proinsulin.

To determine if GLP-1 analogues have islet promoting function, pancreatic perfusion experiments can also be performed. The rat pancreatic in situ perfusion test is an improved method based on the method of Penhos, J, C. et al. (Penhos, J, C., et al., Diabetes, 1969, 18: 733-738). In the pancreas of fasting male Charles River rats weighing 350g-600g, intraperitoneal injection of pentobarbital (Eli Lilly and Co, 160ng/kg), anesthesia, ligation of kidney, adrenal gland, stomach and low position The blood vessels of the colon. Except for the duodenum and descending colon and the rectum, which are about 4 cm, the entire intestine loses its function. Therefore, only a small portion of the intestine is perfused, thereby minimizing the effects of the immune response between the glucagon-like peptide and the intestinal material. The perfusate was a modified Kreba-Ringer bicarbonate buffer containing 4% T70 dextran and 0.2% bovine serum albumin (fragment V), also bubbled with 95% O ₂ and 5% CO ₂ . An extracorporeal looper with a 4-channel ball bearing pump (Buchler polystatic, Buchler Instruments Division, Nuclear-Chicago Corp) was used, as well as a three-way valve that controls the flow of perfusate. The perfusion method is operated, monitored and analyzed by the method of Weir, GC et al. (Weir, GC, et al., J. Clin. Investigat. 1974, 54: 1403-1412).

The method of treatment using a compound of the invention may also be combined with a second or more pharmaceutically acceptable substance, for example, selected from the group consisting of an anti-diabetic agent, an anti-obesity agent, an appetite regulating drug, an antihypertensive drug, a therapeutic and/or a diabetic Or a concomitant syndrome drug, a drug that treats and/or prevents obesity or the accompanying syndrome. Specific examples of such pharmaceutically active substances are: insulin, sulfonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, high-efficiency dipeptidyl peptidase-IV (DPP- IV) Inhibitors, inhibitors of liver damage enzymes associated with gluconeogenesis and/or glycogen stimulation, glucose uptake modulators, compounds that modify lipid metabolism such as antihyperlipidemics such as HMG CoA inhibitors (statins) , Gastric Inhibitory Polypeptides (GIP Analogs), Compounds that Reduce Food Intake, RXR Agonists, and ATP-Dependent β-Cell Potassium Channel Drugs; Cholestyramine Colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol , dextrothyroxine, neteglinide, repaglinide; beta-blockers, such as aprenolol, atenolol, thiophene Timolol, pindolol , propranolol and metoprolol, angiotensin converting enzyme (ACE) inhibitors, such as benazepril, captopril, Elapaliril, fosinopril, lisinopril, alatriopril, quinapril and ramipril, calcium channel blockers such as nifedipine Nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil and alpha-blockers For example, doxazosin, urapidil, prazosin, and terazosin; cocaine amphetamine regulated transcript (CART) agonists, neuropeptides Y (neuropeptide Y, NPY) antagonist, PYY agonist PYY2 agonist, PYY4 agonist, mixed PPY2/PYY4 agonist, melanometric corticosteroid 4 (MC4) agonist, progesterone antagonist, Tumor necrosis factor (TNF) An agonist, a corticotropin releasing factor (CRF) agonist, a corticotropin releasing factor binding protein (CRF BP) antagonist, a urocortin agonist, a β3 agonist, Melanocyte-stimulating hormone (MSH) agonist, melanocyte-concentrating hormone (MCH) antagonist, cholestyrintokinin (CCK) agonist, serotonin reuptake inhibitor , serotonin and norepinephrine reuptake inhibitors, mixed serotonin and norepinephrine-initiated compounds, serotonin (5HT) agonists, bombesin agonists, galanin antagonists, auxin, Auxin releasing compound, thyreotropin releasing hormone (TRH) agonist, UCP 2 uncoupling protein (UCP) or UCP 3 modulator, leptin agonist, DA agonist (bromo ergot Bromocriptin, doprexin), lipase/amylase inhibitor, retinoid X receptor (RXR) modulator, TR beta agonist Histidine H3 antagonists, gastrin inhibitory peptide agonists or antagonists (Gastric Inhibitory Polypeptide analogs, the GIP analogs), gastrin (Gastrin) and gastrin analogs. Treatments using the compounds of the invention may also be combined with surgical-surgical procedures that affect glucose levels and/or body fat balance, such as gastric banding surgery or gastric bypass surgery.

It is to be understood that any suitable compound of the invention in combination with one or more of the above compounds and optionally one or more pharmaceutically active substances is also contemplated as being within the scope of the invention.

The structure, features and other objects of the present invention will be further described in the following detailed description of the preferred embodiments of the present invention. It is not the only restriction on patent applications.

detailed description

Abbreviation comparison table

R.t: room temperature;

H ₂ O: water;

CH ₃ CN: acetonitrile;

DMF: N,N-dimethylformamide;

HBTU: O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate;

Fmoc: 9-fluorenylmethoxycarbonyl;

Boc: tert-butoxycarbonyl;

OtBu: tert-butyl ester;

tBu: tert-butyl;

Trt: trityl;

Pmc: 2,2.5,7,8-pentamethylchroman-6-sulfonyl;

Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl;

ivDde): 1-(4,4-dimethyl-2,6-dioxocyclohexylene)3-methylbutyl;

Mtt: 4-methyltrityl;

Mmt: 4-methoxytrityl;

DCM: dichloromethane;

TIS: triisopropyl decane;

TFA: trifluoroacetic acid;

Et ₂ O: diethyl ether;

NMP: N-methylpyrrolidone;

HOAt: 1-hydroxy-7aza-1H-benzotriazole;

HOBt: 1-hydroxybenzotriazole;

DIC: N,N-diisopropylcarbodiimide.

Synthesis of Q

Q represented by formula (II) is commercially available and is conveniently prepared by the literature or by a variety of methods familiar to those skilled in the art. In 2010, Oishi et al. reported a general synthetic route for the synthesis of compounds of formula (II) (S. Oishi etc., J. Chem. Soc., Perkin Trans. 1, 2001, 2445), in which X, Y and R ₃ are Hydrogen (H), as shown in Scheme 1.

plan 1

Q represented by formula (II) is commercially available or conveniently prepared using methods known in the literature or by a variety of methods familiar to those skilled in the art. A general synthetic route for the synthesis of compounds of formula (II) is shown in Scheme 2, wherein X is fluorine (F), Y and R ₃ are hydrogen (H). The key starting material 4 is commercially available and is known in the literature (T. Narumi et al., Tetrahedron, 2008, 64, 4332).

Scenario 2

Q represented by formula (IIIa) is commercially available and is conveniently prepared in the literature or by a variety of methods familiar to those skilled in the art. A general synthetic route for the synthesis of compounds of formula (IIIa) is shown in Scheme 3, wherein X is trifluoromethyl (CF ₃ ), Z is nitrogen (N), and Y, R ₁ , R ₂ and R ₃ are all hydrogen. (H). The key starting material, 3,3,3-trifluorol-nitropropen 6, is commercially available and is known in the literature. The Aza-Michael addition reaction of glutamic acid diester to 3,3,3-trifluoro-nitropropane 6 is controlled by stereochemistry (M. Molteni et al., Org. Lett., 2003, 5, 3887).

Option 3

The Q represented by the formula (IIIb) is commercially available, and is conveniently prepared by a method known in the literature or by various methods familiar to those skilled in the art. A general synthetic route for the synthesis of compounds of formula (IIIb) is shown in Scheme 4, wherein X is trifluoromethyl (CF ₃ ), Z is nitrogen (N), R ₂ is alkyl, Y, R ₁ and R ₃ All are hydrogen (H). Starting material 10 is commercially available and is known in the literature (J. Andre et al., Eur. J. Org. Chem. 2004, 1558). The key steps include the SN ₂ stereotactic problem of triflate 11 and glutamic acid diester 7 substitution reactions (P. O' Shea et al., J. Org. Chem. 2009, 5, 1605). .

Option 4

Furthermore, Q of formula (IIIb) can be prepared by another synthetic route scheme 5 wherein X is trifluoromethyl (CF ₃ ), Z is nitrogen (N), R ₂ is alkyl, Y, R ₁ and R ₃ is hydrogen (H). The key starting material 10 (J. Andre, et al., Eur. J. Org. Chem. 2004, 1558) is oxidized to give trifluoromethylketone 13. The following imine compound can be formed in the presence of a base. The final step involves stereospecific reduction of imine 14 with sodium borohydride or zinc borohydride to give stereoisomers 12 and 15 (G. Huges, et al., Angew Chem. Int. Ed. 2007, 46). , 1839).

Option 5

Furthermore, Q of formula (IIIb) can also be prepared by another synthetic route scheme 6, wherein X is trifluoromethyl, Z is nitrogen, R ₂ is alkyl, and Y, R ₁ and R ₃ are all hydrogen. The polycondensation reaction of the starting material diamine 16 (M. Mandal, et al., J. Am Chem. Soc. 2002, 6538) with acetaldehyde 17 is known to give the imine 18. Next, in the presence of a catalytic amount of Lewis acid, the imine 18 and TMSCN undergo a diastereoselective Strecker-type reaction. The final step involves the hydrolysis of the cyano-containing intermediate to give stereoisomer 12 (F. Huguentt, et al., J. Org. Chem. 2006, 71, 7075).

Option 6

Q represented by formula (IV) is commercially available and is conveniently prepared in the literature or by a variety of methods familiar to those skilled in the art. A general synthetic route for the synthesis of compounds of formula (IV) is shown in Scheme 7, wherein X is oxygen (O), Z is carbon (C), R ₂ is alkyl, and W, Y, R ₁ and R ₃ are Hydrogen (H). The starting material - ketoester 19 is commercially available and is known in the literature (R. Hoffman, et al., J. Org. Chem. 1999, 64: 1558). After alkylation of the β-ketoester 19 with the trifluoromethanesulfonate 20, R _{9 is} decarbonylated and deprotected to give the ketomethylene isoester 21 (R. Hoffman, et al., J. Org) Chem. 1999, 64: 1558; PS Dragovich, et al., J. Med. Chem. 1999, 42: 1203).

Option 7

Q represented by formula (IV) is commercially available and is conveniently prepared in the literature or by a variety of methods familiar to those skilled in the art. Wherein X is oxygen, Z is carbon, R ₂ is an alkyl group, W is fluorine, and Y, R ₁ and R ₃ are all hydrogen. A general synthetic route for the synthesis of compounds of formula (IV) is shown in Scheme 8. The starting material trifluoroalkylated-ketoester 22 is commercially available or can be prepared according to the literature (R. Hoffman, et al., J. Org. Chem. 1999, 64: 1558). Further, after the alkylation reaction of the β-ketoester 22 with the triflate 20, a decarbonylation reaction is carried out to obtain a ketomethylen isoester 23, and then 23 is converted into a corresponding Z-TMS enol ether, fluorinated with Selectfluo, and finally deprotected to give monofluoro ketomethyl isoester 25 (R. Hoffman, et al., J. Org. Chem. 1999, 64: 1558; PS Dragovich, et al., J. Med. Chem. 1999, 42: 1203).

Option 8

General synthetic scheme

The intermediate peptide fragment attached to the MBHA resin can be prepared by solid phase polypeptide chemistry on Applied Biosystems Inc. (ABI) 460A polypeptide synthesizer using MBHA resin (Applied Biosystems) , lot # A1A023, 0.77 mmol/g). The a-amino group of all amino acids is protected with a tert-butoxycarbonyl group. The protection of these amino acids with reactive side chains is as follows: arginine (TOS), lysine (Cl-Z), tryptophan (CHO), glutamic acid (CHex), tyrosine (bromine) -Z), serine (Bzl), aspartic acid (OBzl), threonine (Bzl).

The protected amino acid was activated in dichloromethane (DCM) with 0.5 equivalent of dicyclohexylcarbodiimide (DCC): 1 equivalent of amino acid to obtain a symmetric anhydride of the amino acid. However, arginine residues, glutamic acid residues, and glycine residues are activated by the formation of 1-hydroxybenzotriazole (HOBt) esters of these amino acids (amino acid equivalent ratio 1:1:1: HOBt: DCC Soluble in dimethylformamide (DMF)).

The amino acid residues are sequentially linked from the C-terminus to the N-terminus, ending with a series of deprotection cycles. A coupling cycle includes a free amino acid nucleophilic substitution process of the activated amino acid by the previously coupled amino acid. Deprotection refers to the removal of the N-terminal capping group Boc with anhydrous trifluoroacetic acid (TFA) to produce a free amino group after neutralization with diisopropylethylamine (DIEA).

The amount of synthesis was 0.5 mmol. The functional site concentration on the MBHA-resin was 0.77 mmol/g, and the amount of the resin used was 649 mg. All amino acids were used in a 2-fold molar amount of additional symmetrical anhydride. The C-terminal arginine can be coupled to the MBHA resin according to standard methods. All amino acid residues are double-coupled, ie each amino acid residue can be coupled twice by a resin to ensure complete reaction of the NH ₂ group on the resin. The second coupling occurs before the new amino acid is re-added in the absence of Boc deprotection. This will help all of the free amino groups of the resin react completely. The tryptophan residue is quadruple coupled. After the second coupling of each double coupling cycle, the terminal Boc group can be removed with anhydrous TFA and neutralized with DIEA.

The formazan side chain capping group on the tryptophan residue can be removed with acridine dissolved in DMF prior to excision of the peptide from the resin. The polypeptide-resin was transferred to a 50 mL sintered glass funnel and washed several times with DCM and DMF. Then, 3-5 mL of a 50/50 acridine/DMF solution was added to the above polypeptide resin so that the polypeptide resin was not passed. After 5 min, the acridine/DMF solution was removed in vacuo and then 3-5 mL of acridine/DMF solution was added. After 10 min, the acridine/DMF solution was again removed by vacuum filtration, then 15-20 mL of acridine/DMF solution was added. After 15 min, acridine/DMF was removed and the resulting polypeptide-resin was washed several times with DMF and DCM. Finally, the resulting polypeptide-resin was then placed in a vacuum oven (without heating) and completely dried to remove the solvent.

In addition, the desired polymer-linked peptide bond fragments can be prepared by Fmoc protection. Rink Amide MBHA resin, Fmoc protected amino acid, O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-urea hexafluorophosphate (HBTU)) soluble in N,N-II Methylformamide (DMF), N-methylmorpholine (NMM) is activated, and acridine is deprotected by Fmoc (step 1). If necessary, the lysine Lys (Aloc) group can be selectively deprotected artificially, and the resin is dissolved in 5 mL of CHCl ₃ with 3 equivalents of palladium (triphenylphosphine) (Pd(PPh ₃ ) ₄ ). :NMM: HOAc (18:1:0.5) solution treatment for 2 hours to complete selective deprotection of the lysine group. Then, the resulting resin was washed residue CHC1 ₃ (6X5 mL), washed 20% HOAc solution of DCM (6X5 mL) of, DCM (6X5 mL) was used, and finally washed with DMF (6X5 mL). In some instances, after this synthesis, the compound is synthesized because of the 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) group, acetic acid or 3-maleimidopropionic acid. The implementation of (MPA) is automatically repeated (step 3). Resin cleavage and product separation were carried out with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol system, and then the product was precipitated from dry ice chilled diethyl ether (Et ₂ O) (step 4). The resulting product isolated and purified by preparative HPLC applicable Varian (Rainin) binary HPLC system: 30 to 55% of Phase B [0.045% TFA in H ₂ O solution (A-phase); 0.045% TFA solution in CH ₃ CN (B Phase)] Gradient elution, flow rate 9.5 mL/min, elution time 180 min, detection using Phenomenex Luna 10μ phenyl-hexyl (21mm x 25cm) column, UV detector (Varian Dynamax UVD II) at 214 nm and 254 nm . The purity of the product was determined by RP-HPLC mass spectrometry to be 95%. The RP-HPLC mass spectrometer used a Hewlett Packard LCMS-1100 series spectrometer with a diode array detector and an electrospray ion trap.

Protecting groups are those chemical fragments that are used to protect a polypeptide derivative from its own reaction. These protecting groups include: ethyl fluorenyl, 9-fluorenylmethoxycarbonyl (FMOC), tert-butoxycarbonyl (Boc), benzyloxycarbonyl (CBZ) and similar protective agents. The specific protection groups for amino acids are shown in Table 1.

Preparation of Q-linker-a: (2S,5R)-2-(3-(tert-Butoxycarbonylamino)-buten-1-yl)glutaric acid-5-tert-butyl ester

References: J. Chem. Soc., Perkin Trans. 1, 2001, 2445

370 mg (1 mmol) of (2S,5R)-2-((3-tert-butoxycarbonylamino)buten-1-yl)glutaric acid-5-tert-butyl ester-1-methyl ester was dissolved in 2 mL of methanol The solution was added to a solution of 2 mL (1 M) of lithium hydroxide (LiOH) at room temperature for 1 hour. Most of the solvent was removed by vacuum distillation, and then diluted with water (10 mL) to adjust pH to 5, and the aqueous phase was extracted with ethyl acetate (3×30 ml) to afford 320 mg of the product.

The characterization data are as follows: ¹ H NMR: 5.43 (m, 1 H), 5.33 (dd, J = 15.5, 5.2 Hz, 1H), 4.59 (d, J = 7.6 Hz, 1H), 3.88 (m, 1H), 2.91 ( m, 1H), 2.25 (m, 2H), 1.91-2.04 (m, 1H), 1.67-1.80 (m, 1H), 1.57 (s, 9H), 1.47 (s, 9H), 1.14 (d, J = 6.7 Hz, 3H); LCMS: 358 (M+H) ⁺ .

Preparation of Q-linker-b: (2S,5R)-2-(3-(tert-Butoxycarbonylamino)-2-fluorobuten-1-yl)glutaric acid-5-tert-butyl ester

Step A: Preparation of 2S,5R-2-(3-tert-butoxycarbonylamino-2-fluoro-buten-1-yl)-glutaric acid 1-(S)sulfonamide

References: Tetrahedron, 2008, 64: 4332.

502 mg (1 mmol) of 5-tert-butoxycarbonylamino-4-fluoro-2-(3-hydroxy-propyl)-hexene-3-acid (S) sulphonamide was dissolved in 5 mL of DMF. 2.5 _m mol of dichromate pyridine (PDC) was added, and the reaction solution was stirred at room temperature for 64 hours. The reaction mixture was then diluted with EtOAc (EtOAc)EtOAc. The combined organic layers were dried with MgSO ₄ and evaporated The residue obtained was purified by flash column chromatography to afford </RTI></RTI><RTIgt;

Step B: 2S,5R-2-(3-tert-Butoxycarbonylamino-2-fluoro-buten-1-yl)-glutaric acid 5-tert-butyl ester 1-(S) sulphonamide Preparation

400 mg (0.75 mmol) of 2S,5R-2-(3-tert-butoxycarbonylamino-2-fluoro-buten-1-yl)-glutaric acid 2-(S)sulfonate obtained from Step A The amine was dissolved in 10 mL of dichloromethane and treated with t-butanol (0.5 mL, 10 eq.), DCC (l. The reaction mixture was stirred at room temperature for 24 hours, then diluted with 20 mL of brine and ethyl acetate (3×20 mL). The organic layers were combined, dried over MgSO ₄ and evaporated The residue obtained was purified by flash column chromatography toield </

The characterization data are as follows: ¹ H NMR: 5.33 (m, 1H), 4.54 (m, 1H), 3.88 (m, 1H), 3.37 (s, 2H), 3.23 (m, 1H), 2.25 (m, 2H), 1.91-2.14 (m, 4H), 1.67-1.80 (m, 5H), 1.57 (s, 9H), 1.47 (d, J = 7.6 Hz, 3H), 1.18 (s, 3H), 1.14 (s, 3H) LCMS: 574 (M+H) ⁺ .

Step C: Preparation of 2S,5R-2-(3-tert-butoxycarbonylamino-2-fluoro-buten-1-yl)-glutaric acid 5-tert-butyl ester

The tert-butyl ester obtained in Step B (410 mg, 0.72 mmol) and 50% aqueous H ₂ O ₂ (260 mL, 3.6 mmol) was dissolved in THF-H ₂ O (5:1, 12 mL) LiOH (1 N, 1.44 mL). The resulting mixture was stirred at room temperature for 2 hours. Then, the resulting mixture was adjusted to pH 5, and extracted with ethyl acetate (3 X 15 mL), the combined organic phases were washed with brine, dried over MgSO _4. The resulting solution was evaporated to dryness to dryness crystals crystals crystals

The characterization data are as follows: ¹ H NMR: 5.23 (m, 1H), 4.45 (m, 1H), 3.11 (m, 1H), 2.45 (m, 2H), 2.24 (m, 2H), 1.52 (s, 9H), 1.47 (s, 9H); LCMS: 376 (M+H) ⁺ .

Q-linker-c: 2R, 5R 2-[1-(tert-butoxycarbonylamino-methyl)-2,2,2-trifluoro-ethylamino]-glutaric acid 5-tert-butyl ester preparation

Step A: 2R,5R 2-[1-(tert-Butoxycarbonylamino-methyl)-2,2,2-trifluoro-ethylamino]-glutaric acid 5-tert-butyl ester 1-methyl ester preparation

References: Org. Lett., 2003, 5, 3887.

364 mg (1 mol) of 2-(1-aminomethyl-2,2,2-trifluoro-ethylamino)-glutaric acid 5-tert-butyl ester 1-methyl ester hydrochloride and 260 mg (1.2 mmol) the Boc DCM ₂ O was dissolved in 15mL was added at a temperature of 0 ℃ DIPEA (0.2mL, 1.5mmol) in DCM (1mL) was added. The reaction solution was stirred at room temperature for 6 hours. The resulting mixture was diluted with 30mL ethyl acetate, was washed (brine) with 0.1N hydrochloric acid and brine, the organic phase was dried over MgSO _4. The resulting solution was evaporated to dryness crystals crystals crystals crystals

The characterization data are as follows: ¹ H NM: 4.54 (m, 1H), 4.12 (m, 1H), 3.68 (s, 3H), 3.45 (m, 1H), 3.11 (m, 2H), 2.45 (m, 2H), 2.24 (m, 2H), 1.52 (s, 9H), 1.47 (s, 9H); LCMS: 430 (M+H) ⁺ .

Step B: 2R, 5R Preparation of 2-[1-(tert-Butoxycarbonylamino-methyl)-2,2,2-trifluoro-ethylamino]-glutaric acid 5-tert-butyl ester

The tert-butyl ester obtained in Step A (420 mg, 0.92 mmol) was dissolved in THF-H ₂ O (5:1, 12 mL). The reaction solution was stirred at room temperature for 2 hours. The mixture was adjusted to pH 5 and extracted with ethyl acetate (3×15 mL). The combined organic phases were dried washed (brine) saline over MgSO _4. The resulting solution was evaporated to dryness crystals crystals crystals

The characterization data are as follows: ¹ H NMR: 4.50 (m, 1H), 4.08 (m, 1H), 3.45 (m, 1H), 3.11 (m, 2H), 2.45 (m, 2H), 2.24 (m, 2H), 1.52 (s, 9H), 1.47 (s, 9H); LCMS: 430 (M+H) ⁺ .

Preparation of Q-linker-d:2R-2-(1S,2S-2-tert-butoxycarbonylamino-1-trifluoro-methyl-propylamino)-pentanedioic acid 5-butyl ester

Step A: Preparation of 2S,3S-3-diphenylamino-1,1,1-trifluorobutane-2-trifluoromethanesulfonate

References: Eur. J. Org. Chem. 2004, 1558.

3.23 g (10 mmol) of 2S,3S-3-diphenylamino-1,1,1-trifluoro-butan-2-ol and 1.7 g (16 mmol) of 2,6-lutidine were dissolved in a 25 ml ring In hexane, 4.2 g (15 mmol) of trifluoromethanesulfonic anhydride was added at a temperature of 10 ° C, and the addition rate was controlled to keep the temperature below 10 ° C, and the reaction was continued for 1.5 hours. The resulting reaction mixture was diluted with water (25 mL) and hexane (50ml). The organic phase was washed with 1N hydrochloric acid (2 x 15 mL) and brine (15ml), dried with MgSO _4, the resulting solution was evaporated to dryness under reduced pressure to give the corresponding triflate (4.34g, 96%).

Step B: Preparation of 2S-2-(1S,2S-2-diphenylamino-1-trifluoromethyl-ppropylamino)-glutaric acid 1-phenylmethyl 5-tert-butyl ester

Potassium carbonate (2.08 g, 15 mmol) was added to a solution of the trifluoromethanesulfonate (4.55 g, 10 mmol The resulting mixture was heated to 65-70 ° C for 24 hours. After cooling to room temperature and add water (25ml) and hexane ring (50ml) was diluted and stirred for 10min, layers were separated and the organic phase was washed with 1N HCl (2X15ml) and brine (15ml), dried MgSO _4, the resultant solution under reduced pressure Concentration gave the corresponding ester (5.88 g, 95%).

Step C: Preparation of 2R-2-(1S,2S-2-tert-butoxycarbonylamino-1-trifluoromethylpropylamino)-glutaric acid 5-tert-butyl ester

5.4 g (9 mmol) of Step 2 B obtained 2S-2-(1S,2S-2-diphenylamino-1-trifluoromethyl-propylamino)-pentanedioic acid 1-phenylmethyl ester 5-un The butyl ester was hydrogenated in the presence of 50 mL of methanol and 0.9 g of Pd/C at a pressure of 50 psi for 24 hours. The catalyst was removed by filtration and the filtrate was concentrated in vacuo. The residue was dissolved in 50mL of dichloromethane, treated with a solution (25ml) with Boc ₂ O (2.60g, 12mmol) in dichloromethane at 0 deg.] C, followed by addition of DIPEA (2mL, 15mmol) in dichloromethane ( 10 mL) solution, the mixture was stirred at room temperature for 6 hours. The resulting mixture was diluted with ethyl acetate (60ml), and each with 1N HCl, water, brine, dried MgSO _4, the resulting solution was concentrated under reduced pressure to give the title compound with a foamed isolated by flash chromatography.

Preparation of Q-linker-e: 2S-2-(3S-3-tert-butoxycarbonylamino-2-oxo-butyl)-glutaric acid 5-tert-butyl ester

References: J. Med. Chem. 1999, 42: 1203; Bioorg. Med. Chem. 2005, 13: 5240

387 mg (1 mmol) of 2S-2-(3S-3-t-butoxycarbonylamino-2-oxo-butyl)-glutaric acid 5-tert-butyl ester 1-methyl ester was dissolved in THF-H ₂ O ( To a solution of 5:1, 12 ml), lithium hydroxide (1N, 1.44 ml) was added at a temperature of 0 ° C, and the mixture was stirred at room temperature for 2 hours. Then, the mixture was adjusted to pH 5, and extracted with ethyl acetate (3 X 15ml), the combined organic phases were washed with brine, MgSO ₄ magnesium sulfate, and the resulting solution was evaporated to dryness under reduced pressure to give the title compound foamed acid (362mg, 92%).

The characterization data are as follows: ¹ H NMR: 4.63 (m, 1 H), 4.38 (br, 1H), 2.68 (d, J = 7.6 Hz, 2H), 2.58 (m, 1H), 2.25 (dd, J = 12.3, 7.6 Hz, 2H), 1.92 (m, 2H), 1.49 (s, 9H), 1.47 (s, 9H), 1.41 (d, J = 7.6 Hz, 3H); LCMS: 374 (M+H) ⁺ .

Preparation of Q-linker-f: 2R-2-(1S,3S-3-tert-butoxycarbonylamino-1-fluoro-2-oxo-butyl)-glutaric acid 5-tert-butyl ester

Step A: Preparation of 2S-2-(1S,3S-3-tert-butoxycarbonylamino-1-fluoro-2-oxo-butyl)-glutaric acid 5-tert-butyl ester 1-methyl ester

In a parr shaker unit, 581 mg (1 mmol) of 2S-2-[1S,3S-fluoro-2-oxo-3-(trityl-amino)-butyl]-glutaric acid at 50 psi 5-Benzylmethyl ester 1-methyl ester and 10% Pd/C (100 mg) were subjected to hydrogenation in methanol (25 ml) for 6 hours. The catalyst was removed by filtration over EtOAc (EtOAc)EtOAc. A solution of Boc ₂ O (238 mg, 1.1 mmol) in dichloromethane (2 ml) was added to the above mixture, and the mixture was stirred at room temperature for 6 hours. The mixture was diluted with EtOAc (30 mL). The resulting solution was evaporated <RTI ID=0.0></RTI> EtOAc m. The reaction mixture was stirred for 24 h then diluted with EtOAc EtOAc m. The organic phases were combined and dried over MgSO _4, the solution was evaporated under reduced pressure, the residue was purified by flash column chromatography to give tert-butyl-shaped foam (315mg, 66%).

The characterization data are as follows: ¹ H NMR: 4.81 (m, 1 H), 4.63 (m, 1 H), 4.38 (br, 1H), 3.67 (s, 3H), 2.78 (m, 1H), 2.35 (dd, J = 12.3) , 7.6 Hz, 2H), 2.06 (m, 2H), 1.49 (s, 9H), 1.47 (s, 9H), 1.41 (d, J = 7.6 Hz, 3H); LCMS: 406 (M+H) ⁺ .

Step B: Preparation of 2S-2-(1S,3S-3-tert-butoxycarbonylamino-1-fluoro-2-oxo-butyl)-glutaric acid 5-tert-butyl ester

260 mg (0.65 mmol) of 2S-2-(1S,3S-3-tert-butoxycarbonylamino-1-fluoro-2-oxo-butyl)-glutaric acid 5-tert-butyl ester 1 obtained in Step A The methyl ester was dissolved in THF-H ² O (5:1, 12 mL). Then adjusted to pH 5, and extracted with ethyl acetate (3 X 15ml), the combined organic phases were washed with brine, dried over MgSO _4. The resulting solution was evaporated to dryness to dryness crystall

The characterization data are as follows: ¹ H NMR: 4.81 (m, 1H), 4.63 (m, 1H), 4.38 (br, 1H), 2.78 (m, 1H), 2.35 (dd, J = 12.3, 7.6 Hz, 2H), 2.06 (m, 2H), 1.49 (s, 9H), 1.47 (s, 9H), 1.41 (d, J = 7.6 Hz, 3H); LCMS: 392 (M+H) ⁺ .

Example 1

synthesis

[Q-linker-d8, glutamic acid 22] GLP-1-(7-37)-peptide

step 1

Fmoc-Rink guanamine-MBHA resin

Solid phase polypeptide synthesis of 100 [mu]m of the above peptide analogs was prepared using artificial solid phase polypeptide synthesis and using a Symphony polypeptide synthesizer. The Symphony peptide synthesizer used was: Fmoc-protected Rink Amide MBHA resin; Fmoc-protected amino acid; dissolved in N,N-dimethylformamide (DMF) and N-methylmorpholine (NMM) Activated O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU); and removal of the Fmoc protecting group with hexahydropyridine (Step 1 ). In the product of step 2, the Boc group is cleaved prior to coupling with Fmoc-His(Trt)-OH, and resin cracking and product separation are accomplished using 85% TFA/5% Tis/5% thioanisole/5% phenol. It is then precipitated with dry ice in ice (step 2). Purification of the product by reverse phase preparative chromatography using a Varian (rainnin) preparative binary HPLC chromatography system: gradient with 30-55% phase B (acetic acid solution containing 0.045% TFA) and phase A (aqueous solution containing 0.045% TFA) Elution, flow rate 9.5 mL/min, gradient elution time 180 min, Phenomenex Luna 10 μm phenyl-hexy column, 21 mm × 25 cm, UV detector (Varian Dynamax UVD II), detection wavelength 214 nm and 254 nm, the target peptide was obtained. The purity of the target peptide was >95%, and its purity was determined using RP-HPLC.

MS measured value: 3412, MS theoretical value: 3412.

Example 2

synthesis

[Q-linker-a8-9, glutamic acid 22] GLP-1-(7-37)-peptide

step 1

Fmoc-Rink guanamine-MBHA resin

The same sequence and the same conditions as described in Example 1 were used to synthesize the target GLP-1 analog.

MS found: 1113 (M+3H) ³⁺ , MS ^mp .

Example 3

synthesis

[Q-linker-b8-9, glutamic acid 22] GLP-1-(7-37)-peptide

step 1

Fmoc-Rink guanamine-MBHA resin

The same sequence and the same conditions as described in Example 1 were used to synthesize the target GLP-1 analog.

MS found: 1119 (M+3H) ³⁺ , MS: 1 1 (M+3H) ³⁺ .

Example 4

synthesis

[Q-linker-c8, glutamic acid 22] GLP-1-(7-37)-peptide

step 1

Fmoc-Rink guanamine-MBHA resin

The same sequence and the same conditions as described in Example 1 were used to synthesize the target GLP-1 analog.

MS found: 3398, MS: 3398.

Example 5

synthesis

[Q-linker-e8-9, glutamic acid 22] GLP-1-(7-37)-peptide

step 1

Fmoc-Rink guanamine-MBHA resin

The same sequence and the same conditions as described in Example 1 were used to synthesize the target GLP-1 analog.

MS found: 1118 (M+3H) ³⁺ , MS: 1118 (M+3H) ³⁺ .

Example 6

synthesis

[Q-linker-f8-9, arginine 34] GLP-1-(7-37)-peptide

step 1

Fmoc-Rink guanamine-MBHA resin

The same sequence and the same conditions as described in Example 1 were used to synthesize the target GLP-1 analog.

MS found: 1127 (M+3H) ³⁺ , MS: 1127 (M+3H) ³⁺ .

Example 7

synthesis

N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-c8, arginine 34]GLP-1-(7-37)-peptide

A mixture of [A-linker-c8, Arg34] GLP-1-OH (36 mg, 11 μmol), EDPA (4.0 mg, 30.8 μmol), acetonitrile (260 μl) and water (260 μl) was slowly stirred at room temperature for 5 min. Then, a solution of N ^α -hexadecanyl-glutamic acid N-hydroxysuccinimide-tert-butyl ether (1.8 mg, 3.3 μmol) in acetonitrile (44.2 μl) was added, and the mixture was slowly stirred at room temperature. After 20 minutes in an hour, the reaction was stopped by adding a solution of glycine (1.8 mg, 242 μmol) dissolved in 50% aqueous ethanol. An additional 0.5% aqueous solution containing ammonium acetate (12 ml) and NMP (300 μl) was added to the resulting mixture on Varian 1g C ₈ Mega Bond The cartridge was eluted, the immobilized compound was eluted with 5% acetonitrile (10 ml), and finally eluted with TFA (6 ml) to desorb the sample from the core. The resulting eluate was allowed to stand at room temperature for 2 hours and then concentrated in vacuo. The residue obtained was purified by column chromatography eluting with EtOAc (EtOAc) Protonated molecular ion m / z measured value: 3790 ± 3. Therefore, the molecular weight was 3790 ± 3 amu (theoretical value 3751 amu).

Example 8

synthesis

N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-d8, arginine 34]GLP-1-(7-37)-peptide

The same sequence and the same conditions as described in Example 7 were used to synthesize the target GLP-1 analog.

MS found: 1268 (M+3H) ³⁺ , MS: 1268 (M+3H) ³⁺ .

Example 9

synthesis

N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-e8, arginine 34]GLP-1-(7-37)-peptide

The same sequence and the same conditions as described in Example 7 were used to synthesize the target GLP-1 analog.

MS found: 1250 (M+3H) ³⁺ , MS mp: 1250 (M+3H) ³⁺ .

Example 10

synthesis

N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-f8, arginine 34]GLP-1-(7-37)-peptide

The same sequence and the same conditions as described in Example 7 were used to synthesize the target GLP-1 analog.

MS found: 1256 (M+3H) ³⁺ , MS: 1256 (M+3H) ³⁺ .

Example 11

synthesis

N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-a8-9,arginine 34]GLP-1-(7-37) -peptide;

The same sequence and the same conditions as described in Example 7 were used to synthesize the target GLP-1 analog.

MS found: 1244 (M+3H) ³⁺ , theory MS: 1244 (M+3H) ³⁺ .

Example 12

synthesis

N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-b8-9,arginine 34]GLP-1-(7-37) -peptide

The same sequence and the same conditions as described in Example 7 were used to synthesize the target GLP-1 analog.

MS found: 1250 (M+3H) ³⁺ , MS mp: 1250 (M+3H) ³⁺ .

Example 13

synthesis

N-ε ²⁶ -[(N ^ε -ω-carboxyheptadecanyl)]-[Q-linker-c8,arginine 34]GLP-1-(7-37)-peptide

Synthesis of the target GLP-1 analog using the same sequence and the same conditions as described in Example 7, and replacing the ^{α α} with ω-carboxyheptadecanoic acid 2,5-dioxapyrrolidin-1-yl ester as a starting material - hexadecanolyl-glutamic acid N-hydroxysuccinimide-tert-butyl ether.

MS found: 1239 (M+3H) ³⁺ , MS mp.: 239 (M+3H) ³⁺ .

Example 14

synthesis

N-ε ²⁶ -[(N ^ε -ω-carboxynonanyl)]-[Q-linker-c8,arginine 34]GLP-1-(7-37)-peptide

The synthetic target GLP-1 analogue was prepared using the same sequence and the same conditions as described in Example 7, and replacing N ^α with ω-carboxyheptadecanoic acid 2,5-dioxapyrrolidin-1-yl ester as a starting material. Hexadecyl-glutamic acid N-hydroxysuccinimide-tert-butyl ether.

MS found: 1249 (M+3H) ³⁺ , MS calc.: 1249 (M+3H) ³⁺ .

Example 15

synthesis

[Q-linker-d8]GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂

[A-linker-d8] GLP-1-(7-37)-cysteine-alanine-NH ₂ (36 mg, 11 μmol) was dissolved in 50 mmol/L Tris-HCl buffer (36 mL) The mixture was reacted for 3 hours at room temperature with 2 mol excess of 20 KDa mPEG-SPA (adjusted from 7.5 to 9.0 with 50 mmol/L Tris-HCl buffer). Monoethylene glycol GLP-1 cross-linking was carried out by reverse-phase high performance liquid chromatography (RP-HPLC) with X-tera C ₁₈ (4.6 X 250 mm, 5 μm, Waters, Milford, MA) at room temperature. Detection and purification. The mobile phase consisted of an aqueous solution of 0.1% TFA (phase A) and a 0.1% solution of TFA in acetonitrile (phase B). The mobile phase eluted with a linear gradient of 30-60% B phase, a flow rate of 1 mL/min, an elution time of 20 min, and UV absorbance at a wavelength of 215 nm. The HPLC components corresponding to the respective peaks were separately collected, purified with nitrogen, and lyophilized.

Example 16

synthesis

[Q-linker-c8]GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂

The synthetic target GLP-1 analog was used in the same sequence and the same conditions as described in Example 13, and 20KDa mPEG-SPA was used.

Example 17

synthesis

[Q-linker-a8]GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂

The synthetic target GLP-1 analog was used in the same sequence and the same conditions as described in Example 13, and 20KDa mPEG-SPA was used.

Example 18

synthesis

[Q-linker-b8]GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂

The synthetic target GLP-1 analog was used in the same sequence and the same conditions as described in Example 13, and 20KDa mPEG-SPA was used.

Example 19

synthesis

[Q-linker-e8]GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂

The synthetic target GLP-1 analog was used in the same sequence and the same conditions as described in Example 13, and 20KDa mPEG-SPA was used.

Example 20

synthesis

[Q-linker-f8]GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂

The synthetic target GLP-1 analog was synthesized using the same sequence and the same conditions as described in Example 13, and 20KDa mPEG-SPA was used.

Example 21

In vitro stability of DPP-IV

An equal amount of the internal purified GLP-1 analog (100 μL, 5 nmol/L) was pretreated with triethylamine hydrochloride buffer (10 mmol/L; pH 7.4). Then DPP-IV (5 mU, 900 μL) was added and the resulting solution was incubated at 37 °C. At each incubation time point, 100 μL of the reaction solution was taken out from the reaction system, and then 5 μL of 10% (v/v) TFA was added to terminate the reaction. Each of the obtained samples was analyzed by the described MALDI-TOF MS and RP-HPLC.

Example 22

cAMP formation in a cell line expressing a cloned human GLP-1 receptor To demonstrate the efficacy of a GLP-1 derivative, the GLP-1 derivative was tested to stimulate cAMP formation in a cell line expressing a cloned human GLP-1 receptor. ability. EC ₅₀ is calculated using the dose-response curves.

In this radioimmunoassay, NIT-1 cells, an islet β-cell line that establishes a transgenic NOD/Lt mouse, are used. The assay was performed in a 96-well microplate with a total volume of 140 μL. The buffer used was 50 mmol/L Tris-HCl, pH 7.4, with 1 mmol/LEGTA, 1.5 mmol/L MgSO ₄ , 1.7 mmol/L ATP, 20 mM GTP, 2 mmol/L 3-isobutyl-1- Methylxanthine, 0.01% Tween-20 and 0.1% human serum albumin. After the compound for the agonist activity test was dissolved and diluted in a buffer, membrane preparation was carried out. The resulting mixture was incubated at 37 ° C for 2 h. The reaction was stopped by adding 25 μl of 0.05 mol/L HCl. After the sample was diluted 10 times, cAMP was analyzed by proximity scintillation analysis.

Example 23

Antihyperglycemic activity of GLP-1 analogues

Spontaneous diabetic db/db mice were divided into 4 groups (n=5) and fasted for 16 hours.

The mice were then intraperitoneally injected with normal saline, 100 μg/kg GLP-1 (7-36) guanamine, and 100 μg eq/kg of [Q-linker-c8, glutamic acid 22] GLP-1. -(7-37)-peptide and dose of 100 μg eq/kg of N-ε ²⁶ -[γ-L-glutenyl (N-α-hexadecanyl)]-[Q-linker-d8, arginine 34] GLP-1-(7-37)-peptide, administered with glucose solution at a dose of 1 g/kg after 10 min of injection.

Blood samples were taken and tested for blood glucose levels at -10 min, 0 min, 10 min, 20 min, 30 min, 60 min, 90 min, 120 min, and 180 min.

Comparison of GLP-1 (7-36) guanamine, [Q-linker-c8, glutamic acid 22] GLP-1-(7-37)- by calculating the area under the blood glucose level-time curve within 0-180 min Peptide and N-ε ²⁶ -[γ-L-glutamine (N-α-hexadecanyl)]-[Q-linker-d8, arginine 34]GLP-1-(7-37)- The peptide inhibits the effect of elevated blood sugar.

The AUC value of the GLP-1(7-36) indole group was 25165±4463 mg-min/dl, which was 27.8% lower than the AUC value of the saline group (34864+4774 mg.min/dl).

However, [Q-linker-c8, glutamic acid 22] GLP-1-(7-37)-peptide and N-ε ²⁶ -[γ-L-glutenyl (N-α-hexadecanyl)]- [Q-linker-d8, arginine 34] The AUC values of GLP-1-(7-37)-peptide were 14470+5700 mg-min/dl and 17520+2484 mg-min/dl, respectively (down 58.5, respectively). % and 49.7%).

These results indicate that [Q-linker-c8, glutamic acid 22] GLP-1-(7-37)-peptide and N-ε ²⁶ -[γ compared to GLP-I(7-36) decylamine -L-glutamyric acid (N-α-hexadecanyl)]-[Q-linker-d8, arginine 34] GLP-1-(7-37)-peptide has a markedly enhanced inhibition of blood sugar Horizontal activity.

In summary, the present invention can achieve the above functions and purposes, so the present invention should meet the requirements of the patent application, and apply in accordance with the law.

Claims (22)

A GLP-1 analogue represented by the formula (I), a composition thereof or a pharmaceutically acceptable salt thereof, Xaa ₇ -Q-Gly-Thr-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Tyr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ -Ala-Ala-Xaa ₂₆ -Xaa ₂₇ -Phe-Ile-Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -B (I) It is: Xaa ₇ is natural or non-natural, selected from L-histidine, D-histidine, dea-histidine, 2-amino-histidine, β -hydroxy-histidine, high An amino acid of histidine, α-fluoromethyl-histidine or α-methyl-histidine; Q is selected from the following linkers (II), (III) or (IV): Wherein R _{1 is} selected from H or (C ₁ -C ₆ )alkyl; R _{2 is} selected from H or (C ₁ -C ₆ )alkyl or; R ₃ is H; and X is selected from H, F, OH, CF ₃ or O; Y is selected from H, OH, F or (C ₁ -C ₆ )alkyl; Z is selected from N, C, O or S; wherein, when Z is selected from N, O or S, W is not Exist; when Z is C, W is selected from H or F; Xaa ₁₄ is natural or non-natural, amino acid selected from serine or histidine; Xaa ₁₆ is natural or non-natural, selected from valine, Lai Amino acid of leucine or leucine; or Xaa ₁₆ is a lysine linked to TU; Xaa ₁₈ is a natural or non-natural amino acid selected from serine, arginine or lysine; Xaa ₂₂ is natural or non- A natural amino acid selected from the group consisting of glycine, aminoisobutyric acid or glutamic acid; Xaa ₂₃ is glutamine; Xaa ₂₆ , Xaa ₂₇ , Xaa ₃₄ , Xaa ₃₅ and Xaa ₃₆ are natural or non-natural, selected from glycine An amino acid of lysine, arginine, leucine, aspartame or aminoisobutyric acid; or Xaa ₂₆ is a lysine linked to TU; T is selected from γ -glutamic acid, β -alanine Acid, γ -aminobutyric acid or HOOC(CH ₂ ) _n COOH, wherein n is selected from 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27; or T is selected from Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Only T is an amino acid selected from γ -glutamic acid, β -alanine or γ -aminobutyric acid, or T is selected from Where is present, wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; U is selected from C ₈ -C ₂₀ fatty acids, when Q is a linker (II) or (IV), B is selected from glycine, glycosamine or from 2 to 5 natural or unnatural amino acids and must have one The amino acid is a peptide fragment consisting of cysteine; when Q is a linker (III), B is selected from 2 to 5 natural or unnatural amino acids and must have a peptide fragment consisting of a cysteine. The GLP-1 analogue, composition or pharmaceutically acceptable salt thereof according to claim 1, wherein Xaa ₂₆ is natural or non-natural, selected from the group consisting of glycine, lysine, and fine Amino acid, leucine and aspartame or α-aminoisobutyric acid without TU, B is a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and one amino acid is cysteine. A GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof according to claim 2, wherein the peptide fragment is cysteine-serine-glycine or cysteine - Alanine or methoxypolyethylene glycol maleimide linked to cysteine. A GLP-1 analogue represented by the formula (V), a composition thereof, or a pharmaceutically acceptable salt thereof, Wherein R _{1 is} selected from H or (C ₁ -C ₆ )alkyl; R _{2 is} selected from H or (C ₁ -C ₆ )alkyl or; R ₃ is H; and X is selected from H, F, OH, CF ₃ or O; D is Gly-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Thr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ -Ala-Ala-Xaa ₂₆ -Xaa ₂₇ -Phe- Ile-Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -B; Xaa ₇ is natural or non-natural, selected from L-histidine, D-histidine, deamination-histamine Amino acid of acid, 2-amino-histidine, β -hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl-histidine; Xaa ₁₄ is natural or non- a natural amino acid selected from serine or histidine; Xaa ₁₆ is a natural or non-natural amino acid selected from the group consisting of valine, lysine or leucine; or Xaa ₁₆ is a lysine linked to TU; Xaa ₁₈ is a natural or non-natural amino acid selected from the group consisting of serine, arginine or lysine; Xaa ₂₂ is a natural or non-natural amino acid selected from glycine, aminoisobutyric acid or glutamic acid; Xaa ₂₃ is Glutamine; Xaa ₂₆ , Xaa ₂₇ , Xaa ₃₄ , Xaa ₃₅ and Xaa ₃₆ are natural or non-natural, selected from glycine, lysine, arginine An amino acid of leucine, aspartame or aminoisobutyric acid; or Xaa ₂₆ is a lysine linked to TU; T is selected from γ -glutamic acid, β -alanine, γ -aminobutyric acid or HOOC(CH ₂ ) _n COOH, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, _{16, 17} , 18, ₁₉ , 20, 21, 22, 23, 24, 25, 26 or 27; or T is selected from Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; U is only an amino acid selected from γ -glutamic acid, β -alanine or γ -aminobutyric acid at T, or T is selected from T When present, wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; U is selected from C ₈ -C ₂₀ fatty acids; when Q is a linker (II) or (IV), B is selected from glycine, glycine or from 2 to 5 natural or unnatural amino acids and must have one The amino acid is a peptide fragment consisting of cysteine; when Q is a linker (III), B is selected from peptide fragments consisting of 2 to 5 natural or unnatural amino acids and having one amino acid being cysteine. A GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof, according to claim 4, wherein Xaa ₂₆ is natural or non-natural, selected from the group consisting of glycine, lysine, and fine Amino acid, leucine and aspartame or α-aminoisobutyric acid without TU, B is a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and one amino acid is cysteine. A GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof, according to claim 4, wherein the peptide fragment is cysteine-serine-glycine, cysteine - Alanine or methoxypolyethylene glycol maleimide linked to cysteine. The GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof, according to claim 4, wherein R ₁ and R ₂ are each selected from H or CH ₃ and R ₃ is H, Or R ₁ is CH ₃ , R ₂ and R ₃ are both H; or both R ₁ and R ₃ are H and R ₂ is CH ₃ . A GLP-1 analogue represented by the formula (VI), a composition thereof, or a pharmaceutically acceptable salt thereof, Characterized in that R _{1 is} selected from H or (C ₁ -C ₆ )alkyl; R _{2 is} selected from H or (C ₁ -C ₆ )alkyl or; R ₃ is H; Y is selected from H, OH, F Or (C ₁ -C ₆ )alkyl; D is +Gly-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Thr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ -Ala-Ala-Xaa _26- Xaa ₂₇ -Phe-Ile-Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -B; Xaa7 is natural or non-natural, selected from L-histidine, D-histidine An amino acid of deamination-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl-histidine; Xaa ₁₄ is a natural or non-natural amino acid selected from serine or histidine; Xaa ₁₆ is a natural or non-natural amino acid selected from the group consisting of valine, lysine or leucine; or Xaa ₁₆ is linked Lysine of TU; Xaa ₁₈ is a natural or non-natural amino acid selected from serine, arginine or lysine; Xaa ₂₂ is natural or non-natural, selected from glycine, aminoisobutyric acid or glutamic acid amino acid; Xaa ₂₃ is _{glutamine; Xaa 26, Xaa 27, Xaa} 34, Xaa 35 and Xaa ₃₆ are natural or unnatural, selected from glycine, lysine Acid, arginine, leucine, amino isobutyric acid or asparagine amino; or Xaa ₂₆ is connected to a TU-lysine; glutamic acid γ- T is selected from, [beta] -alanine, gamma] - aminobutyric acid or HOOC(CH ₂ ) _n COOH, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ₁₆ 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27; or T is selected from Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; U is only an amino acid selected from γ-glutamic acid, β-alanine or γ-aminobutyric acid at T, or T is selected from T When present, wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; U is selected from C ₈ -C ₂₀ fatty acids; B is selected from peptide fragments consisting of 2 to 5 natural or unnatural amino acids and having one amino acid being cysteine. A GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof, according to claim 8, wherein Xaa ₂₆ is natural or non-natural, selected from the group consisting of glycine, lysine, and fine Amino acid, leucine and aspartame or α-aminoisobutyric acid without TU, B is a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and one amino acid is cysteine. A GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof, according to claim 8, wherein the peptide fragment is cysteine-serine-glycine, cysteine - Alanine or methoxypolyethylene glycol maleimide linked to cysteine. A GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof, according to claim 8, wherein R _{1 is} selected from H or (C ₁ -C ₆ )alkyl; R ₂ It is selected from H or (C ₁ -C ₆ )alkyl; R ₃ is H; and Y is selected from H or (C ₁ -C ₆ )alkyl. A GLP-1 analogue represented by the formula (VII), a composition thereof, or a pharmaceutically acceptable salt thereof, Wherein R _{1 is} selected from H or (C ₁ -C ₆ )alkyl; R _{2 is} selected from H or (C ₁ -C ₆ )alkyl or; R ₃ is H; Y is selected from H, OH, F or (C ₁ -C ₆ )alkyl; W is selected from H or F; D is Gly-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Thr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ - Ala-Ala-Xaa ₂₆ -Xaa ₂₇ -Phe-Ile-Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -B; Xaa ₇ is natural or non-natural, selected from L-histidine , D-histidine, dea-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α-methyl- An amino acid of histidine; Xaa ₁₄ is a natural or non-natural amino acid selected from serine or histidine; Xaa ₁₆ is a natural or non-natural amino acid selected from the group consisting of proline, lysine or leucine; Or Xaa ₁₆ is a lysine linked to TU; Xaa ₁₈ is a natural or non-natural amino acid selected from the group consisting of serine, arginine or lysine; Xaa ₂₂ is natural or non-natural, selected from glycine and amino amino acid or glutamic acid; Xaa ₂₃ is _{glutamine; Xaa 26, Xaa 27, Xaa} 34, Xaa 35 and Xaa ₃₆ are natural or unnatural, selected from glycine Lysine, arginine, leucine, amino isobutyric acid or asparagine amino; or Xaa ₂₆ is connected to a TU-lysine; glutamic acid γ- T is selected from, [beta] -alanine , γ-aminobutyric acid or HOOC(CH ₂ ) _n COOH, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27; or T is selected from Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; Only T is an amino acid selected from γ-glutamic acid, β-alanine, γ-aminobutyric acid, or T is selected from T When present, wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; U is selected from C ₈ -C ₂₀ fatty acids; B is selected from glycine, glycosamine or a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and having one amino acid being cysteine. The GLP-1 analogue, composition or pharmaceutically acceptable salt thereof according to claim 12, wherein the Xaa ₂₆ is natural or non-natural, selected from the group consisting of glycine, lysine, and fine Amino acid, leucine and aspartame or α-aminoisobutyric acid without TU, B is a peptide fragment consisting of 2 to 5 natural or unnatural amino acids and one amino acid is cysteine. The GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof, according to claim 12, wherein the peptide fragment is cysteine-serine-glycine, cysteine - Alanine or methoxypolyethylene glycol maleimide linked to cysteine. The GLP-1 analogue, composition or pharmaceutically acceptable salt thereof according to claim 12, wherein R ₃ is H; Y is H or F. A GLP-1 analogue represented by the formula (VIII), a composition thereof, or a pharmaceutically acceptable salt thereof, Xaa7 is natural or non-natural, selected from the group consisting of L-histidine, D-histidine, dea-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine An amino acid of α-fluoromethyl-histidine or α-methyl-histidine; Q is selected from the following linkers (II), (III) or (IV): Wherein R _{1 is} selected from H or (C ₁ -C ₆ )alkyl; R _{2 is} selected from H or (C ₁ -C ₆ )alkyl or; R ₃ is H; and X is selected from H, F, OH, CF ₃ or O; Y is selected from H, OH, F or (C ₁ -C ₆ )alkyl; Z is selected from N, C, O or S; wherein, when Z is N, O or S, W does not exist When Z is C, W is selected from H or F; Xaa ₁₄ is natural or non-natural, amino acid selected from serine or histidine; Xaa ₁₆ is natural or non-natural, selected from valine and lysine An amino acid of acid or leucine; Xaa ₁₈ is a natural or non-natural amino acid selected from the group consisting of serine, arginine or lysine; Xaa ₂₂ is natural or non-natural, selected from glycine, aminoisobutyric acid or valley Amino acid of amino acid; Xaa ₂₃ is glutamine; Xaa ₂₇ , Xaa ₃₄ , Xaa ₃₅ and Xaa ₃₆ are natural or non-natural, selected from glycine, lysine, arginine, leucine, aspartame An amino acid of an amine or aminoisobutyric acid; T is selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid or HOOC(CH ₂ ) _n COOH, wherein n is selected from 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 Or T is selected from Wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; U is only an amino acid selected from γ-glutamic acid, β-alanine, γ-aminobutyric acid at T, or T is selected from T When present, wherein k is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; U is selected from C ₈ -C ₂₀ fatty acids, and B is selected from glycine or glycine. A GLP-1 analogue represented by the formula (IX), a composition thereof or a pharmaceutically acceptable salt thereof, Xaa ₇ -Q-Gly-Thr-Phe-Thr-Xaa ₁₄ -Asp-Xaa ₁₆ -Ser-Xaa ₁₈ -Tyr-Leu-Glu-Xaa ₂₂ -Xaa ₂₃ -Ala-Ala-Xaa ₂₆ -Xaa ₂₇ -Phe-Ile-Ala-Trp-Leu-Val-Xaa ₃₄ -Xaa ₃₅ -Xaa ₃₆ -Gly-Xaa ⁿ -Xaa ⁿ⁺¹ -Xaa ⁿ⁺² -Xaa ⁿ⁺³ -Xaa ⁿ⁺⁴ -Cys ^(PEG) -Xaa ^m -Xaa ^m+1 -Xaa ^m+2 -Xaa ^m+3 -Xaa ^m+4 IX where: Xaa ₇ is natural or non-natural, selected from the group consisting of L-histidine, D-histidine, dea-histidine, 2-amino-histidine, β -hydroxy-histidine, high histamine An amino acid of acid, α-fluoromethyl-histidine or α-methyl-histidine; Q is selected from the following linkers (II), (III) and (IV): Wherein R _{1 is} selected from H or (C ₁ -C ₆ )alkyl; R _{2 is} selected from H or (C ₁ -C ₆ )alkyl or; R ₃ is H; and X is selected from H, F, OH, CF ₃ or O; Y is selected from H, OH, F or (C ₁ -C ₆ )alkyl; Z is selected from N, C, O or S; wherein, when Z is N, O or S, W does not exist When Z is C, W is selected from H or F; Xaa ₁₄ is natural or non-natural, amino acid selected from serine or histidine; Xaa ₁₆ is natural or non-natural, selected from valine and lysine Acid or leucine amino acid; or Xaa ₁₆ is a lysine linked to TU; Xaa ₁₈ is a natural or non-natural amino acid selected from serine, arginine or lysine; Xaa ₂₂ is natural or unnatural An amino acid selected from the group consisting of glycine, aminoisobutyric acid or glutamic acid; Xaa ₂₃ is glutamine; Xaa ₂₆ , Xaa ₂₇ , Xaa ₃₄ , Xaa ₃₅ and Xaa ₃₆ are natural or non-natural, selected from glycine, An amino acid of lysine, arginine, leucine, aspartame or aminoisobutyric acid; or Xaa26 is a lysine linked to TU; Xaa ⁿ , Xaa ⁿ⁺¹ , Xaa ⁿ⁺² , Xaa ^{n +3} , Xaa ⁿ⁺⁴ , Xaa ^m , Xaa ^m+1 , Xaa ^m+2 , Xaa ^m+3 and Xaa ^m+4 together form 1, 2, 3 or 4 a natural or non-natural amino acid; that is, Xaa ⁿ , Xaa ⁿ⁺¹ , Xaa ⁿ⁺² , Xaa ⁿ⁺³ , Xaa ⁿ⁺⁴ , Xaa ^m , Xaa ^m+1 , Xaa ^m+2 , Xaa ^m+3 and Xaa ^m+4 form a fragment of 2 to 5 amino acids with a cysteine linked to methoxypolyethylene glycol maleimide. The GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof according to any one of claims 1-17, which is selected from the group consisting of: [Q-linker-d8, glutamic acid 22] GLP -1-(7-37)-peptide; [Q-linker-a8-9, glutamic acid 22] GLP-1-(7-37)-peptide; [Q-linker-b8-9, glutamine Acid 22] GLP-1-(7-37)-peptide; [Q-linker-c8, glutamic acid 22] GLP-1-(7-37)-peptide; [Q-linker-e8-9, Glutamate 22] GLP-1-(7-37)-peptide; [Q-linker-f8-9, arginine 34] GLP-1-(7-37)-peptide; N-ε ²⁶ -[ γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-c8, arginine 34]GLP-1-(7-37)-peptide; N-ε ²⁶ - [γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-d8, arginine 34] GLP-1-(7-37)-peptide; N-ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-e8, arginine 34]GLP-1-(7-37)-peptide; N-ε ^26- [γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-f8, arginine 34]GLP-1-(7-37)-peptide; N- ε ²⁶ -[γ-L-glutamyl (N-α-hexadecanyl)]-[Q-linker-a8-9, arginine 34]GLP-1-(7-37)-peptide ;N-ε ²⁶ -[ γ -L-glutamyl (N-α-hexadecanyl)]-[Q-linker-b8-9, arginine 34]GLP-1-(7-37 )-peptide; N-ε ²⁶ -[(N ^Ε -ω-carboxyheptadecanyl)]-[Q-linker-c8,arginine 34]GLP-1-(7-37)-peptide; N-ε ²⁶ -[(N ^ε -ω-carboxyl 19-mercapto)]-[Q-linker-c8, arginine 34]GLP-1-(7-37)-peptide; [Q-linker-d8]GLP-1-(7-37)- Cysteine ^(PEG) -aminolevulinic acid-NH ₂ ; [Q-linker-c8] GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ ; [Q-linker-a8-9]GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ ;[Q-linker-b8-9]GLP-1- (7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ ; [Q-linker-e8-9]GLP-1-(7-37)-cysteine ^(PEG) - Aminolevulinic acid-NH ₂ ; [Q-linker-f8-9] GLP-1-(7-37)-cysteine ^(PEG) -aminolevulinic acid-NH ₂ . A pharmaceutical composition comprising the GLP-1 analogue according to any one of claims 1 to 18, a composition thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant. The pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is suitable for gastrointestinal administration. A GLP-1 analogue, a composition thereof, or a pharmaceutically acceptable salt thereof, for the preparation or treatment of hyperglycemia, type II diabetes, impaired glucose tolerance, type I diabetes, obesity, hypertension, syndrome X, Dyslipidemia, cognitive impairment, arteriosclerosis, myocardial infarction, coronary heart disease, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcer drugs, drugs for delaying or preventing deterioration of type 2 diabetes, and for reducing Use of a food intake, a drug that reduces beta cell apoptosis, increases islet beta cell function, increases beta-cell mass, and/or restores glucose sensitivity to β-cells, wherein the GLP-1 analog is The GLP-1 analogue according to any one of claims 1 to 18. A pharmaceutical composition for treating or preventing hyperglycemia, type II diabetes, impaired glucose tolerance, type I diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitive impairment, arteriosclerosis, myocardial infarction, coronary artery Heart disease, stroke, inflammatory bowel syndrome, dyspepsia and gastric ulcer drugs, drugs for delaying or preventing deterioration of type 2 diabetes, and for reducing food intake, reducing beta cell apoptosis, and increasing islet β Cell function, increase β-cell mass and/or restore glucose to β-fine The use of a cell for the sensitivity of a drug, wherein the GLP-1 analogue is a GLP-1 analogue as described in any one of claims 1-18.