Opioid peptides and pharmaceutical composition

 

(57) Abstract:

Opioid peptides of formula (I): X-R1-R2-R3-Q-R4-N(Y)Z and its analogues with peripheral analgesic action, where X Is N or C1-6-alkyl; Y, Z - N, cyclic aralkyl and C1-6-alkyl; R1- tersely balance, 2',6'-dimethyl tersely balance, or the equivalent; R3, R4the remainder of the aromatic amino acids (other designations, see p. 1 claims). 2 S. and 19 C.p. f-crystals, 3 ill., table 1.

Prototypes of the invention

Many endogenous peptides mammals and amphibians are associated with specific opioid receptors and cause analgesic response, similar to the effect of classical narcotic opiates. It is shown that the higher animals coexist many different types of opioid receptors. For example, see W. Martin and others, J. Pharmacol. Exp. Ther., 197, p. 517 (1975); and J. Lord and others, Nature (London), 257, p. 495 (1977). Identify three different types of opioid receptors. The first shows the morphing tool to peptides such as enkephalins. The second shows increased selectivity to morphine and other polycyclic alkaloids. The third k shows the same affinity to any group of the above ligands, primasia effects, receptors, apparently, dealing with behavioral effects, although the pain can also be indirect - and k-receptors.

Each opioid receptor in the interaction with opiate cause specific biological response, one of a kind for this type of receptor. When the opiates activate more than one receptor, the biological response of each receptor in this effect produced side effects. With the introduction of opiate less specific and selective opiate can give greater possibility of increased side effects.

In the prototypes of opiates, opioid peptides and their analogues, or do not show specificity and selectivity to that type of receptor or receptors with which they are associated. The principal site of analgesic action of opioids is a Central nervous system (CNS). Well-known narcotic analgesics are usually hydrophobic and, therefore, easily penetrate the lipid membranes such as the blood-brain barrier. Due to this physical ability analgesics tend to interact with opioid receptors within the Central nervous system in the brain. However, they do not necessarily sopockie effects.

Opiates can cause serious and potentially fatal side effects. Non-specific interactions with receptors of the Central nervous system cause side effects such as respiratory depression, stamina, the ability of physical dependence and sudden withdrawal syndrome. Cm. K. Budd, In International Encyclopedia of Pharmacology and Therapeutics; N. E. Williams and H. Wilkinson, Eds., Pergammon: (Oxford), 112, p. 51 (1983). Therefore, I believe that opioid analgesics, acting mainly through opioid receptors in the peripheral nervous system, do not cause such unwanted side effects as the effects caused by opioid analgesics acting on the Central nervous system. Opioid peptides of the present invention mainly act on the peripheral nervous system and therefore overcome some of the adverse effects characteristic of normal opiates, mainly through prevention of unwanted side effects.

To date, one of the known classes of agents providing peripheral analgesic effect, are non-steroidal anti-inflammatory agents such as aspirin, ibuprofen and Ketorolac. These agents do not interact with opioid is analgesics do not have centrally mediated effects, but may cause other side effects such as ulceration of the gastrointestinal tract. It is an object of the present invention is to provide peptides such as opioids, which have a peripheral effect, but essentially do not have undesirable side effects caused by conventional perifericheskie acting analgesics.

Recently, the prototype was shown that drugs such as opiates have significant peripheral analgesic activity. Cm. A. Barber and R. Gottschlich, Med. Res. Rev., 12, p. 525 (1992) and C. Stein, Anasth. Play mode display., 76, p. 182 (1993). For differences between peripheral and Central analgesic responses as pharmacological probes use of Quaternary salts known centrally acting opioid alkaloids. Quaternary salts of patentable opiates have a permanent positive charge and demonstrate a limited passage of blood-brain barrier. Cm. T. W. Smith and others , Life Sci, 31, p. 1205 (1982); T. W. Smith and others, Int. J. Tiss. Reac., 7, p. 61 (1985); B. B. Lorenzetti and S. H. Ferreira, Braz. J. Med. Biol. Res., 15, p. 285 (1982); D. R. Brown, and L. I. Goldberg, Neuropharmacol., 24, p. 181 (1985); G. Bianchi and other Life Sci, 30, p. 1875 (1982); and J. Russel and others, Eur. J. Pharmacol. , 78, p. 255 (1982). Received strongly polar analogs of enkephalins and dermorphin that preserve prot and others, Br. J. Pharmacol., 93, p. 85 (1988); G. W. Hardy and others, J. Med. Chem. , 32, p. 1108 (1989). On the contrary, in the prototypes believe that lipophilic opioid peptides easier to pass the blood-brain barrier. Surprisingly, opioid peptides of this invention are highly lipophilic, but slightly pass the blood-brain barrier.

In contrast to conventional opiates, opioid peptides are hydrophobic. Their hydrophobicity tends to increase the rate of their removal from the body of a mammal. Hydrophobicity increases the ability of the peptides to cross the epithelial barrier. Despite this it is shown that the introduction of opioid peptides in the body of a mammal affects the Central nervous system. Therefore, effort has been directed at improving the absorption properties of these compounds. Scientists have tried to reduce the penetration of peptides into the Central nervous system, especially if prolonged effect on the body these drugs can cause harmful side effects or even be toxic.

I believe that non-polar peptides are easier in the Central nervous system by crossing the blood-brain barrier than polar peptides. Published that TAPP (H-Tyr-D-Ala-Popean Peptide Symposium, 1988). In rebuttal by the applicants of the present invention discovered that this tetrapeptide TAPP (H-Tyr-D-Ala-Phe-Phe-NH2) does not act centrally. This result is shown by the lack of analgesic action even at doses of 100 mg/kg in the test with hot plate in mice (mouse hot-plate test). This test is standard and known in the art. The test verifies chemical drugs, which are centrally mediated analgesic response.

Used in this application, the term "specificity" refers to a special or unconditional binding of opiate or opioid peptide with individual opioid receptor compared with other opioid receptor. The specificity of opioid peptide indicated by the inhibition constant of binding of Ki. The term "selectivity" refers to the ability of the opiate or opioid peptide to distinguish one among multiple opioid receptors and to only interact with a single receptor. The selectivity of opioid peptide relative to the receptor is indicated by the ratio of the constants of inhibition of binding. For example, to determine the selectivity can use the ratio of the constants of inhibition of binding of Ki/Ki. It is more preferred ligand to interact with the receptor in comparison with the receptor. It is known that the analogue of the usual opioid peptide H-Tyr-D-Ala-Gly-Phe - (NMe)-Gly-ol (DAGO) is one of the most-selective analogs of opioid peptides. The peptide yields a value of Ki/Ki1050. On the other hand, Leu-enkephalin gives a value of Ki/Ki0.2. This value reflects a marked affinity for the receptor relative to the receptor.

For pharmacological application of the peptide must have certain properties. First, the peptide must be resistant to proteoliticheskogo destruction. Secondly, the peptide should cause increased biological reaction. Thirdly, the peptide must be safe for humans. Fourth, the peptide must be such that it can be obtained in large enough quantities for clinical studies on toxicity, and later for use on a commercial basis. In this case, the required properties are also lower lipid solubility and large solubility in water in order to prevent the penetration of the peptide across the blood-brain barrier and to ensure the rapid elimination of excess injected peptide and its metabolites. In addition, for peptides, it is desirable to achieve a selective and specific active">

There is a need to peptides that act on one specific opioid receptor, in particular the receptor. It is desirable to find peptides with lower lipid solubility than the well-known opiates, so not to destroy the blood-brain barrier. In addition, peptides with high polarity usually more soluble in aqueous medium with physiological pH, which increases their excretion and excretion of their metabolites.

Summary of the invention

The present invention provides new compounds that are selective and specific relative to one opioid receptor. The present invention provides peptides, which show the preferred selectivity and specificity to receptors. The present invention also provides peptides that primarily interact with opioid receptors on peripheral nerve endings and to a minor extent pass the blood-brain barrier. Therefore, the present invention reduces the severity and number of side effects compared to conventional opiates and opioid peptides described to date.

The following formula (1) represents the soy is H, and C1-6-alkyl;

Y and Z independently are selected from the group consisting of H, cyclic aralkyl and C1-6-alkyl;

R1is tirocinium balance, 2',6'-dimethylthiazolium residue or an analogue or derivative;

R3is an aromatic amino acid;

R4is the residue of an aromatic amino acid;

R2is an amino acid having an R-configuration, provided that when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are H, and R3is phenylalanine, R4is not unsubstituted phenylalanine or phenylalanine, substituted 4NO2or 4N3;

when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are H, and R4is phenylalanine, R3is not unsubstituted phenylalanine or phenylalanine, substituted 4NO2;

when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are H, and R4is 1'-naphtylamine, R3is not 1'-naphthylamines or 2'-naphtylamine; and

when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are H, R3and R4each

The invention also provides pharmaceutically acceptable compositions comprising these peptides, for use in anesthesia.

The invention also provides the use of these peptides for the production of peripheral analgesics for pain relief.

The invention also provides the use of peptides of the formula H-Tyr-D-Ala-Phe-Phe-NH2for the production of peripheral analgesics for pain relief.

Tables and figures

Table 1 lists the in vivo and in vitro activity of hydrophobic tetrapeptide, related dermorphin.

In Fig.1 shows the change with time of the analgesic actions of morphine (10 mg/kg) (Fig. 1A) and typical of tested compounds (Fig. B: BCH2463; C: BCH2462; D: BCH2687).

In Fig. 2 shows a diagram of the dependence of dose-response for BCH2463 in research convulsions caused by familienaam, which is injected mice subcutaneously, ED50= 0.5 mg/kg over 20 min after injection.

In Fig. 3 shows the comparative change in the time of analgesic action BCH1774 and BCH2463 in research convulsions caused by familienaam, which is injected mice subcutaneously.

Description of the invention

In the description and form of the NIN; Cle - cycloleucine; Gln - glutamine; Gly is glycine; His - histidine; Hph - homophenylalanine; Aib - aminoadamantane acid; Chl - cycloviolacin; Cis(Bzl) - cysteine (benzyl); Dmt - 2',6'-dimethylthiazol; Glu - glutamic acid; GPI the ileum of the Guinea pig; Ile is isoleucine; Leu - leucine; Nle - norleucine; Phe is phenylalanine; Phg - phenylglycine; Ser - serine; Trp: tryptophan; Met - methionine; MVD - seminal duct of the mouse; Nva - Norvaline; Pro - Proline; Thr - threonine; Tyr, tyrosine; Nal - 1' - or 2'-nafcillin; PBQ - phenyl-para-benzoquinoline; Tic tetrahydroisoquinoline-3-carboxylic acid; TAPP - H-Tyr-D-Ala-Phe-Phe-NH2; TSPP - H-Tyr-D-Ser-Phe-Phe-NH2.

Used here, the terms "amino acid" and "aromatic amino acid" includes naturally occurring amino acids, as well as not naturally occurring amino acids, their derivatives and analogues, usually used by professionals in the field of chemical synthesis and chemistry of peptides. Also included are analogs TAPP, where phenylalanine is para-substituted in position 4 nitro - or azido residue. The list is not naturally occurring amino acids can be found in "The Peptide", so 5, 1983, Academic Press, Chapter 6, D. C. Roberts and F. Vellaccio, this monograph include here by reference. Examples of aromatic amino acids include tyrosine, tryptophan, phenylglycine amino acids include phenylalanine, substituted aromatic ring, for example, the groups CH3C2H5, F, Cl, Br, NO2, OH, SH, CF3, CN, COOH and CH2COOH or substituted on carbon lower alkyl radicals, OH, SH or benzene group. The aromatic ring can have several deputies. Aromatic amino acids can also include aromatic carbocycle type phenylglycine, where the aromatic ring is substituted phenylglycine groups CH3C2H5, F, Cl, Br, NO2, OH, SH, CF3, CN, COOH and CH2COOH. These examples are only to illustrate the invention, but any way its limit.

The term "ED50" (ED50as shown in table 1 for study convulsions caused PBQ, defined as the dose of a drug, which includes 50% reduction convulsions observed in comparison with control. The term "ED50"used in experiments with hot plate, defined as the dose required to double the increase of the latent response compared to the control samples and determined by parallel analysis of performance.

The term "likeness inserted amide bond" means a bond, in which the cat inhibiting the binding.

The term "Ki/Ki" denotes the value that can be used to measure the selectivity. This ratio represents the ratio of the abilities of opioid peptides to bind to - and-receptors.

The term "R-configuration" refers to a three-dimensional arrangement of substituents around a chiral element. The basic system to denote the absolute configuration is based on a system of priorities, which is well known in the art and briefly described below. Each group attached to the chiral center, indicate the number according to the priorities. Configuration is defined as "R" if the transition from the highest priority group to a lower priority groups eye follows the direction of clockwise.

The term "residue", applied to the amino acid refers to a radical derived from the corresponding amino acids by removal of the hydroxyl carboxyl group and one amino hydrogen.

The compounds of the present invention are compounds represented by the formula (1):

< / BR>
and their derivatives and analogues, where X is chosen from the group consisting of H and C1-6-alkyl;

Y and Z independently are selected from the group consisting silnym residue or an analogue or derivative;

R3is an amino acid residue selected from the group consisting of aromatic amino acid;

R4is the residue of an aromatic amino acid;

R2is an amino acid having an R-configuration, provided that when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are H, and R3is phenylalanine, R4is not unsubstituted phenylalanine or phenylalanine, substituted 4NO2or 4N3;

when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are H, and R4is phenylalanine, R3is not unsubstituted phenylalanine or phenylalanine, substituted 4NO2;

when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are H, and R4is 1'-naphtylamine, R3is not 1'-naphthylamines or 2'-naphtylamine; and

when R2is D-alanine, R1is tirocinium residue and X, Y and Z are H, R3and R4everyone is tryptophan; and

Q is an amide bond or likeness inserted amide bond.

Preferred are compounds represented is inane, represented by formula (1), and their derivatives and analogues, where

R2is an amino acid residue having the R-configuration, provided that when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are H, R3and R4different and selected from the group consisting of phenylalanine and tryptophan.

Other preferable are compounds represented by the formula (1), and their derivatives and analogues, where

Q is an amide bond or likeness inserted amide linkages of the formula Q1-Q2where Q1selected from the group consisting of CH2, CHOH, C=O, C=S & CH=, and Q2selected from the group consisting of CH2, NH, S, SO, SO2O and CH=, provided that when Q1is CH=, Q2is CH=.

In addition, preferred are compounds represented by the formula (1), and their derivatives and analogues, where

Y and Z are H;

R3and R4independently are aromatic amino acids, and

R2is an amino acid having an R-configuration, with the proviso that when R1is tirocinium residue, and R2is D-alanine, R3and R4different and selected from the group consisting of Finlay (1), and their derivatives and analogues, where

R2is an amino acid having an R-configuration, provided that R2is not D-alanine, and R3and R4are phenylalaninamide remains.

More preferred are compounds represented by the formula (1), and their derivatives and analogues, where R1is tirocinium the remainder, R2selected from the group consisting of D-Norvaline, D-serine and D-arginine;

R3and R4are phenylalanine residues; and

Q is a peptide bond.

More preferable are compounds represented by the formula (1), and their derivatives and analogues, where

X is H,

Y and Z independently are selected from the group consisting of H, aralkyl and C1-6-alkyl;

R1is tirocinium balance, 2',6'-dimethylthiazolium residue or an analogue or derivative;

R3is an aromatic acid,

R4independently selected from the group consisting of aromatic and aliphatic amino acids; and

R2is an amino acid residue having the R-configuration, provided that when R2is D-alanine, R1is tirocinium residue, and Y and Z are H, R1-Q2where Q1selected from the group consisting of CH2, CHOH, C=O, C=S & CH=, and Q2selected from the group consisting of CH2, NH, S, SO, SO2O and CH=, provided that when Q1is CH=, Q2is CH=.

More preferable are compounds represented by the formula (1), and their derivatives and analogues, where X is H, Y and Z are H;

R1is tirocinium balance, 2',6'-dimethylthiazolium residue or an analogue or derivative;

R3and R4independently are aromatic amino acids;

R2is an amino acid having an R-configuration, provided that when R2is D-alanine and R1is tirocinium the remainder, R3and R4independently selected from the group consisting of phenylalanine and tryptophan, but they are not the same, Q is an amide bond or likeness inserted amide linkages of the formula Q1-Q2where Q1selected from the group consisting of CH2, CHOH, C=O, C=S & CH=, and Q2selected from the group consisting of CH2, NH, S, SO, SO2O and CH=, provided that when Q1is CH=, Q2
R1is tirocinium balance, 2',6'-dimethylthiazolium residue or an analogue or derivative;

R2is an amino acid having an R-configuration, provided that R2is not alanine,

R3and R4are phenylalanine residues;

Q is an amide bond or likeness inserted amide linkages of the formula Q1-Q2where Q1selected from the group consisting of CH2, CHOH, C=O, C=S & CH=, and Q2selected from the group consisting of CH2, NH, S, SO, SO2O and CH=, provided that when Q1is CH=, Q2is CH=.

Most preferred are compounds represented by the formula (1), and their derivatives and analogues, where X is H; Y and Z are H;

R1is tirocinium the remainder, R2selected from the group consisting of D-Norvaline, D-serine and D-arginine; R3and R4are phenylalanine residues; and

Q is a peptide bond.

Preferred compounds I and the preferred compounds II of the present invention are given in the end of the description.

The best currently known way to realize this image is as peripheral analgetika connection TAPP H-Tyr-D-Ala-Phe-Phe-NH2.

As ligands of opioid receptors and analgesic agents total received and investigated a large number of tetrapeptides on the basis of General formula (1). These compounds are listed in table 1 together with corresponding constants of inhibition of binding and selectivity for receptors.

2', 6'-dimethylthiazol (Dmt) can be replaced in opioid peptide compounds tyrosine. Experiments show that substitution of Dmt on the tyrosine at position R1in the General formula 1 (the first amino acid residue) increases the potential of opioid peptide relative to the receptor by two orders of magnitude. The selectivity to the receptor increases when the connection comprises a Dmt in the R1. This substitution causes a corresponding shift in the ratio of the constants of inhibition of binding, reflecting increased selectivity for receptors.

Many of the compounds listed in table 1, demonstrate good binding with receptors, but are weak analgesic effect in the study of convulsions in mice (mouse writhing assay). This anomaly may be due to rapid proteolysis, quick cleanse or both reasons. For example, when T rapid degradation in 15-30 minutes Among the peptides listed in table 1, three preferred compounds, in addition to the TAPP have an increased analgesic effect in vivo. These three compounds H-Tyr-Nva-Phe-Phe-NH2(BCH2462), H-Tyr-D-Ser-Phe-Phe-NH2(BCH2463) and H-Tyr-D-Arg-Phe-Phe-NH2(BCH2687). It is shown that BCH2462, BCH2463 and BCH2687 demonstrate peripheral analgesic effect. When using these peptides Central analgesic actions are not observed, even at doses of 100 mg/kg in the test on the mouse hot-plate.

As shown in table 1, the value of U50for TAPP (BCH1774) is 1.4. Corresponding values for H-Tyr-D-Nva-Phe-Phe-NH2(BCH2462), H-Tyr-D-Ser-Phe-Phe-NH2(BCH2463) and H-Tyr-D-Arg-Phe-Phe-NH2(BCH2687) is equal to 2.7, 0.5 and 0.5 respectively. Values U50for the remaining compounds in table 1 is greater than these values. Although the value of the ED50for BCH2813 is only 0.15, found that this compound acts centrally at doses of 40 mg/kg in test hot-plate.

The results show that the compounds BCH1774, BCH2462 and BCH2463 still subjected to proteolysis, but have a greater half-life and, therefore, are more effective analgesics. In Fig. 3 compare the duration of analgesia in vivo caused BC is tiraumea action BCH1774 takes longer than BCH2463, indicating, probably, several accelerated proteolysis BCH2463 compared to BCH1774 in vivo.

In Fig. 1A-D shows the influence of morphine, BCH2463 (TSPP), BCH2462 (TNPP) and BCH2687 mice when assessing the reaction of the mice in the test with hot plate. As shown in Fig. 1A, the reaction time of the mice treated with 10 mg/kg of morphine is about 17 C. the reaction time of the mice treated with 100 mg/kg BCH2463 (Fig. 1B) is approximately 9 compared to the control value of about 7 C. These results indicate that while morphine inhibits painful thermal stimulus, BCH2463 - no; but BCH2463 is a potential analgesic agent, as shown by inhibition of chemically-induced convulsions (Fig. 2). The reaction time of the mice treated BCH2462 and BCH2687 (Fig.1C, 1D) is approximately 8, which shows a similar result as for BCH2463.

Investigated the effects of inhibition of proteolytic metabolism BCH2463 inhibitor DL-tiofana and metabolic disintegration BCH2463 mediated boundary renal membrane (brush border kindney membranes). The obtained data suggest that the kidney may be the main place of cleansing and metabolism to connect BCH2463. In Fig.2 it is clear that the endopeptidase enzyme EC24-11, the kidney brush border kidney extract).

And BCH1774 (TAPP) and BCH2462 (TNPP) produce a lethal effect on mice with the introduction of the dose 1-5 mg/kg intravenously. In contrast BCH2463 (TSPP), surprisingly, has no lethal action at doses up to 20 mg/kg intravenously. In addition, the peptides safe when introduced subcutaneously (s. c. ) at doses over 100 mg/kg Therefore, the desired method of administration of these compounds is subcutaneous administration. Thus, the structural example shown BCH1774 can be modified, while maintaining the exception of the Central nervous system even at doses above 100 mg/kg subcutaneously, and you can minimize harmful toxicity when administered intravenously. Thus, BCH2463 is not lethal to mice at doses of at least above 20 mg/kg (intravenously).

Pharmaceutically suitable salts of the peptides of the present invention can be obtained in the usual way, by adding acid such as hydrochloric, Hydrobromic, phosphoric, acetic, fumaric, salicylic, citric, lactic, almond, wine, axalingua, methansulfonate and other suitable acids, known to specialists.

The present invention also provides pharmaceutical compositions. Suitable compositions contain farmatsevticheskii suitable carrier or diluent.

The present invention also provides a method of pain control in animals such as mammals, including humans. The method includes the introduction phase the patient pharmaceutically effective amount of the peptide of formula 1 or its pharmaceutically suitable salts. You can also use the above-described pharmaceutical compositions.

The following examples are provided to better describe the invention. These examples are given only for illustrative purposes and in no way limit the invention.

Examples

Opioid activity of the peptides evaluated in vitro using the drug longitudinal muscle of the ileum of Guinea pigs (GPI), and their analgesic activity determined in rodents in vivo in a model of convulsions caused PBQ (peripheral activity), and in the test with hot plate (Central activity). Antagonism anesthesia peripheral opioid antagonist N-methylalanine and comparing the activity with convulsions and in tests with hot plate show that the analgesic effect is mainly mediated at the periphery. On the peripheral anesthesia indicates a high potential for observing convulsions and low potential in the test with th the CSOs and peripheral analgesia. The experimental conditions see in the work of Sigmund and others, Proc. Soc. Exp. Biol. Med., 95, p. 729 (1957), which is incorporated herein by reference. Central anesthesia, are determined by inhibition of the reaction of mice on a hot plate. The experimental conditions, see the work of G. Woolf, and A. Macdonald, J. Pharmacol. Exp. Ther., 80, p. 300 (1944), which is incorporated herein by reference. Studies that determine the binding of opioid receptors (or receptor), as well as GPI and MVD estimates were made in the experimental conditions described in the work of Schiller and others, Biophis. Res. Commun., 85, p. 1322 (1975), which is incorporated herein by reference.

Compounds of the present invention receives, using solid phase synthesis described in General terms below and, as a rule, well-known specialists.

Example 1. Solid-phase synthesis of opioid peptides

Synthetic peptides gain, using Rink resin*1[4-(2',4'-acid-Fmoc-aminomethyl)-phenoxy resin] (Novabiochem of Advanced Chemtech) and the corresponding C-terminal N-Fmoc-L-amino acid residue of each synthesized peptide (*trade mark).

All L - and D-amino acids (Novabiochem of Advanced Chemtech) have their Fmoc-protected (9-fluorenyl-methyloxycarbonyl) alpha group and wizarat-butyl ester (OtBu) for aspartic acid and glutamic acid; tert-butyloxycarbonyl (tBoc) for lysine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc) for arginine, trityl (trt) for cysteine.

Not containing dimethylamine dimethyl formamide (DMF) (Anachemia) is treated with activated molecular sieves 4A. Piperidine (Advanced Chemtech) used without further purification. DCC (dicyclohexylcarbodiimide) and HOBt (hydroxybenzotriazole) obtained from Fluka and Advanced Chemtech, respectively.

Solid phase synthesis of peptides is carried out manually on Rink resin*2(*trade mark). The download is about 0.6 mmole/g Condensation of the peptide is carried out using:

1) the reaction of 2 equivalents of each Fmoc-amino acid, HOBt and DCC in DMF for 1-4 h at room temperature;

2) repeated interaction 1 equivalent of each Fmoc-amino acid, HOBt and DCC;

3) acetylation of 20% (volume/volume) (CH2CO)2O/DMC for 1 h at room temperature;

4) removing the N-Fmoc-protection: 20% (volume/volume) piperidine in DMF for 25 minutes

Remove the side of the protective groups (tBu, Boc, Trt, Pmc) and cleavage of the peptide from the resin is carried out at the influence of a cocktail containing TFA:

TFA/Anisole/DCM = 55/5/40 (volume/volume) for 90 min at room temperature under N2. The peptide is precipitated from di who chromatography (HPLC) on a column with reversed phase with gradient elution, using 0.06% TFA/H2O and 0.06% TFA/acetonitrile.

Example 2. Research hot plate

Determination of analgesic activity

For the test using male mouse CD#1 weighing from 20 to 25, the Mice are weighed, labeled and divided into groups of 10 animals.

Usually mice do subcutaneous injection of the compound (or standard, or environment), the injected volume is 0.1 ml per 10 g mouse (10 ml/kg). If you use antagonist, such as Nalaxone or N-methyl-levallorphan (N-methyl-Levallorphan, its injected intraperitoneally 20 min before the introduction of the connection (or standard, or environment). Injected volume is 0.1 ml per 10 g of mouse. The dose of the antagonist is equal to 10 ml/kg

For each mouse assess the reaction time in hot plate. The temperature of the hot plate (Sorel, model DS37) establish 55oC. For mouse watch, noting the signs of discomfort, such as licking of the paws or shake, escape (jumps off the plate) or a shiver. Reaction time (in sec) note when you receive one of these signs. Each mouse watch for a maximum period of 30 s to prevent tissue damage to the paws. Mice can be observed after various time intervals after the introduction of the policy research and the LASS="ptx2">

For each time index, the average response time of the control group multiplied by 1.5. The response time of each treated mouse compared with medium control 1.5". If the reaction time is less than the average control 1.5", believe that the mouse is not received anesthesia. If the reaction time is above average control 1.5", it is considered that the mouse is provided with an analgesic effect. The number of mice who have experienced anesthesia, the group determines the percentage of analgesia data connection for this indicator. If the percentage of pain less than 30%, the connection is considered inactive.

Example 3. The study convulsions

Determining the number of dislocations

For the test using male mouse CD#1 weighing from 18 to 22, the Mice are weighed, labelled. Intraperitoneally injected to mice 0.02% solution of finishinga, 0.3 ml/20 g weight. Consider the number of dislocations over a period of 15 min after injection. Finishined injected subcutaneously at intervals of 5, 20 or 60 min after administration of the compounds (or environment, or standard). Finishined administered orally every 60 min after administration of the compounds (or environment, or standard).

0.02% solution of finishinga - (2-phenyl-1,4-benzoquinone (Sigma) is prepared as posologia and pre-fueling (not to boil), slowly added to 95 ml of distilled water. The solution finishinga constantly protect from light and every day to test prepare a new solution. Before using the solution finishinga is recommended to wait 2 hours

Testing can be carried out on 5 mice at the same time. Each group usually consists of 10 mice. If apply antagonist, such as naloxone (naloxone reversibility), it injected intraperitoneally 20 min before the compounds (or environment, or standard).

1. Opioid peptides of General formula I

< / BR>
and their derivatives and analogues with peripheral analgesic effect, where X = hydrogen; Y and Z = hydrogen; R1is tirocinium balance, 2',6'-dimethylthiazolium residue or an analogue or derivative; R3- the remainder of the aromatic amino acids; R4- the remainder of the aromatic amino acids; R2a balance of amino acids, having the R - configuration, or selected from the group consisting of aminoadamantane acid, cyclopropylalanine, cycloviolacin, cycloleucine provided that when R1is tirocinium the remainder, R2is - alanine, X, Y and Z are hydrogen, and R3is phenylalanine, R4is not unsubstituted phenyl what SUB>2
is D-alanine, X, Y and Z are hydrogen and R4is phenylalanine, R3is not unsubstituted phenylalanine or phenylalanine, substituted 4NO2; when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are hydrogen and R4is 1'-naphtylamine, R3is not 1'-naphthylamines or 2'-naphtylamine; and when R1is tirocinium the remainder, R2is D-alanine, X, Y and Z are H, R3and R4are not tryptophan; and Q is an amide bond, and at the same time excluded

H-Tyr-D-Phe-Phe-Phe-NH2< / BR>
H-Tyr-D-NMePhe-Phe-Phe-NH2< / BR>
H-Tyr-D-Tic-Phe-Phe-NH2< / BR>
2. The compound of formula I under item 1, wherein R2is an amino acid residue having the R-configuration, provided that when R1is tirocinium the remainder, R2is D-alanine, Y and Z are H, R3and R4different and selected from the group consisting of phenylalanine and tryptophan.

3. Connection on p. 2 and its pharmaceutical derivatives for the production of peripheral analgesics to relieve pain.

4. The compound of formula I on p. 1, wherein Y and Z are H; R3and R4illegal is provided that when R1is tirocinium the remainder, R2is D-alanine, R3and R4different and selected from the group consisting of phenylalanine and tryptophan.

5. Connection on p. 4 and its pharmaceutical derivatives for the production of peripheral analgesics to relieve pain.

6. The compound of formula I on p. 4, wherein R2is an amino acid having an R-configuration, provided that R2is not D-alanine, and R3and R4are remnants of phenylalanine.

7. Connection on p. 4, wherein R1is tirocinium the remainder, R2selected from the group consisting of D-Norvaline, D-serine and D-arginine; R3and R4are phenylalaninamide residues; and Q is an amide bond.

8. Connection on p. 1, characterized in that it is chosen from the group consisting of

H-Tyr-Aib-Phe-Phe-NH2< / BR>
H-Tyr-D-Nle-Phe-Phe-NH2< / BR>
H-Tyr-D-Ala-Phe-2'-Nal-NH2< / BR>
H-Tyr-D-Ala-D-Phe-Phe-NH2< / BR>
H-Tyr-D-Ala-Phe(4NO2)-Phe(4NO2)-NH2< / BR>
H-Tyr-D-Ala-Phe-Tic-NH2< / BR>
H-Tyr-D-Ala-Phe-1'-Nal-NH2< / BR>
H-Tyr-D-Ala-Trp-Phe-NH2< / BR>
H-Tyr-D-Ala-Phe-Trp-NH2< / BR>
H-Tyr-D-Nle-Phe-Trp-NH2< / BR>
H-Tyr-D-Nle-Phe-2'-Nal-NH2< / BR>
H-Tyr-D-Nle-Trp-Phe-NHB>2< / BR>
H-Tyr-D-Ser-Phe-Phe-NH2< / BR>
H-Tyr-D-Val-Phe-Phe-NH2< / BR>
H-Tyr-D-Leu-Phe-Phe-NH2< / BR>
H-Tyr-D-Ile-Phe-Phe-NH2< / BR>
H-Tyr-D-Abu-Phe-Phe-NH2< / BR>
H-Tyr-Chl-Phe-Phe-NH2< / BR>
H-Tyr-Cle-Phe-Phe-NH2< / BR>
H-Tyr-D-Arg-Phe-Phe-NH2< / BR>
H-Tyr-D-Cys-Phe-Phe-NH2< / BR>
H-Tyr-D-Thr-Phe-Phe-NH2< / BR>
H-DMT-D-Ser-Phe-Phe-NH2< / BR>
H-Tyr-D-Ala-Phe-Phg-NH2- salt triperoxonane acid

H-Tyr-D-Arg-Phe-Hgh-NH2- salt of bis-triperoxonane acid

H-DMT-D-Ala-Phe-Phe-NH2- triperoxonane acid

H-D-DMT-D-Ala-Phe-Phe-NH2- salt triperoxonane acid

H-Tyr-D-Ala-Phe-Hph-NH2- salt triperoxonane acid

H-Tyr-D-Ala-Phe-Cys(Bzl)-NH2- salt triperoxonane acid

H-Tyr-D-Arg-Phg-Phe-NH2- salt of bis-triperoxonane acid

H-Tyr-D-Arg-Hph-Phe-NH2- salt of bis-triperoxonane acid

H-Tyr-D-Ala-Phe-Phe(pf)-NH2- salt triperoxonane acid

H-Tyr-D-Ala-Phe-D-Phe(pf)-NH2- salt triperoxonane acid

H-Tyr-D-Ala-Hph-Phe-NH2- salt triperoxonane acid

H-Tyr-D-Met-Phe-Phe-NH2- salt triperoxonane acid

H-Tyr-D-Arg-Phe-D-Phe-NH2- salt of bis-triperoxonane acid

H-Tyr-D-Ala-Phg-Phe-NH2- salt triperoxonane acid

H-Tyr-D-Ala-D-Phg-Phe-NH2- salt triperoxonane acid

H-Tyr-D-Arg-Phe-Phe(pf)-NH2inania under item 1, characterized in that the said compound is H-Tyr-D-Nva-Phe-Phe-NH2.

10. Connection on p. 1, characterized in that the said compound is H-Tyr-D-Ser-Phe-Phe-NH2.

11. Connection on p. 1, characterized in that the said compound is H-Tyr-D-Arg-Phe-Phe-NH2.

12. Connection PP.8 - 11 and its pharmaceutical derivatives for the production of peripheral analgesics to relieve pain.

13. The compound of formula I under item 1 for the production of peripheral analgesics to relieve pain.

14. Connection on p. 13, where the specified connection H-Tyr-D-Ala-Phe-Phe-NH2or its analogs, or a pharmaceutical derivative intended for the production of peripheral analgesics to relieve pain.

15. Connection on p. 14, characterized in that the analogue selected from 4-Tyr-D-Ala-Phe-Phe(4-NO2)NH2and H-Tyr-D-Ala-Phe-Phe(4-N3)-NH2.

16. Pharmaceutical composition having analgesic activity, comprising an effective amount of at least one compound of formula I under item 1, in a mixture with a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to p. 16, comprising an effective amount of at least the song on p. 16, with peripheral analgesic activity, comprising an effective amount of at least one connection on p. 2 in a mixture with a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to p. 16, characterized in that, in addition, includes an effective amount of at least one connection on p. 4 in a mixture with a pharmaceutically acceptable carrier.

20. The pharmaceutical composition according to p. 17, further comprising at least another therapeutically active agent.

21. The pharmaceutical composition according to paragraphs.18 and 19, additionally comprising an effective amount of at least one therapeutically active agent.

 

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FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

FIELD: organic chemistry, medicine.

SUBSTANCE: invention represents ligands MC-4 and/or MC-3 of the formula (I): , wherein X means hydrogen atom, -OR1, -NR1R1' and -CHR1R1' wherein R1 and R1' are taken among the group: hydrogen atom, (C1-C6)-alkyl and acyl; (1) each R2 is taken independently among the group: hydrogen atom, (C1-C6)-alkyl; or (2) (a) R2 bound with carbon atom that is bound with X and Z1 and substitute R5 can be optionally bound to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; or (b) R2 bound with carbon atom that is bound with ring Ar can be bound with R7 to form ring condensed with ring Ar; each among Z1, Z2 and Z3 is taken independently from the following groups: -N(R3e)C(R3)(R3a)-, -C(R3)(R3a)N(R3e)-, -C(O)N(R3d)-, -N(R3d)C(O)-, -C(R3)(R3a)C(R3b)(R3c)-, -SO2N(R3d)- and -N(R3d)SO2- wherein each among R3, R3a, R3b and R3c, R3d, R3e when presents is taken independently among hydrogen atom and (C1-C6)-alkyl; p is a whole number from 0 to 5 wherein when p above 0 then R4 and R4' are taken among hydrogen atom, (C1-C6)-alkyl and aryl; R5 represents 5 substitutes in phenyl ring J wherein each R5 is taken among hydrogen atom, hydroxy-, halogen atom, thiol, -OR12, -N(R12)(R12'), (C1-C6)-alkyl, nitro-, aryl wherein R12 and R12' are taken among hydrogen atom and (C1-C6)-alkyl; or two substitutes R5 can be bound optionally to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; q = 0, 1, 2, 3, 4 or 5 wherein when q above 0 then R6 and R6' are taken among hydrogen atom and (C1-C6)-alkyl; Ar is taken among the group consisting of phenyl, thiophene, furan, oxazole, thiazole, pyrrole and pyridine; R7 are substitutes at ring Ar wherein each R7 is taken among hydrogen, halogen atom, -NR13R13', (C1-C6)-alkyl and nitro- wherein R13 and R13' are taken among hydrogen atom and (C1-C6)-alkyl; r is a whole number from 0 to 7 wherein when r is above 0 then R8 and R8' are taken among hydrogen atom and (C1-C6)-alkyl; B is taken among -N(R14)C(=NR15)NR16R17, -NR20R21, heteroaryl ring and heterocycloalkyl ring wherein R14-R17, R20 and R21 are taken independently among hydrogen atom and (C1-C6)-alkyl; s = 0, 1, 2, 3, 4 or 5 wherein when s is above 0 then R and R9' are taken among hydrogen atom and (C1-C6)-alkyl; R10 is taken among the group consisting of optionally substituted bicyclic aryl ring and optionally substituted bicyclic heteroaryl ring; D is taken among hydrogen atom, amino- and -C(O)R11 wherein R11 is taken among the following group: hydroxy-, alkoxy-, amino-, alkylamino-, -N(R19)CH2C(O)NH2 wherein R19 represents (C1-C6)-alkyl, -NHCH2CH2OH and -N(CH3)CH2CH2OH, or its isomers, salts, hydrates or biohydrolysable ester, amide or imide.

EFFECT: valuable medicinal properties of compounds.

18 cl, 107 ex

FIELD: medicine.

SUBSTANCE: peptide of the following formula: X-Pro-Gly-P, where X = Thr-Lys-Pro-Arg-; Lys-pro-Arg-; pro-Arg-; Arg-, being of untiulcerous activity. They should be applied at intraperitoneal injection at the dosage of 0.58-3.20 mcM g/kg for preventing and treating ulcers of gastro-intestinal tract.

EFFECT: higher efficiency and prophylaxis.

4 dwg, 5 ex

FIELD: medicine, chemistry of peptides, amino acids.

SUBSTANCE: invention relates to novel biologically active substances. Invention proposes the novel composition comprising peptides of the formula: H-Arg-Gly-Asp-OH and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys. The composition shows ability to inhibit proliferative activity of mononuclear cells, to induce suppressive activity and their ability for secretion of cytokines TNF-1β (tumor necrosis factor-1β) and IL-10 (interleukin-10 ).

EFFECT: simplified method for preparing composition, valuable medicinal properties of composition.

4 cl, 16 tbl, 9 ex

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