New dipeptidyl peptidase iv inhibitors and uses thereof as hypotensive agent

FIELD: medicine, pharmaceuticals.

SUBSTANCE: disclosed is application of dipeptidyl peptidase inhibitors (DPIV-inhibitors) particularly isoleucyl-tiazolidine as active ingredient of pharmaceutical hypotensive composition for mammalian suffering from diabetes. Disclosed is stabilizing of systolic blood pressure in diabetic fats and lowering levels thereof from 170 mmHg (control animals without isoleucyl-tiazolidine) to 150 mmHg.

EFFECT: new dipeptidyl peptidase inhibitors useful in pharmaceutical hypotensive composition.

9 cl, 9 dwg, 11 tbl, 20 ex

 

The technical field

The present invention relates to inhibitors of the activity of dipeptidylpeptidase IV and dipeptidyl peptidase IV-like enzymes and, in particular, to pharmaceutical compositions containing the above compounds, and the use of these compounds for lowering blood pressure in mammals and (treatment) related disorders.

Prior art

The dipeptidyl peptidase IV (DPIV) is a serine protease that it N-terminal dipeptides from a peptide chain containing preferably a Proline residue in the penultimate position. Although the biological role of DPIV systems mammals is not fully installed, I believe that it plays an important role in the metabolism of neuropeptides, activation of T cells and the penetration of HIV (HIV) in lymphoid cells.

The present invention provides a new use of DPIV-inhibitors for preventing and treating conditions mediated by inhibition of DPIV and DPIV-like enzymes, in particular for lowering blood pressure levels and treating related disorders, and pharmaceutical compositions, for example, are useful for inhibiting DPIV and DPIV-like enzymes, and a method of inhibiting the activity of the above enzymes.

This invention relates to a method of treatment, in particular to a method of reducing levels of cu is reentered pressure in mammals and to compounds and compositions for use in the above method. The dipeptidyl peptidase IV (DPIV; EC 3.4.14.5; CD26) is a serine proteinase that cleave peptide bonds following Proline (to a lesser extent after alanine, after serine or after glycine), which is expressed in several tissues, including epithelial cells and a subset of leukocytes. In addition, it is associated with the membrane ectopeptidases, which shows its activity in the extracellular domain.

Examples of low molecular weight inhibitors dipeptidylpeptidase IV are tools, such as: derivative tetrahydroisoquinoline-3-carboxamide, N-substituted 2-cyanopropyl and-pyrrolidine, N-(N'-substituted glycyl)-2-cyanopyrrolidines, N-(substituted glycyl)thiazolidine, N-(substituted glycyl)-4-cyanothiophene, amino-acyl-borono-preliminary, cyclopropyl-condensed pyrrolidine and heterocyclic compounds. Inhibitors dipeptidylpeptidase IV described in U.S. patent 6380398, U.S. patent 6011155; U.S. patent 6107317; U.S. patent 6110949; U.S. patent 6124305; U.S. patent 6172081; international publication WO 95/15309,international publication WO 99/61431, WO 99/67278, international publication WO 99/67279, the patent DE 19834591, international publication WO 97/40832, the patent DE 196 16 486 C 2, international publications WO 98/19998, WO 00/07617, WO 99/38501, WO 99/46272, WO 99/38501, WO 01/68603, WO 01/40180, WO 01/81337, WO 01/81304, WO 01/55105, WO 02/02560 and WO 02/14271, described here refer to what are stated in their entirety, especially in respect of the above inhibitors, their definitions, applications and receive them.

The term DPIV-like enzymes relates to a structurally and/or functionally DPIV/D26-related enzyme proteins (Sedo &Malik, Dipeptidyl peptidase IV-like molecules: homologous proteins or homologous activities? Biochimica et Biophysica Acta 2001 36506: 1-10). Essentially, this small group, as it was revealed, releases H-Xaa-Pro Dipeptides and H-Xaa-Ala-Dipeptide from the N-terminal oligo - or polypeptides. They show a common feature, namely, that they act in a Pro-position, as well as Ala, Ser, Thr or other amino acids with small hydrophobic side chains, such as Gly or Val. Hydrolytic activity is located in the following Pro > Ala, Ser, Thr " Gly, Val. The same proteins were available in such small quantities that you can install only the post-Pro and post-Ala cleavage. While proteins DPIV, DP II, FAPa (Seprase), DP 6, DP 8 and DP 9 of structurally related and show high homology sequences, attractin is extraordinary functional DPIV-like enzyme, characterized by similar activity and a similar pattern of inhibition.

Other DPIV-like enzymes are disclosed in international patent publications WO 01/19866, WO 02/04610, WO 02/34900 and W0 02/31134. WO 01/19866 reveals a new human dipeptidylpeptidase (DPP8) with structural and functional the nd similarities with DPIV and fibroblast-activating protein (FAP). In international publication WO 02/34900 describes the new dipeptidyl peptidase 9 (DPP9), the structure of the amino acid sequence which is substantially similar to aminoacidopathies DPIV and DPP8. In international publication WO 02/31134 revealed three DPIV-like enzyme, DPRP1, DPRP2 and DPRP3. Sequencing showed that DPRPI identical DPP8, as disclosed in international publication WO 01/19866 that DPRP2 identical DPP9 and DPRP3 identical K1AA1492, as disclosed in international publication WO 02/04610.

High blood pressure (hypertension) is an asymptomatic condition, and this abnormally high pressure in the arteries increases the risk of problems such as stroke, aneurysm, heart failure, heart attack and kidney damage. For many people, the word "hypertension" means excessive tension, nervousness or stress. In medical terms, however, hypertension refers to the condition of high blood pressure regardless of the cause of its manifestation. It is called the "silent killer"because hypertension usually does not cause symptoms directly related to the disease, for many years, up until not damaged vital organ. High blood pressure is defined as systolic pressure at rest, which on average is 140 mm Hg or more, dia is tonicheskoe pressure at rest, which on average is equal to 90 mm Hg or more, or both. High blood pressure usually both systolic and diastolic pressures are elevated.

As a secondary action of diabetes is nerve damage, which regulate blood pressure and digestion. This leads to fluctuations in blood pressure; complications associated with swallowing, and change the function of the gastrointestinal tract with bouts of diarrhea. In addition, as a secondary effect of diabetes in some areas of the tissue of the arterial wall leads to the formation of atherosclerotic plaques that can block arteries of large and medium sizes in heart, brain, legs and penis. The walls of small blood vessels are damaged to such an extent that the vessels do not transport oxygen properly and can prevent the leakage.

Other definitions and classification of high blood pressure are presented in the Merck Manual of Medical Information-Home Edition, Merck & Co., 2000. In those cases, when systolic and diastolic blood pressure of the person correspond to the different categories for classification of blood pressure using a higher category. For example, 160/92 classified as hypertension stage 2, and 180/120 classified as hypertension (stage 4). Optimal CROs is anoe pressure, which minimizes the risk of problems associated with cardiovascular disease, below 120/80 mm Hg. However, unusually low pressuresshould be taken into account.

CategorySystolic blood pressureDiastolic blood pressure
Normal blood pressureBelow 130 mm HgBelow 85 mm Hg
High blood pressure130-13985-89
Hypertension stage 1 (mild)140-15990-99
Hypertension stage 2 (moderate)160-179100-109
Hypertension stage 3 (severe)180-209110-119
Hypertension stage 4 (very severe)210 or higher120 or higher

If a person has high blood pressure, corresponding to stage 3 (severe), or it is constant and is not treated, you experience symptoms such as: headache, fatigue, nausea, vomiting, shortness of breath, restlessness, and blurred vision due to brain, eyes, heart and kidneys. Sometimes people with high blood pressure there comes a state of drowsiness and even coma develops, call the major swelling of the brain. This condition, called hypertensive encephalopathy, requires emergency treatment.

The lack of systematic treatment of high blood pressure in humans increases the risk of heart disease (such as heart failure or heart attack), kidney failure and stroke at an early age. High blood pressure is the most important risk factor for stroke. In addition, it is one of the three major risk factors for developing a heart attack (myocardial infarction)that people can adjust; the other two factors are Smoking and high cholesterol.

Disclosure of inventions

The present invention provides a new application of DPIV-inhibitors of formula 1 to 12, and their corresponding pharmaceutically acceptable additive salts of acids to lower blood pressure or treat related disorders in mammals.

Reduced expression of ectopeptidases DPIV and lack of DPIV-like activity in the mutant F344 rats lacking enzymatic activity and expression of DPIV, leads to lower blood pressure. Were tested mutant as compared to the F344 lacking enzymatic activity of DPIV, and wild lines of typeF344. Chronic intragastric infusion isoleucyl-cyanopyrrolidine TPA and isoleucyl-thiazo is one fumarata using osmotic minnasota for two weeks reduced blood pressure in rats in a dose-dependent manner. Thus, blood pressure is reduced by chronic treatment with various inhibitors of DPIV (isoleucyl-thiazolidine fumarate; isoleucyl-cyanopyrrolidine TPA), which suggests the manifestation of similar protective action by two different DPIV-inhibitors/ligands. Perhaps isoleucyl-thiazolidine fumarate and isoleucyl-cyanopyrrolidine TPA protect against high blood pressure by increasing the levels of substrates DPIV, which indirectly mediate the appropriate action.

The present invention relates to a new method in which the reducing activity of the enzyme dipeptidylpeptidase (DPIV or D26) or activity of DPIV-like enzymes in the blood of mammals specific effectors of the enzyme leads to reduced degradation of endogenous or exogenous input insulinotropic peptides (incretins), a peptide hormone secreted by the stomach/glucose-dependent insulinotropic polypeptide 1-42 (GIP1-42) and glucagon-like Peptide-1 7-36 amide(GLP-17-36)(or analogues of these peptides). The decrease in the concentration of these peptides or their analogues, resulting from their degradation DPIV and DPIV-like enzymes, will be accordingly reduced or delayed.

As a result of increasing the stability of endogenous or exogenous input of incretins is whether their analogues, due to the reduction of DPIV activity, their insulinotropic steps are increased, which leads to a pronounced stimulation of insulin secretion from pancreatic islets of Langerhans and more rapid removal of glucose from the blood. As a result, the glucose tolerance increases.

As a consequence, the metabolic abnormalities associated with diabetes, including abnormalities of carbohydrate and lipid metabolism, glycosuria and diabetic ketoacidosis, and chronic changes, such as: microvascular and macrovascular disease, polyneuropathy and diabetic retinopathy, which are a result of prolonged elevated concentrations of circulating glucose, prevented or alleviated and, in particular, reduced high blood pressure levels.

The present invention represents a new approach to the reduction of elevated concentrations of blood glucose and elevated blood pressure levels. This is a simple, commercially viable and usable for treatment, especially of human diseases that are caused by elevated or extraordinary levels of glucose in the blood and/or blood pressure.

BRIEF DESCRIPTION of DRAWINGS

A deeper understanding of the present invention can be obtained by referring to the accompanying drawings, where:

Figure 1 shows MALDI-TOF analysis of DPIV-kata is isirimah hydrolysis GIP 1-42(a) and GLP7˜36(b) and inhibition of hydrolysis isoleucyl-thiazolidine.

2shows HPLC analysis of the presence in the serum metabolites of GLP-1 in the presence of an inhibitor of DPIV, isoleucyl-thiazolidine,in vivo.

3demonstratesthe impact ofDPIV-inhibitor, isoleucyl-thiazolidine on various parameters in blood undead. (i.d.)-glucosestimulated rats.

4demonstrates the effect of chronic oral treatment of obese (fa/fa) VDF rats Zucker DPIV-inhibitor, isoleucyl-thiazolidine on the levels of fasting blood glucose within 12 weeks of taking the drug.

Figure 5 demonstrates the effect of chronic treatment of obese (fa/fa) VDF rats Zucker DPIV-inhibitor, isoleucyl-thiazolidine on systolic blood pressure within an 8-week drug administration (systolic blood pressure measured using an overlay method cuffs on the tail).

6demonstrates dose-dependent reduction of glucose levels in blood of diabetic Zucker rats after oral administration of 5 mg/kg, 15 mg/kg, 50 mg/kg of M.L. glutaminetaurine and placebo, respectively;

7 shows dose-dependent reduction of glucose levels in blood of diabetic Zucker rats after oral administration of 5 mg/kg, 15 mg/kg, 50 mg/kg M.L., glutamylcysteine the a and placebo, respectively;

Fig shows the chemical structure of pyroglutamyl-thiazolidine, product degradation, detected after oral administration of glutamylcysteine to Wistar rats; and

Fig.9shows the chromatogram of the extract of rat plasma obtained after oral administration of glutamylcysteine obese Zucker rats. Peak at 2.95 and min represents glutamylcysteine and peak at 6,57 min represents Pyroglutamate.

A detailed description of the invention

The aim of the present invention is to develop a simple and new method of reducing the level of glucose in the blood and/or blood pressure, which decreases the activity of the enzyme dipeptidylpeptidase (DPIV or D26) or activity of DPIV-like enzymes in the blood of mammals induced by effectors of the enzyme, resulting in reduced degradation of endogenous (or exogenous input) insulinotropic peptides (incretins), a peptide hormone secreted by the stomach/glukozooksidasa insulinotropic polypeptide 1-42 (GIP1-42) and glucagon-like peptide-amide 1 (7-36) (GLP-17-36) (or analogues of these peptides). In accordance with this, the decrease in the concentration of these peptides or their analogues, resulting from their degradation DPIV and DPIV-like enzymes, will decrease or slow down.

The present invention is based n the unexpected finding, that reduction of the enzymatic activity of dipeptidylpeptidase IV (DPIV or D26) or activity of DPIV-like enzymes in the body of mammalsin vivoleads to increase glucose tolerance and decrease high blood pressure.

The authors found that:

1. The decreased activity of dipeptidylpeptidase IV (DPIV or D26) or DPIV-like enzymes leads to increased stability glucosestimulated endogenous secreted or exogenous input of incretins (or their equivalents) with the ensuing result that the introduction of effectors DPIV or DPIV-like proteins can be used to regulate the degradation of incretins participating in circulation.

2. Enhanced biological stability of incretins (or their analogues) leads to change in response to insulin.

3. The increased stability of circulating incretins caused by the reduction of dipeptidylpeptidase IV (DPIV or D26) or DPIV-like enzymes, leads to a subsequent change in insulin disposal (removal) of glucose, indicating that glucose tolerance can be enhanced by the application of DPIV-effectors.

4. High blood pressure is reduced.

Accordingly, this invention relates to the use of effectors activity dipeptidylpeptidase IV (DPIV or DPIV-like enzymes to reduce elevated levels chap the goats in the blood and/or blood pressure, such as the levels found in mammals, demonstrating clinically inappropriate basal and post-dining hyperglycemia. Application in accordance with the invention, in particular, is characterized by the introduction of effector activity of DPIV or DPIV-like enzymes to prevent or alleviate the pathological abnormalities of the metabolism of mammals, such as glycosuria, hyperlipidemia, diabetic ketoacidosis, diabetic retinopathy, and diabetes. In another preferred embodiment, the invention relates to a method of reducing elevated levels of blood glucose in mammals, such as the levels found in a mammal, demonstrating clinically inappropriate basal and post-dining hyperglycemia, comprising the administration to a mammal, in need of such treatment, a therapeutically effective amount of effector activity dipeptidylpeptidase IV (DPIV or DPIV-like enzymes.

In another preferred embodiment, the invention relates effector activity dipeptidylpeptidase IV (DPIV or DPIV-like enzymes used in the method of reducing elevated glucose levels in the blood and/or blood pressure in mammals, such as the levels found in mammals, demonstrating clinically nesootvetstvuyushih the basal and post-dining hyperglycemia.

The proposed effectors of DPIV and DPIV-like enzyme according to the present invention can be used in pharmaceutical compositions as inhibitors of enzymes, substrates, pseudosubstance, inhibitors of DPIV gene expression, binding proteins or antibodies, proteins, enzymes target or as a combination of these various compounds, which reduce the concentration of DPIV protein and DPIV-like protein or enzyme activity in mammals. Effectors according to the invention are, for example, inhibitors of DPIV, such as derivatives of dipeptides or dipeptide mimetics, such as elavilmigraine, solicitation and pseudospectral N-poured-prolyl, O-benzoyl-hydroxylamine. Such compounds are known from the literature [DEMUTH, H-U., Recent developments in the irreversible inhibition of serine and cysteine proteases.J. Enzyme Inhibition3, 249 (1990)] or they can be synthesized according to the methods described in the literature.

The method according to the invention represents a new approach to reducing the high concentration of glucose circulating in the blood of mammals, and to decrease high blood pressure levels.

The present invention relates to the field of inhibition of dipeptidylpeptidase IV (DPIV) and in particular to a new use of inhibitors of the activity of DPIV and DPIV-like enzymes to reduce high blood pressure levels or (treatment) related the data with these disorders in mammals, and pharmaceutical compositions containing the above compounds.

Unlike other proposed methods in this field the present invention provides an orally available therapy with low molecular weight inhibitors dipeptidylpeptidase IV. The present invention represents a new approach to reducing blood pressure levels and treating related disorders in mammals. This approach user-friendly, commercially viable and suitable for use in the treatment scheme, especially related to human diseases.

Based on the data, the study of the role of DPIV expression and enzymatic activity of DPIV in the regulation of blood pressure according to the invention has allowed to establish that oral administration of DPIV inhibitors decrease blood pressure levels.

The purpose of the present invention is to develop inhibitors dipeptidylpeptidase IV and/or ligands, which would have a high bioavailability. In another preferred embodiment, the present invention provides inhibitors of DPIV, which have a precisely predictable activity in the target tissue.

Examples of orally available funds with low molecular weight are prodrugs of stable and unstable inhibitors dipeptidylpeptidase IV total fo the mules A-B-C, where A represents an amino acid, B is a chemical bond between A and C or amino acid and is unstable or stable inhibitor dipeptidylpeptidase IV, respectively. Such compounds are described in international patent publications WO 99/67278 and WO 99/67279, the disclosure of which regarding a provision of the definition, use, and receipt of prodrugs here reference in their entirety. This particularly applies to the detailed description of the definitions of a, b and C.

The present invention relates to a new method in which the reduction of enzyme activity, dipeptidylpeptidase (DPIV or CD26) or activity of DPIV-like enzymes, or where the binding of the specific ligand DPIV have beneficial effects in mammals, induced by effectors of the enzyme and leads as a causal consequence of lowering blood pressure in a mammal. In the mammals, with high blood pressure will benefit from treatment with inhibitors of the activity of DPIV and DPIV-like enzymes.

The method and the use according to the present invention includes the prevention of high blood pressure or decrease in blood pressure and related disorders in an animal, including humans, by inhibiting DPIV, or activities of related enzymes, using the inhibitor or landuseplanning enzymes. In most circumstances, the oral administration of a DPIV inhibitor may be preferable.

Further the present invention is illustrated referring to the following examples, focusing on action in reducing blood pressure and glucose levels in the blood caused by reduction of DPIV-like activity and/or binding.

In one illustrative embodiment, the embodiment of the present invention relates to the use of the dipeptide-like compounds and compounds such dipeptide compounds that are formed from amino acids and thiazolidinone or pyrrolidino group, and their salts, referred to hereinafter as the dipeptide-like compounds. Preferably the amino and a group of thiazolidine or pyrrolidine linked by an amide bond.

Particularly suitable for this purpose according to the invention are dipeptide compounds in which the amino acid is preferably selected from natural amino acids, such as leucine, valine, glutamine, glutamic acid, Proline, isoleucine, asparagine and aspartic acid.

Dipeptide-like compounds used according to the invention, show at concentrations (dipeptide compounds) 10 µm reduced activity dipeptidylpeptidase IV or activities DPIV-like enzymes, at least 10%, especially at least 40%. Often required to reduce the activity of at least 60% or at least 70%. Preferred effectors can also show a decrease in the activity of up to 20% or 30%.

The preferred compounds are N-poured-prolyl, O-benzoyl-hydroxylamine, alanyl-pyrrolidin, isoleucyl-thiazolidin as well as their optical isomers L-Hello-isoleucyl-thiazolidine, L-Treo-isoleucyl-pyrrolidin and their salts, especially salts of fumaric acid, and L-Hello-isoleucyl-pyrrolidin and its salts. Particularly preferred compounds are glutaminergic and glutamylation formulas 1 and 2:

Other preferred compounds are presented in Table 1.

Salt of the dipeptide-like compounds can be in a molar ratio of dipeptide(-equivalent) component to the component of salt is 1:1 or 2:1.

This salt is, for example, (Ile-Thia)2fumaric acid.

Table 1: Patterns additional dipeptide compounds

The effector
H-Asn-pyrrolidin
H-Asn-thiazolidin
H-Asp-pyrrolidin
H-Asp-thiazolidin
H-Asp(NHOH)-pyrrolidin
H-Asp(NHOH)-thiazolidin
H-Glu-pyrrolidin
H-Glu-thiazolidin
H-Glu(NHOH)-pyrrolidin
H-Glu(NHOH)-thiazolidin
H-His-pyrrolidin
H-His-thiazolidin
H-Pro-pyrrolidin
H-Pro-thiazolidin
H-Ile-azetidin
H-Ile-pyrrolidin
H-L-ALLO-Ile-thiazolidin
H-Val-pyrrolidin
H-Val-thiazolidin

In another preferred variant of the embodiment of the present invention is the use of peptide compounds of formula 3, are useful for competitive modulation of dipeptidyl peptidase IV catalysis:

where A, B, C, Dand E represent independently any amino acid including proteinogenic amino acids, non-proteinogenic amino acids, L-amino acids and D-amino acids, and where E and/or D may be missing.

Additional conditions in relation to formula (3):

A represents an amino acid, excluding the D-amino acid,

B represents an amino acid selected from Pro, Ala, Ser, Gly, Hyp, azetidin-(2)-CT is about acid and pipecolinic acid,

C represents any amino acid except Pro, Hyp, azetidin-(2)-carboxylic acid, pipecolinic acid and except N-alkylated amino acids, e.g. N-methylvaline and sarcosin,

D represents any amino acid or is absent, and

E represents any amino acid or is absent

or:

C represents any amino acid except Pro, Hyp, azetidin-(2)-carboxylic acid, pipecolinic acid other than N-alkyl amino acids, for example, N-methylvaline and sarcosine, and also D-amino acids;

D represents theany amino acid selected from thePro, Ala, Ser, Gly, Hyp, azetidin-(2)-carboxylic acid and pipecolinic acid, and

E represents any amino acid except Pro, Hyp, azetidin-(2)-carboxylic acid, pipecolinic acid and except N-alkylated amino acids, e.g. N-methylvaline and sarcosin.

Examples of amino acids that can be used in the present invention are L and D-amino acids, N-methyl-amino acids;Hello-andtree-formIle and Thr, which can, for example, be a-, I - ω-amino acids, among which the a-amino acids are preferred.

Examples of amino acids for the claims and descriptions are:

aspartic acid (Asp), glutamic acid (Glu), arginine (Arg), lysine (Lys), histidine (His), glycine (Gly), serine (Ser) and cysteine (Cys), threonine (Thr, asparagine (Asn), glutamine (Gln), tyrosine (Tyr), alanine (Ala), Proline (Pro), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tryptophan (Trp), hydroxyproline (Hyp), beta-alanine (beta-Ala), 2-aminooctanoic acid (Aoa), azetidin-(2)-carboxylic acid (Aces), pipecolinate acid (Pip), 3-aminopropionic acid, 4-aminobutyric acid, etc., alpha aminoadamantane acid (Aib), sarcosine (Sar), ornithine (Orn), citrulline (Cit), homoarginine (Har),t-bucillamine (t-butyl-Ala),t-butylglycol (t-butyl-Gly), N-methylisoleucine (N-MeIle), phenylglycine (Phg), cyclohexylamine (Cha), norleucine (Nle), cysteine acid (Cya) and methionine sulfoxide (MSO), Acetyl-Lys, modified amino acids, such as phosphoryl-serine (Ser(P)), benzyl-serine (Ser(Bzl)) and phosphoryl-tyrosine (Tyr(P)), 2-aminobutyric acid (Abu), aminoacylation (AECys), carboxymethylcysteine (Cmc), dehydroalanine (Dha), dehydroamino-2-butyric acid (Dhb), carboxyglutamic acid (Gla), homoserine (Hse), hydroxylysine (Hyl), CIS-hydroxyproline(CISHyp), TRANShydroxyproline(TRANSHyp),isovaline (Iva), Pyroglutamate acid (Pyr), Norvaline (Nva), 2-aminobenzoic acid (2-Abz), 3-aminobenzoic acid (3-Abz), 4-aminobenzoic acid (4-Abz), 4-(aminomethyl)benzoic acid (Amb), 4-(aminomethyl)cyclohexanecarbonyl acid (4-Amc), Penicillamine (Pen), 2-amino-4-cyanomelana acid (Cba), cycloalkane carbon is the new acid.

Examples ω-amino acids are, for example: 5-Ara(aminosalicilova acid), 6-Ahx (aminohexanoic acid), 8-Aoc (aminooctanoic acid), 9-Anc (aminofenola acid), 10-Adc (mindcanvas acid), 11-Aun (aminoundecanoic acid), 12-Ado (aminododecanoic acid).

Additional amino acids are: intergrity (Igl), indolin-2-carboxylic acid (Idc), octahedron-2-carboxylic acid (Oic), diaminopropionic acid (Dpr), diaminobutane acid (Dbu), nafcillin (1-Nal), (2-Nal), 4-aminophenylalanine (Phe(4-NH2)), 4-benzylpenicillin (Bpa), diphenylalanine (Dip), 4-brompheniramine (Phe(4-Br)), 2-chlorophenylalanine (Phe(2-Cl)), 3-chlorophenylalanine (Phe(3-Cl)), 4-chlorophenylalanine (Phe(4-Cl)), 3,4-chlorophenylalanine (Phe(3,4-Cl2)), 3-forfinally (Phe(3-F)), 4-forfinally (Phe(4-F)), 3,4-forfinally (Phe(3,4-F2)), pentacarbonyliron (Phe(F5)), 4-guanidinopentanoic (Phe(4-guanidino)), homophenylalanine (hPhe), 3-itfinally (Phe(3-J)), 4-itfinally (Phe(4-J)), 4-methylphenylamine (Phe(4-Me)), 4-nitrovanillin (Phe-4-N02)), biphenylene (Bip), 4-phosphonomethylglycine (Pmp), cyclohexylglycine (Ghg), 3-pyridinylamino (3-Pal), 4-pyridinylamino (4-Pal), 3,4-dihydropyran (A-Pro), 4-metoprolol (Pro(4-keto), tiopronin (Thz), isonicotinoyl acid (Inp), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), propargylglycine (Pra), 6-hydroxyisoleucine (NU(6-OH)), somatropin (hTr), 3 iodotyrosine (Tyr(3-J)), 3,5-diiodotyrosine (Tyr(3,5-J2)), d-methyl-tyrosine (Tyr(Me)), 3-N02-tyrosine (Tyr(3-N02)), phosphotyrosine (Tyr(PO3H2)), allylglycine, 1-aminoindan-1-carboxy acid, 2-aminoindan-2-carboxy acid (Aic), 4-amino-methylpyrrole-2-carboxylic acid (Py), 4-amino-pyrrolidin-2-carboxylic acid (Abpc), 2-aminotetralin-2-carboxylic acid (Atc), diaminooctane acid (Gly(NH2)), diaminobutane acid (Dab), 1,3-dihydro-2H-isoindol-carboxylic acid (Disc), homecollection (hCha), homophenylalanine (hPhe or Hof),TRANS-3-phenyl-azetidin-2-carboxylic acid, 4-phenyl-pyrrolidin-2-carboxylic acid, 5-phenyl-pyrrolidine-2-carboxylic acid, 3-pyridylamine (3-Pya), 4-pyridylamine (4-Pya), stellaland, tetrahydroisoquinoline-1-carboxylic acid (Tiq), 1,2,3,4-tetrahydronaphthalen-3-carboxylic acid (Tpi), I-(2-thienyl)alanine (Tha).

Other substitutions of amino acids, the amino acids encoded in the genetic code, can also be included in peptide compounds within the scope of the invention and can be classified within this General scheme.

The term of proteinogenic amino acids means a-amino acids derived from natural proteins. The term non-proteinogenic amino acids refers to all other amino acids that are the building blocks of ordinary natural proteins.

The resulting PE the Chida can be synthesized as in the form of a free C-terminal acid, and the form of the C-terminal amide. Peptides with a free acid or amides may vary by modifications of their side chains. Such modifications of the side chain include, for example, but not limited to, education of homoserine, education pyroglutamic acid, disulfide bond formation, deliciousa residues asparagine or glutamine, methylation,t-butylation,t-butyloxycarbonyl, 4-methylbenzylamine, toonsylvania, teokratichna, benzyloxypyrrolidine, 4-nitrophenylamino, benzyloxycarbonylamino, 2-nitrobenzylamine, 2-nitrosodiphenylamine, 4-toluensulfonate, pentacarbonyliron, diphenylmethylsilane, 2-chlorobenzylidenemalononitrile, 2,4,5-trichloropropane, 2-bromobenzyloxycarbonyl, 9-fluorenylmethoxycarbonyloxy, triphenylmethane, 2,2,5,7,8-pentamethylchroman-6-sulfonylamine, hydroxylation, oxidation of methionine, formirovanie, acetylation, Anileridine, benzylidene, benzoylation, trichoroethylene, carboxylation aspartic acid or glutamic acid, phosphorylation, sulfation, containerbase, picolinate a pentose, deoxyhexose, hexosamine, hexoses or N-acetylhexosamines, farnesiana, aristolochiaceae, biotinyl the Finance, palmitoylation, staromlinovka, geranylgeranylation, glutathionylation, 5'-aminosilicone, ADP-ribosylating, modification of the N-glycolylneuraminic acid, N-acetylneuraminic acid, pyridoxal-phosphate, lipoic acid, 4'-phosphopantetheine, or N-hydroxysuccinimide.

In the compounds of the formula (3) amino acid part A, B, C, D, and E respectively attached to the adjacent part of the amide bonds in the normal way according to the standard nomenclature so that the amino end (N-terminal) amino acids (peptide) is depicted on the left (N-end) and carboxyl-end of the amino acids depicted on the right (From the end).

Prior to the present invention as peptide substrates Proline-specific serine protease of dipeptidylpeptidase IVin vitrowere known asthe tripeptides Diprotin A (Ile-Pro-Ile), Diprotin B (Val-Pro-Leu) and Diprotin C (Val-Pro-Ile). The authors it has been unexpectedly found that the compounds disclosed here above and below, act as substrates dipeptidylpeptidase IVin vivoin mammal and in pharmacological doses lower blood pressure and alleviate the pathological abnormalities of metabolism in mammals, such as glucosuria, hyperlipidaemia, metabolic acidosis and diabetes, through competitive catalysis.

Particularly preferred compounds of the present invention, which is used as a module is the tori dipeptidylpeptidase IV and DPIV-like enzymes, include such compounds which exhibit Kithe values for binding DPIV, effective in the inhibition of DPIVin vivoafter intravenous(i.v.) and/or oral (p.o.) introduction to Wistar rats.

Other preferred compounds are peptidylglycine formula 4:

where A is selected from

X1is H or acyl, or oxycarbonyl group, including all amino acid or peptide residues,

X2represents H, -(CH)n-NH-C5H3N-Y n=2-4 or C5H3N-Y (divalently the rest of pyridil) and Y is selected from H, Br, Cl, I, NO2or CN,

X3represents H or phenyl or pyridyl, unsubstituted or substituted one, two or more alkyl, alkoxy, halogen, nitro, cyano or carboxy groups,

X4represents H or phenyl or pyridyl, unsubstituted or substituted one, two or more alkyl, alkoxy, halogen, nitro, cyano or carboxy groups,

X5represents H or alkyl, alkoxy or phenyl,

X6represents H or alkyl;

for n=1

X is selected from H, OR2, SR2, NR2R3N+R2R3R4where

R2means acyl residues, which are unsubstituted or substituted one, two or more alkyl is m, cycloalkyl, aryl or heteroaryl, or denotes any amino acid and peptide residues, or alkyl residues, which are unsubstituted or substituted one, two or more alkilani, cycloalkyl, arinami and heteroaryl residues,

R3denotes alkyl and acyl function, where R2and R3can be part of one or more ring structures of unsaturated and saturated carbocyclic or heterocyclic structures

R4means alkyl residues, where R2and R4or R3and R4can be part of one or more ring structures of saturated and unsaturated carbocyclic or heterocyclic structures;

for n=0

X is chosen from:

where B denotes O, S, NR5where R5represents H, alkylidene or acyl,

C, D, E, F, G, H are independently selected from unsubstituted and substituted alkyl, oxyalkylene, thioalkyl, aminoalkyl, carbonrally, acyl, carbamoyl, aryl and heteroaryl residues; and

for n=0 and n=1

Z is selected from H, or C1-C9the alkyl branched or single chain or With2-C9alkenyl branched or single chain, With3-C8cycloalkyl,5-C7cycloalkenyl, aryl - or heteroaryl residue, or a side chain selected from all side chains of all natural amino acids or their derivatives.

In addition, according to the present invention are disclosed compounds of the formula 5, 6, 7, 8, 9, 10 and 11, including all stereometry and their pharmaceutically acceptable salts, and they can all be used:

where R1is H, a branched or straight1-C9alkyl, branched or straight2-C9alkenyl,3-C8cycloalkyl,5-C7cycloalkenyl, aryl or heteroaryl or a side chain of a natural amino acid or its derivative,

R3and R4selected from H, hydroxy, alkyl, alkoxy, aryloxy, nitro, cyano or halogen,

A represents H or ISOStAR carboxylic acid functional group selected from CN, SO3H, CONHOH, P03R5R6, tetrazole, amide, complex, ester, anhydride, thiazole, and imidazole,

B is selected from

where R5represents H, -(CH)n-NH-C5H3N-Y-c n=2-4 and C5H3N-Y (divalently the rest of pyridil) with Y=H, Br, CL, I, NO2or CN,

R10is H, acyl, oxycarbonyl or the balance of amino acids,

W represents H or phenyl or pyridyl, unsubstituted or substituted one, two or more alkyl, alkoxy, halogen, nitro, cyano or carboxy groups,

W1represents H, alkyl, alkoxy or phenyl,

Z is H is whether phenyl or pyridyl, unsubstituted or substituted one, two or more alkyl, alkoxy, halogen, nitro, cyano or carboxy residues,

Z' is H or alkyl,

D represents a cyclic C4-C7alkyl, C4-C7alkenyl, which may be unsubstituted or substituted one, two or more groups, or cyclic (4-7)-membered heteroalkyl or cyclic (4-7)-membered heteroalkyl,

X2represents O, NR6N+(R7)2or S,

X3to X12independently selected from CH2, CR8R9, NR6N+(R7)2, O, S, SO and SO2including all saturated or unsaturated structures

R6, R7, R8, R9independently selected from H, branched or straight C1-C9of alkyl, branched or straight C2-C9alkenyl, C3-C8cycloalkyl, C5-C7cycloalkenyl, aryl or heteroaryl,

under the following conditions:

Formula 6: X6represents CH, if A is not H,

Formula 7: X10is C if a is not H,

Formula 8: X7represents CH, if A is not H,

Formula 9: X12is C if A is not H,

Throughout the description and claims, the expression "acyl" may mean1-20acyl residue, predpochtitel the o 1-8the acyl residue and especially preferred With1-4acyl residue, "cycloalkyl" can be C3-12cycloalkenyl residue, preferably4With5or6cycloalkenyl balance, "carbocyclic" can mean3-12carbocyclic residue, preferably4With5or6carbocyclic residue. "Heteroaryl" denotes aryl residue, where 1 to 4, preferably 1, 2 or 3 ring atoms are replaced by heteroatoms such as N, S or O. "Heterocycle" means cycloalkyl balance, where 1, 2 or 3 ring atoms are replaced by heteroatoms such as N, S or O. "the Peptides" choose from dipeptides to Decapeptide, preferably dipeptides, tripeptides, tetrapeptides and pentapeptides. Amino acids for the formation of "peptides" can be selected from the amino acids listed above.

Because of the wide distribution of protein in the body and a variety of mechanisms, involving DPIV, DPIV activity and DPIV-related proteins, systemic therapy (interline or parenteral) DPIV-inhibitors may cause several unwanted side effects.

In addition, the task to be solved is the development of compounds that could be used for targeted impact on locally manifested pathophysiological and eziologicheskie processes. The invention, in particular, is to provide a locally limited inhibition of DPIV or DPIV-similar activity with the aim of purposeful intervention in the regulation of activity locally active substrates.

This task is solved in accordance with the invention using compounds of General formula (12)

where A represents an amino acid having at least one functional group in the side chain,

B is a chemical compound covalently associated with at least one functional group of the side chain And,

C represents thiazolidin, pyrrolidin, cyanopyrrolidine, hydroxyproline, digitopolis or piperidino group associated with And amide bond.

The connection can, for example, be used to lower blood pressure, acting on the DPIV or DPIV-like enzymes of the endothelium of blood vessels.

In accordance with a preferred embodiment of the invention, pharmaceutical compositions are used comprising at least one compound of General formula (12) and at least one customary adjuvant acceptable to the scene.

PreferablyAis the a-amino acid, especially of natural a-amino acid having one, two or more functional groups in the side chain, n is edocfile threonine, tyrosine, serine, arginine, lysine, aspartic acid, glutamic acid or cysteine.

PreferablyInrepresents an Oligopeptide with chain length up to 20 amino acids, polyethylene glycol having a molar mass of up to 20000 g/mol, optionally substituted organic amine, amide, alcohol, acid, or an aromatic compound having from 8 to 50 atoms C.

Throughout the text of the description and claims, the expression "alkyl" means1-50alkyl group, preferably6-30alkyl group, especially With8-12alkyl group; for example, the alkyl group may be methyl, ethyl, sawn, ISO-propyl or bucilina group. The expression "Ala", for example, in the expression "alkoxy" and the expression "alkane", for example, in the expression "alkanoyl" means "alkyl"; the aromatic compounds are preferably substituted or optionally unsubstituted phenyl, benzyl, naphthyl, biphenyl or anthracene groups, which preferably have at least 8 C atoms; the expression "alkenyl" can mean2-10alkenylphenol group, preferably2-6alkenylphenol group, which has the double bond(s) in any desired location and may be substituted or unsubstituted; the expression "quinil" can be C2-10alkylamino group, preferably/sub> With2-6alkylamino group, which has a triple bond(s) in any desired location and may be substituted or unsubstituted; the expression "substituted" or Deputy may indicate any desired substitution of one or more, preferably one or two, alkyl, alkenyl, quinil, mono - or multivalent acyl-, alkanoyl, alkoxyalkyl or alkoxyalkyl group; the above substituents may in turn have one or more (but preferably not to have) alkyl, alkenyl, quinil, mono - or multivalent acyl, alkanoyl, alkoxyalkyl or alkoxyalkyl groups in as side groups; organic amines, amides, alcohols or acids, each of which has from 8 to 50 C atoms, preferably from 10 to 20 C atoms, can have the formula (alkyl)2N - or alkyl-NH-, -CO-N(alkyl)2or-CO-NH(alkyl), -alkyl-OH, or-alkyl-COOH.

Despite the extended function of the side chain, the compounds of formula (12) can still bind with the active center of the enzyme dipeptidylpeptidase IV and of analogous enzymes, however, they cannot be transported further active peptide Transporter PepT1. Received reduced or very limited transportiruetes according to the invention leads to a local or targeted inhibition activity of DPIV and DPIV-like enzyme.

Connection is by means of formula (12) or other compounds and prodrugs, used in accordance with the invention, may be present or used, respectively, in the form of racemates or in the form of enantiomerically pure compounds, preferably L-Treo-or L-Hello-form in respect of part a of the formula (12).

By introducing branches in the side chain, for example, containing more than seven carbon atoms, it is possible accordingly to achieve a sharp reduction in transportability (see Example 12). The examples in Table 12.1 show that with increasing spatial size of the side chains is reduced transportability substances. By spatial and steric increase the size of the side chains, for example, an over-sized atomic group monosubstituted phenyl radical, radical hydroxylamine or amino acid residue in accordance with the invention to modify or suppress transportiruetes target substances.

According to the invention, the compounds of formula (12) inhibit the activity of DPIV or DPIV-like enzyme in the body of a mammal site-specific manner. In accordance with this effective impact on the local physiological and pathophysiological conditions (inflammation, psoriasis, arthritis, autoimmune diseases, allergies, cancer, metastasis, blood pressure in the endothelium of blood vessels), thus significantly reducing on the face-to-face action.

Preferred compounds of the formula (12) are compounds where oligopeptides have a length of chain from 3 to 15 amino acids, in particular from 4 to 10, and/or polyethylene glycols have a molecular weight of at least 250 g/mol, preferably at least 1500 g/mol and up to 15000 g/mol, and/or optionally substituted organic amines, amides, alcohols, acids or aromatic compounds have at least 12 C atoms and preferably up to 30 atoms C.

Compounds of the present invention can be converted into additive salt of the acid and used in the form of these salts, especially pharmaceutically acceptable additive salts of the acid. Pharmaceutically acceptable salt usually takes the form in which the basic side chain amino acids protonated inorganic or organic acid. Representative organic or inorganic acid include hydrochloric, Hydrobromic, perchloro, sulphuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, maleic, tartaric, citric, benzoic, almond, methansulfonate, hydroxyethanesulfonic, benzosulfimide, oxalic acid, Paveway, 2-naphthalenesulfonate, p-toluensulfonate, cyclohexanesulfamic, salicylic, some saccharine or triperoxonane acids the. All forms of pharmaceutically acceptable additive salts of acids are included in the scope of the present invention.

Because of the close relationship between the free compounds and the compounds in the form of their salts, whenever in this context, mention the connection is assumed and the corresponding salt, provided that in the circumstances it is possible or advisable.

In addition, the present invention includes within its scope prodrugs of the compounds of the present invention. In General, such prodrugs are functional derivatives of the compounds of the present invention, which can easily becomein vivothe desired therapeutically active compound. Accordingly, in these cases, the use of the present invention is to capture and treat various disorders described herein, variants (versions) of prodrugs of one or more of the inventive compounds, which becomein vivoin the above-mentioned specific compound after their introduction to the subject. Conventional procedures for the selection and obtaining the appropriate derivative prodrugs described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985 and patent applications DE 19828 113 and DE 19828 114, which are here referred to.

In the case when the inventive compounds or prodrugs have at least one chiral center, they who may accordingly exist as enantiomers. In the case where the compounds or prodrugs possess two or more chiral centers, they may additionally exist as diastereomers. It should be borne in mind that all of the above isomers or mixtures thereof are included in the scope of the present invention. In addition, some of the crystalline forms of the compounds or prodrugs may be in the form of polymorphic modifications, and it is assumed that such modifications are also covered by the present invention. In addition, some of the compounds may form a solvate with water (i.e. hydrates) or common organic solvents, and such solvate, as expected, also not beyond the scope of this invention.

The compounds, including their salts, can also be obtained in the form of their hydrates, or do they include other solvents used for crystallization.

As mentioned above, the compounds or prodrugs of the present invention and their corresponding forms, pharmaceutically acceptable additive salts of the acids are useful for inhibiting the activity of DPIV and DPIV-like enzymes.The ability of the compounds and prodrugs of the present invention and their respective forms of the pharmaceutically acceptable additive salts of acids to inhibit the activity of DPIV and DPIV-like enzymes may be PR is demonstrated, using the test activity of DPIV to determine the values of Kiand values IC50in vitroas described in examples 7 and 8.

The ability of the compounds of the present invention, and their corresponding pharmaceutically acceptable forms of the additive salts of the acid to inhibit DPIVin vivoit can be demonstrated that the introduction of their oral or intravascular by the Wistar rats as described in example 11. Compounds of the present invention inhibit the activity of DPIVin vivoafter their introduction to Wistar rats as oral and vnutrisosudisto.

DPIV is present in a wide range of organs and tissues of mammals, such as the brush border of the epithelial tissue of the intestine (Gutschmidt S et al., "In situ" measurement of protein content in the area of the brush border along the villi of the jejunum in rats and its correlation with the activities of the four enzymes. Histochemistry 1981, 72 (3), 467-79), endocrine epithelium, hepatocytes, renal tubules, endothelium, myofibroblast (A.C. Feller et al., A monoclonal antibody detecting dipeptidylpeptidase IV in human tissue. Virchows Arch. A. Pathol. Anat. Histopathol. 1986; 409 (2):263-73), nerve cells, lateral membranes of certain surface epithelia, such as the fallopian tubes, uterus and vesicular gland, luminale the cytoplasm, such as the epithelium, vesicular glands, and mucous cells of the duodenal glands (S. Hartel et al., Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemitry and activity histochemistry. Histochemistry 1988; 89 (2): 151-61), reproductive organs, for example, hostelry Department of epididymis and capsules (cauda epididymis and ampulla), the seminal vesicles and their secrets (Agrawal & Vanha-Perttula, Dipeptidyl peptidases in bovine reproductive organs and secretions. Int. J. Androl. 1986, 9 (6): 435-52). In human serum has two molecular forms of dipeptidylpeptidase (Krepela, E. et al., Demonstration of two molecular forms of dipeptidyl peptidase IV in normal human serum. Physiol. Bohemoslov. 1983, 32 (6): 486-96). Serum high-molecular form DPIV is expressed on the surface of activated T cells (Duke-Cohan J.S. et al., Serum high molecular weight dipeptidyl peptidase IV (CD26) is similar to a novel antigen, DP-PT-L released from activated T cells. J. Immunol. 1996, 156 (5): 1714-21).

The compounds and prodrugs of the present invention and their corresponding forms, pharmaceutically acceptable salts of the acid is able to inhibit DPIVin vivo.In one embodiment of the present invention all molecular forms, homologs and epitopes from all tissues and organs of mammals, and also those that have not been revealed, as expected, included in the scope of this invention.

Among the rare group of Proline-specific proteases, as originally believed, DPIV is the only membrane-bound enzyme, specific Proline as the penultimate residue at the amino end of the polypeptide chain. However, recently identified other molecules, structurally non-homologous DPI, but bearing the corresponding enzymatic activity. To DPIV-like enzymes, which have been identified at the present time include, for example, the fibroblast-activating protein a, dipeptidyl peptidase IV I, dipeptidylpeptidase-like protein, N-acetylated a-linked acidic dipeptidase, latent cell prolinamide, dipeptidyl peptidase II, attractin and dipeptidyl peptidase IV related protein (DPP 8), and they are described in a review article Sedo &Malik (Sedo &Malik, Dipeptidyl peptidase V-like molecules: homologous proteins or homologous activities? Biochimica et Biophysica Acta 2001 36506: 1-10). Other DPIV-like enzymes are disclosed in international publications WO 01/19866, WO 02/04610 and WO 02/34900. In WO 01/19866 revealed a new human dipeptidylpeptidase (DPP8), which has structural and functional similarities with DPIV, and fibroblast-activating protein (FAP). The dipeptidyl peptidase IV-like enzyme, described in WO 02/04610, well known in this field. In the database, gene Bank of this enzyme is registered as KIAA1492. In another preferred embodiment of the present invention all molecular forms, homologs and epitopes of proteins, including the activity of DPIV-like enzyme from all tissues and organs of mammals, and also those structures that have not been revealed, as expected, included in the scope of this invention.

The ability of compounds and Proletarsk the present invention, and their corresponding pharmaceutically acceptable forms of the additive salts of the acid to inhibit DPIV-like enzymes can be demonstrated, using the test of enzyme activity to determine the values of Kiin vitroas described in example 9. There were determined the values of Kicompounds of the present invention in relation to pork dipeptidylpeptidase II glutaminetaurine as Ki= charged 8.52·10-5M ± 6,33·10-6M and glutamylcysteine as Ki= 1,07·10-5M ± 3,81·10-7M.

In another variant embodiment of the compounds and prodrugs of the present invention and their corresponding forms, pharmaceutically acceptable additive salts of the acids have only a low (if any present) inhibitory activity against non-DPIV and non-DPIV-like Proline-specific enzymes. As described in example 10, in the case of glutamylcysteine and glutaminetaurine, no inhibition of dipeptidylpeptidase I and proletarisation was not detected. Regarding prolidase both compounds showed appreciable lower efficiency compared with DPIV. Were determined the values of the IC50regarding prolidase for glutamylcysteine as IC50> 3 mm for glutaminetaurine as IC50= 3,4·10-4M ± 5,63·10-5.

The present invention provides a method for prevention or treatment of a condition mediated by modulation ofactivityDPIV or DPIV-beneath the aqueous enzyme the subject, if necessary, of such treatment, which includes the introduction of any of the compounds of the present invention or pharmaceutical compositions based on the amount and regimen, a therapeutically effective for the treatment of the above-mentioned condition. In addition, the present invention includes the use of the compounds and prodrugs of the present invention, and their corresponding pharmaceutically acceptable additive salts of acids to obtain drugs for prevention or treatment of a subject condition mediated by modulation of the activity of DPIV. Connection, you can enter any patient in the usual way of introduction, including, but not limited to, intravenous, oral, intramuscular, intradermal, parenteral route of administration, and combinations thereof.

In another illustrative embodiment, the embodiment of the present invention provides formulations of the compounds of formulas 1 to 12, and their corresponding pharmaceutically acceptable additive salts of acids for pharmaceutical compositions.

The term "subject"used herein refers to an animal, preferably a mammal, most preferably the person who was the object of treatment, observation or experiment.

Used herein, the term "therapeutically effective amount" means such amount of the active with the organisations or pharmaceutical means, which causes the biological or medical response system tissue, animal or human, which is the goal of the researcher, veterinarian, doctor or other Clinician, and this reaction includes the endowment of the symptoms of the disease or condition to be treated.

Used herein, the term "composition", as suggested, covers the product, including the claimed compounds in therapeutically effective amounts, as well as any product that is directly or indirectly the result of combinations of the claimed compounds.

To obtain pharmaceutical compositions used in the present invention, one or more compounds of formula 1-12 or their respective pharmaceutically acceptable prodrugs or additive salts of acids as the active ingredient is thoroughly mixed with a pharmaceutical carrier to obtain a homogeneous mixture in accordance with conventional methods of preparing pharmaceutical compositions,and this media can take many forms depending on what dosage form of the drug is required for the selected route of administration, such as oral or parenteral route, such as intramuscular. To obtain the compositions for oral dosage form can be any of the usual pharmaceutically what environments. Thus, for liquid oral preparations, such as, for example, suspensions, elixirs and solutions, suitable carriers and additives can mainly include water, glycols, oils, alcohols, flavouring tools, preservatives, dyes and the like; for solid oral preparations such as, for example, powders, capsules, gel capsules and tablets, suitable carriers and additives include starches, sugars, diluents, granulating tools, lubricants, binders, dezintegriruetsja tools, etc. because of the ease with the introduction of tablets and capsules represent the most advantageous oral dosage unit form and in this case, use solid pharmaceutical carriers. If necessary, the tablets may be coated with a sugar coating or intersolubility coating in the usual ways. In the case of parenteral preparations, the media usually includes sterile water for other purposes, such as to facilitate dissolution or for preservation, may be included and other components.

In addition, you can get injectable suspension, and in this case can be used appropriate liquid carriers, suspendresume funds and other Pharmaceutical compositions, which are here referred to, usually contain per dosage unit, for example, tablet, capsule, powder, injection, Paul is th teaspoon etc., this amount of active ingredient that is needed to deliver an effective dose as described above. Pharmaceutical compositions, which are here referred to, usually contain per dosage unit, for example, tablet, capsule, powder, injection, full teaspoon and the like, from about 0.01 mg to about 1000 mg (preferably from about 5 to about 500 mg) and can be administered in a dosage of from about 0.1 to about 300 mg/kg of body weight per day (preferably from 1 to 50 mg/kg / day). However, the dosage can vary depending on the needs of patients, the severity of the condition to be treated, and the specific compounds to be applied. Can be used daily or introduction or post-periodic dosing. Usually the dosage is regulated by the attending physician, based on the characteristics of the individual patient, his/her condition and the expected therapeutic effect.

Preferably these compositions are presented in dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, auto device for injection or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration of putaminal or insufflation. An alternative composition may be presented in a form suitable for administration once a week or once a month; for example, an insoluble salt of the active compound, such as decanoate salt, can be used with receiving a depot preparation for intramuscular injection. For solid compositions such as tablets, the principal active ingredient is thoroughly mixed with a pharmaceutical carrier, e.g. conventional components for tableting, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to obtain a solid pre-composition containing a homogeneous mixture of the compounds of the present invention or its pharmaceutically acceptable salt. At the mention of these above pre-compositions as homogeneous, it is meant that the active ingredient is ideally dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective dosage forms such as tablets, pills and capsules. Specified solid pre-composition can then be subdivided into unit dosage forms of the type described above containing from about 0.01 to about 1000 mg, preferably from about 5 to about 500 mg of the asset the CSO component of the present invention.

The tablets or pills of the new compositions can be with a purpose covered or otherwise prepared to provide a dosage form, the advantage of which resulted prolonged action. For example, the tablet or pill may include an inner dosage and an outer dosage component, the latter serves as a shell covering the first. Two components can be separated intersolubility layer, which serves to prevent disintegration in the stomach and allows you to penetrate the intact inner component into the duodenum or contributes to its slow release. Various substances can be used for the above intersolubility layers or coatings, such substances include a number of polymeric acids with such substances as shellac, cetyl alcohol and cellulose acetate.

These liquid forms in which the new compositions of the present invention can be advantageously incorporated for administration orally or by injection include aqueous solutions, in an appropriate manner flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and analogues of the major pharmaceutical excipients. Suitable dispersing or suspendresume means for aqueous suspensions include synthetic or natural resins, such as tragakant, gum Arabic, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin.

In those cases where the means of obtaining the compounds according to the invention lead to a mixture of stereoisomers, these isomers may be separated by conventional methods such as preparative chromatography. The compound can be obtained in racemic form, or individual enantiomers may be obtained either enantiospecific synthesis or by separation. The connection can, for example, be separated into their enantiomers components by conventional means, such as the formation of diastereomeric pairs in salt formation with an optically active acid such as (-)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-l-tartaric acid, followed fractionated crystallization and regeneration of the free base. In addition, the compounds can be separated by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary substances. Alternative compounds can be separated using HPLC column filled with chiral sorbent.

In time the I any of the methods of obtaining the compounds of the present invention may be required or may be desirable to use protect sensitive or reactive groups on any of the examined molecules. This can be achieved using conventional protective groups such as the groups described in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; and T.W. Greene &P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons,1991,included here as reference. Protective groups can be removed at a convenient subsequent stage using known in the field methods.

Method of treating conditions modulated by dipeptidyl peptidase IV and DPIV-like enzymes described in the present invention can also be implemented usingpharmaceutical composition comprising one or more compounds identified here, and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain from about 0.01 mg to 1000 mg, preferably from about 5 to about 500 mg, of the compounds (compounds)and can be cooked in any form suitable for the selected method of administration.Carriers include necessary and inert pharmaceutical excipients, including, but not limited to, binders, suspendresume tools, lubricants, flavorings, sweeteners, preservatives, dyes and coatings. Compositions suitable for oral administration include solid forms, such as pills, tablets, Carley, capsules (each including formulations of immediate release, normalized by the straps release and extended release), granules and powders; and liquid forms such as solutions, syrups, elixirs, emulsions and suspensions. The forms used for parenteral administration include sterile solutions, emulsions and suspensions.

Mainly compounds of the present invention can be administered as a single daily dose or the total daily dosage may be injected parts, in small doses, two, three or four times a day. In addition, the compounds of the present invention can be introduced in intranasal form via topical application of suitable intranasal fillers or using transdermal pads (patches), well known to experts in the field.With the introduction of using a transdermal delivery system, the dosage should be continuous rather than intermittent throughout the dosage regimen medicines, and in accordance with this, the active substance will require changes in order to achieve the desired therapeutic effect.

In the case of oral administration in the form of tablets or capsules more preferably, when the active ingredient of medicines can be mixed with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. in Addition, if you or the hat the mixture may also include suitable binders; lubricants, dezintegriruetsja tools and dyes. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or betalactams, corn sweeteners, natural and synthetic gums, such as gum Arabic, tragakant or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and other compounds known in the field.

Suitable liquid forms include flavored suspendresume or dispersing agents such as the synthetic and natural gums, for example tragakant, acacia, methylcellulose and the like For parenteral administration requires sterile suspensions and solutions. In the case of intravenous use isotonic preparations, which usually contain suitable preservatives.

In addition, the compounds of the present invention can be introduced in the form of liposomal delivery systems, such as small adnyamathanha vesicles, large adnyamathanha and mnogokanalnye vesicles. Liposomes can be obtained from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholine using the method is, described in this field.

Compounds of the present invention can be also associated with soluble polymers, acting as agents, capable of directional transport drugs. Such polymers can include polyvinylpyrrolidone, copolymer Piran, polyhydroxybutyrates, polyhydroxyethylmethacrylate or polyethylenepolyamine, replaced by a Palmitoyl residue. In addition, the compounds of the present invention can be associated with a class of biodegradable polymers used for controlled release of drugs, for example polaktinova acid, poly-Exelon-caprolacton, polyhydroxybutyric acid, polychaetes, Polyacetals, policyidreference, polycyanoacrylate and sewn or amphipatic block copolymers of hydrogels.

The compounds of this invention can be enter in the composition of any of the above compositions in accordance with schemes of medicine established in this area, whenever you want treatment to address violations.

The daily dosage of the products may vary in a wide range from 0.01 to 1,000 mg per day for an adult. In the case of oral administration, the compositions are preferably provided in the form of tablets containing the x 0.01, 0,05, 0,1, 0,5, 1,0, 2,5, 5,0, 10,0, 15,0, 25,0, 50,0, 100, 150, 200, 250, 500 and 1000 mg of the active ingredient, so that you symptomatically, adjust the dose to the patient who undergoes treatment. An effective quantity of a drug is usually provided at the dose level of from about 0.1 mg/kg to about 300 mg/kg of body weight per day. Preferably, the dosage level is in the range from about 1 to about 50 mg/kg of body weight per day. Connections can be taken under the scheme from 1 to 4 times a day.

The optimal dose, subject to the introduction, can be easily defined by the experts in this field and they usually vary depending on the specific compound, the route of administration, the content of active ingredient in the drug, bioavailability, due to the way the introduction and progression of the disease state. In addition, when adjusting doses should take into account factors relating to the specific patient to be treated, including the patient's age, weight, diet and time of administration.

The compounds or compositions of the present invention can be taken before a meal, for example 1 hour, 30, 15 or 5 minutes before eating or drinking while you are eating or after eating.

When taken during meals compounds or compositions of the present invention can be added to foods or can be taken as a separate drug f RMI, as explained above.

EXAMPLES

Example 1: Synthesis of dipeptide-like compounds

1.1. Total synthesis of salt isoleucyl-thiazolidine

The BOC-protected amino acid BOC-Ile-OH is placed in ethyl acetate and the resulting mixture is cooled to about -5°C. dropwise at a constant temperature type N-metamorphosis, the acid chloride pavlinovoi acid (plant laboratory scale) or neodecanoate (installation polusovetskogo scale) is added dropwise at a constant temperature. The reaction mixture is stirred for several minutes to activate. Successively added dropwise N-methylmorpholine (laboratory scale) and thiazolidine hydrochloride (lab scale) add thiazolidin (polusovetskaya scale). Processing in the laboratory perform the usual way, using salt solutions, when installed polusovetskogo scale mixture of clear solutions of NaOH and CH3COOH.

The removal of the protective group Boc carried out using a mixture of HCl/dioxane (laboratory scale) or H2S04(polusovetskaya scale). In the laboratory hydrochloride is crystallized from a mixture of EtOH/simple ether.

When installed polusovetskogo scalethe free amine receive, adding a mixture of NaOH/NH3.Fumaric acid is dissolved in hot ethanol, is added dropwise free amine and (Ile-Thia)2fumarate (M =520,71 gmol-1) is deposited. Analysis of isomers and enantiomers carry out electrophoresis.

1.2. The synthesis of the free base of glutaminetaurine

The acylation

N-Benzyl-oxycarbonyl (2,02 g, 7,21 mmol) is dissolved in 35 ml THF and cooled to -15°C. To this mixture add CAIBE (isobutylparaben) (0,937 ml, 7,21 mmol) and 4-methylmorpholine (0,795 ml, 7,21 mmol) and the solution stirred for 15 minutes, the Formation of the mixed anhydride control using TLC (TLC) (eluent: CHCl3/MeOH: 9/1). After heating up to -10°C add pyrrolidine (0,596 ml, 7,21 mmol). The mixture allow to warm to room temperature and stirred over night.

Processing

The formed precipitate is filtered off and the solvent is evaporated. The oil obtained is dissolved in ethyl acetate (20 ml) and washed with a saturated solution of sodium hydrosulphate, then saturated sodium bicarbonate solution, water and saturated salt solution. The organic layer is separated, dried and evaporated. The resulting product is controlled for purity TLC (eluent: CHCl3/MeOH: 9/1).

Yield: 1.18 g, waxy solid

Splitting

1.18 g of the obtained solid Z-protected compound was dissolved in 40 ml of absolute ethanol. To the solution add approx. 20 mg of Pd on coal (10%, FLUKA) and the suspension is shaken in the atmosphere of hydrogen for 3 hours The course of the reaction is controlled by using TLC (eluent: CHCl3/MeOH: 9/1). After completion of the reaction the solvent is removed, obtaining the free base.

Yield: 99%

Purity control using TLC: a mixture of n-butanol/AcOH/water/ethyl acetate. 1/1/1/1, Rf= 0,4. The identity of the product of the reaction is controlled by the NMR method.

1.3. Synthesis glutamylcysteine hydrochloride

1.4. The acylation

N-t-Butyl-oxycarbonyl (2.0 g, 8,12 mmol) dissolved in 5 ml of THF and cooled to -15°C. To this mixture CAIBE (isobutylparaben) (1,06 ml of 8.12 mmol) and 4-methylmorpholine (0,895 ml of 8.12 mmol) and the solution stirred for 15 minutes, the Formation of the mixed anhydride control TLC (eluent: CHCl3/MeOH: 9/1). After heating up to -10°C add the equivalent of a 4-methylmorpholine (0,895 ml of 8.12 mmol) and thiazolidine hydrochloride (1,02 g, 8,12 mmol). The mixture allow to warm to room temperature and stirred over night.

Processing

The formed precipitate is filtered off and the solvent is evaporated. The oil obtained absorb chloroform (20 ml) and washed with a saturated solution of sodium hydrosulphate, and then saturated sodium bicarbonate solution, water and saturated salt solution. The organic layer is separated, dried and evaporated. The resulting product analyzed for purity using TLC (eluent: CHCl 3/MeOH: 9/1).

Yield: 1.64 g, solid

Splitting

640 mg of the obtained solid BOC-protected compound was dissolved in 3.1 ml ice mixture of HCl in dioxane (12,98M, 20 equivalents) and leave on ice. The course of the reaction is controlled by using TLC (eluent: CHCl3/MeOH: 9/1). After completion of the reaction the solvent is removed and then the resulting oil is dissolved in methanol and again evaporated. The resulting oil is dried over phosphorus oxide (V) and triturated twice with diethyl ether. Purity control by using HPLC (HPLC).

Output: 0,265 g

Purity control by using HPLC. The identity of the product of the reaction is controlled by the NMR method.

1.4. Synthesis glutaminergic hydrochloride

The acylation

N-t-Butyl-oxycarbonyl (3.0 g, 12,18 mmol) dissolved in 7 ml of THF and cooled to -15°C. To this mixture CAIBE (isobutylparaben)) (1.6 ml, 12,18 mmol) and 4-methylmorpholine (1.3 ml, 12,18 mmol) and the solution stirred for 15 minutes, the Formation of the mixed anhydride control TLC (eluent: CHCl3/MeOH: 9/1). After heating up to -10°C was added 1 equivalent of pyrrolidine (10 ml, 12,18 mmol). The mixture allow to warm to room temperature and stirred over night.

Processing

The formed precipitate is filtered off and the solvent you arevut. The oil obtained absorb chloroform (20 ml) and washed with a saturated solution of sodium hydrosulphate, then saturated sodium bicarbonate solution, water and saturated salt solution. The organic layer is separated, dried and evaporated. The resulting product is controlled using TLC (eluent: CHCl3/MeOH: 9/1).

Yield: 2.7 g, solid

Splitting

2.7 g of the obtained solid substance was dissolved 13.0 ml ice mixture of HCl in dioxane (12,98M, 20 equivalents) and leave on ice. The course of the reaction is controlled by using TLC (eluent: CHCl3/MeOH: 9/1). After completion of the reaction the solvent is removed and the resulting oil absorb methanol and again evaporated. The resulting oil is dried over phosphorus oxide (V) and triturated twice with diethyl ether.

Output: 980 mg

Purity control by using HPLC. Identichnosti product of the reaction is controlled by analysis of the NMR method.

Example 2: Chemical analysis of selected dipeptide compounds

2.1. Determination of the melting point

The melting point was determined on the microscope, equipped with a heating platform from Leica Aktiengesellschaft, values are given without correction, or installing DSC (HeumannPharma).

2.2. Optical rotation

The values of the rotation of the plane of polarization of light recorded at different wavelengths n the Polarimeter 341" or above from Perkin-Elmer.

2.3. The measurement conditions for mass spectroscopy

Mass spectra recorded by the method of electrospray ionization (ESI) on devices API 165" or "API 365" PE Sciex. The operation is carried out, using the appropriate concentration of c=10 µg/ml of the substance dissolved in a mixture of MeOH/H20 to 50:50, with 0.1% HCO2H, the introduction of samples carried out using a spray pump (20 µl/min). The measurements were carried out with the registration of positive ions [M+H]+the ESI voltage U=W.

2.4. Results

2.4.1. The study isoleucyl-thiazolidin fumarata (isomer)

SubstanceTPL (°)CE (min)MS[a]H20
L-threo-IT*F150DSC160203-10,7

(405 nm)
D-threo-IT*F147158203not defined.
L-ALLO-IT*F145-6154203-4,58

(380 nm)
D-ALLO-IT*f144-61502034,5

(380 nm)

IT*F = isoleucyl-thiazolidine fumarate

Data of NMR and HPLC podvergaut identichnosti the substances

2.4.2 Study of other salts isoleucyl-thiazolidine

IT*SolM (gmol-1)TPL (°C)
Succinate522,73116
Tartrate352,41122
Fumarate520,71156
hydrochloride238,77169
Phosphate300,32105

Example 3: Synthesis of Xaa-Pro-Yaa tripeptides

All syntheses carried out on the peptide synthesizer SP 650 (Labortec AG), using Fmoc/Twi strategy. Protected amino acids were supplied by Novabiochem or Bachem, triperoxonane acid (TFA) were purchased from Merck, triisopropylsilane (TIS) was purchased from Fluka.

Preloaded with Fmoc-Yaa-Wang resin (2.8 g, degree of substitution of 0.57 mmol/g) remove the protection using a mixture of 20% piperidine/N,N-dimethylformamide (DMF), DMF). After washing DMF solution of 2 EQ. (1.1 g) Fmoc-Pro-OH was dissolved in DMF (12 ml solvent per gram of resin). Add 2 EQ. (1.04 g) of 2-(1H - benzotriazol-1-yl)-1,1,3,3-tetramethyluronium of tetrafluoroborate (TBTU) and 4 EQ. (1,11 ml) N,N-diisopropylethylamine (DIEA, DIEA) and placed in the reaction vessel. The mixture is shaken at room temperature for 20 minutes Then cycle accession repeat. After subsequent washing DMF dichloromethane, isopropanol and diethyl e the IR of the obtained Fmoc-Pro-Ile-Wang resin is dried and then is divided into six parts to attach the last derived amino acids.

Fmoc-protective group is removed as described above. After that, 0.54 mmol of the BOC-amino acid, 0.54 mmol TBTU and to 0.108 mmol DIEA (DIEA) in DMF shaken for 20 min. Cycle accession repeat. Finally, the resin-peptide is washed and dried as described above.

Peptide otscheplaut from the resin using a mixture of triperoxonane acid (TFA, TFA) for 2.5 h, contains the following sinks: TPA/H2O/triisopropylsilane (TIS)=9,5/0,25/0,25.

The outputs of the crude peptides was 80-90%, on average. The crude peptide is purified by means of HPLC on a column of Nucleosil C18 (7 μm, 250*21,20 mm, 100 A)using a linear gradient of 0.1% TFA/H2O with increasing concentration of 0.1% TFA/acetonitrile (5% to 65% over 40 min) at a flow rate of 6 ml/min

Pure peptide is produced by lyophilization, identify methods of electrospray mass spectrometry and HPLC.

3.1. Results Identification of tripeptides Xaa-Pro-Yaa after chemical synthesis

PeptideWeight (expect.)Weight (exp.)1< / br>
[M+H+]
HPLC k'2
Abu-Pro-Ile313,4314,0the 5.7
Cha-Pro-Ile381,52382,010,4
Nva-Pro-Ile327,43 328,26,82
Phg-Pro-Ile361,44362,27,9
NIe-Pro-Ile341,45342,28,09
Pip-Pro-Ile338,56340,06,5
Thr-Pro-Ile329,4330,05,12
Trp-Pro-Ile414,51415,29,85
Phe-Pro-Ile375,47376,28,96
Ser-Pro-Ile315,37316,35,24
Ser(P)-Pro-Ile395,37396,03,35
Tyr(P)-Pro-Ile471,47472,35,14
Val-Pro-Val313,4314,05,07
Ile-Pro-Val327,43328,56,41
Ile-Pro-Hello-Ile341,4342,07,72
Val-Pro-Hello-Ile327,4328,56,51
Tyr-Pro-Hello-Ile391,5392,07,02
2-amino octanoic acid-Pro-Ile369,5370,210,63
Ser(Bzl)-Pro-Ile405,49406,0 9,87
Orn-Pro-Ile342,42343,1to 3.73
Tic-Pro-Ile387,46388,08,57
Aze-Pro-Ile311,4312,4of 5.29
Aib-Pro-Ile313,4314,0the 5.25
t-butyl-Gly-Pro-Ile341,47342,17,16
Ile-Hyp-LEU356,45358,2to 6.57
t-butyl-Gly-Pro-Val327,4328,46,32
t-butyl-Gly-Pro-Gly285,4286,33,74
t-butyl-Gly-Pro-Ile-amide340,47341,37,8
t-butyl-Gly-Pro-D-Val327,4328,67,27
t-butyl-Gly-Pro-t-butyl-Gly341,24342,5a 9.09
Ile-Pro-t-butyl-Gly341,47342,366,93
Val-Pro-t-butyl-Gly327,4328,155,98

1[M+H+] were identified by means of electrospray mass spectrometry in the registration mode, the positive ions.

2RP-HPLC conditions:

column: LiChrospher 100 P 18 (5 μm), 125 x 4 mm

detection (UV), 214 MMO

gradient elution system: acetonitrile (atsn, ACN)/H20 (0,1% TFA) from 5% atsn up to 50% for 15 min,

flow: 1 ml/min

k'=(tr-to)/to

to=1,16 min

t-butyl-Gly means:

Ser(Bzl) and Ser(P) denote benildean and fosfomycin, respectively. Tyr(P) means phosphorilation.

Example 4: Synthesis of peptidoglicanov

H-Val-Pro-OMe*HCl2

BOC-Val-OH (3.00 g, of 13.8 mmol) dissolved in 10 ml THF and cooled down to -15°C. To this mixture CAIBE (isobutylparaben, 1.80m, is 13.8 mmol) and NMM (1,52 ml of 13.8 mmol) and the solution stirred until then, until the formation of the mixed anhydride. The mixture is then brought to -10°and added NMM (1,52 ml of 13.8 mmol), and then H-Pro-OMe*HCl (to 2.29 g of 13.8 mmol). The mixture allow to reach room temperature and left over night. After removal of the solvent and conventional treatment have received ester1without additional okharakterizovanie. Ester1dissolved in a mixture of HCl/HOAc (5 ml, 6N) and leave at 0°up until the removal of the BOC-group is complete. Then the solvent is removed and the oil obtained is treated with diethyl ether, obtaining white solid2.

Yield: 2.5 g, 80%

Z-Ala-Val-Pro-OMe3

Z-Ala-OH (3.5 g, 15.7 mmol) and2(4,18 g,15.7 mmol) was processed in the same way as mentioned above for1,getting3in the form of a white solid.

Output: 4.2V g, 64%

Z-Ala-Val-Pro-OH4

3(4,2 g 9.6 mmol) is dissolved in 30 ml of a mixture water/acetone (1/5 V/V) and add to 11.6 ml of NaOH (1N). After completion of the reaction, the organic solvent is removed by evaporation and the resulting solution was diluted with 15 ml of a saturated solution of NaHCO3. The mixture is then extracted three times with 10 ml ethyl ester of acetic acid. After that, the pH of the solution was adjusted to 2 by adding HCl (15% in water). The resulting mixture was extracted three times with 30 ml ethyl ester of acetic acid. The organic layer is separated and washed three times with saturated salt solution, dried (Na2SO4) and evaporated.

Yield: 3.5 g, 87%

Z-Ala-Val-Pro-CH2-Br5

4(2.00 g, 4.76 mmol) was dissolved in 15 ml of THF and turn into a mixed anhydride (see compound 1)using CAIBE (0,623 ml, 4.76 mmol) and NMM (0,525 ml, 4.76 mmol). The formed precipitate is filtered and cooled down to -15°C. Then the solution is added dropwise in an argon atmosphere add diazomethane (23,8 mmol in 30 ml simple ether). After keeping the mixture for 1 h at 0°type of 1.27 ml of HBr (33% in AcOH) and the solution stirred for 30 min at room temperature. After that add the 70 ml simple ether and the mixture is washed with 20 ml of water. The organic layer is separated and dried (Na2SO4) and evaporated.

Yield (crude): 1.8 g, 80%

Z-protected acyloxymethyl

Acid (2 EQ.) dissolved in DMF (DMF) and added an equimolar amount of KF. Suspension give the opportunity to mix at room temperature for 1 h Then add bromethalin (1 EQ.) and the solution is allowed the opportunity to mix throughout the night. After that, the solvent is removed in vacuum and the oil obtained is dissolved in chloroform and washed with a saturated solution of salt. Then the organic layer is separated, dried (Na2SO4) and the solvent is removed. The product was then purified column chromatography using silica gel and a mixture of heptane/chloroform as eluent.

Z-Ala-Val-Pro-CH2O-C(O)-CH36

Acetic acid (230 μl, 4.02 mmol), KF (0,234 g, 4.02 mmol),5(1,00 g, a 2.01 mmol).

Output: 0,351 g, 36%

Z-Ala-Val-Pro-CH2O-C(O)-Ph7

Benzoic acid (0,275 g, 2.25 mmol), KF (0,131 mg, 2.25 mmol),5(0.56 g, 1.13 mmol).

Yield: 0.34 g, 56%

Unprotect

Z-protected compound was dissolved in a mixture of HBr/AcOH and stirred. After the reaction is complete, add a simple ether, the resulting white precipitate filtered and dried.

H-Ala-Val-Pro-CH2O-C(O)CH3* HBr8

6(0,351 g, 0.73 mmol)

Output: 0,252 g, 98%

H-Ala-Val-Pro-CH2O-C(O)Ph*Hr 9

7(0.34 g, to 0.63 mmol)

Output: 0,251 g, 99%

Example 5: Synthesis of cycloalkylation

Vos-isoleucinol2

Oxalicacid (714 μl, of 8.28 mmol) dissolved in 10 ml of dry dichloromethane and cooled to -78°C. Then added dropwise DMSO (DMSO) (817 μl, of 8.28 mmol). The solution is stirred for 20 min at -78°C. Then add1(1,00 g, 4.6 mmol) and the mixture is stirred for 20 minutes then add TFA (2,58 ml, 18.4 mmol) and the mixture allow to reach room temperature. The mixture is diluted with a mixture of hexane/ethyl acetate (2/1 V/V)and add 10 ml HCl (10% in water). The organic layer is separated and the aqueous phase is extracted with 20 ml of methylene chloride. The organic layers are combined and washed with saturated salt solution, then with water and then dried. The product was then purified column chromatography using silica gel and a mixture of heptane/chloroform as eluent.

Yield: 0.52 g, 52%

tert-Butyl N-1-[cyclopentyl(hydroxy)methyl]-2-methyl-BUTYLCARBAMATE3

2(0.52 g, 2,42 mmol) dissolved in 10 ml of dry THF and cooled down to 0°C. add cyclopentylamine (1,45 ml of 2M solution). After completion of the reaction, add 2 ml of water and the solution is neutralized by adding aqueous HCl. Then add methylene chloride and the organic layer separated and dried (Na2SO ). After evaporation the resulting oil is used without further okharakterizovanie.

tert-Butyl N-[1-(cyclopentanecarbonyl)-2-methylbutyl]carbamate4

3(0,61 g of 2.15 mmol) is treated like a1.Oxalicacid (333 μl, a 3.87 mmol), DMSO (382 μl, 5.37 mmol), TFA (1.2 ml, 8,59 mmol).

Output: 0,180 g, 30%

1-cyclopentyl-3-methyl-1-oxo-2-phenteramin chloride5

4(0.18 g, to 0.63 mmol) dissolved in 2 ml of HCl (7N in dioxane). After completion of the reaction, the solvent is removed and the resulting oil purified column chromatography on silica gel, using gradient elution with a mixture of chloroform/methanol/water. The resulting oil triturated with simple ether.

Output: to 0.060 g, 54%

Example 6: Synthesis of DPIV inhibitor with a modified side chain

6.1.Synthesis of BOC-glutamylcysteine (BOC-Glu-Thia)

Reaction (interaction) of the BOC-Glu(OMe)-OH with Thia*HCl according to the method (see section 6.4 for methods), hydrolysis of the BOC-Glu(OMe)-Thia according to method G.

6.1.1Analytical data for Boc-Glu-Thia

ConnectionEmpirical formula

Mr;

The method of synthesis;

Output
MS [M+H]+TLC: Rf/system

TPL
[a]20D Concentration; SolventElemental analysis (expect/

found) %
HPLC

Rt[min]/

system
Boc-Glu-ThiaWith13H22N2O5S 318,38;

B+G;

62%
319,5

0,52/A10,42/B1< / br>
115-1180
-3,1;

c=1; methanol
C:49,04/48,89

H:6,96/6,82

N:8,80/8,59
13,93 /A2

1Thin-layer chromatography

A: chloroform/methanol90:10

System B: benzene/acetone/acetic acid 25:10:0,5

System C: n-butanol/EA/acetic acid/H2O1:1:1:1

2The HPLC conditions for the separation of:

Column: Nucleosil C-18, 7 MK, 250 mm x 21 mm

Eluent: isocratic elution, 40 % atsn/water/0.1% of TFA

Flow rate: 6 ml/min

λ=220 nm

6.2. Vos-glutamylcysteine with a modified side chain

BOC-Glu-Thia change in position of function γ-carboxylic acid by the introduction of radicals with different chain length. Radicals attached via their amino groups with the formation of amide linkages with the function γ-carboxylic acid, used different methods of joining, depending on the (nature) of the radical. Below terminal of amine component, which is attached to the BOC-Glu-Thia indicating used for this method:

Amino componentMethods of joining

Outputs
The polyethylene glycol amine (Mr≈8000)C93%
H-Gly-Gly-Gly-OHD+E49%
H-Gly-Gly-Gly-Gly-Gly-OHD+E86%

In 2 cases, purification of the reaction products differ from the General description of synthesis.

BOC-Glu(Gly5)-Thia

The product precipitates from the mixture already under stirring over night; in the future it is filtered off and washed with 0,1N HCl and with copious quantities of water and then dried over P4010in a vacuum.

BOC-Glu(PEG)-Thia

In contrast to General procedure educt for the synthesis dissolved in 500-fold excess DMF. After completion of the reaction, DMF is completely removed in vacuo, and the residue is dissolved in a large quantity of methanol. After adding a simple ether with the formation of the upper layer, the product precipitates together with unreacted PEG. Fine cleaning is carried out by separation using preparative HPLC on a gel-filtration column (Pharmazia, Sephadex G-25, 90 mm, 260 mm-100 mm). The separation conditions: eluent: water; flow rate: 5 ml/min; λ=220 nm.

6.2.2.Data synthesis for modified in the side chain Boc-glutamylcysteine

ConnectionEMPI is practical formula

Mr;

Output
MS [M+H]+TLC/Rf/ system

TPL
[a]20D

Concentration

Solvent
Elemental analysis

(Expect to./

found)%
HPLC

Rt[min]/ system
BOC-Glu-(Gly3)-ThiaWith19H31N5O8S; 489,54;

49%
490,5C:46,62

H:6,38

N:14,31
BOC-Glu-(Gly5)-ThiaC23H37N7O10S

603,64

86%
604,5;

0,09/S;

different.

from 202°
not determined.C:45,76/45,60

H:6,18/6,11

N:16,24/16,5
11,93/A2
BOC-Glu-(PEG)-Thia93%≈8000

(emphasis on a lot)

52-53°
Not determined.not determined.not determined.

2Conditions of HPLC separation:

Column: Nucleosil C-18, 7 MK, 250 mm x 21 mm

Eluent: isocratic elution, 40% atsn/water/0.1% of TFA

Flow rate: 6 ml/min

λ=220 nm

6.3.Glutamylcysteine with a modified side chain

N-terminal BOC protective group otscheplaut from the compounds described in Table 6.2.2, using method F. Substances, modified Gly-derived, purified by separation of the preparation is effective HPLC and are available in the form of trifurcation. H-Glu(PEG)-Thia purified on a gel-filtration column in the same way as the Boc-protected precursor.

6.3.1.Data synthesis for modified in the side chain of glutamylcysteine

ConnectionEmpirical formula;

Mr< / br>
Output
MS [M+H]+< / br>
TLC/Rf/ system

TPL
[a]20D

Concentration

Solvent
Elemental analysis (expect to./found)%HPLC

Rt[min]/ system
H-Glu(Gly3)-Thia *TPAC16H24N5O8SF3< / br>
503,45

94%
503,45

0,32/C

91-940
+4,1

c=1

methanol
C:38,17/37,56

H:4,80/4,78

N:13,91/13,43
7,84/C3
H-Glu(Gly5)-Thia*TPAC20H30N7O10SF3< / br>
617,55

98%
617,55

0,25/C

105-107°
not determined.C:38,90/38,82

H:4,90/4,79

N:15,88/15,39
by 8.22/C3
H-Glu(PEG)-Thia *HCl92%≈8000

(emphasis on a lot)
not determined.not determined.not determined.

3Conditions of HPLC separation:

Column: Nucleosil C-18, 7 MK, 250 mm x 21 mm

Eluent: atsn/water/0.1% of TFA

Gradient: 20%Atsn 90% atsn for 30 min

Flow rate: 6 ml/min

λ=220 nm

not determ. - not determined or not determined

6.4. General methods of synthesis

Method A: Connection with the formation of the peptide bond by the method of mixed anhydrides using CFIBE as the activating reagent

10 mmol of the amino acid or peptide with an N-protected end was dissolved in 20 ml of absolute THF. The solution is cooled to -15° ± 2°C. With stirring in each case successively added 10 mmol NMM and 10 mmol of isobutyl ether of Harborview acid, strictly maintaining the prescribed temperature range. After approximately 6 min add 10 mmol aminocompounds. In those cases, when aminocompounds is salt, then to the reaction mixture add an additional 10 mmol NMM. Then the reaction mixture was stirred for 2 h in the cold and over night at room temperature.

The reaction mixture is concentrated using a rotary evaporator, absorbs in EA (ethyl acetate, EA), washed with 5% solution KH2SO4, a saturated solution of NaHCO3and saturated NaCl solution and dried over NaSO4. After removal of solvent in vacuo, the compound is recrystallized from a mixture of EA/pentane.

Method B: Attach with education is the Finance of the peptide bond by the method of mixed anhydrides, using the acid chloride pavlinovoi acid as the activating reagent

10 mmol of the amino acid or peptide with an N-protected end was dissolved in 20 ml of absolute THF. The solution is cooled to 0°C. With stirring in each case successively added 10 mmol NMM and 10 mmol of acid chloride pavlinovoi acid, strictly maintaining the set temperature interval. After approximately 6 minutes and the mixture is cooled to -15°and as soon as this was achieved low temperature, add 10 mmol aminocompounds. In those cases, when aminocompounds is salt, then to the reaction mixture add an additional 10 mmol NMM. Then the reaction mixture was stirred for 2 h in the cold and over night at room temperature.

Further treatment is carried out in the same way as in Method A.

Method C: Connection with the formation of the peptide bond, using TBTU as the activating reagent

10 mmol of the amino acid or peptide with an N-protected end and 10 mmol aminocompounds with protected end was dissolved in 20 ml of absolute DMF (DMF). The solution is cooled to 0°C. With stirring in each case successively added 10 mmol DIPEA and 10 mmol TBTU. The reaction mixture was stirred for 1 h at 0°and then over night at room temperature is. DMF completely removed in vacuum, and the product is treated as described in Method A.

Method D: Synthesis of activated complex ester (ester of N-hydroxysuccinimide)

10 mmol of the amino acid or peptide with an N-protected end and 10 mmol of N-hydroxysuccinimide dissolved in 20 ml of absolute THF. The solution is cooled to 0°and with stirring, add 10 mmol of dicyclohexylcarbodiimide. The reaction mixture is stirred for another 2 h at 0°and then over night at room temperature. The obtained N,N'-dicyclohexylmethane filtered off and the solvent is removed in vacuum and the remaining after the product is recrystallized from a mixture of EA/pentane.

Method E: Attach (with education) amide linkages, using esters of N-hydroxysuccinimide

10 mmol aminocompounds with unprotected-end is introduced into a solution of NaHCO3(20 mmol in 20 ml water). At room temperature and under stirring, slowly added dropwise 10 mmol of ester N-hydroxysuccinimide with N-protected end, dissolved in 10 ml of dioxane. Stirring of the reaction mixture is continued overnight and then the solvent is removed in vacuum.

Further treatment is carried out in the same way as in Method A.

Method F: Cleavage of the BOC-protective group

3 ml of a mixture of 1,1N HCl/glacial acetic is acid ( Method F1)or 3 ml of a mixture of 1,1N HCL/dioxane(Method F2) or3 ml of 50% TFA in DHM (DCM)(Method F3)add to1 mmol of the BOC-protected amino acid - pyrrolidin, thiazolidin or peptide. Splitting at CT is controlled by means of TLC. After completion of the reaction (approximately 2 hours) compound precipitated in the form of hydrochloride, using absolute diethyl ether, and was isolated by suction and dried over P4About10in a vacuum. Using a mixture of methanol/simple ether, the product is recrystallized or periostat.

Method G: Hydrolysis

1 mmol of methyl (complex) ester of the peptide dissolved in 10 ml of acetone and 11 ml 0,1M NaOH solution and stirred at room temperature. The progress of hydrolysis is controlled by using TLC. After the reaction, the acetone removed in vacuo. The remaining aqueous solution is acidified using concentrated solution KH2SO4up until the pH reaches 2-3. Then the product is extracted several times using EA (EA); United an ethyl acetate fraction was washed with saturated NaCI solution and dried over Na2SO4and the solvent is removed in vacuum. Carry out crystallization from a mixture of EA/pentane.

Example 7: determination of Ki

To determine the Kiuse dipeptidyl peptidase IV, isolated from porcine kidney, with specific activity against glycylvalyl-4-n is Taanilinna 37.5 u/mg and the concentration of enzyme of 1.41 mg/ml initial solution.

Mixture for analysis:

100 μl of the test compound in the concentration range 1·10-5M -1·10-8M respectively mixed with 50 μl glycylvalyl-4-nitroaniline at various concentrations (0.4 mm, 0.2 mm, 0.1 mm, 0.05 mm) and 100 µl of HEPES (40 mm, pH 7,6; ionic strength =0,125). Analytical mixture is pre-incubated at 30°C for 30 min After pre-incubation, add 20 ál of DPIV (1:600 diluted) and supervise the development of yellow color, caused by the release of 4-nitroaniline, at 30°and λ=405 nm for 10 min using a spectrophotometer to read the tablets (HTS7000 plus, Applied Biosystems, Weiterstadt, Germany).

Values of Kicalculated using Graphit version 4.0.13, 4.0.13 and 4.0.15 (Erithacus Software Ltd, UK),

7.1.Results-Ki inhibition of DPIV

ConnectionKi[Ml
H-Asn-pyrrolidin1,20*10-5
H-Asn-thiazolidin3,5*10-6
H-Asp-pyrrolidin1,4*10-5
H-Asp-thiazolidin2,9*10-6
H-Asp(NHOH)-pyrrolidin1,3*10-5
H-Asp(NHOH)-thiazolidin8,8*10-6
H-Glu 2,2*10-6
H-Glu-thiazolidin6,1*10-7
H-Glu(NHOH)-pyrrolidin2,8*10-6
H-Glu(NHOH)-thiazolidin1,7*10-6
H-His-pyrrolidin3,5*10-6
H-His-thiazolidin1,8*10-6
H-Pro-pyrrolidin4,1*10-6
H-Pro-thiazolidin1,2*10-6
H-Ile-azetidin3,1*10-6
H-Ile-pyrrolidin2,1*10-7
H-L-threo-Ile-thiazolidin8,0*10-8
H-L-aIIo-Ile-thiazolidin1,9*10-7
D-threo-isoleucyl-thiazolidin-fumarateno inhibition
D-allo-isoleucyl-thiazolidin-fumarateno inhibition
H-L-threo-Ile-thiazolidin-succinate5,1*10-8
H-L-threo-Ile-thiazolidin-tartrate8,3*10-8
H-L-threo-Ile-thiazolidin-fumarate8,3*10-8
H-L-threo-Ile-thiazolidine hydrochloride7,2*10-8
H-L-threo-Ile-thiazolidin-phosphate1,3*10-7
H-Val-pyrrolidin4,8*10-7
H-Val-thiazolidin2,7*10-7
Diproton Aof 3.45*10-6
Diproton B2,24*10-5
Nva-Pro-Ile6,17*10-6
Cha-Pro-Ileof 5.99*10-6
Nle-Pro-Ile9,60*10-6
Phe-Pro-Ile1,47*10-5
Val-Pro-Valof 4.45*10-6
Ile-Pro-Valthe 5.25*10-6
Abu-Pro-Ileis 8.75*10-6
Ile-Pro-allo-Ile5,22*10-6
Val-Pro-allo-Ile9,54*10-6
Tyr-Pro-allo-Ile1,82*10-5
AOA-Pro-Ileof 1.26*10-5
t-butyl-Gly-Pro-Ile3,10*10-6
Ser(Bzl)-Pro-Ile2,16*10-5
Aze-Pro-Ile2,05*10-5
t-butyl-Gly-Pro-Valis 3.08*10-6
Gln-Pyrrof 2.26*10-6
Gln-Thia1,21*10-6
Val-Pro-t-butyl-Gly1,96*10-5
t-butyl-Gly-Pro-Gly1,51*10-5
Ile-Pro-t-butyl-Gly1,89*10-5
t-butyl-GIy-Pro-IleNH2the ceiling of 5.60*10-6
t-butyl-Gly-Pro-D-Valto 2.65*10-5
t-butyl-Gly-Pro-t-butyl-Gly1,41*10-5
Ile-cyclopentolate6,29*10-6
t-butyl-Gly-cyclohexylethane2,73*10-4
Ile-cyclohexylethaneof 5.68*10-5
Val-cyclopentolate1,31*10-5
Val-Pro-ketone4,76*10-8
Val-Pro-acyloxymethyl1,05*10-9
Val-Pro-benzoylmethyleneare 5.36*10-10
Val-Pro-benzothiazolylthioto 3.73*10-8
H-Glu-Thia6,2*10-7
H-Gly(NHOH)-Thia1,7*10-6
H-Glu(Gly3)-Thia1,92*10-8
H-Glu(Gly5)-Thiato 9.93*10-8
H-Glu(PEG)-Thia3,11*10-6

t-butyl-Gly means:

Ser(Bzl) and Ser(P) denote benzyl-serine and phosphoryl-serine, respectively, Tyr(P) means phosphoryl-tyrosine.

Example 8: determination of the IC50-Values

100 ál of inhibitor solution is mixed with 100 μl buffer (HEPES pH 7.6) and 50 µl of substrate (Gly-Pro-pNA, final concentration 0.4 mm) and the pre is sustained fashion incubated at 30° C. the Reaction is initiated by adding 20 μl of purified pork DPIV. The formation of pNA product is determined at 405 nm for 10 min using HTS 7000Plus spectrophotometer for reading plates (Perkin Elmer), and calculate the slope. The final concentration of inhibitor ranged between 1 mm and 30 nm. To calculate the IC50using GraFit version 4.0.13 (Erithacus Software).

8.1.Results- The determination of the values ofIC50

ConnectionIC50[M]
Isoleucyl thiazolidine fumarate1,28*10-7
Diproton A4,69*10-6
Diproton B5,54*10-5
Phg-Pro-Ile1,54*10-4
Nva-Pro-Ile2,49*10-5
Cha-Pro-Ile2,03*10-5
Nle-Pro-Ile2,19*10-5
Ser(P)-Pro-Ile0,012
Tyr(P)-Pro-Ile0,002
Phe-Pro-Ile6,20*10-5
Trp-Pro-Ile3,17*10-4
Ser-Pro-Ile2,81*10-4
Thr-Pro-Ileof 1.00*10-4
Val-Pro-Val1,64*10-5
Ile-Pro-Valof 1.52*10-5
Abu-Pro-Ile3,43*10-5
Pip-Pro-Ile0,100
Ile-Pro-aIIo-Ile1,54*10-5
Val-Pro-aIlo-lle1,80*10-5
Tyr-Pro-alIo-Ile6,41*10-5
AOA-Pro-Ile4,21*10-5
t-butyl-Gly-Pro-Ile9,34*10-6
Ser(Bzl)-Pro-Ile6,78*10-5
Tic-Pro-Ile0,001
Orn-Pro-Ile2,16*10-4
Gln-Thia5,27*10-6
Aze-Pro-Ile7,28*10-5
Ile-Hyp-LEU0,006
t-butyl-Gly-Pro-Valto 1.38*10-5
Gln-Pyrr1,50*10-5
Val-Pro-t-butyl-Glyto 6.75*10-5
t-butyl-Gly-Pro-Gly5,63*10-5
Ile-Pro-t-butyl-Gly8,23*10-5
t-butyl-Gly-Pro-IleNH2to 2.29*10-5
t-butyl-Gly-Pro-D-Val1,12*10-4
t-butyl-Gly-Pro-t-butyl-Glyat 2.45*10-5
Aib-Pro-Ileno inhibition
Ile-cyclopentolate3,82*10-5
t-butyl-Gly-cyclohex keton 2,73*10-4
Ile-cyclohexylethane2,93*10-4
Val-cyclopentolate4,90*10-5
Val-cyclohexylethane0,001
Val-Pro-ketone5,79*10-7
Val-Pro-acyloxymethyl1,02*10-8
Val-Pro-benzoylmethylene1,79*10-8
Val-Pro-benzothiazolylthioto 1.38*10-7

t-butyl-Gly means:

Ser(Bzl) and Ser(P) denote benzyl-serine and phosphoryl-serine, respectively, Tyr(P) means phosphoryl-tyrosine

Example 9: Inhibition of DPIV-like enzymes dipeptidylpeptidase II

DP II (3.4.14.2) releases the N-terminal dipeptides from oligopeptides, if N is not fully protonated (McDonald, J,K., Ellis, S, & Reilly, T,J., 1966,J, Biol, Chem,,241, 1494-1501). Pro and Ala in P1-position are preferred residues. The enzymatic activity of described as DPIV-like activity, but DP II has a pH optimum in the acidic region. Used enzyme isolated from porcine kidney.

Analysis:

100 μl of glutaminetaurine or glutamylcysteine in the concentration range 1·10-4M - 5·10-8M mixed with 100 μl of buffer solution (40 mm HEPES, pH 7,6, of 0.015% Brij, 1 mm DTT), 50 μl of a solution is islaenelmediodelmar (5 mm) and 20 μl of porcine DP II (250-fold dilution in buffer solution). The fluorescence measurement is carried out at 30°and λexcitation=380 nm, λradiation=465 nm for 25 min using reader tablet (HTS7000plus, Applied Biosystems, Weiterstadt, Germany). Values of Kicalculated using Graphit version 4.0.15 (Erithacus Software, Ltd., UK) and determine for glutaminetaurine Ki=charged 8.52·10-5M ± 6,33·10-6M and glutamylcysteine Ki=1,07·10-5M ± 3,81·10-7M.

Example 10: Enzymes cross-interactions

Glutaminergic and glutamylation were tested for their effectiveness cross-interactions in relation to dipeptidylpeptidase I shed oligopeptides and prolidase.

Dipeptidylpeptidase I (DP I, cathepsin C)

DP I or cathepsin C represents lysosomal a cysteine proteinase that it dipeptides from the N-end of their substrates (Gutman, H. R. & Fruton, J.S., 1948,J. Biol: Chem., 174,851-858).It is classified as a cysteine proteinase. Used enzyme purchased from Qiagen (Qiagen GmbH, Hilden, Germany). To obtain a fully active enzyme, the enzyme was diluted 1000 times in MES buffer pH of 5.6 (40 mm MES, 4 mm DTT, 4 mm KCI, 2 mm EDTA, of 0.015% Brij) and pre-incubated for 30 min at 30°C.

Analysis:

50 μl of glutaminetaurine or glutamylcysteine in the concentration range 1·10-5M - 1·10-7M mix x μl of a mixture of buffer-enzyme. Mixture for analysis pre-incubated at 300C for 15 minutes After pre-incubation, add 100 ál gestibility-β-nitroaniline (2·10-5M) and supervise the development of the yellow color is caused by the release of β-nitroaniline at 30°and λexcitation=380 nm, λradiation=465 nm for 10 min using reader tablet (HTS7000plus, Applied Biosystems, Weiterstadt, Germany).

The values of the IC50calculated using Graphit version 4.0.15 (Erithacus Software Ltd, UK). No inhibition of enzyme activity DP I glutaminebinding or glutamylcysteine was not found.

Proletarisation (POP)

Proletarisation is endoproteinase serine type, which it the peptides at the N-terminal part of the Xaa-Pro communications (Walter, R., Shiank, H., Glass, J. D., Schwartz, I.L. & Kerenyi, T.D., 1971,Science,173, 827-829). Substrates are peptides with molecular weight up to 3000 Da. Used by the enzyme was human proletarisation. Recombinant expression was carried out inE. coliunder standard conditions, as described elsewhere relating to this area.

Analysis:

100 μl of glutaminetaurine or glutamylcysteine in the concentration range 1·10-4M-5·10-8M mixed with 100 μl of buffer solution (40 mm HEPES, H 7,6, of 0.015% Brij, 1 mm DTT) and 20 μl of a solution POP. Mixture for analysis pre-incubated at 30°within 15 minutes After pre-incubation, add 50 ál of solution pillpropranolol-4-nitroaniline (0,29 mm) and supervise the development of the yellow color is caused by the release of 4-nitroaniline at 30°and λ=405 nm for 10 min using reader tablet (Sunrise, Tecan Crailsheim, Germany). The values of the IC50calculated using Graphit version 4.0.15 (Erithacus Software, Ltd., UK). No inhibition activity POP glutaminebinding or glutamylcysteine was not found.

Prolidase (X-Pro dipeptidase)

Prolidase (EC 3.4.13.9) was first described by Bergmann & Fruton (Bergmann, M. & Fruton, JS, 1937,J. Biol. Chem.189-202). Prolidase releases the N-terminal amino acid of the Xaa-Pro dipeptides and has a pH optimum between 6 and 9.

Prolidase from pig kidney (ICN Biomedicals, Eschwege, Germany) was dissolved (1 mg/ml) in the analytical buffer (20 mm NH4(CH3C00)2, 3 mm MnCl2pH of 7.6). To obtain a fully active enzyme solution incubated for 60 min at room temperature.

Analysis:

450 ál of glutaminetaurine or glutamylcysteine in the concentration range 5·10-3M - 5·10-7M mixed with 500 μl of buffer solution (20 mm NH4(CH3C00)2, pH 7.6) and 250 ál of Ile-Pro-OH (0.5 mm in the mixture for analysis is). Mixture for analysis pre-incubated at 30°within 5 minutes After pre-incubation add 75 ál prolidase (1:10 diluted in buffer for analysis) and provide measurements at 30°and λ=220 nm for 20 min using UV/Vis photometer, UV1 (Thermo breakers, Cambridge, UK).

The values of the IC50calculated using version Graphit 4.0.15 (Erithacus Software, Ltd., UK). They were IC50>3 mm for glutamylcysteine and glutaminetaurine IC50= 3,4·10-4M± 5,63·10-5M

Example 11: determination of the inhibitory activity of the compounds of the invention in respect of DPIV after intravascular and oral administration to Wistar rats

Animals

Rats male Wistar (ShOe: Wist(Sho)) weighing between 250 and 350 g buy from Tierzucht Schönwalde (Schönwalde, Germany).

Conditions

Animals were kept in cages, one individual in a cage, under normal conditions with controlled temperature (22±2° (C) when the cycle of light/dark 12/12 h (light from 06:00 to noon). With access to standard pellets food (ssniff® Soest, Germany) and tap water, acidified with HCl, if desired.

The insertion of a catheter into the carotid artery

Later ≥ one week of adaptation in these conditions to Wistar rats in the carotid artery implanted catheters under about who she anesthesia (VNP. injection of 0.25 ml/kg of body weight Rompun® [2 %], BayerVital, Germany and 0.5 ml/kg of body weight Ketamin 10, Atarost GmbH & Co., Twistringen, Germany). Animals provide an opportunity to recover within one week. The catheter was washed with a mixture of heparin-saline solution (100 IU/ml) three times per week. In the case of dysfunction of the catheter, introducing a second catheter in the contralateral carotid artery relevant rats. One week after the second surgery the specified animal is administered in the study. In the case of dysfunction of the second catheter animal removed from the study. Pick up a new animal, as the experiments continue in a planned sequence, starting at least 7 days after implantation of the catheter.

Scheme of the experiment

Rats with intact function of boats administered placebo (1 ml of physiological solution, 0,154 mol/l) or test compound by oral and intravascular (intraarterial) by.

After fasting over night 100 μl samples of heparinized arterial blood taken at -30, -5, and 0 minutes Prepare a fresh solution of the test compound in 1.0 ml of physiological solution (0,154 mol/l) and injected at time 0 or orally through a feeding tube for artificial nutrition (75 mm; Fine Science Tools, Heidelberg, Germany) or by intravascular. If oral is doing in arterial catheter is injected additional volume of 1 ml of saline. In the case of intra-arterial injection catheter immediately washed with 30 μl of saline and orally given through a feeding tube an additional 1 ml of physiological solution.

After application of placebo or test substances selected samples of arterial blood 2,5; 5; 7,5; 10, 15, 20, 40, 60 and 120 min of the catheter in the carotid artery in rats, in consciousness and natural state. All blood samples were collected in a cooled ice Eppendorf tubes (Eppendorf-Netheler-Hinz, Hamburg, Germany)filled with 10 ál of buffer - 1M sodium citrate (pH 3.0), for determining the activity of DPIV in the plasma. The Eppendorf tubes, immediately centrifuged (12000 rpm for 2 min, Hettich Zentrifuge EBA 12, Tuttlingen; Germany). The fraction of plasma stored on ice until analysis or frozen at -20°until analysis. All plasma samples were labeled with the following information:

- code number

room animal

- date of sampling

the time of sampling

Analytical methods

Mixture for analysis to determine the activity of DPIV in plasma contained 80 μl of the reagent and 20 μl of plasma sample. Kinetic measurement of the formation of the yellow product - 4-nitroaniline from the substrate glycylvalyl-4-nitroaniline was carried out at 390 nm for 1 min at 30°after pre-incubation for 2 min at the same temperature. The DPIV activity was expressed in IU/min/p>

Statistical methods

Statistical evaluation and graphs were performed using PRISM® 3.02 (GraphPad Software, Inc.). All parameters are presented as generally accepted in the descriptive statistics definitions, including the average (value) and (standard deviation).

11.1.Results-in vivo inhibition of DPIV tmax

STRUCTUREDose

(mg/kg)
undead.(%)oral (%)
Gln-Pyrr1008067
Gln-Thia1008871
Diproton A10073no inhibition
Diproton B10050no inhibition
Tyr(P)-Pro-Ile10037no inhibition
t-butyl-Gly-Pro-Ile1007128
t-butyl-Gly-Pro-Val1007225
Ala-Val-Pro-acyloxymethyl1008986
Ala-Val-Pro-benzoylmethylene1009776
Ile-cyclopentolate100/td> 3415

Example 12: Effect of modified in the side chain of glutamylcysteine as smoothly-transported DPIV-inhibitors

Were synthesized modified in the side chain glutamylcysteine, having a structure H-Glu(X)-Thia, at the same time as X using polyethylene glycol and glycine oligomers with different lengths of chains (see Method And sample description of the synthesis). Were investigated the binding characteristics of these derivatives and their transportiruetes peptide Transporter PepT1.

Unexpectedly, it was found that modification of the side chain change the characteristics of the binding of compounds with DPIV only to a small extent. In contrast, the ability of the inhibitors to be transported peptide Transporter is greatly reduced by modification of their side chain.

Therefore, modified in the side chain inhibitors of DPIV or DPIV-like enzymes are well suited to achieve the targeted inhibition of DPIV in a specific location (the target organ) of a living organism.

12.1.Results: Transportiruetes selected DPIV inhibitors

Connection< / br>
amino acid - thiazolidine
EC50(mm)1Imax(nA) 2
H-Ile-Thia

H-Glu-Thia
0,98

1,1
25 ± 8

35 ± 13
modified in the side chain glutamylcysteine
H-Gly(NHOH)-Thia

H-Glu(Gly3)-Thia

H-Glu(Gly5)-Thia

H-Glu(PEG)-Thia
3,18

8,54

> 10

> 10
42 ± 11

not determined.3< / br>
not determined.3< / br>
not determined.3

1The effective concentration of compounds that inhibit the binding of3H-D-Phe-Ala (80 mm) withcells of P. pastoris,expressing PepT1, 50% (value EC50).

2Transport characteristics in PepT1-expressing the oocytes of X. leavis - point-to-point method of measuring the potential difference, I=internal air currents generated by transport

Example 13: catalyzed Inhibition of DPIV hydrolysis of incretins GIP1-42and GLP-17-36in vitro

You can suppressin vitrohydrolysis of incretins caused by DPIV and DPIV-like enzyme activity using purified enzyme or aggregated human serum (figure 1).

According to the present invention complete suppression catalyzed by enzyme hydrolysis of both peptide hormones is achieved byin vitroincubation of 30 mm GIP1-42or 30 mm GLP-17-36and 20 mm solicitation (1A), reversible DPIV-inhib the Torah, 20% of the mixed serum at pH 7.6 and 30°C for 24 h (1b and 1C, both upper spectrum). Synthetic GIP1-42(5 mm) and synthetic GLP-17-36(15 μm) were incubated with human serum (20%)0.1 mm TRICINE Buffer at pH 7.6 and 30°C for 24 h Sample incubation analyzed mixtures (in the case of GIP1-422.5 pcmall in the case of GLP-17-367.5 pgmol) were collected at different time intervals. The samples were subjected to cocrystallization, using as matrix 2',6'-dihydroxyacetophenone, and were analyzed by MALDI-TOF mass spectrometry. Spectra (Fig. 1) show the accumulation of 250 individual laser pulses on the sample.

(1b) the Signalm/z4980,1±5,3 corresponds DPIV-substrate GIP1-42(M4975,6) and the signal massm/z4745,2±5,5 corresponds to the product GIP3-42(M4740,4),released under the influence of DPIV.

(1c) the Signalm/z3325,0±1,2 corresponds DPIV-substrate GLP-17-36(M3297,7) and the signal massm/z3116,7±1,3 corresponds to the product of GLP-19-36(M3089,6), released under the influence of DPIV.

In the control trials containing no inhibitor, incretin was almost completely degraded (fig.1b and 1c, both the lower spectrum).

Example 14:Inhibition of degradation of GLP17-36DPIV-inhibitor, isoleucyl-thiazolidine,in vivo.

Analysis of the metabolism of PR is native incretins (in this case, GLP-1 7-36in circulation (circulation) rats in the presence or absence of DPIV-inhibitor isoleucyl-thiazolidine (EXT. vessel. injection of 1,5M inhibitor in 0.9% saline solution) and in the control. No degradation insulinotropic peptide hormone GLP-17-36does not occur when the concentration of 0.1 mg/kg of the inhibitor isoleucyl-thiazolidine the treated animals (n=5) during the course of the experiment (figure 2).

In order to analyze the metabolites of incretins in the presence and absence of a DPIV inhibitor, test and control animals received additional undead. the injection of 50-100 PM(pM)125I-GLP-17-36(specific activity of about 1 µci/PM) after 20 min after the initial undead. the introduction of the inhibitor and/or injection of saline. Blood samples were collected after 2 to 5 min of the incubation time and the plasma was extracted using 20% acetonitrile. Further peptide extract was separated on RP-HPLC. Multiple fractions of the eluate were collected between 12-18 min and read on γ-counter. Data are presented as number of counts per minute (cpm) relative to the maximum.

Example 15:Modulation of the insulin response and reduce the levels of glucose in the blood after EXT. vessel. the introduction of a DPIV inhibitor, isoleucyl-thiazolidinein vivo.

The figure presents the response of circulating (blood) glucose and insulin on in radiodetalei ( ed.) the introduction of glucose to rats in the presence or absence of isoleucyl-thiazolidine (0.1 mg / kg). There is a more rapid decrease in the concentration of circulating glucose in animals that received DPIV-effectors, compared with untreated animals. The observed effect is dose-dependent in nature and is reversible after cessation of infusion of 0.05 mg/min DPIV-inhibitor isoleucyl-thiazolidine on 1 kg of body weight of the rat. Unlike I.D. glucosestimulated animals after EXT. vessel. the introduction of the same amount of glucose control animals subjected to processing inhibitor, nothing comparable to the above effect was not observed. Figure 3 presents the above dependencies, showing the inhibitor-dependent changes in selected plasma parameters: A - DPIV activity, B - insulin levels in the plasma, C - glucose in plasma.

Example 16: Effect of chronic treatment of obese rats Zucker on the level of fasting blood glucose during the 12-week oral administration of the drug.

Chronic application of DPIV-inhibitor isoleucyl-thiazolidine leads to a dramatic decrease and normalization of the level of fasting blood glucose in selected animal models of diabetic rats (figure 4).

Animals.

Six pairs odnopolnyh fat rats-SAMC is in (fa/fa)VDF Zucker were randomly allocated to either the control group or in the group to be processed (isoleucyl-thiazolidine fumarate), body weight 440 g (age 11±0.5 weeks). Animals were kept for one cycle 12 cswe/dark (light from 6 : 00 to noon), with free access to standard diet to rats and water.

The daily Protocol control and the introduction of medicines

Group to be processed, received 10 mg/kg isoleucyl-thiazolidin fumarata using oral probe twice a day (8:00 to noon and 5:00 in the afternoon) for 100 days, while control animals received parallel dose filler consisting of a 1% solution of cellulose. Every two days was assessed by body weight, glucose levels in the morning and evening and consumption of food and water. Blood samples for glucose determination were taken from the tail and the concentration was determined using a glucose analyzer SureStep (Lifescan Canada Ltd., Burnaby).

The Protocol for monthly assessments of glucosetolerance

Every four weeks from the beginning of the experiment was performed oral glucose tolerance test (PGT, OGTT): animals were subjected to fasting after 17 h of dosing and received 1 g/kg oral glucose. This period of time equivalent to ˜12 periods of the half-life of circulating isoleucyl-thiazolidin fumarata.

Por what measures 17: Effect of chronic oral treatment among obese Zucker rats DPIV-inhibitor, isoleucyl-thiazolidine on systolic blood pressure

Chronic application of DPIV-inhibitor, isoleucyl-thiazolidin fumarata leads to stabilization sistolicheskogo blood pressure in selected animal models of diabetic rats (figure 5).

Animals

Six pairs (same litter) obese rats-males(fa/fa)VDF Zucker were randomly allocated to either the control group or in the group to be processed (isoleucyl-thiazolidine fumarate), body weight 440 g (age 11±0.5 weeks). Animals were kept for one cycle 12 cswe/dark (light from 6 : 00 to noon), with free access to standard diet to rats and water.

The daily Protocol control and the introduction of medicines

Group to be processed, received 10 mg/kg isoleucyl-thiazolidin fumarata using oral probe twice a day (8:00 to noon and 5:00 in the afternoon) for 100 days, while control animals received parallel dose filler consisting of a 1% solution of cellulose. Systolic blood pressure was measured weekly using the overlay method cuffs on the tail).

The test animals (n=5 rats male Wistar, 200-225 g) were first received 1,5M isoleucyl-thiazolidine in 0.9% saline solution (or the same amount net of 0.9% saline (¦ ) (control group n=5). The test group was additionally subjected to infusion of an inhibitor of 0.75 M/min for 30 min experimental time (*).The control group was exposed during the same time interval infusion of 0.9% saline solution containing no inhibitor. At the initial moment of time t=0, all animals were injectedVND.(i.d.) the dose of glucose 1 g/kg (40% solution of dextrose, wt./vol.). Blood samples were collected from all test animals in 10-minute intervals. Glucose was analyzed using whole blood (analyzer Lifescan One Touch II), while the activity of DPIV and insulin concentrations were analyzed in plasma. Radioimmunoassay analysis of insulin sensitive in the range of 10 and 160 IU/ml [PEDERSON, R. A., BUCHAN, A.M.J., ZAHEDI-ASH, S., CHEN, C.B. & BROWN, J.CReg. Peptides,3, 53-63 (1982)]. The DPIV activity was assessed spectrophotometrically[DEMUTH, H.-U. and HEINS, J., On the catalytic Mechanism of Dipeptidyl Peptidase IV. inDipeptidyl Peptidase IV (CD26) in Metabolism and the Immune Response(B. Fleischer, Ed.) R.G. Landes, Biomedical Publishers, Georgetown, 1-35 (1995)]. All data are presented as mean ± SSO (average standard deviation).

Example 18: a Study of the effects of oral administration of increasing doses of glutaminetaurine on fat rats Zucker

Animals

N=30 rats-males line Zucker (fa/fa), mean age of 11 weeks (5-12 weeks), the average body weight of 350 g (150-400 g) were for upline from Charles River (Sulzfeld, Germany).

After delivery the rats were kept for >12 weeks up until almost all the fat rat line Zucker was not detected features explicit diabetes. A group of N=8 animals was tested three increasing doses of glutaminetaurine in comparison with placebo (saline solution).

Conditions

Animals were kept in cages, one individual in a cage, in standardized conditions with controlled temperature (22±2° (C) when the cycle of light/dark 12/12 h (light from 06:00 to noon). Access to standard sterile granular food (ssniff®Soest, Germany) and tap water, acidified with HCl, was free.

The insertion of a catheter into the carotid artery

Fat rat line Zucker age 24-31 (average: 25 weeks) weeks, adapted to the conditions of detention were properly prepared for research.

Obese Zucker rats in the carotid artery implanted catheter under General anesthesia (VNP. injection of 0.25 ml/kg of body weight Rompun®[2 %], BayerVital, Germany and 0.5 ml/kg of body weight Ketamin 10, Atarost GmbH & Co., Twistringen, Germany). Animals provide an opportunity to recover within one week. The catheter was washed with a mixture of heparin-saline solution (100 IU/ml) three times a week.

Scheme of the experiment

Groups of N=8 fat rats Zucker centuries is completed with placebo (1 ml of physiological solution, 0,154 mol/l) or increasing doses of glutaminetaurine (5, 15 and 50 mg/kg body weight). 375 mg of glutaminetaurine dissolved in 1000 μl of DMSO (E. Merck, Darmstadt; Germany [Dimethyl sulfoxide p.a.]). Add 10 ml of saline and 1 ml aliquots, each containing 34,09 mg of glutaminetaurine, stored at -20°C. For the preparation of test substance dose-dependent aliquots diluted in physiological solution.

After fasting through the night obese Zucker rats were given a placebo or the test substance orally using a feeding tube (15 G, 75 mm; Fine Science Tools, Heidelberg, Germany) at -10 minutes Oral glucose tolerance test (PGT, OGTT) with 2 g glucose/kg M.L. (40% solution, B. Braun Melsungen, Melsungen, Germany) was applied on ±0 min using a second nutrient probe. Samples of venous blood from the tail vein were collected at -30 min,-15 min, ±0 min and at 5, 10, 15, 20, 30, 40, 60, 90 and 120 min in 20-µl glass capillaries, which were placed in a standard test tube filled with 1 ml of the solution to determine the level of glucose in the blood.

All blood samples were labeled with the following information:

- code number

room animal

- date of sampling

the sampling time

Analytical methods

Glucose levels were determined using the glucose oxydase method (Super G Glucose analyzer; Dr. Müller Gerätebau, Freital, Germany)

statisticheskie methods

Statistical evaluation and graphs were performed using PRISM® 3.02 (GraphPad Software, Inc.). All parameters are presented as generally accepted in the descriptive statistics definitions, including the average (value).

The effect of treatment on glucose tolerance

Diabetic rats line Zucker, subjected to treatment with placebo, showed a strong deviation in the direction of increased levels of glucose in the blood that indicates intolerance to glucose, which is characteristic of patients with pronounced diabetes. Introduction 5 mg of glutaminetaurine/kg M.L. led to a limited increase of glucosetolerance in diabetic Zucker rats. A significant reduction in elevated levels of glucose in the blood and increase glucosetolerance was achieved after administration of 15 mg/kg and 50 mg of glutaminetaurine/kg M.L. (see Fig.6).

Example 19:Study of the effect of oral administration of increasing doses of glutamylcysteine on fat rats Zucker

Animals:

N=30 rats-males line Zucker (fa/fa), mean age of 11 weeks (5-12 weeks), the average body weight of 350 g (150-400 g) were purchased from Charles River (Sulzfeld, Germany).

After delivery the rats were kept for >12 weeks up until almost all the fat rat line Zucker was not detected features explicit diabetes. A group of N=8 animals was subjected to what spatariu three increasing doses of glutamylcysteine in comparison with placebo (saline solution).

Conditions

Animals were kept in cages, one individual in a cage, in standardized conditions with controlled temperature (22±2° (C) when the cycle of light/dark 12/12 h (light with06:00 to noon). Access to standard sterile granulirovannogo food (ssniff® Soest, Germany) and tap water, acidified with HCl, was free.

The insertion of a catheter into the carotid artery

Fat rat line Zucker age 24-31 (average: 25 weeks) weeks, adapted to the conditions of detention were properly prepared for research.

Obese Zucker rats in the carotid artery implanted catheters under General anesthesia (VNP. injection of 0.25 ml/kg of body weight Rompun®[2 %], BayerVital, Germany and 0.5 ml/kg of body weight Ketamin 10, Atarost GmbH & Co., Twistringen, Germany). The animals were provided the opportunity to recover within one week. The catheters were flushed with a mixture of heparin-saline solution (100 IU/ml) three times a week.

Scheme of the experiment

Groups of N=8 among obese Zucker rats were given a placebo (1 ml of physiological solution, 0,154 mol/l) or increasing doses of glutamylcysteine (5, 15 and 50 mg/kg body weight). The appropriate number of glutamylcysteine was dissolved in 1000 μl of saline.

After fasting through the night obese Zucker rats enter the placebo or the test substance orally with the help of nutrient probe (15 G, 75 mm; Fine Science Tools, Heidelberg, Germany) at -10 minutes Oral glucose tolerance test (PGT, OGTT) with 2 g glucose/kg M.L. (40% solution, B. Braun Melsungen, Melsungen, Germany) was applied on ±0 min using a second nutrient probe. Samples of venous blood from the tail vein were collected at -30 min,-15 min, ±0 min and at 5, 10, 15, 20, 30, 40, 60, 90 and 120 min in 20-µl glass capillaries, which were placed in a standard test tube filled with 1 ml of solution to determine the level of glucose in the blood.

All blood samples were labeled with the following information:

- code number

room animal

- date of sampling

the sampling time

Analytical methods

Glucose levels were determined using the glucose oxydase method (Super G Glucose analyzer; Dr. Müller Gerätebau, Freital, Germany)

Statistical methods

Statistical evaluation and graphs were performed using PRISM® 3.02 (GraphPad Software, Inc.). All parameters are presented as generally accepted in the descriptive statistics definitions, including the average (value).

The effect of treatment on glucose tolerance:

Diabetic Zucker rats subjected to treatment with placebo, showed a strong deviation in the direction of increased levels of glucose in the blood that indicates intolerance to glucose, which is characteristic of regions with a pronounced diabetes. Introduction 5 mg/kg, 15 mg/kg and 50 mg CH is amenitieseven/kg M.L. led to dose-dependent decrease in elevated levels of glucose in the blood and increase glucosetolerance in diabetic rats Zucker (see Fig.7).

Example 20:In vivoinactivation of glutamylcysteine after oral administration to Wistar rats

Animals/layout of the experiment

Glutamylcysteine administered to Wistar rats orally as described in example 9.

Analytical methods

After application of placebo or glutamylcysteine in rats, in consciousness and in their natural state, take samples of arterial blood from a catheter implanted in the carotid acteria, 2,5;5; 7,5; 10, 15, 20, 40, 60 and 120 min to detect the formation of degradation products of glutamylcysteine.

For analysis, for discharge from the plasma of interest connections use a simple solid-phase extraction on C18 cartridges. The extracts are analyzed using the combined method of analysis - liquid chromatography with reversed phase column (Lichrospher 60 RP Select B, in combination with condemnee mass spectrometry, operating in mode APCI (chemical ionization at atmospheric pressure), the positive ions. For a quantitative evaluation using the method of internal standard.

Results

After oral administration of glutamylcysteine rats is m Wistar was detected degradation of the connection. Using LC/MS, the product of degradation could be defined as Pyroglutamate. Cm. Fig and 9.

1. The use of at least one inhibitor of the enzymatic activity of dipeptidylpeptidase (DPIV) as an active ingredient for a pharmaceutical composition for lowering blood pressure levels in a mammal with diabetes.

2. The use according to claim 1, where the inhibitor is a compound formed from amino acids and thiazolidinone or pyrrolidino group, or its salt.

3. The use according to claim 2 where the compound is chosen from the group consisting of L-threo-isolatin1encoding, L-ALLO-solicitation, L-ALLO-isolatin1encoding, L-glutamylcysteine, L-glutaminetaurine, L-glutamic acid-thiazolidine, L-glutamic acid-pyrrolidine, amaniparadise, N-poured prolyl-O-benzylhydroxylamine and their salts.

4. The use according to claim 1, where the inhibitor is peptidoglycan represented by the General formula

including all stereometry, and its pharmaceutically acceptable salt,

where a is selected from

and X1represents H or an acyl or oxycarbonyl group or the residue of an amino acid or peptide

X2represents H, -(CH)n-NH-C5H3N-Y n=2-4 or C5H3N-Y (divalently the rest of pyridil) and Y is selected from H, Br, Cl, I, NO2or CN,

X3represents H or phenyl or pyridyl, unsubstituted or substituted one, two or more alkyl, alkoxy, halogen, nitro, cyano or carboxy groups,

X4represents H or phenyl or pyridyl, unsubstituted or substituted one, two or more alkyl, alkoxy, halogen, nitro, cyano or carboxy groups,

X5represents H or alkyl, alkoxy or phenyl,

X6represents H or alkyl;

for n=1

X is selected from H, OR2, SR2, NR2R3N+R2R3R4where

R2means acyl residues, which are unsubstituted or substituted one, two or more alkyl, cycloalkyl, aryl or heteroaryl residues, or denotes any amino acid and peptide residues, or alkyl residues, which are unsubstituted or substituted one, two or more alkilani, cycloalkyl, arinami and heteroaryl residues,

R3denotes alkyl and acyl function, where R2and R3can be part of one or more ring structures of saturated and nenasyshchennosti or heterocyclic structures

R4means alkyl residues, where R2and R4or R3and R4can be part of one or more ring structures of saturated and unsaturated carbocyclic or heterocyclic structures;

for n=0

X is selected from

where In the means O, S, NR5where R5represents H, alkylidene or acyl,

C, D, E, F, G, H are independently selected from unsubstituted and substituted Akilov, oxyalkyl, thioalkyl, aminoalkyl, carbonylation, atilov, carbarnoyl, aryl and heteroaryl residues; and

for n=0 and n=1

Z is selected from H, C1-C9the alkyl branched or single chain or With2-C9alkenyl branched or single chain, With3-C8cycloalkyl,5-C7cycloalkenyl, aryl - or heteroaryl residue, or a side chain selected from all side chains of natural amino acids.

5. The use according to claim 1, where the inhibitor is derived aminoketone represented by the General formula 5, 6, 7, 8, 9, 10 and 11, including all stereometry, and their pharmaceutically acceptable salt,

where R1represents H, a branched or straight C1-C9alkyl residue, a branched or straight C2-C9Elke the ilen residue, With3-C8cycloalkyl-From5-C7cycloalkenyl-, aryl - or heteroaryl residue or side chain of natural amino acids

R3and R4independently selected from H, hydroxy, alkyl, alkoxy, aryloxy, nitro, cyano or halogen,

And represents H or ISOStAR carboxylic acid functional group selected from CN, SO3N, CONHOH, RHO3R5R6, tetrazole, amide, complex, ester, anhydride, thiazole and imidazole;

Selected from

where R5represents H, -(CH)n-NH-C5H3N-Y n=2-4 and C5H3N-Y (divalently the rest of pyridil) with Y=H, Br, Cl, I, NO2or CN,

R10represents H, acyl, oxycarbonyl or the balance of amino acids

W represents H or phenyl or pyridyl, unsubstituted or substituted one, two or more alkyl, alkoxy, halogen, nitro, cyano or carboxy groups,

W1represents H, alkyl, alkoxy or phenyl,

Z represents H or phenyl or pyridyl, unsubstituted or substituted one, two or more alkyl, alkoxy, halogen, nitro, cyano or carboxy groups,

Z' represents H or alkyl,

D isone cyclic With 4-C7alkyl, C4-C7alkanniny residue, which may be unsubstituted or substituted one, two or more alkyl groups, or cyclic (4-7)-membered heteroalkyl or cyclic (4-7)-membered heteroalkyl the rest,

X2represents O, NR6N+(R7)2or S,

X3to X12independently selected from CH2, CR8R9, NR6N+(R7)2, O, S, SO and SO2including all saturated and unsaturated structures

R6, R7, R8, R9independently selected from H, branched or straight C1-C9alkyl residue, a branched or straight2-C9alkenylphenol balance With3-C8cycloalkyl balance With5-C7cycloalkenyl residue, aryl or heteroaryl residue,

under the following conditions:

formula 6: X6represents CH, if a is not H,

formula 7: X10is C if a is not H,

formula 8: X7represents CH, if a is not H,

formula 9: X12is C if a is not N.

6. The use according to claim 1, where the inhibitor of the enzymatic activity of DPIV represented by the General formula.

including all art is remery and their pharmaceutically acceptable salts, where

But is α-amino acid, in particular, natural α-amino acid having at least one functional group in the side chain, selected from the group consisting of threonine, tyrosine, serine, arginine, lysine, aspartic acid, glutamic acid or cysteine,

In is a chemical compound covalently bound at least one functional group of the side chain And, in particular

Oligopeptide having a chain length up to 20 amino acids, or

polyethylene glycol having a molar mass of up to 20000 g/mol,

optionally substituted organic amine, amide, alcohol, acid, or an aromatic compound having from 8 to 50 C atoms, and

With is thiazolidin, pyrrolidin, cyanopyrrolidine, hydroxyproline, digitopolis or piperidino group associated with And through amide bond.

7. The use according to any one of the preceding paragraphs, where the pharmaceutical composition includes a pharmaceutically acceptable carrier or diluent and a therapeutically effective amount of the indicated inhibitor or its pharmaceutically acceptable acid additive salt.

8. The use according to any one of the preceding paragraphs to lower levels of blood pressure in mammals, having blood pressure greater than 140 nm Hg, DG is, at least one inhibitor is administered periodically.

9. The use according to any one of the preceding paragraphs, where the pharmaceutical composition is presented in a form for oral administration.



 

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