Novel analogues of glucagon-like peptide, composition and method of use

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to novel analogues of a glucagon-like peptide, and can be used in medicine to activate insulin expression in mammals and to treat sugar diabetes.

EFFECT: obtained peptide derivatives have a peptide imitating linker and provide prolonged action when treating diabetes and other diseases associated with insulinotropic peptides, gastrointestinal functions and glucagon levels.

25 cl, 23 ex

 

AREA of TECHNOLOGY

The present invention relates to new analogues glucagon-like peptide and compositions useful for the activation of the expression of insulin in mammals and for the treatment of diabetes. In particular, these peptide derivatives provide long term action in the treatment of diabetes and other diseases associated with insulinotropic peptides, gastrointestinal function and the activities associated with levels of glucagon.

Background of the INVENTION

Endocrine secretion of pancreatic islets is controlled by a complex control mechanism, managed not only to bring the fluid metabolites, such as glucose, amino acids and catecholamines, but also local paracrine effects. The main hormones of the pancreatic islets insulin, glucagon and somatostatin - interact with certain types of pancreatic cells (cells A, B and D, respectively) to regulate the secretory response. Although insulin secretion is controlled mainly by the level of blood glucose, somatostatin inhibits glukozooksidasa insulin secretion. In addition to the regulation of insulin secretion within the pancreatic islets, there is evidence that supports the existence of insulinotropic factors in the intestine. This and�retinova concept comes from the observation that the food intake or enteral glucose infusion caused a greater stimulation of insulin release compared with the same amount of energy (glucose) injected intravenously (Elrick, H., et al., J Clin. Endocrinol. Metab., 24, 1076-1082, 1964; McIntyre, N., et al., J Clin. Endocrinol. Metab., 25, 1317-1324, 1965). Therefore, it was postulated that obtained from the intestine signals, stimulated by the oral intake of nutrients orally, are effective stimulators of insulin secretion, is responsible for the augmentation of insulin release in the case when the energy is introduced through the intestine, compared with the parenteral route (Dupre, J., et al., Diabetes. 15, 555-559, 1966). Despite the fact that some neurotransmitters and hormones bowel possess incretin-like activity, important evidence from immunological studies, studies with antagonists and studies using knockouts indicate that glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide (GLP)-1 are basic peptides responsible for the basic amount of insulin secretion, stimulated by nutrients. The observation that the patient with diabetes mellitus type 2 demonstrates a significant decrease in secretory insulin release stimulated by food, is the basis of the interest in determining s�Lee et release defective incretin or resistance to the action of incretin the pathophysiology of β-cell dysfunction in subjects with diabetes.

Glucagon-like peptide-1 (GLP-1) was first identified in 1987 as incretin-based hormone, a peptide secreted in the intestine after ingestion. GLP1 is secreted by L-cells of the intestine after proteolytic processing of a 160-amino-acid protein precursor, preproglucagon. The splitting of preproglucagon first gives GLP-1, a 37-amino acid peptide GLP-1(1-37)HE that is weak. Subsequent cleavage at position 7 leads to the formation of biologically active GLP-1(7-37)IT. Approximately 80% is synthesized in L-cell GLP-1(7-37)HE amidines with C-end after removal of the terminal glycine residue. The biological effects and metabolic turnover of the free acid GLP-1(7-37), HE and amide GLP-1(7-37)NH2indistinguishable.

It is known that GLP-1 stimulates the secretion of insulin, causing glucose uptake by cells, which reduces the level of glucose in blood serum (Mojsov, S., et al., J Clin. Invest., 79, 616-619, 1987; Kreymann, B., et al., Lancet ii,1300-1304, 1987; Orskov, C., et al., Endocrinology, 123, 2009-2013, 1988). Emergency intracerebroventricularly injection of receptor agonists GLP-1 or GLP-1 causes a temporary reduction in food intake (Turton, M. D., et al., Nature, 379, 60-72, 1996), whereas longer intracerebroventricularly or parenteral administration of agonists of the receptor GLP-1 is associated with weight loss in some studies (Meeran, K., et al., Endocrinology, 140, 244-250, 1999; Davies, H. R. Jr.,Real trap. Res., 6, 147-156, 1998; Szayna, M., et al., Endocrinology, 141, 1936-1941, 2000; Larsen, P. J., et al., Diabetes, 50, 2530-2539, 2001). In this area known for its numerous analogues of GLP-1, demonstrating insulinotropic action. These analogs include, for example, GLP-1(7-36), Gln9-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). Derivatives of GLP-1 include, for example, salts with addition of acids, carboxylate salts, lower alkyl esters and amides (W091/11457; EP0733644; U.S. Patent 5512549).

Much of the activity of GLP-1 in preclinical experiments, was also demonstrated in human studies. Infusion of GLP-1(7-36)NH2 the normal subject-person stimulated insulin secretion, significantly reduced levels of fasting blood glucose after glucose administration or ingestion (Orskov, C, et al., Diabetes, 42, 658-661, 1993; Qualmann, C., et al., Acta.Diabetol, 32, 13-16, 1995).

The peptide-based GLP-1 has generated great prospects as an alternative to insulin therapy in patients with diabetes mellitus, for which no progress has been made in the treatment of sulfonylurea (Nauck, M. A., et al., Diabetes Care, 21, 1925-1931, 1998). GLP-1 stimulates insulin secretion, but only during the period of hyperglycemia. The safety of GLP-1 compared with insulin enhanced by this property of GLP-1 and the observation that the amount of insulin secreted is proportional to the magnitude of hyperglycemia. In addition, GLP-1 therapy $ �to cause the release of insulin by the pancreas and presystemic the action of insulin in the liver. This leads to lower circulating levels of insulin in the periphery compared with the levels in subcutaneous injections of insulin. GLP-1 slows gastric emptying, which is preferable because it prolongs the absorption of nutrients over a long period of time, reducing postprandial glucose peak. Several reports suggest that GLP-1 can increase insulin sensitivity in peripheral tissues such as muscle and adipose tissue and the liver tissue. Finally, it was shown that GLP-1 is a potential regulator of appetite.

therapeutic potential of GLP-1 and its analogues is increasing even more, if we consider its use in patients with diabetes mellitus type 1. Several studies have shown the effectiveness of native GLP-1 in the treatment of insulin-dependent diabetes mellitus (IDDM). Similarly, for patients with non-insulin-dependent diabetes mellitus (insd) GLP-1 is effective to reduce fasting hyperglycemia due to his glucagonostatic properties. Additional studies showed that GLP-1 also reduces the range of postprandial blood glucose in IDDM, likely due to the delayed emptying of the stomach. These observations suggest that GLP-1 may be useful for the treatment of both IDDM and INCD.

However, biologists�die half-life of native GLP molecules-1, which is determined by the activity of dipeptidyl-peptidase IV (DPP IV) is fairly short. For example, the biological half-life of GLP-1(7-37), this represents only 3 to 5 minutes (U.S. patent 5118666). A steady decrease in the concentration of glucose in the blood is observed only when continuous infusion, as shown by studies in which GLP-1 was administered by intravenous infusion during the course duration 24 hours (Larsen, J.; et al. Diabetes Care, 24, 1416-1421, 2001). The enzyme DPP-IV, a serine protease that hydrolyzes preferentially the peptide after the penultimate NH2-terminal Proline (XAA-Pro) or alanine (XAA-Ala-) (Mentlein, R., Regul. Pept., 85, 9-25, 1999) have been shown to rapidly metabolizes GLP-1 in vitro. Therefore, the peptide-based GLP-1 prolonged action, which are resistant to DPP IV, may have great therapeutic potential for the treatment of diabetes.

Description of the INVENTION

The present invention provides new analogues of GLP-1, which have increased activity compared to native GLP-1 and completely resistant to hydrolysis by DPP-IV.

The invention includes compounds with the General formula I:

Xaa7-Q-Gly-Thr-Phe-Thr-Xaa14Asp Xaa16-Ser-Xaa18-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Xaa27-Phe-Ile-Ala-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-B

I

or its pharmaceutically acceptable salt, where:

Xaa7is a natural or non natural amino acid selected from the group consisting of L-His, D-histidine, denominalization, 2-aminoguanidine, β-hydroxycytidine, homocysteine, α-formetiketten and α-methylhistidine;

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

,

where

R1represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy;

R2represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy;

R3represents hydrogen, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2;

X represents hydrogen, fluorine, hydroxy, trifluoromethyl or oxygen;

Y represents hydrogen, hydroxyl, fluorine or (C1-C6)alkyl;

Z represents nitrogen, carbon, oxygen, or sulfur;

In the case when Z represents a nitrogen, oxygen or sulfur, W is absent; When Z is carbon, W represents hydrogen or fluorine.

Xaa14is a natural or not naturally occurring amino acid selected from the group consisting of serine and histidine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa16is prirodno� or not naturally occurring amino acid, selected from the group consisting of valine, lysine and leucine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups. Or Xaa16is a lysine linked with T-U.

Xaa18is a natural or not naturally occurring amino acid selected from the group consisting of serine, arginine and lysine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa22and Xaa23are natural or not naturally occurring amino acids selected from the group consisting of glycine, Aib and glutamic acid, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa26, Xaa27, Xaa34, Xaa35and Xaa36are natural or not naturally occurring amino acids selected from the group consisting of glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid), where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups. Or Xaa26is a lysine linked with T-U.

B is selected from the group consisting of glycine, NH2and OH that represent amide formuale free acid end of the amino acid? or

in the case where Xaa26is a natural or not naturally occurring amino acid selected from the group consisting of glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid) and is not associated with T-U, B is preferably selected from peptide fragments consisting of two to five natural or not naturally occurring amino acids and one amino acid must be cysteine, such as, as non-limiting examples, the cysteine-serine-glycine or cysteine-alanine, and cysteine linked .

T is selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid HOOC(CH2)nCOOH;

n is 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;

T is selected from the group consisting of

,

and where k is selected from the group 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 present only in the case when T is an amino acid selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid; or when T is selected from the group consisting of

,

where k is selected from the group 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 a fatty acid with length of 8 to 20 carbons;

E�e one preferred compound is the compound shown by the Formula V:

,

where

R1represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy.

R2represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy;

R3represents hydrogen, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2.

X represents hydrogen, fluorine, hydroxy, trifluoromethyl or oxygen.

D represents Gly-Phe-Thr-Xaa14Asp Xaa16-Ser-Xaa18-Thr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Xaa27-Phe-Ile-Ala-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-B.

Xaa7is a natural or not naturally occurring amino acid selected from the group consisting of L-His, D-histidine, denominalization, 2-aminoguanidine, β-hydroxycytidine, homocysteine, α-formetiketten and α-methylhistidine;

Xaa14is a natural or not naturally occurring amino acid selected from the group consisting of serine and histidine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Haa16is a natural or not naturally occurring amino acid selected from the group consisting of valine, lysine and leucine, where one or Bo�her of carbon atoms specified amino optionally substituted by one or more alkyl groups. Or Xaa16is a lysine linked with T-U.

Xaa18is a natural or not naturally occurring amino acid selected from the group consisting of serine, arginine and lysine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa22and Xaa23are natural or not naturally occurring amino acids selected from the group consisting of glycine, Aib and glutamic acid, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa26, Xaa27, Xaa34, Xaa35and Xaa36are natural or not naturally occurring amino acids selected from the group consisting of glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid), where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups. Or Xaa26is a lysine linked with T-U.

B is selected from the group consisting of glycine, NH2and OH that represent the amide form or free acid end of the amino acid, or

in the case where Xaa26is a natural or not naturally occurring amino acid selected from the group, with�standing from glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid) and is not associated with T-U, B is preferably selected from peptide fragments consisting of two to five natural or not naturally occurring amino acids and one amino acid must be cysteine, such as, as non-limiting examples, the cysteine-serine-glycine or cysteine-alanine, and cysteine linked .

T is selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid HOOC(CH2)nCOOH;

n is 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 the group consisting of

,

where k is selected from the group 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 present only when T is an amino acid selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid; or when T is selected from the group consisting of

,

where k is selected from the group 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 a fatty acid with length of 8 to 20 carbons.

As a preferred technical solution,

R3represents hydrogen or forms a 5-8-membered ring with R1or R2.

As a preferred technical solution, X represents hydrogen, fluorine or trifluoromethyl.

As a preferred technical solution, R1, R2and R3represent hydrogen or methyl.

As a preferred technical solution, R1is methyl, R2and R3represent hydrogen.

As a preferred technical solution, R1and R3represent hydrogen and R2is methyl.

As a preferred technical solution, R3forms a 5-8-membered ring with R1and R2represents hydrogen; or R3forms a 5-8-membered ring with R2and R1represents hydrogen.

The most preferred compounds are compounds as shown in Formula VI,

,

where

R1represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy.

R2represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy.

R3represents hydrogen, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2.

Y represents hydrogen, hydroxyl, fluorine or (C1-C6)alkyl.

D represents Gly-Phe-Thr-Xaa14 Asp Xaa16-Ser-Xaa18-Thr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Xaa27-Phe-Ile-Ala-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-B.

Xaa7is a natural or not naturally occurring amino acid selected from the group consisting of L-His, D-histidine, denominalization, 2-aminoguanidine, β-hydroxycytidine, homocysteine, α-formetiketten and α-methylhistidine;

Xaa14is a natural or not naturally occurring amino acid selected from the group consisting of serine and histidine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups;

Haa16is a natural or not naturally occurring amino acid selected from the group consisting of valine, lysine and leucine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups. Or Xaa16is a lysine linked with T-U.

Xaa18is a natural or not naturally occurring amino acid selected from the group consisting of serine, arginine and lysine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa22and Xaa23are natural or not meet�concern in the nature of amino acids, selected from the group consisting of glycine, Aib and glutamic acid, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa26, Xaa27, Xaa34, Xaa35and Xaa36are natural or not naturally occurring amino acids selected from the group consisting of glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid), where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups. Or Xaa26is a lysine linked with T-U.

B is selected from the group consisting of glycine, NH2and OH that represent the amide form or free acid end of the amino acid, or

in the case where Xaa26is a natural or not naturally occurring amino acid selected from the group consisting of glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid) and is not associated with T-U, B is preferably selected from peptide fragments consisting of two to five natural or not naturally occurring amino acids and one amino acid must be cysteine, such as, as non-limiting examples, the cysteine-serine-glycine or cysteine-alanine, and cysteine linked monomethoxypolyethylene�maleimid.

T is selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid HOOC(CH2)nCOOH;

n is 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 the group consisting of

,

where k is selected from the group 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 present only when T is an amino acid selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid; or when T is selected from the group consisting of

,

where k is selected from the group 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 a fatty acid with length of 8 to 20 carbons.

As a preferred technical solution, R3represents hydrogen.

As a preferred technical solution, Y represents hydrogen or (C1-C6)alkyl.

Another preferred compounds are compounds as shown by Formula VII

,

where

R1represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy.

R2represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy.

R3is a nitric acid�, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2.

Y represents hydrogen, hydroxyl, fluorine or (C1-C6)alkyl.

W represents hydrogen or fluorine.

D represents Gly-Phe-Thr-Xaa14Asp Xaa16-Ser-Xaa18-Thr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Xaa27-Phe-Ile-Ala-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-B.

Xaa7is a natural or not naturally occurring amino acid selected from the group consisting of L-His, D-histidine, denominalization, 2-aminoguanidine, β-hydroxycytidine, homocysteine, α-formetiketten and α-methylhistidine;

Xaa14is a natural or not naturally occurring amino acid selected from the group consisting of serine and histidine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Haa16is a natural or not naturally occurring amino acid selected from the group consisting of valine, lysine and leucine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups. Or Xaa16is a lysine linked with T-U.

Xaa18is a natural or not naturally occurring amino acid, selected and� group, consisting of serine, arginine and lysine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa22and Xaa23are natural or not naturally occurring amino acids selected from the group consisting of glycine, Aib and glutamic acid, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa26, Xaa27, Xaa34, Xaa35and Xaa36are natural or not naturally occurring amino acids selected from the group consisting of glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid), where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups. Or Xaa26is a lysine linked with T-U.

B is selected from the group consisting of glycine, NH2and OH that represent the amide form or free acid end of the amino acid, or

in the case where Xaa26is a natural or not naturally occurring amino acid selected from the group consisting of glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid) and is not associated with T-U, B is preferably selected from peptide fragments, �Octasic from two to five natural or not naturally occurring amino acids, and one amino acid must be cysteine, such as, as non-limiting examples, the cysteine-serine-glycine or cysteine-alanine, and cysteine linked .

T is selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid HOOC(CH2)nCOOH;

n is 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 the group consisting of

,

where k is selected from the group 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 present only when T is an amino acid selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid; or when T is selected from the group consisting of

,

where k is selected from the group 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 a fatty acid with length of 8 to 20 carbons.

As a preferred technical solution, R3represents hydrogen or forms a 5-8-membered ring with R1or R2;

As a preferred technical solution, Y represents hydrogen or fluorine;

As a preferred technical solution, W represent hydrogen or fluorine.

Another preferred compound PR�astavliaut a connection as shown by Formula VIII

or a pharmaceutically acceptable salt, where:

Xaa7is a natural or not naturally occurring amino acid selected from the group consisting of L-His, D-histidine, denominalization, 2-aminoguanidine, β-hydroxycytidine, homocysteine, α-formetiketten and α-methylhistidine;

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

,

where

R1represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy.

R2represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy.

R3represents hydrogen, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2.

X represents hydrogen, fluorine, hydroxy, trifluoromethyl or oxygen.

Y represents hydrogen, hydroxyl, fluorine or (C1-C6)alkyl.

Z represents nitrogen, carbon, oxygen or sulfur.

When Z represents a nitrogen, oxygen or sulfur, W is absent. If Z is carbon, W represents hydrogen, fluorine.

Xaa14is a natural or not naturally occurring amino acid selected from the group consisting of serine and histidine, where one or more carbon atoms of the specified amino acid�you optionally substituted with one or more alkyl groups.

Haa16is a natural or not naturally occurring amino acid selected from the group consisting of valine, lysine and leucine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa18is a natural or not naturally occurring amino acid selected from the group consisting of serine, arginine and lysine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups

Xaa22and Xaa23are natural or not naturally occurring amino acids selected from the group consisting of glycine, Aib and glutamic acid, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa27, Xaa34, Xaa35and Xaa36are natural or not naturally occurring amino acids selected from the group consisting of glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid), where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

B is selected from the group consisting of glycine, NH2and OH that represent the amide form or free acid conc�howl amino acids.

T is selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid HOOC(CH2)nCOOH;

n is 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 the group consisting of

,

where k is selected from the group 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 present only when T is an amino acid selected from the group consisting of γ-glutamic acid, β-alanine, γ-aminobutyric acid; or when T is selected from the group consisting of

,

where k is selected from the group 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 a fatty acid with length of 8 to 20 carbons;

Another preferred compounds are compounds as shown by Formula (IX

Xaa7-Q-Gly-Thr-Phe-Thr-Xaa14Asp Xaa16-Ser-Xaa18-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Xaa27-Phe-Ile-Ala-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-Gly-Xaan-Xaan+1-Xaan+2-Xaan+3-Xaan+4-Cys(PEG)-Xaam-Xaam+1-Xaam+2-Xaam+3-Xaam+4

IX

or a pharmaceutically acceptable salt, where:

Xaa7is a natural or not naturally occurring amino acid selected from the group consisting of L-His, D-histidine, de�of aminoguanidine, 2-aminoguanidine, β-hydroxycytidine, homocysteine, α-formetiketten and α-methylhistidine;

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

,

where

R1represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy.

R2represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy.

R3represents hydrogen, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2.

X represents hydrogen, fluorine, hydroxy, trifluoromethyl or oxygen.

Y represents hydrogen, hydroxyl, fluorine or (C1-C6)alkyl.

Z represents nitrogen, carbon, oxygen or sulfur.

In the case when Z represents a nitrogen, oxygen or sulfur, W is absent. If Z is carbon, W represents hydrogen, fluorine.

Xaa14is a natural or not naturally occurring amino acid selected from the group consisting of serine and histidine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Haa16is a natural or not naturally occurring amino acid selected from the group consisting of valine, lysine and leucine, where one or more carbon atoms are indicated� amino optionally substituted by one or more alkyl groups.

Xaa18is a natural or not naturally occurring amino acid selected from the group consisting of serine, arginine and lysine, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa22and Xaa23are natural or not naturally occurring amino acids selected from the group consisting of glycine, Aib and glutamic acid, where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

Xaa26, Xaa27, Xaa34, Xaa35and Xaa36are natural or not naturally occurring amino acids selected from the group consisting of glycine, lysine, arginine, leucine and asparagine, Aib (α-aminoadamantane acid), where one or more carbon atoms of the specified amino optionally substituted by one or more alkyl groups.

All together Xaan, Xaan+1, Xaan+2, Xaan+3, Xaan+4, Xaam, Xaam+1, Xaam+2, Xaam+3, Xaam+4can be one, or two, or three, or four amino acids selected from natural or synthetic amino acids. In another group of characters Xaan, Xaan+1, Xaan+2, Xaan+3, Xaan+4, Xaam, Xaam+1, Xaam+2, Xaam+3That Xa m+4together with cysteine to form fragments from two to five amino acid, and cysteine associated with .

To give the reader understanding of the compounds covered by the invention, is further provided with the following compounds according to the invention:

- [Q-linker-d8,Glu22]GLP-1-(7-37)-peptide;

- [Q-linker-a8-9,Glu22]GLP-1-(7-37)-peptide;

- [Q-linker-b8-9,Glu22]GLP-1-(7-37)-peptide;

- [Q-linker-c8,Glu22]GLP-1-(7-37)-peptide;

- [Q-linker-e8-9,Glu22]GLP-1-(7-37)-peptide;

- [Q-linker-f8-9,Arg34]GLP-1-(7-37)-peptide;

- N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-c8,Arg34]GLP-1-(7-37)-peptide;

- N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-d8,Arg34]GLP-1-(7-37)-peptide;

- N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-e8-9,Arg34]GLP-1-(7-37)-peptide;

- N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-f8-9,Arg34]GLP-1-(7-37)-peptide;

- N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-a8-9,Arg34]GLP-1-(7-37)-peptide;

- N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-b8-9,Arg34]GLP-1-(7-37)-peptide;

- N-ε26-[(Nε-ω-carboxypropanoyl)]-[Q-linker-c8,Arg34]GLP-1-(7-37)-peptide;

- N-ε26-[(Nε-ω-carboxyrhodamine)]-[Q-linker-c8,Arg34]GLP-1-(7-37)-peptide;

- [Q-linker-d8]GLP-1-(7-37)-Cys(PEG)-Ala-NH2;

- [Q-linker-c8]GLP-1-(7-37)-Cys(PEG)-Ala-NH2;

- [Q-linker-a8-9]GLP-1-(7-37)-Cys(PEG)-Ala-NH2;

- [Q-linker-b8-9]GLP-1-(7-37)-Cys(PEG)-Ala-NH2 ;

- [Q-linker-e8-9]GLP-1-(7-37)-Cys(PEG)-Ala-NH2;

- [Q-linker-f8-9]GLP-1-(7-37)-Cys(PEG)-Ala-NH2.

The key point of the present invention is the replacement of the amide bond Ala8amino end of GLP-1, which represents a site of recognition DPP-IV that mimic the peptide bond by linkers. Linkers that mimic the peptide bond, represent a classical approach in drug discovery, by simulating the connection of a natural peptide that retains the ability to interact with biological targets and has the same biological effect (Curr Chem Bio, 12, 292-296, 2008). On the basis of the same principle, analogues of GLP-1, modified mimics the peptide bond linkers, should retain the same biological activity and have a long term of as insulinotropic agents.

Compounds according to the invention can have one or more asymmetric centers, such as the a-linker in Formula I. Such compounds may be presented in one or more stereoisomeric forms. These compounds can be, for example, racemate, optically active forms, or enantiomerically enriched mixtures of stereoisomers. Where required, separate the enantiomers, i.e., optically active forms, can be obtained using known methods, e.g. by asymmetric synthesis, by �Intesa from optically active starting materials or by separating racemates. Division of racemates can be achieved using traditional methods, such as, for example, crystallization in the presence of a separating agent; derivatization with enantiomeric purity or enriched separating reagent, followed by separation of the desired isomer; or chromatography, using, for example, a chiral HPLC column.

The term "polypeptide" and "peptide" as used herein means a compound composed of at least five constituent amino acids connected by peptide bonds. Components of amino acids may belongs to the group of amino acids encoded by the genetic code, and they may represent a natural amino acids that are not encoded by the genetic code, as well as synthetic amino acids. Natural amino acids that are not encoded by the genetic code are, for example, γ-carboxyglutamate, ornithine, phosphoserine, D-alanine and D-glutamine. Synthetic amino acids comprise amino acids manufactured by chemical synthesis, i.e. D-isomers of amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib (α-aminoadamantane acid), Abu (α-aminobutyric acid), Tie (tert-butylglycol), β-alanine, 3-aminomethylbenzoic acid, Anthranilic acid.

22 proteogenic amino acids are:

Alanine, ar�Yining, asparagine, aspartic acid, cysteine, cystine, glutamine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, Proline, serine, threonine, tryptophan, tyrosine, valine.

Thus, Napoleona amino acid is a molecule which can be incorporated into the peptide by peptide bonds, but not which proteogenic amino acid. Examples are γ-carboxyglutamate, ornithine, phosphoserine, D-amino acids such as D-alanine and D-glutamine, synthetic neprotivlenie amino acids containing amino acids manufactured by chemical synthesis, i.e. D-isomers of amino acids encoded by the genetic code such as D-alanine and D-leucine, Aib (α-aminoadamantane acid), Abu (α-aminobutyric acid), Tie (tert-butylglycol), 3-aminomethylbenzoic acid, Anthranilic acid, deteministic, beta-analogs of amino acids, such as β-alanine, etc. D-histidine, deteministic, 2-aminoguanidine, β-hydroxycytidine, homocystein, Nα-acetylcystein, α-formetiketten, α-methylhistidine, 3-pyridylamine, 2-pyridylamine or 4-pyridylamine, (1-aminocyclopropane)carboxylic acid, (1-aminocyclopent)carboxylic acid, (1-aminocyclopent)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocyclopent)carboxylic acid, or (1-a�initlocale) carboxylic acid;

Amino acid sequence of GLP was published by several researchers (Lopez, L. C. et al., Proc. NAT'l Acad. Sci. USA 80, 5485-5489, 1983; Bell, G. I., et al., Nature 302:716-718(1983); Heinrich, G., et al, Endocrinol, 115:2176-2181 (1984)). The structure of mRNA preproglucagon and the corresponding amino acid sequence is well known. Was characterized by proteolytic processing of the gene product predecessor, proglucagon, glucagon and two insulinotropic peptide. As used herein, the entry of GLP-1(1-37) refers to a polypeptide GLP-1 having all amino acids from 1 (N-end) to 37 (C-end). Similarly, GLP-1(7-37) refers to a polypeptide GLP-1 having all amino acids from 7 (N-end) to 37 (C-end). Similarly, GLP-1(7-36) refers to a polypeptide GLP-1 having all amino acids from 7 (N-end) to 36 (C-end).

The present invention also provided a pharmaceutical composition comprising a compound of the present invention in combination with one or more pharmaceutically acceptable carriers, diluents or excipients.

The principle of solid-phase synthesis of polypeptides are well known in this area and can be found in the basic texts in the area such as Dugas, H. and Penney, C., Bioorganic Chemistry (1981) Springer-Verlag, New York, page 54-92; Merrifield, J. M., Chem. Soc., 85, 2149, 1962, and Stewart and Young, Solid Phase Peptide Synthesis, page 24-66, Freeman (San Francisco, 1969).

Example�, the peptide fragment according to the invention can be synthesized using the technique of solid phase used in the peptide synthesizer Applied Biosystems 430 (Applied Biosystems, Inc., 850 Lincoln Centre Drive, foster city, CA 94404), and synthetic cycles supplied by Applied Biosystems. Boc-protected amino acids and other reagents are commercially available from Applied Biosystems and other sellers of chemical products. Boc-chemistry sequences, using protocols dual attach, apply at the initial stage of the u-methylbenzhydrylamine resins for producing C-terminal carboxamides. For producing C-terminal acids can be used corresponding PAM-resin. Asp, Gln and Arg connect using a pre-prepared hydroxyl benzotriazole esters.

Another object of the present invention is the provision of a pharmaceutical composition comprising a compound of the present invention, which is present in a concentration from 0.1 mg/ml to 25 mg/ml, and where said composition has a pH from 3.0 to 9.0. The composition may further comprise a buffer system, preservative(s), tonicity agent(s), chelating agent(s), stabilizers and surfactants. In one embodiment the pharmaceutical composition is an aqueous composition, i.e. a composition comprising water. Such a composition typically is a p�the target or suspension. In an additional embodiment the pharmaceutical composition is an aqueous solution. The term "aqueous composition" means a composition that contains at least 50% mass/mass of water. Similarly, the term "aqueous solution" means a solution containing at least 50% weight/weight of water, and the term "aqueous suspension" is defined as a suspension containing at least 50 mass%/mass of water.

In another embodiment of the pharmaceutical composition is a lyophilized composition, in which the doctor and the patient adds solvents and/or diluents before use.

In another embodiment of the pharmaceutical composition is a dried composition (e.g. freeze-dried or spray-dried) ready for use without any prior dissolution.

In another aspect the invention relates to a pharmaceutical composition, comprising an aqueous solution of the compounds according to the present invention, and a buffer, where the specified compound is present in a concentration from 0.1 mg/ml or above, and where said composition has a pH from about 3.0 to about 9.0 to. In another embodiment the pH of the composition is from about 7.0 to about 9.5. In another embodiment, the implementation of�of bretania pH of the composition ranges from approximately 3.0 to approximately 7,0. In another embodiment the pH of the composition is from about 5.0 to about 7.5. In another embodiment the pH of the composition ranges from about 7.5 to about 9.0 to. In another embodiment the pH of the composition ranges from about 7.5 to about 8.5. In another embodiment the pH of the composition is from about 6.0 to about 7.5.

In another embodiment the pH of the composition is from about 6.0 to about 7.0 is. In another embodiment the pH of the composition is from 8.0 to 8.5.

In an additional embodiment, the buffer is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, onesemester sodium phosphate, dibasic sodium phosphate, sodium phosphate and Tris(gidroksimetil)aminomethane, bicine, Tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the invention.

In an additional embodiment, the composition further includes a pharmaceutically acceptable preservative. In an additional embodiment, the preservative SEL�be beneficial to 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 and thimerosal, bronopol, benzoic acid, imagemotion, chlorhexidine, dehydroacetate sodium, chlorocresol, ethyl-p-hydroxybenzoate, benzethonium, chlorphenesin (3P-chlorphenoxamine-1,2-diol) or mixtures thereof. In a variant implementation of the preservative is phenol or m-cresol. In an additional embodiment, the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In an additional embodiment, the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In an additional embodiment, the preservative is present in a concentration of 5 mg/ml to 10 mg/ml. In an additional embodiment, the preservative is present in a concentration of 10 mg/ml to 20 mg/ml. Each of these specific preservatives constitutes an alternative embodiment of the invention. The use of a preservative in pharmaceutical compositions is well known to specialists in this field. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In an additional embodiment, the composition also VC�uchet isotonic agent. In an additional embodiment, the isotonic agent is selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, amino acids (e.g. L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), alditol (for example, glycerol (glycerine), 1,2-PROPANEDIOL (propylene glycol), 1,3-PROPANEDIOL, 1,3-butanediol), polyethyleneglycol (e.g. PEG400), or mixtures thereof. In a variant implementation of the isotonic agent is a glycol. You can use any sugar such as mono-, di - or polysaccharides, or water soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na. In one embodiment, the implementation of additive sugar is a sucrose. Sugar alcohol is defined as a C4-C8-hydrocarbon, having at least one-HE group, and includes, for example, mannitol, sorbitol, Inositol, indicate, dulcet, xylitol and Arabic. In one embodiment, the implementation of the sugar alcohol additive is a mannitol. Sugar and sugar alcohols mentioned above can be used separately or in combination. Certain restrictions on your use of�creating the number does not exist, provided that the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely impact on the stabilizing effects achieved using the methods of the invention. In one embodiment of the implementation, the concentration of the sugar or sugar alcohol is in the range from about 1 mg/ml to about 150 mg/ml. In an additional embodiment, the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In an additional embodiment, the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In an embodiment, the isotonic agent is present in a concentration of 5 mg/ml to 7 mg/ml. In an additional embodiment, the isotonic agent is present in a concentration of 8 mg/ml to 24 mg/ml. In an additional embodiment, the isotonic agent is present in a concentration of 25 mg/ml to 50 mg/ml. Each of these specific isotonic agents constitutes an alternative embodiment of the invention. The use of an isotonic agent in pharmaceutical compositions is well known to specialists in this field. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In an additional embodiment, the composition also on�denotes complexing agent. In an additional embodiment the complexing agent is selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid and aspartic acid, and mixtures thereof. In an additional embodiment the complexing agent is present in a concentration from 0.1 mg/ml to 5 mg/ml. In an additional embodiment the complexing agent is present in a concentration from 0.1 mg/ml to 2 mg/ml. In an additional embodiment the complexing agent is present in a concentration of 2 mg/ml to 5 mg/ml. Each of these specific complexing agents constitutes an alternative embodiment of the invention. The use of complexing agent in pharmaceutical compositions is well known to specialists in this field. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In an additional embodiment, the composition also contains a stabilizer. The use of a stabilizer in pharmaceutical compositions is well known to specialists in this field. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In particular, the compositions according to the invention are stabilized liquid pharmaceutical com�osili, whose therapeutically active components include a polypeptide that possibly demonstrates the formation of aggregates during storage in liquid pharmaceutical formulations. By "education units" means the physical interaction between the polypeptide molecules, which leads to the formation of oligomers, which may remain soluble, or large visible aggregates that precipitate from the solution. "During storage" means a liquid pharmaceutical composition or composition which, when prepared, is not immediately administered to a subject. Preferably, after preparation, it is packaged for storage, either in liquid form, frozen, or dried form for subsequent recovery in liquid form or in another form suitable for administration to a subject. By "dried form" is meant a liquid pharmaceutical composition and the composition is dried either by freeze drying (i.e., lyophilization; see, for example, Williams and Polli (1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) in Spray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1 169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11: 12-20), or drying of air (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser (1991) Biopharm. 4:47-53). The formation of aggregates of the polypeptide during storage of the Jew�Oh pharmaceutical composition can adversely affect biological activity of that polypeptide, leading as a result to loss of therapeutic efficacy of the pharmaceutical composition. In addition, the formation of aggregates can cause other problems, such as blockage of tubing, membranes, or pumps in the case where the polypeptide-containing pharmaceutical composition is administered using an infusion system.

The pharmaceutical compositions according to the invention may additionally contain a number of amino acid base sufficient to decrease the formation of aggregates of the polypeptide during storage of the composition. By "amino acid base" means an amino acid or combination of amino acids, where any given amino acid is present either in free base form or in its salt form. In the case of combinations of amino acids, all amino acids can be present in the form of the free base can be present in their salt forms, or some of them may be present in the form of the free base, while others are in their salt forms. In one embodiment, the amino acids used in the preparation of the compositions according to the invention, are amino acids carrying a charged side chain, such as arginine, lysine, aspartic acid and glutamic acid. Any stereoisomer (i.e., L, D or �rity) a particular amino acid (e.g., methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof) or combinations of these stereoisomers, may be present in the pharmaceutical compositions according to the invention, provided that a certain amino acid is present either in its free base form or in its salt form. In one embodiment, the implementation uses the L-stereoisomer. The composition of the invention can also be with analogues of these amino acids. By "amino acid analogue" means derived from existing in the nature of the amino acid that brings about the desired effect of reducing the formation of aggregates of the polypeptide during storage of the liquid pharmaceutical compositions according to the invention. Suitable analogues of arginine include, for example, aminoguanidine, ornithine and N-monoethyl L-arginine, suitable analogs of methionine include ationen and buthionine and suitable cysteine analogues include S-methyl-L-cysteine. Like other amino acids, analogues amino acids included in the composition either in the form of the free base or in its salt forms. In an additional embodiment, amino acids and analogues amino acids are used in a concentration that is sufficient to prevent or delay aggregation of the protein. Additional options�ante embodiment of the invention can be added methionine (or other sulphuric amino acids and analogues amino acids) to inhibit oxidation of methionine residues to methanesulfonamide in case when the polypeptide acting as therapeutic agent is a polypeptide comprising at least one methionine residue susceptible to such oxidation. By "inhibition" refers to the minimum accumulation of varieties of oxidized methionine over time. Inhibition of oxidation of methionine leads to greater conservation of the polypeptide in its proper molecular form. You can use any stereoisomer of methionine (L or D) or combinations thereof. The amount that should be added should be an amount sufficient to inhibit oxidation of methionine residues, so that the number of methanesulfonic is acceptable to regulatory factors. Typically, this means that the composition contains not more than about 10% to about 30% of methanesulfonamide. In most cases this can be achieved by adding methionine, so that the ratio of added methionine to methionine residues ranges from about 1:1 to about 1000:1 , such as from 10:1 to about 100:1.

In an additional embodiment, the composition further comprises a stabilizer selected from the group of polymers with high molecular weight or low molecular weight compounds. In an additional embodiment the article�beliator chosen from polyethylene glycol (e.g., PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydrocellulose or derivatives thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulphur-containing substances as monothioglycerol, thioglycolic acid and 2-methylthioethyl, and different salts (e.g. sodium chloride). Each of these specific stabilizers constitutes an alternative embodiment of the invention.

Pharmaceutical compositions may also contain additional stabilizing agents, which further increase the stability of their therapeutically active polypeptides. Of particular interest stabilizing agents of the present invention include, as non-limiting examples, methionine and EDTA, which protect the polypeptide against oxidation of methionine, and nonionic surfactant, which protects the polypeptide against aggregation associated with freeze-thawing or mechanical shearing.

In an additional embodiment, the composition further includes a surfactant. In another embodiment the pharmaceutical composition comprises two different surfactants. The term "surfactant" as used herein refers to any molecules or ions, �contain a quiet water-soluble (hydrophilic) part, head and fat-soluble (lipophilic) segment. Surfactants accumulate preferentially at the interface, while the hydrophilic part is oriented to water (hydrophilic phase), and the lipophilic part to an oil or hydrophobic phase (i.e. glass, air, oil, etc.). The concentration at which surfactants begin to form micelles is known as the critical micelle concentration or CMC. In addition, surfactants reduce surface tension of the liquid. Surfactants also known as amphipathicity connection. The term "detergent" is a synonym used for surfactants in General.

Anionic surfactants can be selected from the group: chenodeoxycholic acid, sodium salt of chenodeoxycholic acid, halyevoy acid, dehydrocholic acid, deoxycholic acid, methyl ester of deoxycholic acid, digitonin, digitoxigenin, N,N-dimethyldodecylamine N-oxide, docusate sodium, nitroglycerindiscount acid, glycocholic acid hydrate, glycometabolic acid monohydrate, sodium salt glycometabolic acid, sodium salt glycometabolic acid, glycometabolic acid 3-sulfatonitrata salt, glycols�kalavai acid ethyl ester, sodium salt of N-lauroylsarcosine, sodium salt N-lauroylsarcosine, N-lauroylsarcosine, N-lauroylsarcosine, lithium dodecyl sulfate, iodine solution, 1-octanesulfonic acid sodium salt, 1-octanesulfonic acid sodium salt, 1-butanesulfonate sodium, 1-decanesulfonate sodium, 1-dodecanesulfonate sodium, 1-heptanesulfonate sodium, 1-heptanesulfonate sodium, 1-nonsulfate sodium, 1-propanesulfonate sodium monohydrate, 2-bromoethanesulfonate sodium, it consists of sodium hydrate, the bile of an ox or sheep, it consists of sodium hydrate, holeta sodium, desoxycholate sodium, sodium dodecyl sulfate, sodium dodecyl sulfate, hexanesulfonate sodium, octisalate sodium, pentanesulfonate sodium, taurocholate sodium, taurochenodeoxycholate acid sodium salt, taurodeoxycholic acid sodium salt monohydrate, tauralaukio acid 3-sulfatization salt, tauroursodeoxycholic acid sodium salt, Trizma®of dodecyl sulfate, DSS (docusate sodium, CAS registry [577-1 1-7]), docusate calcium, CAS registry [128-49-4]), docusate potassium, CAS registry [7491-09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate), dodecylphosphocholine (FOS-choline-12), decylphosphonic (FOS-choline-10), nonlipophilic (FOS-choline-9), dipalmitoylphosphatidyl acid, kapilavatthu sodium and/or ursodeoxycholic acid.

Cation�s surfactants can be chosen from the group: , benzylaniline, , , , dimethyldioctadecylammonium, dodecyltrimethylammonium, dodecyltrimethylammonium, dodecyltrimethylammonium, , hexadecyltrimethylammonium, hexadecyltrimethylammonium, polyoxyethylene(10)-N-tallow-1,3 - diaminopropane, transhipped and/or trimethyl(tetradecyl)ammoniumbromide.

Nonionic surfactants can be chosen from the group of: BigCHAP, Bis(]), block-copolymers, such as block copolymers of polyethylenoxide/polypropyleneoxide such as poloxamer, poloxamer 188 and poloxamer 407, Brij®35, Brij®56, Brij®72, Brij®76, Brij®92V, Brij®97, Brij®58P, Cremophor®EL, ether, N-decanoyl-N-methylglucamine, n-decanoyl-N-methylglucamide, alkylpolyglycoside, ethoxylated castor oil, geptaetilenglikolya ether, ether, ether, ether, ether, ether, hexamethylendiamine�tradizionale ether, lgepal CA-630, lgepal CA-630, Methyl-6-0-(N-heptylcarbinol)-beta-D-glucopyranosid, ether, N-nonanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, octamethyltetrasiloxane ether, ether, ether, ether, ether, octyl-β-D - glycopyranoside, pentamethylenetetrazol ether, ether, ether, ether, ether, pentamethylenetetrazol ether, polyethylenepolypropylene ether, polyethylene glycol ether W-1, polyoxyethylene 10 tridecylalcohol ether, polyoxyethylene 100 stearate, polyoxyethylene 20 isohexadecane ether, ether polyoxyethylene 20, polyoxyethylene 40 stearate, polyoxyethylene 50 stearate, polyoxyethylene 8 stearate, polyoxyethylene bis(imidazolylidene), polyoxyethylene 25 propilenglikolstearat, saponin from Quillaja bark, Span®20, Span®40, Span®60, Span®65, Span®80, Span®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, �IPA NP-40, tergitol, type NP-7, tergitol, type NP-9, tetradecyl-β-D-maltoside, tetraethylethylenediamine ether, ether, ether, triethyleneglycoldinitrate ether, Triethylenetetramine ether, ether, triethyleneglycoldinitrate ether, ether, Triton CF-21 , CP Triton-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, Triton®X-100, Triton®X-114, Triton solution®X-165, Triton solution®X-305, Triton®X-405, Triton®X-45, Triton®X-705-70, TWEEN®20, TWEEN®40, TWEEN®60, TWEEN®6, TWEEN®65, TWEEN®80, TWEEN®81, TWEEN®85, tyloxapol, sphingophospholipids (sphingomyelin), and sphingoglycolipids (ceramides, gangliosides), phospholipids and/or n-undecyl β-D - glycopyranoside.

Zwitterionic surfactants can be chosen from the group of: CHAPS, CHAPSO, 3-(decollimation)propanesulfonate inner salt, 3-(dodecyldimethylammonium)propanesulfonate inner salt, 3-(dodecyldimethylammonium)propanesulfonate inner salt, 3-(N,N-dimethyldiallylammonium)propanesulfonate, 3-(N,N-dimethylethanolamine)propanesulfonate, 3-(N,N-dimethylammonio)propanesulfonate inner salt -(N,N-dimethylphenethylamine)propanesulfonate, N-alkyl-N,N-dimethylammonio-1-propanesulfonate, 3-jalamid-1 propyltrimethylammonium-1-propanesulfonate, dodecylphosphocholine, myristoyltransferase, Zwittergent 3-12 (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent 3-10 (3-(decollimation)-propanesulfonate inner salt), Zwittergent 3-08 (3-(octylammonium)propanesulfonate), glitserofosfolipidy (lecithins, Catalina, phosphatidyl serine), glyceroglycolipids (galactopyranoside), alkyl, alkoxyl (alkyl ether), alkoxy(alkyl ether)-derivatives of lysophosphatidyl and phosphatidylcholines, for example, eurologic and myristoylated derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, and modifications of the polar group heads, which is a choline, ethanolamine, phosphatidic acid, serine, threonine, glycerol, Inositol, lysophosphatidylserine and lysophosphatidylcholine, acylcarnitines and derivatives, Nbeta-acylated derivatives of lysine, arginine or histidine, or derivatives of lysine or arginine with acylated side chain, the Nbeta-acylated derivatives of dipeptides comprising any combination of lysine, arginine or histidine and a neutral or acidic amino acid, Nbeta-acylated derivative of a Tripeptide comprising any combination of a neutral amino acid and two charged amino acids, or power�surface-active agent can be chosen from the group of imidazoline derivatives, long-chain fatty acids and salts of this C6-Ci2(e.g. oleic acid and Caprylic acid), N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic (alkyl-aryl-sulfonates) monovalent surfactants, palmitoylethanolamide-L-serine, lysophospholipids (for example, 1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine or threonine), or mixtures thereof.

The term "alkyl-polyglucoside" as used herein refers to linear or branched C5-C20-alkyl, -alkenyl or-valkininkai chain, which is substituted one or more glucoside parts such as maltose, saccharide, etc. Options for the implementation of these alkyl-polyglucosides include C6-18-alkylpolyglucoside. Specific embodiments of these alkylpolyglucosides include chains with an even number of carbons, such as alkyl chains (C6, C8, C10, C12, C14, C16, C18and C20. Specific embodiments of glucosidic parts include paranoid, glycopyranosyl, maltose, maltotriose and sucrose. In embodiments of the invention to an alkyl group attached less than 6 glucosidic parts. In embodiments of the invention to an alkyl group attached less than 5 glucosidic parts. In�the options of implementation of the invention to an alkyl group attached less than 4 glucosidic parts. In embodiments of the invention to an alkyl group attached less than 3 glycosidic parts. In embodiments of the invention to an alkyl group attached less than 2 glucosidic parts. Specific embodiments of alkylpolyglucosides represent Alkylglucoside, such as n-decyl β-D-glycopyranoside, decyl β-D-maltobiose, dodecyl β-D-glucopyranosid, n-dodecyl β-D-maltoside, n-dodecyl β-D-maltoside, n-dodecyl β-D-maltoside, tetradecyl β-D-glycopyranoside, decyl β-D-maltoside, hexadecyl β-D-maltoside, decyl β-D-maltotrioside, dodecyl β-D-maltotrioside, tetradecyl β-D-maltotrioside, hexadecyl β-D-maltotrioside, n-dodecyl-sucrose, n-decyl-sucrose, nanocapacitors, monolaurate, monomerization and monopalmitate. The use of surface-active substances in pharmaceutical compositions is well known to specialists in this field. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In an additional embodiment, the composition further comprises protease inhibitors such as EDTA (ethylenediaminetetraacetic acid) and benzamidine HCl, and you can also use other available protease inhibitors. The use of protease inhibitor is particularly useful in pharmaceutical compositions, comprising winters�genes of proteases, to inhibit the autocatalysis.

It is possible that peptide in the pharmaceutical composition of the present invention may include other components. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, blocking agents, tone modifiers, chelating agents, metal ions, fatty fillers, proteins (e.g. human serum albumin, gelatin or proteins) and zwitterion (e.g., an amino acid such as betaine, lysine, arginine, glycine, lysine and histidine). Such additional ingredients, of course, should not adversely affect the overall stability of the pharmaceutical composition of the present invention.

Pharmaceutical compositions containing a compound in accordance with the present invention, can be administered to the patient in need of such treatment at several sites, for example, in local areas, for example, skin and mucosal, areas which avoid absorption, for example, the introduction into the artery, vein, heart, and plots which involve absorption, for example, the introduction into the skin, under the skin, in muscle or in the abdomen.

The introduction of pharmaceutical compositions in accordance with the invention can be realized patients in need of such treatment, using several routes of administration, for example, lingual sublingual, buccal, in the mouth, oral, in the stomach and intestines, nazalnam, pulmonary, for example, in the bronchioles and alveoli or a combination thereof, epidermal, dermal, transdermal, vaginal, rectal, ophthalmic, for example, through the conjunctiva, realnum and parenteral.

The compositions of the present invention can be applied in several dosage forms, for example, in the form of solutions, suspensions, emulsions, microemulsions, serial emulsions, foams, ointments, pastes, plasters, ointments, tablets, coated tablets, rinses, capsules, for example hard gelatin capsules and soft gelatin capsules, suppositories, rectal capsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal ointments, injection solution, solutions, prepared on the spot, for example, the preparation of the gel in place, setting in place, precipitation, crystallization in place, an infusion solution, and implants. The composition of the invention may also be mixed with, or attached, for example through covalent, hydrophobic and electrostatic interactions, media, pharmaceuticals, drug delivery and improved drug delivery in a�x further enhance the stability of the compounds of the present invention, increase bioavailability, increase solubility, decrease adverse effects, achieve chronotherapy well known to specialists in this field, and increasing the compliance of patients, or any combination thereof. Examples of carriers, drug delivery system and advanced drug-delivery systems include, as non-limiting examples of polymers, for example cellulose and derivatives, polysaccharides, for example dextran and derivatives, starch and derivatives, polyvinyl alcohol, acrylate and methacrylate polymers, polylactic and polyglycol acid and block copolymers, polyethylene glycols, carrier proteins, for example albumin, gels, for example, the system thermorelay, for example block co-polymeric systems well known to specialists in this field, micelles, liposomes, microspheres, nanoparticles, liquid crystals and dispersions, phase L2 and dispersion of this, known to experts in the field of phase behavior in lipid-water systems, polymeric micelles, compound emulsion, samoemulgirutisa, semimicroanalysis the cyclodextrins and derivatives thereof, and dendrimers.

The compositions of the present invention are useful in the preparation of solids, semi-solid substances, powders and solutions for pulmonary administration of the compounds of infusion�him to the invention, using, for example, metered dose inhaler, dry powder inhaler and a nebulizer, all devices are well known to specialists in this field. The compositions of the present invention is particularly useful in the formation of controlled, sustained, prolonged delivery systems and delivery systems with delayed and sustained release of the drug. More specifically, as non-limiting examples, the compositions are useful in the formation of systems of parenteral controlled release and sustained release (both systems leading to multiple to reduce the number of injections), well known to specialists in this field. Even more preferably, the system of controlled release and sustained release is administered subcutaneously. Without limiting the scope of the invention, useful examples of the controlled release system and compositions are hydrogels, oily gels, liquid crystals, polymeric micelles, microspheres, nanoparticles. How to create controlled release systems useful for compositions of the present invention include, as non-limiting examples, crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion, homogenization p�and high pressure, encapsulation, spray drying, encapsulation, coacervation, phase separation, solvent evaporation to produce microspheres, extrusion and supercritical fluid processes. General reference is made to Handbook of Pharmaceutical Controlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences vol. 99: Protein Formulation and Delivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000). Parenteral administration can be performed using subcutaneous, intramuscular, intraperitoneal or intravenous injection using a syringe, optionally, the syringe like a pen. Alternatively, parenteral administration can be performed using an infusion pump. Another option is the composition, which can be a solution or suspension, or powder for the introduction of compounds of the present invention in the form of a nasal or pulmonal liquid or powder spray. As a further option, the pharmaceutical composition containing the compound according to the invention, can also be adapted to transdermal administration, e.g. by needle-free injection or from a patch, optionally iontophoretic patch, or insertion through the mucous membrane, for example, buccal. Compounds of the present invention can be injected with the pulmonary means in the filler as a solution, suspension or dry powder using any investigtion devices suitable for pulmonary drug delivery. Non-limiting examples include three main types of aerosol-forming devices for pulmonary drug delivery, and may include jet or ultrasonic nebulizers, metered-dose inhalers or dry powder inhalers (see Yu J, Chien YW. Pulmonary drug delivery: Physiologic and mechanistic aspects. Crit Rev Ther Drug Carr Sys 14(4) (1997) 395-453).

On the basis of a standardized methodology for testing the aerodynamic diameter (daof a particle is defined as the geometric equivalent diameter of the reference standard spherical particles of unit density (1 g/cm3). In the simplest case, for spherical particles, darefers to the reference diameter (d) as a function of the square root of the density ratio, as described in:

Modifications to this relationship occur for non-spherical particles (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" well described and known in the field under consideration (cp. 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 mean effective aerodynamic diameter (MMEAD) are used interchangeably, represent statistically� parameters and empirically describe the size of aerosol particles, in relation to their potential deposition in the lungs, regardless of the actual shape, size and density (Ms. 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). MMAD is usually calculated by the measurement conducted with the use of the inertial separator, a tool that measures the inertial behavior of particles in the air. In an additional embodiment, the implementation, the composition can be translated into aerosol using any known technologies aerosolization, such as aerosol spraying, to achieve a MMAD of aerosol particles less than 10 microns, more preferably, from 1 to 5 μm, and most preferably from 1 to 3 μm. The preferred particle size is based on the most effective size for drug delivery to the deep lung, where the protein is optimally absorbed (cp. Edwards DA, Ben-Jebria A, Langer A, Recent advances in pulmonary drug delivery using large, porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385).

Deposition in the deep lung pulmonary formulations containing the compound of the present invention can optionally be further optimized by using modifications of the inhalation techniques, such as, as non-limiting examples: slow inhalation flow (e.g. 30 l/min), breath holding and the timing of activation.

The term "stable composition" relative�GSI to a composition with enhanced physical stability, increased chemical stability or increased physical and chemical stabilities.

The term "physical stability" of the protein composition, as used herein, refers to the propensity of the protein to form biologically inactive and/or insoluble aggregates of the protein as a result of exposure to the protein thermo-mechanical stresses and/or interaction with interfaces and surfaces that are destabilizing, such as hydrophobic surfaces and interfaces. Physical stability of the aqueous protein formulations is evaluated by visual inspection and/or measurement of turbidity after storage of the composition, placed in suitable containers (e.g. cartridges or vials) to mechanical/physical stress (e.g. agitation) at different temperatures for different periods of time. Visual inspection of the compositions is carried out in a strong focused light on a dark background. The turbidity of the composition is characterized by visual assessment, ranking the degree of turbidity, for example, on a scale from 0 to 3 (composition showing no turbidity corresponds to a visual score 0, and the composition showing visual turbidity in daylight corresponds to visual score 3). The composition is classified physically unstable in relation�AI protein aggregation, if it shows visual turbidity in daylight. Alternatively, the turbidity of the composition can be evaluated by simple turbidity measurement, well known to specialists in this field. Physical stability of the aqueous protein formulations can also be evaluated by using a spectroscopic agent or probe of the conformational status of the protein. The probe is preferably a small molecule, which binds non-native conformer of the protein. One little example-molecular spectroscopic probe the structure of protein is thioflavin So Thioflavin T is a fluorescent dye that is widely used for the detection of amyloid fibrils. In the presence of fibrils, and possibly also other protein configurations, thioflavin T gives rise to a new high excitation at 450 nm and enhanced emission at about 482 nm when bound to fibrillar protein form. Unrelated, thioflavin T, in essence, is not fluorescent at these wavelengths.

Other small molecules can be used as probes of changes in protein conformation from the native state to the state than native. For example, probes for "hydrophobic patches", which is associated mainly with exposed guide�ofonime the patches of protein. Hydrophobic patches, as a rule, are sunk in the tertiary structure of the protein in its native state, but become exposed as the protein begins to unfold or denature. Examples of such little-molecular spectroscopic probes are aromatic, hydrophobic dyes, such as anthracene, acridine phenanthrolin or similar. Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids such as phenylalanine, leucine, isoleucine, methionine and valine or similar.

The term "chemical stability" of the protein composition, as used herein, refers to chemical covalent changes in the protein structure leading to formation of products of chemical degradation with potentially lower biological activity and/or potential increased immunogenic properties compared to the native structure of the protein. Various products of chemical degradation can occur depending on the type and nature of the native protein and the environment, the effects of which the protein is exposed. Elimination of chemical degradation, most likely, will not be able to completely avoid, and increasing the number of products of chemical degradation is often observed during storage� and application of protein composition, as is well known to specialists in this field. Most proteins have a tendency to dezaminirovanie, the process in which the amide group of the side chain residues of glutamine or asparagine is hydrolysed with formation of free carboxylic acids. Other ways of degradation involves the formation of transformed products with high molecular weight, where two or more protein molecules are covalently linked together through parametervalue and/or disulfide interactions leading to formation of covalently bound dimer, oligomer and polymer degradation product (Stability of Protein Pharmaceuticals, Ahem. T. J. & Manning, M. C, Plenum Press, New York 1992). Oxidation (e.g., methionine residue) is another option chemical degradation. The chemical stability of protein formulations can be assessed by measuring the amount of the products of chemical degradation at different time points after exposure to different environmental conditions (the formation of degradation products can often be accelerated by, e.g., increasing temperature). The amount of each individual degradation product is often determined by separation of the products of degradation depending on the size of the molecule and/or charge using various chromatography methods (for example, size exclusion HPLC size�s and/or RP-HPLC).

Thus, as mentioned above, the "stable composition" refers to a composition with increased physical stability, increased chemical stability or increased physical and chemical stability. In most cases, the composition must be stable during use and storage (in accordance with the recommended conditions of use and storage) prior to the expiry date.

In one embodiment the pharmaceutical composition containing the compound of the present invention is stable for more than 6 weeks of usage and for more than 3 years of storage.

In another embodiment, the pharmaceutical composition containing the compound of the present invention is stable for more than 4 weeks of usage and for more than 3 years of storage. In an additional embodiment the pharmaceutical composition containing the compound of the present invention is stable for more than 4 weeks of usage and for more than two years of storage.

In yet another embodiment, the pharmaceutical composition containing the compound of the present invention is stable for more than 2 weeks of usage and for more than two years of storage.

In another aspect, the present invention relates to the use of compounds in accordance with the invention for the preparation of medicines.

The present invention also includes a salt-form analogues of GLP-1. An analogue of GLP-1 according to the invention may be sufficiently acidic or sufficiently basic to react with any of a number of inorganic bases, and inorganic acids to form salts. Acid, commonly used for formation of salts with the addition of the acid are inorganic acids such as hydrochloric acid, Hydrobromic acid, itestosterone acid, sulfuric acid, phosphoric acid and the like, and organic acids such as p-toluensulfonate acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include sulfate, hydrogen sulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, fumarate, maleate, Butin-1,4-diet, hexyn-1,6-diet, benzoate, chlorobenzoate, mediashout, dinitrobenzoate, hydroxybenzoate methoxybenzoate, phthalate, sulfonate, killshot, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, salt of mandelic acid and the like. Preferred acid-additive salts are salts formed with mineral acids such as hydrochloric acid and Hydrobromic acid, and especially hydrochloric acid.

Basically additive salts include salts derived from inorganic bases, such as hydroxides of ammonium or alkali or alkaline earth metals, carbonates, bicarbonates and the like. Such bases useful in preparing the salts of the present invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate and the like. Particularly preferred salt forms are analogues of GLP-1. Of course, when the compounds of the present invention are used for therapeutic purposes, these compounds can also be in the form of salt, but the salt must be pharmaceutically acceptable.

Modified analogues of GLP-1 according to the invention find numerous applications, including use as agents for treatment of diabetes, sedatives, agents for the treatment of diseases nerve�th system, applying the induction of anxiolytic action on the Central nervous system, the application to activate the Central nervous system, used for postoperative treatment and as a means for the treatment of insulin resistance.

A. Treatment of diabetes mellitus

Modified analogues of GLP-1 according to the invention, as a rule, normalize hyperglycemia through dependent insulinotropic mechanisms. As such, the modified analogues of GLP-1 are useful as primary agents for the treatment of diabetes mellitus type II and as extension agents for the treatment of diabetes mellitus type I.

The use of an effective amount of modified analogs of GLP-1 as a means for the treatment of diabetes has an advantage over non-modified GLP-1 as a more effective tool. As modified analogues of GLP-1 are more stable in vivo for effective treatment you can enter a smaller number of molecules. The present invention is particularly suitable for the treatment of patients with diabetes, both type I and type II, since the effect of the peptides depends on the concentration of glucose in the blood and, thus, the risk of hypoglycemic side effects are significantly reduced compared with the risk in the use of modern methods of treatment.

The present invention also provides a method of treating sah�rnogo of diabetes in the individual, where the method includes providing the number of modified analogs of GLP-1, sufficient to treat diabetes; where the composition comprises a modified analogue of GLP-1.

B. Treatment of diseases of the nervous system

Modified analogues of GLP-1 according to the invention also find use as sedatives. In one aspect of the invention, provided is a method of calming the subject of a mammal with impaired, leading to enhanced activation of the Central or peripheral nervous system, with the use of modified analogs of GLP-1 to the subject in an amount sufficient to provide a sedative and anxiolytic action on the subject. Modified analogues of GLP-1, you can enter intracerebroventricular, orally, subcutaneously, intramuscularly or intravenously. Such methods are useful for the treatment and improvement of conditions of the nervous system such as anxiety, a disorder of movements, aggression, psychosis, seizures, panic attacks, hysteria and a sleep disorder.

In a similar aspect, the invention includes a method of increasing activity of a subject mammal, comprising administering modified analogs of GLP-1 to a subject in an amount sufficient to provide catalytic effects on the subject. Preferably, the subject has a condition, which leads to with�iginio activation of the Central or peripheral nervous system. Modified analogues of GLP-1 are used, in particular, for treating or alleviating depression, schizoaffective disorders, sleep apnea, attention deficit disorder with poor concentration, memory loss, forgetfulness, and narcolepsy, to name a few of the States in which the excitation of the Central nervous system may be useful.

Modified analogues of GLP-1 according to the invention can be used to induce arousal for the treatment or alleviation of depression, schizoaffective disorders, sleep apnea, attention deficit disorder with poor concentration, memory loss, forgetfulness, and narcolepsy. therapeutic efficacy of treatment by modified analogues of GLP-1 can be controlled through interviews with the patient to assess their condition, by means of psychological/neurological testing or improve the symptoms associated with these conditions. For example, by means of monitoring the prevalence of narcoleptic attacks can evaluate the treatment of narcolepsy. As another example: the effect of modified analogs of GLP-1 on the ability of the subject to concentration or memory capabilities can be tested using any of a number of diagnostic tests are well known to specialists in this field.

C. Postoperative treatment

Mod�truth analogues of GLP-1 according to the invention can be used for postoperative treatment. The patient needs to be modified analogues of GLP-1 according to the present invention is about 1 to 16 h before surgery on the patient during surgery on the patient and after the operation on the patient for a period of time not more than 5 days.

Modified analogues of GLP-1 according to the present invention is administered in about sixteen hours to about one hour before your surgery. Period of time before the operation, during which the compound used in the present invention should be administered in order to reduce the catabolic effects of insulin resistance depends on several factors. These factors generally known to any doctor and include, most importantly, fast whether the patient or receives glucose in the form of infusion or beverage, or any other form of nutrition during the preparatory period before the operation. Other important factors include gender, weight and age of the patient, the severity of any inability to regulate the level of blood glucose, the underlying causes of inability to regulate the level of glucose in the blood, the anticipated severity of the injury caused by surgery, the route of administration and bioavailability, stability in the body, composition, and activity of compounds. The preferred time interval during which it is necessary to start the introduction of modified analogs of GLP-1, used�used in the present invention, is from about one hour to about ten hours before surgery. The preferred interval for the beginning of the introduction is from two hours to eight hours before surgery.

Insulin resistance after a certain type of surgery, elective abdominal surgery, is the deepest the first post-operative day, lasts at least five days and can take up to three weeks to normalize. Thus, the postoperative patient may need in the introduction of modified analogs of GLP-1 used in the present invention within a certain period of time after the trauma from the surgery, which will depend on factors, starving the patient or receives glucose in the form of infusion or beverage, or any other form of nutrition after surgery, as well as, as non-limiting examples, the sex, weight and age of the patient, the severity of any inability to regulate the level of glucose in the blood, leading causes of inability to regulate the level of glucose in the blood, the severity of the alleged injury, caused by surgical operation, the method of administration and bioavailability, stability in the body, composition and activity of the compound introduced. The preferred duration of the introduction connected�th, used in the present invention, is not more than five days after the operation.

D. Treatment of insulin resistance

Modified analogues of GLP-1 according to the invention can be used for the treatment of insulin resistance, regardless of their use in the postoperative treatment. Insulin resistance can be caused by a decrease in the binding of insulin to receptors on the surface of cells or a change in intracellular metabolism. The first type is characterized as reduced sensitivity to insulin, as a rule, can be overcome by increasing the concentration of insulin. The second type, which is characterized as a weakening of the response to insulin, cannot be overcome due to the large amount of insulin. Insulin resistance after trauma can be overcome with the help of doses of insulin, which is proportional to the degree of insulin resistance, and, thus, it is obvious that it is caused by decreased sensitivity to insulin.

Dose modified analogs of GLP-1, effective to normalize the level of glucose in the blood of the patient will depend upon a number of factors that include, as non-limiting examples, gender, weight and age of the patient, the severity of any inability to regulate the level of blood glucose, the underlying causes of inability to regulate the level �lukosi in the blood, the alleged severity of the injury caused by surgery, the route of administration and bioavailability, stability in the body, composition, and activity.

The ability of analogs of GLP-1 to stimulate insulin secretion can be determined by the provision of an analog of GLP-1 for cultured animal cells, such as cell line RIN-38 insulinoma rats, and control the release of immunologically reactive insulin (IRI) on Wednesday. Alternatively, you can inject an analogue of GLP-1 in animal and control the level of immunological reactive insulin (IRI) in plasma.

The presence of IRI detected by radioimmunoassay, which can specifically detect insulin. You can use any radioimmunoassay capable of detecting the presence of IRI; one such analysis is a modification of the method Albano,J. D. M. et al., Acta Endocrinol, 70: 487-509 (1972). In this modification uses a phosphate/albumin buffer with pH 7.4. The incubation is prepared with the consecutive addition of 500 μl of phosphate buffer, 50 µl perfoirmance sample or insulin standard rats in perfusate, 100 μl of antisera against insulin (Wellcome Laboratories; dilution 1:40000) and 100 ál of [125I] insulin, with a total volume of 750 μl in a disposable glass test tube 10 x 75 mm. After incubation for 2-3 days at 4ºC free insulin from�elaut from associated with insulin antibody by coal separation. The sensitivity analysis is 1-2 uU/ml to measure the release of IRI in the cell culture medium of cells grown in tissue culture, in proinsulin preferably include a radioactive label. Although you can use any radioactive label capable of labeling a polypeptide, to obtain labeled proinsulin is preferable to use the3H-leucine.

Determining whether an analogue of GLP-1 insulinotropic properties, can also be performed using pancreatic infusion. In situ analysis of the isolated pancreas perfusion rats is a modification of the method Penhos, J, C., et al., Diabetes, 18: 733-738 (1969). Hungry male albino rats line Charles river weighing 350-600 g anastasiopolis by intraperitoneal injection of Amytal Sodium (Eli Lilly and Co, 160 ng/kg). The blood vessels of the kidneys, adrenal glands, stomach and the lower portion of the colon was ligated. The entire intestine was removed, with the exception of about four centimeters of the duodenum and the descending colon and rectum. Thus, only a small portion of the intestine is perfusion that minimizes the potential impact of intestinal substances with glucagon-like immunoreactivity. The perfusate is a modified bicarbonate buffer kreba-ringer with 4% dextran T70 and 0.2% bovine BBQ cyberotic�wow albumin (fraction V), and with blowing 95% O2and 5% CO2. Used non-pulsating flow, 4-channel roller-bearing pump (Buchler polystatic, Buchler Instruments Division, Nuclear-Chicago Corp.), and the transition from one source of a perfusate to another is carried out by switching the 3-way tap. The way in which perfusion is performed, control and analysis, follows the way Weir, G. C., et at. J. Clin. Investigat. 54: 1403-1412 (1974), which is incorporated herein by reference.

Treatment a compound in accordance with the present invention can also be combined with a second or more pharmacologically active substances, e.g. selected from antidiabetic agents, anti-obesity agents that regulate appetite, antihypertensive agents, agents for the treatment and/or prevention of complications caused by or associated with diabetes and agents for the treatment and/or prevention of complications and disorders caused by or associated with obesity. Examples of these pharmacologically active substances are: insulin, sulfonylureas, biguanides, meglitinides, glucosidase inhibitors, glucagon antagonists, inhibitors of DPP-IV (dipeptidyl peptidase-IV) inhibitors of liver enzymes involved in stimulation of gluconeogenesis and/or glycogenosis, modulators of glucose uptake, compounds modifying the lipid metabolism such as hypogl�khimicheskie agents, as inhibitors of HMG CoA (statins) that inhibit the polypeptides of the stomach (GIP analogs), compounds that reduce the digestibility of food, RXR agonists and agents acting on the ATP-dependent potassium channel of β-cells, cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin, pravastatin, simvastatin, probucol, dextrothyroxine, nateglinide, Repaglinide; β-blockers, such as alprenolol, atenolol, pindolol, timolol, propranolol and metoprolol, ACE (angiotensin converting enzyme) inhibitors such as benazepril, captopril, enalapril, fosinopril, lisinopril, elatioris, quinapril and ramipril, calcium channel blockers, such as nifedipine, felodipine, nicardipine, isradipine, nimodipine, diltiazem and verapamil, and α-blockers, such as doxazosin, urapidil, prazosin and terazosin; agonists CART (cocaine-amphetamine-regulated transcript), antagonists of NPY (neuropeptide Y), PYY agonist, agonists PYY2, agonists PYY4, mixed agonists PPY2/PYY4, MC4 agonists (melanocortin 4), antagonists of orexin, agonists of TNF (tumor necrosis factor), an agonist of CRF (corticotropin releasing factor)CRF antagonists BP (a protein that binds corticotropin releasing factor) agonists urocortin, β3 agonists, agonists, MSH (melanocyte-stimulating hormone), antagonists of MCH (melanocyte-concentrating hormone) agonists, CCK (cholecystokinin), inhibitors of serotonin popcornopolis, inhibitors of serotonin and norepinephrine re-uptake, mixed serotonin and noradrenergic compounds, 5HT agonists (serotonin), bombesin agonists, Galanin antagonists, growth hormone; compounds which release growth hormone; TRH agonists (thyrotropin releasing hormone), modulators, UCP 2 or 3 (release protein 2 or 3), leptin agonists, DA agonists (bromocriptine, depressin), inhibitors of lipase/amylase, RXR modulators (receptor retinoid X), TR agonists β; antagonists of histamine H3 agonists or antagonists of inhibitory polypeptides of the stomach (GIP analogs), gastrin and gastrin analogues. Treatment a compound in accordance with the present invention can also be combined with surgery - surgery which affects the level of glucose and/or lipid homeostasis, such as gastric banding or gastric bypass.

It should be understood that any suitable combination of the compounds in accordance with the invention with one or more of the aforementioned compounds and optionally one or more additional pharmacologically active substances, is considered in the framework of the present invention.

The present invention is further illustrated by the following examples, which, however, should not be construed as limiting the scope of protection. The features disclosed in the above description and in the trail�actual operation examples may constitute, individually or in any combination, the material for realizing the invention in various forms.

As an illustration, the following examples are given that help to describe how to make and use various embodiments of the invention. These examples in no way imply a limitation of the scope of the invention.

EXAMPLES

Abbreviations used:

r.t: Room temperature;

DIPEA: diisopropylethylamine;

H2O: water;

CH3CN: acetonitrile;

DMF: N,N dimethylformamide;

HBTU: 2-(1H-benzotriazole-1-yl-)-1,1,3,3 tetramethyluronium hexaflurophosphate;

Fmoc: 9 H-fluoren-9-ylmethoxycarbonyl;

BOC: tert-butyloxycarbonyl;

OtBu: tert-butyl ether;

-tBu: tert-butyl Trt: triphenylmethyl;

Pmc: 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl;

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

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

Mtt: 4-medicrity;

Mmt: 4-methoxytrityl;

DCM: dichloro methane;

TIS: triisopropylsilane;

TFA: trifluoroacetic acid;

Et2O: diethyl ether;

NMP: 1-methyl-pyrrolidin-2-he;

HOAt: 1-hydroxy-7-asobancaria;

HOBt: 1-hydroxy-benzotriazole;

DIC: diisopropylcarbodiimide.

Synthesis Q

Q as, for example, those of formula II are commercially available, known in the literature �can be conveniently prepared in various ways, known to specialists in this field. One common method of synthesis of formula II, where X, Y, and R3represent hydrogen, which has been described (S. Oishi etc., J. Chem. Soc, Perkin Trans. 1, 2001, 2445), shown in Scheme 1.

Q as, for example, those of formula II are commercially available, known in the literature or may be conveniently prepared in various ways known to specialists in this field. One common method of synthesis of formula II, where X represents a fluorine, and Y and R3represent hydrogen, shown in Scheme 2. Key source material 4 is commercially available, known in the literature (T. Narumi et al., Tetrahedron, 2008, 64, 4332)

Q as, for example, those of formula IIIa are commercially available, known in the literature or may be conveniently prepared in various ways known to specialists in this field. One common method of synthesis of formula III, where X represents trifluoromethyl, Z represents nitrogen, Y, R1, R2and R3represent hydrogen, shown in Scheme 3. Key source material 3,3,3-trifluoro-nitropropene 6 is commercially available, known in the literature. Aza-Michael-addition of the diester of glutamic acid to 3,3,3-trifluoro-nitro�rapino 6 is carried out stereocamera way (M. Molteni et al., Org. Lett., 2003, J, 3887).

Q as, for example, those of the formula IIIb are commercially available, known in the literature or may be conveniently prepared in various ways known to specialists in this field. One common method of synthesis of formula III, where X represents trifluoromethyl, Z is a nitrogen, R2represents alkyl, Y, R1and R3represent hydrogen, shown in Scheme 4. The initial material 10 is commercially available, known in the literature (J. Andre et al., Eur. J. Org. Chem. 2004, 1558). Key stage assumes stereospetsifichno substitution of SN2 triflate 11 diester of glutamic acid 7 (P. O'shea et al., J. Org. Chem. 2009, 5, 1605).

Alternatively, Q as, for example, those of formula IIIb, prepare with the help of another synthetic method, where X represents trifluoromethyl, Z represents a nitrogen; R2represents alkyl, Y, R1and R3represent hydrogen, shown in Scheme 5. The key starting material 10 (J. Andre et al., Eur. J. Org. Chem. 2004, 1558) is oxidized with the formation of trifluoromethylation 13. The subsequent formation of imina conclude the presence of the base. The final stage involves stereospetsifichno reducing imina 14 borhydride sodium� or borhydride zinc, with the formation of the desired miasteniceskih isomers 12 and 15 And (G. Hughes et al., Angew Chem. Int. Ed. 2007, 46, 1839).

Alternatively, Q as, for example, those of formula IIIb, prepare with the help of another synthetic method, where X represents trifluoromethyl, Z represents a nitrogen; R2represents alkyl, Y, R1and R3represent hydrogen, shown in Scheme 6. Condensation is known to the source material of the diamine 16 (M. Mandal et al., J. Am Chem. Soc. 2002, 6538) the aldehyde 17 gives Yiming 18. Subsequent diastereoselective reaction imina Strecker-type 18 with TMSCN conclude the presence of a catalytic amount of a Lewis acid. The final stage involves the hydrolysis of the cyano intermediate with the formation of diastolicheskogo 12 isomer A (F. Huguentt et al., J. Org. Chem. 2006, 71, 7075).

Q as, for example, those of formula IV are commercially available, known in the literature or may be conveniently prepared using various methods known to experts in this field. One General method of synthesis of compounds with formula IV, where X represents oxygen; Z is carbon; R2represents alkyl, W, Y, R1and R3represent hydrogen, shown in Scheme 7. The starting material β-ketoester 19 is commercial access�th, known in the literature (R. Hoffman et al., J. Org. Chem. 1999, 64, 1558). Alkylation of β-keeeper 19 triflate 20, followed by decarboxylation and removing the protective group, R9to provide geometrization 21 (R. Hoffman et al., J. Org. Chem. 1999, 64, 1558, P. S. Dragovich et al., J. Med. Chem. 1999, 42, 1203).

Q as, for example, those of formula IV, where X represents oxygen; Z is carbon; R2represents alkyl, W represents fluorine; Y, R1and R3represent hydrogen, are commercial available, known in the literature or may be conveniently prepared in various ways known to specialists in this field. One General method of synthesis thereof of the formula IV shown in Scheme 8. The source material trailerbuy β-ketoester 22 is commercially available, or prepared according to the literature (R. Hoffman et al., J Org. Chem. 1999, 64, 1558). Alkylation of β-keeeper 22 triflate 20, followed by decarboxylation to give geometrization 23, then 23 is converted to the corresponding Z-TMS enlever and foryouth Selectfluor, and the final release gives monitorcommunity 25 (R. Hoffman et al., J. Org. Chem. 1999, 64, 1558, P. S. Dragovich et al., J. Med. Chem. 1999, 42, 1203).

Total

Intermediate peptide fragment associated with MBHA-resin can producira�ü using solid-phase peptide chemistry on a peptide synthesizer Applied Biosystems (ABI) 460A using MBHA-resin (Applied Biosystems, Inc., lot No. A1A023, 0.77 mmol/g). All amino acids have their α-amino group protected by tert-butyloxycarbonyl (t-Boc) group. Any reactive side chains are protected as follows: Arg (Tos); Lys (Cl-Z); Trp (CHO); Glu (CHex); Tyr (Br-Z), Ser (Bzl); Asp (OBzl); Thr (Bzl).

Protected amino acids activated in dichloromethane (DCM) half of the equivalent dicyclohexylcarbodiimide (DCC) at the equivalent amino acid c the formation of the symmetric anhydride of the amino acid. However, residues of arginine, glutamine and glycine activate in the formation of 1-hydroxybenzotriazole (HOBt) esters of these amino acids (1:1:1 equivalents of amino acid, HOBt and DCC in dimethylformamide (DMF)).

The remains are connected in series from the C-end N-end series of loops attaching and removing the protective groups. The cycle of accession consists of activated amino acids that undergo nucleophilic substitution free primary amine previously attached amino acid. The removal of the protective groups is the replacement of N-terminal blocking group Boc anhydrous trifluoroacetic acid (TFA). This will generate a free amino group after neutralizing diisopropylethylamine (DIEA).

The scale of synthesis is 0.5 mmol. The concentration of functional sites on MBHA-resin was 0.77 mmol/g, was used 649 mg of resin. A two-fold molar excess of symmetric�th anhydride used for all amino acids. C-terminal arginine attached to MBHA-resin using a standard Protocol. All balances are twice United. That is, each residue is connected with the resin twice to ensure complete reaction of NH2groups on the resin. Second connection stage is carried out without removing the protective group Boc before adding amino acids. This helps to ensure that all tree-based amino resin is completely reacted. The tryptophan residue is connected to four times. After the second stage of accession of each cycle dual attach terminal Boc-group was removed with anhydrous trifluoroacetic acid and neutralized with DIEA.

Formyl group, a blocking side chain of the tryptophan residue, is removed by piperidine in DMF prior to the separation of the peptide from the resin. After the transfer of the peptidyl-resin in the funnel sintered glass with a volume of 50 ml of it were washed repeatedly with DCM and DMF. Then 3-5 ml of a solution of piperidine/DMF (50/50) was added to the peptide resin, so that it just cover. After 5 minutes, the piperidine/DMF is removed under vacuum and add 3-5 ml piperidine/DMF. After 10 minutes, the piperidine/DMF was again removed by vacuum filtration and add 15-20 ml piperidine/DMF. After 15 minutes, the piperidine/DMF is removed and the peptidyl-resin was washed with DMF several times, and then DCM. The peptidyl-resin is then placed in a vacuum oven (no heat) to remove Rast�of orites.

Alternatively, the desired resin-bound peptide fragment can also be prepared by using Fmoc-protection. Resin Rink Amide MB HA, Fmoc-protected amino acids, O - benzotriazole-1-yl-N,N,N',N'-tetramethyl-uronium hexaflurophosphate (HBTU) in a solution of N,N-dimethylformamide (DMF) and activation of N-methyl morpholine (NMM), and removal of the protective Fmoc group by piperidine (stage 1). If necessary, the selective removal of the protective group of the Lys(Aloc) was performed manually and was completed by treating the resin with a solution of 3 equivalents of Pd (PPh3)4dissolved in 5 ml of CHCl3: NMM:Or (18:1:0,5) for 2 h (step 2). The resin then was washed with CHCl3(6×5 ml), 20% Or in DCM (6×5 ml), DCM (6×5 ml) and DMF (6×5 ml). In some cases, the synthesis was re-automated for the addition of one AEEA (aminoethoxyethanol acid) group, adding acetic acid or adding 3-maleinimide acid (MPA) (stage 3). Cleavage from the resin and the product selection was performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation in cold dry ice with Et2O (Stage 4). The products were purified by preparative reversed-phase HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of 30-55% B (0.045 percent TFA in H2O (A) and to 0.045% TFA in CH3CN (B)) over 180 min at 9.5 ml/min using column Phenomenex Luna 10μ phenyl-hexil, 21 mm × 25 cm and UV detector (Varian Dynamax UVD II) PR� 214 and 254 nm. Purity was determined as 95% by using reversed-phase-HPLC mass spectrometry using a spectrometer Hewlett Packard LCMS-1100 series, equipped with a diode array detector and using electro ionization-injection.

Protective groups are chemical functional groups used to protect a peptide derived from interaction with themselves. Such protective groups include acetyl, fluorenylmethoxycarbonyl (FMOC), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (CBZ) and the like. Specific protected amino acids are presented in Table 1.

TABLE 1
NATURAL AMINO acids AND THEIR ABBREVIATIONS
NAME3-letter abbreviation1-letter abbreviationprotected amino acids
AlanineAlaAFmoc-Ala-OH
ArginineArgRFmoc-Arg (pbf)-OH
AsparagineAsn NFmoc-Asn (Trt)-OH
Aspartic acidAspDFmoc-Asp (tBu)-OH
CysteineCysCFmoc-Cys (Trt)-OH
Glutamic acidGluEFmoc-Glu (tBu)-OH
GlutamineGlnQFmoc-Gln(Trt)-OH
GlycineGlyGFmoc-Gly-OH
HistidineHisHFmoc-His(Trt)-OH
IsoleucineIleIFmoc-Ile-OH
LeucineLeuLFmoc-Leu-OH
LysineLysKFmoc-Lys (Mtt)-OH
IU�of Jonas MetMFmoc-Met-OH
PhenylalaninePheFFmoc-Phe-OH
ProlineProPFmoc-Pro-OH
SerineSerSFmoc-Ser (tBu)-OH
ThreonineThrTFmoc-Thr (tBu)-OH
TryptophanTrpWFmoc-Trp(Boc)-OH
TyrosineTyrYBoc-Tyr(tBu)-OH
ValineValVFmoc-Val-OH

Q-linker-a: Obtaining 2S,5R-2-(3-tert-butoxycarbonylamino-booth-1-enyl)-glutaric acid 5-tert-butyl ether

2S,5R-2-(3-tert-butoxycarbonylamino-booth-1-enyl)-glutaric acid 5-tert-butyl ester 1-methyl ester (J. Chem. Soc, Perkin Trans 1, 2001, 245) (370 mg, 1 mmol) in methanol (2 ml) was treated with LiOH (1M, 2 ml) at room temperature for 1 hour. Most of the solvent is evaporated under vacuum, diluted with water (10 ml) and the pH adjusted to 5, and the aqueous layer was extracted with ethyl acetate (3 × 30 ml) to obtain specified in the header of the product as a film (320 mg, 90%).

1H NMR δ 5,43 (m, 1H), is 5.33 (DD, J=15,5, 5,2 Hz, 1H), 4,59 (d, J=7,6 Hz, 1H), 3,88 (m, 1H), of 2.91 (m, 1H), 2,25 (m, 2H), 1,91-2,04 (m, 1H), 1,67-of 1.80 (m, 1H), of 1.57 (s, 9H), of 1.47 (s, 9H), 1.14 in (d, J=6,7 Hz, 3H). LCMS 358(M++1).

Q-linker-b: Obtaining 2S,5R-2-(3-tert-butoxycarbonylamino-2-fluoro-but-1-enyl)-glutaric acid 5-tert-butyl ether

Step A: Obtaining 2S,5R-2-(3-tert-butoxycarbonylamino-2-fluoro-but-1-enyl)-glutaric acid 1-(S) sultam

To 5-tert-butoxycarbonylamino-4-fluoro-2-(3-hydroxy-propyl)-hex-3-enoeou acid (S) sultam (Tetrahedron, 2008, 64, 4332) (502 mg, 1 mmol) in DMF (5 ml) was added PDC (pyridinediamine, 2.5 mmol) and the resulting solution was stirred at rt for 64 hours. The reaction mixture was diluted with brine (20 ml) and extracted with ethyl acetate (3 × 20 ml). The combined organic extracts are dried over MgSO4and the solvent is evaporated under reduced pressure. The residue is purified using evaporative column to obtain the acid as films (425 mg, 79%) which was used without further purification.

With�In adiya: Receiving 2S,5R-2-(3-tert-butoxycarbonylamino-2-fluoro-but-1-enyl)-glutaric acid 5-tert-butyl ester 1-(S) sultam

2S,5R-2-(3-tert-butoxycarbonylamino-2-fluoro-but-1-enyl)-glutaric acid 2-(S) sultam from step A (400 mg, 0,75 mmol) in dichloromethane (10 ml) was treated with tert-butanol (0.5 ml, 10 perematyvanie), DCC (1.5 mmol) and DMAP (1.5 mmol). The reaction mixture was stirred 24 hours prior to dilution with brine (20 ml), extracted with ethyl acetate (3 × 20 ml). The combined organic extracts are dried over MgSO4and the solvent is evaporated under reduced pressure. The residue is purified using evaporative column to obtain tert-butyl ether as a film (425 mg, 79%).1H NMR δ is 5.33 (m, 1H), of 4.54 (m, 1H), 3,88 (m, 1H), 3,37 (s, 2H), 3,23 (m, 1H), 2,25 (m, 2H), 1.91 a to 2.14 (m, 4H), 1,67-of 1.80 (m, 5H), of 1.57 (s, 9H), of 1.47 (d, J=7,6 Hz, 3H), of 1.18 (s, 3H), 1.14 in (s, 3H). LCMS 574 (M++1).

Stage C: Obtain 2S,5R-2-(3-tert-butoxycarbonylamino-2-fluoro-but-1-enyl)-glutaric acid 5-tert-butyl ether

To a solution of tert-butyl ester from Stage B (410 mg, 0,72 mmol) and aqueous 50% H2O2(260 ml, 3.6 mmol) in THF-H2O (5:1, 12 ml) at 0ºC is added LiOH (1N, 1.44 ml), and the mixture was stirred at room temperature for 2 h. After adjusting the pH to 5, the mixture was extracted with ethyl acetate (3 × 15 ml). The combined organic extracts were washed with brine and dried over MgSO4. The solvent is evaporated under reduced pressure to obtain the corresponding acid as film (262 m�, 95%).1H NMR δ 5,23 (m, 1H), 4,45 (m, 1H), 3,11 (m, 1H), 2,45 (m, 2H), 2,24 (m, 2H), of 1.52 (s, 9H), of 1.47 (s, 9H). LCMS 376 (M++1).

Q-linker-c: Obtaining 2S,5R-2-[1-tert-butoxycarbonylamino-methyl)-2,2,2-Cryptor-ethylamino]-glutaric acid 5-tert-butyl ether

Step A: Obtaining 2S,5R-2-[1-tert-butoxycarbonylamino-methyl)-2,2,2-Cryptor-ethylamino]-glutaric acid 5-tert-butyl ester 1-methyl ester

To a solution of hydrochloride salt 2-(1-Aminomethyl-2,2,2-Cryptor-ethylamine)- glutaric acid 5-tert-butyl ester 1-methyl ester (Org. Lett., 2003, 5, 3887) (364 mg, 1 mol) and Boc2O (260 mg, 1.2 mmol) in dichloromethane (15 ml) at 0ºC was added a solution of DIPEA (0.2 ml, 1.5 mmol) in dichloromethane (1 ml) and the mixture was stirred at room temperature for 6 h. the Mixture was diluted with ethyl acetate (30 ml). The mixture was washed with 0.1 N HCl, brine, and dried over MgSO4. The solvent is evaporated under reduced pressure and then FC, to obtain the corresponding diester, as a film (420 mg, 85%).1H NMR δ of 4.54 (m, 1H), 4,12 (m, 1H), 3,68 (s, 3H), of 3.45 (m, 1H), 3,11 (m, 2H), of 2.45 (m, 2H), 2,24 (m, 2H), of 1.52 (s, 9H), of 1.47 (s, 9H). LCMS 430 (M++1), 330 (M+1-tert-Bu).

Stage b: Obtain 2S,5R-2-[1-tert-butoxycarbonylamino-methyl)-2,2,2-Cryptor-ethylamino]-glutaric acid 5-tert-butyl ether

To a solution of tert-butyl ester from step A (420 mg, 0,92 mmol) in THF-H2O (5:1, 12 ml) p�and 0ºC was added LiOH (1N, Of 1.44 ml), and the mixture was stirred at room temperature for 2 h. After adjusting the pH to 5, the mixture was extracted with ethyl acetate (3 × 15 ml). The combined organic extracts were washed with brine and dried over MgSO4. The solvent is evaporated under reduced pressure to obtain the corresponding acid as film (362 mg, 92%).1H NMR δ 4,50 (m, 1H), 4,08 (m, 1H), of 3.45 (m, 1H), 3,11 (m, 2H), of 2.45 (m, 2H), 2,24 (m, 2H), of 1.52 (s, 9H), of 1.47 (s, 9H). LCMS 430 (M++1), 330 (M+1-tert-Bu).

Q-linker-d: Obtain 2R-2-(1S,2S-2-[1-tert-butoxycarbonylamino-1 trifter-methyl-propylamino)-glutaric acid 5-tert-butyl ether

Step A: 2S,3S-3-dibenzylamino-1-1,1-triptorelin-2-triftormetilfullerenov

To a solution of 2S,3S-3-dibenzylamino-1-1,1-Cryptor-butan-2-ol (Eur. J. Org. Chem. 2004, 1558) (3,23 g, 10 mmol) and 2,6-lutidine (1.7 g, 16 mmol) in c-hexane (25 ml) at-10ºC added triflate anhydride (4.2 g, 15 mmol) at a rate to maintain the temperature <10 ° C, and the reaction was continued for 1.5 h. the Reaction mixture was diluted with water (25 ml) and c-hexane (50 ml). The organic layer was washed with 1N HCl (2 × 15 ml) and brine (15 ml). After drying over MgSO4the solvent is evaporated under reduced pressure to obtain the corresponding triftormetilfullerenov (4,34 g, 96%).

Step B: 2S-2-(1S,2S-2-dibenzylamino-1-trifluoromethyl-propylamino)-glut�world acid 1-benzyl ester 5-tert-butyl ether

Potassium carbonate (2.08 g, 15 mmol) was added to a solution of triflate with stage A (4,55 g, 10 mmol), c-hexane (25 ml) and the Mixture was heated to 65-70ºC for 24 h. the Mixture was cooled to room temperature, diluted with water (25 ml) and c-hexane (50 ml), the mixture is then stirred for 10 min. the Layers were separated, the organic layer was washed with 1N HCl (2 × 15 ml) and brine (15 ml). After drying over MgSO4the solvent is evaporated under reduced pressure to obtain the corresponding ether (5,88 g, 95%).

Step C: 2R-2-(1S,2S-2-tert-butoxycarbonylamino-1 triftormetilfullerenov)-glutaric acid 5-tert-butyl ether

Hydrogenation of a solution of 2S-2-(1S,2S-2-dibenzylamino-1-trifluoromethyl-propylamino)-glutaric acid 1-benzyl ester 5-tert-butyl ether from Stage (B (5.4 g, 9 mmol) complete in methanol (50 ml) and Pd/C (0.9 g) at 50 psi for 24 h. After filtration to remove catalyst and the filtrate was concentrated under vacuum. The residue was dissolved in dichloromethane (50 ml) and treated with Boc2O (2,60 g, 12 mmol) in dichloromethane (25 ml) at 0ºC, and then added a solution of DIPEA (2 ml, 15 mmol) in dichloromethane (10 ml) and the mixture was stirred at room temperature for 6 h. the Mixture was diluted with ethyl acetate (60 ml). The mixture was washed with 0.1 N HCl, brine, and dried over MgSO4. The solvent is evaporated under reduced pressure and then FC, so� to receive specified in the title compound as a film

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

To a solution of 2S-2-(3S-3-tert-butoxycarbonylamino-2-oxo-butyl)-glutaric acid 5-tert-butyl ester 1-methyl ester (J Med. Chem. 1999, 42, 1203; Bioorg. Med. Chem. 2005, 13, 5240) (387 mg, 1 mmol) in THF-H2O (5:1, 12 ml) at 0ºC is added LiOH (1N, 1.44 ml), and the mixture was stirred at room temperature for 2 h. After adjusting the pH to 5, the mixture was extracted with ethyl acetate (3 × 15 ml). The combined organic extracts were washed with brine and dried over MgSO4. The solvent is evaporated under reduced pressure to obtain the corresponding acid as a film (362 mg, 92%).1H NMR δ 4,63 (m, 1H), 4.38 gigabytes (W, 1H), 2,68 (d, J=7,6 Hz, 2H), 2,58 (m, 1H), 2,25 (DD, J=123, 7,6 Hz, 2H), 1,92 (m, 2H), 1,49 (C, 9H), of 1.47 (s, 9H), of 1.41 (d, J=7,6 Hz, 3H). LCMS 374 (M++1), 274 (M+1-tert-Bu).

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

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

2S-2-[1S,3S-fluoro-2-oxo-3-(trityl-amino)-butyl]-glutaric acid 5-benzyl ester 1-methyl ester (581 mg, 1 mmol) and 10% Pd/C (100 mg) in methanol (25 ml) gidrogenit in a Parr shaker at 50 pounds per square du�m for 6 hours. The catalyst was removed by filtration through celite. The filtrate is concentrated. The precipitate is dissolved in dioxane (25 ml) and treated with 1N NaOH (1.2 ml). Boc2O (238 mg, 1.1 mmol) in dioxane (2 ml) was added to the corresponding solution at 0ºC, and the mixture was stirred at room temperature for 6 h. the Mixture was diluted with ethyl acetate (30 ml). The mixture was washed with 0.1 N HC1, brine, and dried over MgSO4. The solvent is evaporated under reduced pressure. The residue was dissolved in dichloromethane (10 ml), treated with tert-butanol (0.5 ml, 10 EQ.), DCC (1.5 mmol) and DMAP (1.5 mmol). The reaction mixture was stirred for 24 hours before diluting with saline (20 ml), extracted with ethyl acetate (3 × 20 ml). The combined organic extracts are dried over MgSO4and the solvent is evaporated under reduced pressure. The residue is purified using a column with evaporative formation of tert-butyl methyl ether, as a film (315 mg, 66%).1H NMR δ to 4.81 (m, 1H), 4,63 (m, 1H), 4.38 gigabytes (W, 1H), to 3.67 (s, 3H), 2,78 (m, 1H), of 2.35 (DD, J=12,3, 7,6 Hz, 2H), of 2.06 (m, 2H), 1,49 (C, 9H), of 1.47 (s, 9H), of 1.41 (d, J=7,6 Hz, 3H). LCMS 406 (M++1), 306 (M+1-tert-Bu).

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

In 2S-2-(1S,3S-3-tert-butoxycarbonylamino-1-fluoro-2-oxo-butyl)-glutaric acid 5-tert-butyl ester 1-methyl ester from step A (260 m�, 0,65 mmol) in THF-H2O (5:1, 12 ml) at 0ºC is added LiOH (1N, 10 ml) and the mixture was stirred at room temperature for 2 h. After adjusting the pH to 5, the mixture was extracted with ethyl acetate (3 × 15 ml). The combined organic extracts were washed with brine and dried over MgSO4. The solvent is evaporated under reduced pressure to obtain the corresponding acid as film (238 mg, 95%).1H NMR δ to 4.81 (m, 1H), 4,63 (m, 1H), 4.38 gigabytes (W, 1H), 2,78 (m, 1H), of 2.35 (DD, J=12,3, 7,6 Hz, 2H), of 2.06 (m, 2H), 1,49 (C, 9H), of 1.47 (s, 9H), of 1.41 (d, J= 7,6 Hz, 3H). LCMS 392 (M++1), 292 (M+1-tert-Bu).

Example 1

Synthesis

[Q-linker-d8,Glu22]GLP-1-(7-37)-peptide

Stage 1

Fmoc-Rink Amide of MBNA resin

Solid-phase peptide synthesis of analogue 100 mcmanamon scale is performed using manual solid-phase synthesis and peptide synthesizer Symphony using Fmoc-protected Rink Amide resin, MVNA, Fmoc-protected amino acids, O-benzotriazole-1-yl-N,N,N',N'-tetramethyl-uronium of hexaflurophosphate (HBTU) in a solution of N,N-dimethylformamide (DMF) and the activation of N-methylmorpholine (NMM) and piperidino removing the protective Fmoc groups (step 1). The BOC-group in the product of step 2 Hatshepsut before attaching Fmoc-His(Trt)-HE. Cleavage of the resin and the allocation �product is performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol, with subsequent precipitation in a cool dry ice with Et2O (Stage 2). The product was then purified using preparative reversed-phase HPLC using a preparative dual HPLC system Varian (Rainin): gradient elution of 30-55% B (0.045 percent TFA in H2O (A) and to 0.045% TFA in CH3CN (V) over 180 min at 9.5 ml/min using column Phenomenex Luna 10µl phenyl-hexyl, 21 mm × 25 cm and UV detector (Varian Dynamax PBX II) at λ 214 and 254 nm, to obtain a peptide with a purity >95% as determined by reversed-phase HPLC.

Maldi-Tof-MS: 3412. Calculated MS: 3412.

Example 2

Synthesis

[Q-linker-A8-9,GIu22]GLP-1-(7-37)-peptide

Stage 1

Fmoc-Rink Amide of MBNA resin

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 1.

LCMS: 1113 (M+3H)3+. Calculated MS: 1113 (M+3H)3+.

Example 3

Synthesis

[Q-linker-b8-9,Glu22]GLP-1-(7-37)-peptide

Stage 1

Fmoc-Rink Amide MBHA resin

The desired GLP-1-analogue synthesized using the same sequence and conditions to�which is described in Example 1.

LCMS: 1119 (M+3H)3+. Calculated MS: 1119 (M+3H)3+.

Example 4

Synthesis

[Q-linker-C8,Glu22]GLP-1-(7-37)-peptide

Stage 1

Fmoc-Rink Amide of MBNA resin

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 1.

Maldi-Tof-MS: 3398. Calculated MS: 3398.

Example 5

Synthesis

[Q-linker-e8-9,Glu22]GLP-1-(7-37)-peptide

Stage 1

Fmoc-Rink Amide MBHA resin

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 1.

LCMS: 1118 (M+3H)3+. Calculated MS: 1118 (M+3H)3+.

Example 6

Synthesis

[Q-linker-f8-9,Arg34]GLP-1-(7-37)-peptide

Stage 1

Fmoc-Rink Amide of MBNA resin

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 1.

LCMS: 1127 (M+3H)3+. Calculated MS: 1127 (MN) 3+.

Example 7

Synthesis

N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-C8,Arg34]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 mmol), acetonitrile (260 ml) and water (260 μl) was carefully shaken for 5 min at room temperature. To the resulting mixture was added a solution of Nα-hexadecanoyl-Glu(ONSu)-OBut, (1.8 mg, 3.3 μmol) in acetonitrile (44,2 μl) and the reaction mixture was gently shaken for 1 h and 20 min at room temperature. The reaction was quenched by adding a solution of glycine (1,8 mg, 242 μmol) in 50% aqueous ethanol (181 µl). Added 0.5% aqueous solution of ammonium acetate (12 ml) and NMP (300 μl) and the resulting mixture was suirable on a Varian cartridge 1g C8 Mega Bond Elut®, immobilizovannoi compound was washed with 5% aqueous acetonitrile (10 ml), and finally liberated from the cartridge by elution TFA (6 ml). The eluate was left alone for 2 hours at room temperature and then concentrated under vacuum. The residue was purified using column chromatography and standard system acetonitrile/TFA. Specified in the title compound (12 mg, 46%) was isolated, and the product was analyzed using PDMS. The m/z value for the protonated molecular ion, as it turned out, was $ 3790±3. The resulting molecular weight was thus�Ohm, 3790±3 atomic mass units (theoretical value 3751 atomic mass unit).

Example 8

Synthesis

N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-d8,Arg34]GLP-1-(7-37)-peptide

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 7.

LCMS: 1268 (M+3H)3+. Calculated MS: 1268 (M+3H)3+.

Example 9

Synthesis

N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-E8-9,Arg34]GLP-l(7-37)-peptide

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 7.

LCMS: 1250 (M+3H)3+. Calculated MS: 1250 (M+3H)3+.

Example 10

Synthesis

N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-f8-9,Arg34]GLP-l(7-37)-peptide

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 7.

LCMS: 1256 (M+3H)3+. Calculated MS: 1256 (M+3H)3+.

Example 11

Synthesis

N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-A8-9,Arg34]GLP-1-(7-37)-peptide

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 7.

LCMS: 1244 (M+3H)3+. Calculated MS: 1244 (M+3H)3+.

When�EP 12

Synthesis

N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-b8-9,Arg34]GLP-l(7-37)-peptide

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 7.

LCMS: 1250 (M+3H)3+. Calculated MS: 1250 (M+3H)3+.

Example 13

Synthesis

N-26-[(Nε-ω-carboxypropanoyl)]-[Q-linker-C8,Arg34]GLP-1-(7-37)-peptide

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 7, and using ω-carboxypropanoyl acid 2,5-dioxopiperidin-1-silt ether as a starting material instead of Nα-hexadecanoyl-Glu(ONSu)-OBut.

LCMS: 1239 (M+3H)3+. Calculated MS: 1239 (M+3H)3+.

Example 14

Synthesis

N-26-[(Nε-ω-carboxyrhodamine)]-[Q-linker-C8,Arg34]GLP-1-(7-37)-peptide

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 7, and using ω-carboxypropanoyl acid 2,5-dioxopiperidin-1-silt ether as a starting material instead of Nα-hexadecanoyl-Glu(ONSu)-OBut.

LCMS: 1249 (M+3H)3+. Calculated MS: 1249 (M+3H)3+.

Example 15

Synthesis

[Q-linker-d8]GLP-1-(7-7)-Cys (peg)-Ala-NH2

A mixture of [A-linker-d8]GLP-1-(7-37)-Cys-Ala-NH2(36 mg, 11 μmol) in 50 mmol/l buffer solution (36 ml) was reacted with 2 mol excess of 20 kDa mPEG-SPA (pH was adjusted from 7.5 to 9.0 50 mmol/l Tris-HCl buffer) at room temperature for 3 h. the Mono-Paglierani GLP-1 conjugates was tested and purified using reversed-phase high-performance liquid chromatography (RP-HPLC) on X-tera C18 (4,6 × 250 mm, 5 m, Waters, Milford, MA) at room temperature. The mobile phase consisted of 0.1% TFA in distilled water (eluent A) and ACN containing 0.1% TFA (eluent B). The mobile phase was run with a linear gradient from 30 to 60% eluent B over 20 min at a speed flow 1 ml/min and UV-absorbing eluent was monitored at 215 nm. HPLC fractions corresponding to the relative peaks were collected separately, purged with nitrogen and liofilizirovanny.

Example 16

Synthesis

[Q-linker-C8]GLP-1-(7-37)-Cys(peg)-Ala-NH2

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 13, using 20 kDa mPEG-SPA.

Example 17

Synthesis

[Q-linker-A8-9]GLP-1-(7-37)-Cys(peg)-Ala-NH2

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 13, using 20 kDa mPEG-SA.

Example 18

Synthesis

[Q-linker-b8-9]GLP-1-(7-37)-Cys(PEG)-Ala-NH2

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 13, using 20 kDa mPEG-SPA.

Example 19

Synthesis

[Q-linker-E8-9]GLP-1-(7-37)-Cys(PEG)-Ala-NH2

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 13, using 20 kDa mPEG-SPA.

Example 20

Synthesis

[Q-linker-f8-9]GLP-1-(7-37)-Cys(peg)-Ala-NH2

The desired GLP-1-analogue synthesized using the same sequence and conditions as described in Example 13, using 20 kDa mPEG-SPA.

Example 21

Stability against DPP-IV in vitro

GLP-1 (100 ál, 5 nmol/l), an equivalent amount of purified on HPLC synthesized analogues of GLP-1 was prepared in the triethylamine-HCl buffer (10 mmol/l; pH 7.4). Added DPP-IV (5 mU, 900 μl) and the solution was incubated at 37°C. At specified time points of the reaction mixture were taken at 100 μl and the reaction was stopped by adding 5 ál of 10% (V/V) TFA. Each sample was analyzed by MALDI-TOF-MS and RP-HPLC as described above.

Example 22

Education camp in cleoc�the first line, expressing the cloned receptor GLP-1 person

In order to demonstrate the effectiveness of GLP-1-derivatives, we tested their ability to stimulate formation of camp in a cell line expressing the cloned GLP-1 receptor human. EC50was calculated from the curve dose-response.

In this radioimmunoassay using NIT-1, line beta cells of the pancreas formed from transgenic mice NOD/Lt. The assay was performed in 96-well tablet for micrometrology in the total volume of 140 μl. Used buffer consisted of 50 mmol/l Tris-HCI, pH 7.4 with addition of 1 mmol/l EGTA, 1.5 mmol/l of MgSO4And 1.7 mmol/l ATP, 20 mm GTP, 2 mmol/l 3-isobutyl-1-methylxanthine, 0.01% of Tween-20 and 0.1% human serum albumin. Compounds designed to test agonist activity were dissolved and diluted in buffer, added to the preparation of membranes and the mixture incubated 2 h at 37°C. the Reaction was terminated by addition of 25 μl of 0.05 mol/l HCl. Before analysis on camp with the help of scintillation contact of the test samples were diluted 10 times.

Example 23

Protivogipergonichesky activity of GLP-1-analogues

Mouse (db/db) with induced diabetes were divided into 4 groups (n=5) and fasted for 16 hours. Intraperitoneally administered physical�ideological solution 100 μg/kg GLP-1(7-36)amide, 100 mg-EQ./kg [Q-linker-C8,Glu22]GLP-1-(7-37)-peptide and 100 μg-EQ./kg N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-d8,Arg34]GLP-1-(7-37)-peptide and 10 minutes after oral was administered 1 g/kg glucose solution. Through -10, 0, 10, 20, 30, 60, 90, 120 and 180 minutes were taken blood samples and determined the level of glucose in the blood. Effects of inhibition of the increase in the level of glucose in the blood GLP-1(7-36)amide, [Q-linker-C8, Glu22]GLP-1-(7-37)-peptide and N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-d8,Arg34]GLP-1-(7-37)-peptide was compared by calculating the area under the time curve of glucose level in the blood for time (0-180 minutes). AUG GLP-1(7-36)amide group was 25165±4463 mg-min/DL, which is a smaller value of 27.8%, compared with the saline group (34864+4774 mg. min/DL). However, the values for [Q-linker-C8,Glu22]GLP-1-(7-37)-peptide and N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-d8,Arg34]GLP-1-(7-37)-peptide was 14470+5700 mg-min/DL and 17520+2484 mg-min/DL, respectively (i.e. 58,5% and 49.7% reduction, respectively). These results show that [Q-linker-C8,Glu22]GLP-1-(7-37)-peptide and N-ε26-[γ-L-glutamyl(N-α-hexadecanoyl)]-[Q-linker-d8,Arg34]GLP-1-(7-37)-peptide have a significantly increased activity of inhibiting glucose in the blood compared to GLP-1(7-36)amide.

1. An analogue of GLP-1 with the Formula I or its pharmaceutically acceptable salt:

Haa7represents L-His, D-histidine, deteministic, 2-aminoguanidine, β-hydroxycytidine, homocystein, α-formetiketten or α-methylhistidine;
Q is a linker II, III or IV:

where
R1represents hydrogen, (C1-C6)alkyl or (C1-C6) alkoxy;
R2represents hydrogen, (C1-C6)alkyl or (C1-C6) alkoxy;
R3represents hydrogen or forms a 5-8-membered ring with R1or R2;
X represents hydrogen, fluorine, hydroxy, trifluoromethyl or oxygen;
Y represents hydrogen, hydroxyl, fluorine or (C1-C6) alkyl;
Z represents nitrogen, carbon, oxygen, or sulfur;
W is absent in the case when Z represents a nitrogen, oxygen or sulfur; W represents hydrogen or fluorine in the case when Z represents carbon;
Xaa14represents a serine, or histidine; and one or more carbon atoms Xaa14optionally substituted with one or more alkyl groups;
Xaa16represents valine, lysine and leucine; and one or more carbon atoms Xaa16optionally substituted with one or more alkyl groups, or
Xaa16is a lysine linked with T-U, where
T is a γ-glutamic Ki�lot, β-alanine, γ-aminobutyric acid, HOOC(CH2)nCOOH orwhere
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U exists and is a fatty acid with length of 8 to 20 carbons only in the case when T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Xaa18represents a serine, arginine or lysine; and one or more carbon atoms Xaa18optionally substituted with one or more alkyl groups;
each of XAA22and Haa23independently represents glycine, Aib or glutamic acid; and one or more carbon atoms as Haa22and Haa22optionally substituted with one or more alkyl groups;
each of XAA26That haa27That haa34That haa35and Haa36independently represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid), and one or more carbon atoms of each of XAA26That haa27That haa34That haa35and Haa36optionally substituted with one or more alkyl groups;
or XAA26is a lysine linked with T-U, where
T p�establet a γ-glutamic acid, β-alanine, γ-aminobutyric acid, HOOC(CH2)nCOOH orwhere
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U exists and is a fatty acid with length of 8 to 20 carbons only in the case when T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
In is a glycine, or NH2or HE, who represent the amide form or free acid end of the amino acid, or
In a is a peptide segment consisting of cysteine and from one to four amino acids, each of which independently represents a serine, glycine, alanine or in the case where XAA26represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid) and is not associated with T-U, where
T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, HOOC(CH2)nCOOH orwhere
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U is present and is a well�RNA acid length of from 8 to 20 carbon just in case when T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

2. An analogue of GLP-1 according to claim 1, where XAA26represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid) and is not associated with T-U; b is a cysteine-serine-glycine-cysteine-alanine or cysteine-.

3. An analogue of GLP-1 with the Formula V or its pharmaceutically acceptable salt:

where
R1represents hydrogen, (C-C6)alkyl or (C1-C6) alkoxy;
R2represents hydrogen, (C1-C6)alkyl or (C1-C6) alkoxy;
R3represents hydrogen, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2;
X represents hydrogen, fluorine, hydroxy, trifluoromethyl or oxygen;
Haa7represents L-His, D-histidine, deteministic, 2-aminoguanidine, β-hydroxycytidine, homocystein, α-formetiketten and α-methylhistidine;
D represents Gly-Thr-Phe-Thr-Xaa14Asp Xaa16-Ser-Xaa18-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Xaa27-Phe-Ile-Ala-Trp-Leu-Val-XAA34-Haa35-Haa36-In;
where
Xaa14represents serine or GIS�idin; and one or more carbon atoms Xaa14optionally substituted with one or more alkyl groups;
Haa16represents valine, lysine and leucine; and one or more carbon atoms Haa16optionally substituted with one or more alkyl groups; or
Haa16is a lysine linked with T-U, where
T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, HOOC(CH2)nCOOH orwhere
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U exists and is a fatty acid with length of 8 to 20 carbons only in the case when T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Xaa18represents a serine, arginine or lysine; and one or more carbon atoms Xaa18optionally substituted with one or more alkyl groups;
each of XAA22and Haa23independently represents glycine, Aib or glutamic acid; and one or more carbon atoms as Haa22and Haa22optionally substituted with one or more alkyl groups;
each of XAA26That haa27 That haa34That haa35and Haa36independently represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid), and one or more carbon atoms of each of XAA26That haa27That haa34That haa35and Haa36optionally substituted with one or more alkyl groups; or
Haa26is a lysine linked with T-U; where
T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, HOOC(CH2)nCOOH orwhere
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U exists and is a fatty acid with length of 8 to 20 carbons only in the case when T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
In is a glycine, or NH2or is HE, which respectively represent the amide form or free acid end of the amino acid; or
In a is a peptide segment consisting of cysteine and from one to four amino acids, each of which independently represents a cysteine, serine, glycine, alanine or in case�AE when XAA26represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid) and is not associated with T-U.

4. An analogue of GLP-1 according to claim 3, where XAA26represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid) and is not associated with T-U; b is a cysteine-serine-glycine-cysteine-alanine or cysteine-.

5. An analogue of GLP-1 according to claim 3, where X represents hydrogen, fluorine or trifluoromethyl.

6. An analogue of GLP-1 according to claim 3, where each of R1, R2and R3independently represent hydrogen or methyl.

7. An analogue of GLP-1 according to claim 3, where R1is methyl and each of R2and R3independently represents hydrogen.

8. An analogue of GLP-1 according to claim 3, where each of R1and R3independently represents hydrogen, a R2is methyl.

9. An analogue of GLP-1 according to claim 3, where R3forms a 5-8-membered ring with R1and R2represents hydrogen; or R3forms a 5-8-membered ring with R2, a R1represents hydrogen.

10. An analogue of GLP-1 with the Formula VI, or its pharmaceutically acceptable salt:

where
R1represents hydrogen, (C1-C6)alkyl or (C1-C6)alkoxy;
R2represents hydrogen, (C1-C6 )alkyl or (C1-C6) alkoxy;
R3represents hydrogen, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2;
Y represents hydrogen, hydroxyl, fluorine or (C1-C6) alkyl;
Haa7represents L-His, D-histidine, deteministic, 2-aminoguanidine, β-hydroxycytidine, homocystein, α-formetiketten or α-methylhistidine;
D represents Gly-Thr-Phe-Thr-Xaa14Asp Xaa16-Ser-Xaa18-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Xaa27-Phe-Ile-Ala-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-In
Xaa14represents a serine, or histidine; and one or more carbon atoms Xaa14optionally substituted with one or more alkyl groups;
Xaa16represents valine, lysine, or leucine; and one or more carbon atoms Haa16optionally substituted with one or more alkyl groups; or
Haa16is a lysine linked with T-U, where
T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, HOOC(CH2)nCOOH or; where
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U exists and is a fatty acid with length of 8 to 20 carbons only to�Yes T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Xaa18represents a serine, arginine or lysine; and one or more carbon atoms Xaa18optionally substituted with one or more alkyl groups;
each of XAA22and Haa23independently represents glycine, Aib or glutamic acid; and one or more carbon atoms as Haa22and Haa23optionally substituted with one or more alkyl groups;
each of XAA26That haa27That haa34That haa35and Haa36independently represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid); and one or more carbon atoms of each of XAA26That haa27That haa34That haa35and Haa36optionally substituted with one or more alkyl groups; or
Haa26is a lysine linked with T-U, where
T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, HOOC(CH2)nCOOH or; where
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U exists and is a fatty acid with length of 8 to 20 carbons only to�when T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
In is a glycine, or NH2or is HE, which respectively represent the amide form or free acid end of the amino acid, or
In a is a peptide segment consisting of cysteine and from one to four amino acids, each of which independently represents a cysteine, serine, glycine, alanine or in the case where XAA26represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid) and is not associated with T-U.

11. An analogue of GLP-1 according to claim 10, wherein XAA26represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid) and is not associated with T-U; b is a cysteine-serine-glycine-cysteine-alanine or cysteine-.

12. An analogue of GLP-1 according to claim 10, where R3represents hydrogen.

13. An analogue of GLP-1 according to claim 10, where Y represents hydrogen or (C1-C6) alkyl.

14. An analogue of GLP-1 with the Formula VII or its pharmaceutically acceptable salt:

where
R1represents hydrogen, (C1-C6) alkyl or (C1-C6) alkoxy;
R2represents hydrogen, (C -C6) alkyl or (C1-C6) alkoxy;
R3represents hydrogen, (C1-C6) alkyl or forms a 5-8-membered ring with R1or R2;
Y represents hydrogen, hydroxyl, fluorine or (C1-C6) alkyl;
W represents hydrogen or fluorine;
Haa7represents L-His, D-histidine, deteministic, 2-aminoguanidine, β-hydroxycytidine, homocystein, α-formetiketten or α-methylhistidine;
D represents Gly-Thr-Phe-Thr-Xaa14Asp Xaa16-Ser-Xaa18-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Xaa26-Xaa27-Phe-Ile-Ala-Trp-Leu-Val-Xaa34-Xaa35-Xaa36-B; where
Haa14represents a serine, or histidine; and one or more carbon atoms Xaa14optionally substituted with one or more alkyl groups;
Xaa16represents valine, lysine, or leucine; and one or more carbon atoms Xaa16optionally substituted with one or more alkyl groups,
or Xaa16is a lysine linked with T-U, where
T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, HOOC(CH2)soon or; where
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U exists and is a fatty acid�the length from 8 to 20 carbon just in case when T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Xaa18represents a serine, arginine or lysine; and one or more carbon atoms Haa18optionally substituted with one or more alkyl groups;
each of XAA22and Haa23independently represents glycine, Aib or glutamic acid; and one or more carbon atoms as Haa22and Haa23optionally substituted with one or more alkyl groups;
each of XAA26That haa27That haa34That haa35and Haa36independently represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid); and one or more carbon atoms of each of XAA26That haa27That haa34That haa35and Haa36optionally substituted with one or more alkyl groups, or
Haa26is a lysine linked with T-U; where
T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, HOOC(CH2)soon or; where
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U exists and is a fatty sour�in length from 8 to 20 carbon just in case when T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
In is a glycine, or NH2or is HE, which respectively represent the amide form or free acid end of the amino acid, or
In a is a peptide segment consisting of cysteine and from one to four amino acids, each of which independently represents a cysteine, serine, glycine, alanine or in the case where XAA26represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid) and is not associated with T-U.

15. An analogue of GLP-1 according to claim 14, where XAA26represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid) and is not associated with T-U; b is a cysteine-serine-glycine-cysteine-alanine or cysteine-.

16. An analogue of GLP-1 according to claim 14, where R3represents hydrogen or forms a 5-8-membered ring with R1or R2.

17. An analogue of GLP-1 according to claim 14, where Y represents hydrogen or fluorine.

18. An analogue of GLP-1 with the Formula VIII or its pharmaceutically acceptable salt:

Haa7represents L-His, D-histidine, desamino�tiden, 2-aminoguanidine, β-hydroxycytidine, homocystein, α-formetiketten or α-methylhistidine;
Q is a linker II, III or IV:

where
R1represents hydrogen, (C1-C6)alkyl or (C1-C6) alkoxy;
R2represents hydrogen, (C1-C6)alkyl or (C1-C6) alkoxy;
R3represents hydrogen, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2;
X represents hydrogen, fluorine, hydroxy, trifluoromethyl or oxygen;
Y represents hydrogen, hydroxyl, fluorine or (C1-C6) alkyl;
Z represents nitrogen, carbon, oxygen, or sulfur;
W is absent in the case when Z represents a nitrogen, oxygen or sulfur; or W represents hydrogen or fluorine in the case when Z is carbon;
Haa7represents L-His, D-histidine, deteministic, 2-aminoguanidine, β-hydroxycytidine, homocystein, α-formetiketten or α-methylhistidine;
Xaa14represents a serine, or histidine; and one or more carbon atoms Xaa14optionally substituted with one or more alkyl groups;
Haa16represents valine, lysine, or leucine; and one or more carbon atoms Xaa16optionally substituted with one or more Alki�ranked groups;
Xaa18represents a serine, arginine or lysine; and one or more carbon atoms Xaa18optionally substituted with one or more alkyl groups;
each of XAA22and Haa23independently represents glycine, Aib or glutamic acid; and one or more carbon atoms as Haa22and Haa23optionally substituted with one or more alkyl groups;
each of XAA27That haa34That haa35and Haa36independently represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid); and one or more carbon atoms of each of XAA27That haa34That haa35and Haa36optionally substituted with one or more alkyl groups;
T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, HOOC(CH2)soon or; where
n is 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;
k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
U exists and is a fatty acid with length of 8 to 20 carbons only in the case when T is a γ-glutamic acid, β-alanine, γ-aminobutyric acid, or

where k is equal to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
In is a glycine, or NH2, �if HE which respectively represent the amide form or free acid end of the amino acid.

19. An analogue of GLP-1 with the Formula IX or its pharmaceutically acceptable salt:

where
Haa7represents L-His, D-histidine, deteministic, 2-aminoguanidine, β-hydroxycytidine, homocystein, α-formetiketten or α-methylhistidine;
Q is a linker II, III or IV:

where
R1represents hydrogen, (C1-C6)alkyl or (C1-C6) alkoxy;
R2represents hydrogen, (C1-C6)alkyl or (C1-C6) alkoxy;
R3represents hydrogen, (C1-C6)alkyl or forms a 5-8-membered ring with R1or R2;
X represents hydrogen, fluorine, hydroxy, trifluoromethyl or oxygen;
Y represents hydrogen, hydroxyl, fluorine or (C1-C6) alkyl;
Z represents nitrogen, carbon, oxygen, or sulfur;
W is absent in the case when Z represents a nitrogen, oxygen or sulfur, W represents hydrogen or fluorine in the case when Z is carbon;
Haa14represents a serine, or histidine; and one or more carbon atoms Xaa14optionally substituted with one or more alkyl groups;
Haa16is�Lin, lysine or leucine; and one or more carbon atoms Xaa16optionally substituted with one or more alkyl groups;
Xaa18represents a serine, arginine or lysine; and one or more carbon atoms Xaa18optionally substituted with one or more alkyl groups;
each of XAA22and Haa23independently represents glycine, Aib or glutamic acid; and one or more carbon atoms as Haa22and Haa23optionally substituted with one or more alkyl groups;
each of XAA26That haa27That haa34That haa35and Haa36independently represents glycine, lysine, arginine, leucine, asparagine, or Aib (α-aminoadamantane acid), and one or more carbon atoms of each of XAA26That haa27That haa34That haa35and Haa36optionally substituted with one or more alkyl groups;
Haan, Xaan+1That haan+2That haan+3That haan+4all together absent or represents a peptide segment of one, two, three or four amino acids; XaamThat haam+1That haam+2That haam+3That haam+4all together absent or represent a peptide segment of one, two, three or four amino acids, provided that the total number of amino acids represented HaanThat haan+1That haan+2That haa n+3That haan+4, XaamThat haam+1That haam+2That haam+3That haam+4is 1, 2, 3 or 4 and the cysteine is associated with .

20. An analogue of GLP-1, which is a
;
;
;
;
;
;
;
;
;
;
;
;
;
;
;
;
; or

21. Pharmaceutical composition for treatment or prevention of hyperglycemia, diabetes type 2 diabetes, disorders of glucose tolerance, diabetes mellitus type 1, obesity, hypertension, dyslipidemia, atherosclerosis and other cardiovascular disease or stomach ulcers, comprising a therapeutically effective amount of an analog of GLP-1 according to any one of claims. 1-20, and a pharmaceutically acceptable filler.

22. Pharmaceutical composition according to claim 21, where the pharmaceutical composition is suitable for parenteral introduction�Oia.

23. The use of an analogue of GLP-1 according to any one of claims. 1-20 for the manufacture of a medicament for the treatment or prevention of hyperglycemia, diabetes type 2 diabetes, disorders of glucose tolerance, diabetes mellitus type 1, obesity, hypertension, dyslipidemia, atherosclerosis and other cardiovascular disease or stomach ulcers.

24. The use of an analogue of GLP-1 according to any one of claims. 1-20 for the manufacture of drugs to slow or prevent the progression of disease in patients with diabetes mellitus type 2.

25. The use of an analogue of GLP-1 according to any one of claims. 1-20 for the manufacture of a medicament for reducing the absorption of food, reduction of apoptosis of β-cells, amplification of β-cell function or increase β-cell mass and/or for restoring glucose sensitivity to β-cells.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology, namely to obtaining GLP-2 analogues, and can be used in medicine for treating GLP-2-associated disorders. The analogues of GLP-2 with agonistic activity with respect to GLP-2 receptors have been obtained.

EFFECT: invention makes it possible to increase resistance to proteases, which provides the lower clearance of the obtained analogues and prolongation of their bioavailability in comparison with native GLP-2.

29 cl, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to prodrug preparations of glucagon superfamily peptides, in which the glucagon superfamily peptide is modified by binding a dipeptide with the peptide of the glucagon superfamily by an amide bond.

EFFECT: produgs, disclosed in the claimed invention, have an increased half-life and transfer into an active form in physiological conditions as a result of a non-enzymatic reaction, caused by chemical instability.

30 cl, 15 dwg, 8 tbl, 16 ex

FIELD: chemistry.

SUBSTANCE: invention refers to gene engineering, more specifically to producing the peptide GLP-1, modified by an oligosaccharide chain, and can be used in medicine for treating or preventing diseases associated with GLP-1. In the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3 two amino acid peptides are substituted by an amino acid modified by a complex bi-antennal oligosaccharide chain, and wherein each of the centres is specified in a group consisting of positions 18, 22, 26, 30, 34 and 36 in the peptide GLP-1 with SEQ ID NO: 2 or SEQ ID NO: 3. The above modified peptide GLP-1 can involve the deletion, substitution or attachment of 1-5 amino acids, except for the amino acids modified by the oligosaccharide chain.

EFFECT: invention enables producing the peptide GLP-1 modified by the oligosaccharide chain, which shows the stronger activity of blood glucose suppression and twice increased half lifetime as compared to GLP-1 with SEQ ID NO: 3.

24 cl, 5 dwg, 6 tbl, 16 ex

Oxyintomoduline // 2542362

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to peptide analogues of oxyintomoduline (OXM, glucagon-37), which can be modified for providing the stability of cleavage and inactivation with dipeptidyl peptidase IV (DPP-IV) for increasing a half-life time in vivo of the peptide analogue alongside with enabling the peptide analogue acting as a double agonist GLP-1/glucagon receptor (GCGR).

EFFECT: peptide analogues are applicable for treating metabolic disorders, such as diabetes and obesity.

16 cl, 16 dwg, 11 tbl, 12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically to a GLP-1 peptide having an attached oligosaccharide chain, and can be used in medicine. Said GLP-1 peptide, having GLP-1 activity, has (a1) one amino acid further attached to the C end (position 37), wherein said attached amino acid is replaced with an amino acid with an attached oligosaccharide chain; or (a2) one or two amino acids, replaced with an amino acid with an attached oligosaccharide chain, where the replacement site is selected from positions 18, 20, 22, 30 and 36, and can further include 1 to 5 amino acid deletions, replacements or inserts, where said oligosaccharide chain contains five or more sugars and is represented by Formula 1. The invention also relates to a pharmaceutical composition and a method of treating or preventing diseases which can be treated or prevented by administering GLP-1, for example diabetes, which comprises use of said GLP-1 peptide having an attached oligosaccharide chain.

EFFECT: invention enables to obtain a GLP-1 peptide having an attached oligosaccharide chain, having improved stability and higher activity in controlling blood sugar level compared to GLP-1.

14 cl, 22 dwg, 10 tbl, 49 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a glucagon-like peptide-1 (GLP-1) analogues presented by general formula I

wherein X represents glycine or glycinamide. The GLP-1 analogue is resistant to the action of dipeptidyl-peptidase IV and thereby has the prolonged half-life in vivo. What is also presented is using the GLP-1 for reducing blood sugar.

EFFECT: preparing the glucagon-like peptide-1.

9 cl, 3 ex, 3 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: present invention provides a method of controlling conditions for site-specific binding of a polypeptide and a non-peptide polymer by controlling pH and alcohol content of the reaction medium.

EFFECT: method is intended to prevent formation of secondary conjugates, wherein a non-peptide polymer binds with a physiologically vital amino acid residue.

15 cl, 36 dwg, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel peptide analogues of oxyntomodulin, their pharmaceutical compositions and their application for treatment and/or prevention of excessive weight, as well as disorders and diseases, accompanying obesity.

EFFECT: peptide finds special application as appetite suppressor and in treatment of obesity.

30 cl, 41 dwg, 19 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: compounds of the present invention are novel peptide analogues of oxyntomodulin (oxm) wherein one or more amino acid residue of the sequence oxm are replaced. The replacement of amino acid residues 15-24 of the peptide oxm either by amino acid residues 968-977 of the α-latroxin peptide (and versions thereof), or by amino acid residues 15-24 of extendin-4 (and versions thereof), or combining the amino acid residues of these sources, and/or the replacement of amino acid residues 27-33 of the peptide oxm by amino acid residues 27-33 of extendin-4, and/or adding the amino acid residues to an C-terminal of the peptide, enables producing a number of the analogues oxm presenting oxm-like activity to reduce food consumption, and according to some other aspects, more evident ability to reduce food consumption.

EFFECT: reduced food consumption.

32 cl, 779 dwg, 106 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining glucagon analogues and can be used in medicine. The peptide amino acid sequence includes a negatively charged amino acid at position 28 and a negatively charged amino acid at position 29 or 30 of the native glucagon sequence (SEQ ID NO: 1). The amino acid sequence can also include up to 7 additional amino acid modifications with respect to SEQ ID NO: 1, which enable the glucagon peptide to retain glucagon activity and improved solubility or stability. The obtained peptide is used to treat hypoglycaemia and for temporary paralysis of the intestinal tract.

EFFECT: invention enables to obtain a glucagon peptide with improved solubility or stability with respect to the native glucagon.

15 cl, 10 dwg, 4 tbl, 15 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to pharmaceutical composition, which contains compound of insulin in concentration, sufficient for supplying therapeutically effective level of insulin compound in blood plasma for at least 3 days. Insulin compound relates to pro-medical compound, which represents insulin conjugate with linker, bound with hydrogel carrier. Also described is suspension, containing pharmaceutical composition of insulin conjugate, method of suspension obtaining, set, including pharmaceutical composition of insulin conjugate and container for introduction of composition.

EFFECT: pharmaceutical composition of insulin conjugate by invention is characterised by the fact that it has pharmacokinetic profile in vivo in fact without release of insulin compound.

26 cl, 9 dwg, 21 ex

FIELD: medicine.

SUBSTANCE: correcting cognitive disorders in the patients suffering arterial hypertension accompanying type 2 diabetes mellitus is ensured by combining a standard drug therapy with administering the preparation Kudesan 60 mg a day throughout two months.

EFFECT: administering Kudesan in the above dose and regimen provides the effective correction of cognitive disorders in the above group of patients in a combination with improving the cardiovascular function and metabolic processes.

2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: correcting increased levels of anxiety and depression in the patients with arterial hypertension accompanying type 2 diabetes mellitus is ensured by combining a standard drug treatment and administering Kudesan 60 mg a day for two months.

EFFECT: method provides the effective correction of anxiodepressive conditions in the given category of patients that in turn enables normalising blood pressure more effectively by reducing the negative psychosomatic effect.

1 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to methods of treating type 2 diabetes, insulin resistance, insulin hyposecretion, obesity, hyperglycaemia and hyperinsulinemia, involving administering an effective amount of an anti-IL-1β antibody or its fragment into an individual, as well as to using the anti-IL-1β antibody or its fragment in preparing a composition applicable for treating the above diseases or conditions.

EFFECT: group of inventions is effective in treating type 2 diabetes mellitus, insulin resistance, insulin hyposecretion, obesity, hyperglycaemia and hyperinsulinemia.

67 cl, 13 dwg, 5 tbl, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel N-containing heteroaryl derivatives of formula I or II or their pharmaceutically acceptable salts, which possess properties of JAK kinase, in particular JAK3, and can be applied for treating such diseases as asthma and chronic obstructive pulmonary disease (COPD). In formulae A represents carbon and B represents nitrogen or A represents nitrogen and B represents carbon; W represents CH or N; R1 and R2, independently represent hydrogen, C1-4alkyl, halogenC1-4alkyl, -CN; R3 represents C1-4alkyl, R9-C1-4alkyl, Cy1, where Cy1 is optionally substituted with one or several substituents R10; R4 represents hydrogen, C1-4alkyl, R12R7N-C0alkyl, where one of R7 and R12 represents hydrogen, and the other represents C1-4alkyl or group R13, which is selected from C1-5alkyl, Cy2-C0alkyl; R5 represents hydrogen; R6 represents hydrogen, C1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl, R12R7N-C1-4alkyl, R16CO-C0alkyl, Cy1; R7 represents hydrogen or C1-4alkyl; R9 represents halogen, -CN, -CONR7R12, -COR13, CO2R12, -OR12, -SO2R13, -SO2NR7R12, -NR7R12, -NR7COR12; R10 represents C1-4alkyl or R9-C0-4alkyl; R11 represents C1-4alkyl, halogen, -CN, -NR7R14; R12 represents hydrogen or R13; R13 represents C1-5alkyl, hydroxyC1-4alkyl, cyanoC1-4alkyl, Cy2-C0alkyl or R14R7N-C1-4alkyl; where Cy2 is optionally substituted with one or several constituents R11; R14 represents hydrogen or C1-4alkyl; R16 represents C1-4alkyl, halogenC1-4alkyl, C1-4alkoxyC1-4alkyl, hydroxyC1-4alkyl or cyanoC1-4alkyl; Cy1 represents monocyclic carbocyclic unsaturated or saturated ring, selected from C3-C6cycloalkyl, phenyl, or saturated monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or partially unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available C atom, and where one or several ring C or S atoms are optionally oxidised with formation of CO or SO2; and Cy2 represents monocyclic carbocyclic unsaturated ring, selected from C3-C6cycloalkyl, or aromatic monocyclic 4-6-membered heterocyclic ring, containing from 1 to 2 heteroatoms, selected from N and S, or unsaturated 10-membered bicyclic heterocyclic ring, containing oxygen atom as heteroatom, which can be substituted with group R11, where said ring is bound with the remaining part of molecule via any available atom C or N.

EFFECT: obtaining novel heteroaryl derivatives.

27 cl, 41 ex

Transdermal plaster // 2553350

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine. Described is matrix layer, suitable for application in plaster for transdermal delivery, aimed at introduction of biologically active compounds, which includes phosphate compound of tocopherol and polymer carrier. Also described is transdermal plaster and method of its production.

EFFECT: plaster makes it possible to efficiency introduce biologically active compounds.

48 cl, 15 tbl, 13 dwg, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and represents clinical nutrition for prevention, treatment or relief of one or several symptoms, associated with impairment of metabolism or its disorder, which contains composition of polysaccharide high-viscosity dietary fibre, including viscous fibre mixture or its complex, consisting of from 48% to 90% in wt % of glucomannan, from 5 to 20 % in wt % of xanthan gum and from 5% to 30% in wt % of alginate, as well as, at least, one macroelement, selected from the group, consisting of protein carbohydrate and fat, where clinical nutrition is composed in order to provide dose of composition of polysaccharide high-viscosity dietary fibre from 20 g/day to 35 g/day for time period, effective for prevention, treatment and relief of one or several symptoms, associated with impairment of metabolism or its disorder.

EFFECT: invention ensures extension of arsenal of means, preventing, relieving or treating one or several symptoms, associated with impairment of metabolism or metabolic disease.

14 cl, 6 ex, 20 tbl, 48 dwg

FIELD: medicine.

SUBSTANCE: patients with diabetic microangiopathy are subjected to an examination which includes: general blood test, blood sugar, general urine analysis, ultrasonic examination of kidneys with the determination of indices of the kidney blood flow (Vmax, Vmin, S/D, PI, RI), basic ophthalmological parameters (vision acuity, examination of eye fundus vessels). Then, the intake of mildly-mineralised hydrocarbonate-chloride-sodium mineral water "Obyhovskaya" directly from the spring under sanatorium conditions is administered. Water is taken in heated to a temperature of 37°C in a dose of 3 ml per 1 kg of body weight 3 times per day 40 minutes before meal, the course constitutes 18 days.

EFFECT: application of the invention makes it possible to normalise the general blood test, blood sugar, general urine analysis, improve the condition of the visual analyser and indices of the kidney blood flow.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to 2-pyridone compounds, represented by general formula [1], , where A represents benzene ring or pyridine ring, X represents structure, represented by general formula [3], V represents single bond or lower alkylene, W represents single bond, ether bond or lower alkylene, which can include ether bond, or their tautomers or stereoisomers.

EFFECT: obtaining pharmaceutically acceptable salts, which possess excellent activating activity with respect to GK and can be applied as medications.

27 cl, 23 tbl, 371 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quinolines substituted by phosphorus-containing group of formula and applicable in medicine, wherein Z represents V1 and V2 are independently specified in hydrogen or halogen; one of R and R` represent phosphorus-containing substitute Q; the other one is specified in hydrogen or methoxyl; wherein the phosphorus-containing substitute Q represents A represents O; L represents C1-6alkyl; J represents NH or C3-6heterocycloalkyl and J is optionally substituted by G3; X is absent or represents -C(=O)-; X is absent or represents C1-6alkyl; each of R1 and R2 are independently specified in C1-6alkyl or C1-6alkoxy; G3 represents C1-6alkyl, R3S(=O)m-, R5C(=O)- or R3R4NC(=O)-; R3, R4 and R5 are independently specified in 3 or C1-6alkyl; m is equal to 0-2.

EFFECT: there are presented new protein kinase inhibitors effective for treating the diseases associated with abnormal protein kinase activity.

20 cl, 42 ex, 8 tbl, 3 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biotechnology, namely to obtaining GLP-2 analogues, and can be used in medicine for treating GLP-2-associated disorders. The analogues of GLP-2 with agonistic activity with respect to GLP-2 receptors have been obtained.

EFFECT: invention makes it possible to increase resistance to proteases, which provides the lower clearance of the obtained analogues and prolongation of their bioavailability in comparison with native GLP-2.

29 cl, 4 tbl

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