RU2146681C1 - Opioid peptides and pharmaceutical composition - Google Patents

Abstract

FIELD: organic chemistry, peptides. SUBSTANCE: invention relates to opioid peptides of the formula (I): X-R₁-R₂-R₃-Q-R₄-N(Y)Z and its analogues where X is H or C_1-6-alkyl; Y, Z - H, cyclic aralkyl and C_1-6-alkyl; R₁ - tyrosyl residue, 2',6'-dimethyltyrosyl residue or their analogue; R₃, R₄ - aromatic amino acid residue (other values see p. 1 of the invention claim). Peptides show peripheral analgetic effect. EFFECT: valuable pharmacological property of peptides. 21 cl, 1 tbl, 6 dwg, 3 ex

Description

Prototypes of the invention
Many endogenous peptides of mammals and amphibians are associated with specific opioid receptors and cause an analgesic response similar to the effect of classical narcotic opiates. In higher animals, many different types of opioid receptors have been shown to coexist. For example, see W. Martin et al., J. Pharmacol. Exp. Ther., 197, p. 517 (1975); and J. Lord et al., Nature (London), 257, p. 495 (1977). Three different types of opioid receptors are identified. The first δ shows a modifying agent for peptides like enkephalins. The second μ demonstrates increased selectivity for morphine and other polycyclic alkaloids. The third k shows the same affinity for any group of the above ligands and predominant affinity for dynorphin (dynorphin). Apparently, mainly μ receptors are more involved than others in analgesic effects, δ receptors seem to deal with behavioral effects, although analgesia can also be mediated by δ and k receptors.

 Each opioid receptor, when interacting with an opiate, produces a specific biological response, one of a kind for this type of receptor. When an opiate activates more than one receptor, the biological response of each receptor is influenced by the side effects produced. When an opiate is administered, a less specific and selective opiate may give a greater chance of an increase in side effects.

 In prototypes, opiates, peptides of the opioid type and their analogues either do not show specificity and selectivity for the type of receptor or receptors with which they are associated. The main site of action of analgesic opioids is the central nervous system (CNS). Well-known narcotic analgesics are usually hydrophobic and therefore easily penetrate 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 are not necessarily associated with a subtype of a homogeneous receptor. This binding causes medically undesirable side effects.

 Opiates can cause serious and potentially fatal side effects. Nonspecific interactions with receptors of the central nervous system cause side effects such as respiratory depression, endurance, the ability of physical dependence and sudden withdrawal syndrome. See K. Budd, In International Encyclopedia of Pharmacology and Therapeutics; N.E. Williams and H. Wilkinson, Eds., Pergammon: (Oxford), 112, p. 51 (1983). Therefore, it is believed that opioid analgesics, acting primarily through opioid receptors in the peripheral nervous system, do not cause such undesirable side effects as effects caused by opioid analgesics acting on the central nervous system. The opioid peptides of the present invention primarily act on the peripheral nervous system and therefore overcome some of the adverse effects characteristic of conventional opiates, mainly by preventing undesirable side effects.

 To date, one of the known classes of agents providing a peripheral analgesic effect are non-steroidal anti-inflammatory agents, such as aspirin, ibuprofen and ketorolac. These agents do not interact with opioid receptors, but it is known that they inhibit cyclooxygenase and weaken the synthesis of prostaglandin. These weak analgesics do not have centrally mediated effects, but can cause other side effects, such as ulceration of the gastrointestinal tract. It is the subject of the present invention to provide peptides similar to opioids that exert a peripheral effect but essentially do not have undesirable side effects caused by conventional peripherally acting analgesics.

 Recently, prototypes have shown that opiate-type drugs have significant peripheral analgesic activity. See A. Barber and R. Gottschlich, Med. Res. Rev., 12, p. 525 (1992) and C. Stein, Anasth. Analg., 76, p. 182 (1993). To distinguish between peripheral and central analgesic responses, quaternary salts of known centrally acting opioid alkaloids are used as pharmacological probes. The quaternary salts of patented opiates have a constant positive charge and exhibit limited passage of the blood-brain barrier. See T.W. Smith et al., Life Sci, 31, p. 1205 (1982); T.W. Smith et al., 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 et al. Life Sci, 30, p. 1875 (1982); and J. Russel et al., Eur. J. Pharmacol. , 78, p. 255 (1982). Strongly polar analogues of enkephalins and dermorphins are obtained, which retain analgesic activity, but show limited penetration into the central nervous system. See R. L. Follenfant et al., Br. J. Pharmacol., 93, p. 85 (1988); G. W. Hardy et al., J. Med. Chem. , 32, p. 1108 (1989). Conversely, prototypes believe that lipophilic opioid peptides more readily cross the blood-brain barrier. Surprisingly, the opioid peptides of this invention are highly lipophilic, but only slightly cross the blood-brain barrier.

 Unlike conventional opiates, opioid peptides are hydrophobic. Their hydrophobicity tends to increase the rate of their elimination from the body of a mammal. Hydrophobicity increases the ability of peptides to cross the epithelial barrier. Despite this, it has been shown that the administration of opioid peptides to the mammalian body affects the central nervous system. Therefore, the maximum effort was aimed 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 exposure to the body with these drugs can cause harmful side effects or even prove toxic.

It is believed that non-polar peptides more easily pass into the central nervous system, crossing the blood-brain barrier than polar peptides. It has been reported that TAPP (H-Tyr-D-Ala-Phe-Phe-NH ₂ ) demonstrates peripheral and central analgesic properties (P. Schiller et al., Proceedings of 20th European Peptide Symposium, 1988). In opposition to the applicants of the present invention, it was found that this TAPP tetrapeptide (H-Tyr-D-Ala-Phe-Phe-NH ₂ ) does not act centrally. This result is shown by the absence of analgesic action even at doses of 100 mg / kg in the mouse hot plate test. This test is standard and is well known in the art. The test checks for chemicals that cause a centrally mediated analgesic reaction.

As used in this application, the term “specificity” refers to the particular or unconditional binding of an opiate or opioid peptide to a single opioid receptor compared to another opioid receptor. The specificity of the opioid peptide is indicated by the K _i binding inhibition constant. The term "selectivity" refers to the ability of an opiate or opioid peptide to distinguish one among several opioid receptors and interact with only one single receptor. The selectivity of the opioid peptide relative to μ receptors is indicated by the ratio of binding inhibition constants. For example, to determine the selectivity, you can use the ratio of the binding inhibition constants K $\genfrac{}{}{0ex}{}{\text{δ}}{\text{i}}$ / K $\genfrac{}{}{0ex}{}{\text{μ}}{\text{i}}$ . This ratio represents the ratio of affinity for μ and δ receptors. A larger value of this ratio indicates a greater preference for the ligand to interact with the μ receptor compared to the δ receptor. It is known that the analogue of the conventional opioid peptide H-Tyr-D-Ala-Gly-Phe (NMe) -Gly-ol (DAGO) is one of the most μ-selective analogues of opioid peptides. The peptide gives a value of K $\genfrac{}{}{0ex}{}{\text{δ}}{\text{i}}$ / K $\genfrac{}{}{0ex}{}{\text{μ}}{\text{i}}$ 1050. On the other hand, Leu-enkephalin gives a value of K $\genfrac{}{}{0ex}{}{\text{δ}}{\text{i}}$ / K $\genfrac{}{}{0ex}{}{\text{μ}}{\text{i}}$ 0.2. This value reflects the apparent affinity for the δ receptor with respect to the μ receptor.

 For pharmacological use, the peptide must have certain properties. First, the peptide must be resistant to proteolytic destruction. Secondly, the peptide should cause an enhanced biological response. Thirdly, the peptide must be safe for humans. Fourth, the peptide must be such that it can be obtained in sufficient quantity for a clinical study of toxicity, and later for use on a commercial basis. In this case, the required properties are also lower lipid solubility and greater solubility in water in order to prevent the peptide from penetrating the blood-brain barrier and to ensure rapid elimination of excess introduced peptide and its metabolites. In addition, it is desirable for peptides to achieve selective and specific receptor binding activity in order to minimize potential side effects.

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

Summary of invention
The present invention provides novel compounds that are selective and specific for a single opioid receptor. The present invention provides peptides that exhibit preferred selectivity and specificity for μ receptors. The present invention also provides peptides that primarily interact with opioid receptors at the peripheral nerve endings and to a small extent cross the blood-brain barrier. Therefore, the present invention reduces the severity and number of side effects compared to the conventional opiates and opioid peptides described to date.

и их производные и аналоги, где X выбирают из группы, состоящей из H и C_1-6-алкила;
Y и Z независимо выбирают из группы, состоящей из H, циклического аралкила и C_1-6-алкила;
R₁ является тирозильным остатком, 2',6'-диметилтирозильным остатком или их аналогом или производным;
R₃ является ароматической аминокислотой;
R₄ является остатком ароматической аминокислоты;
R₂ является аминокислотой, имеющей R-конфигурацию при условии, что когда R₁ является тирозильным остатком, R₂ является D-аланином, X, Y и Z являются H, и R₃ является фенилаланином, то R₄ не является незамещенным фенилаланином или фенилаланином, замещенным 4NO₂ или 4N₃;
когда R₁ является тирозильным остатком, R₂ является D-аланином, X, Y и Z являются H, и R₄ является фенилаланином, то R₃ не является незамещенным фенилаланином или фенилаланином, замещенным 4NO₂;
когда R₁ является тирозильным остатком, R₂ является D-аланином, X, Y и Z являются H, и R₄ является 1'-нафтилаланином, то R₃ не является 1'-нафтилаланином или 2'-нафтилаланином; и
когда R₁ является тирозильным остатком, R₂ является D-аланином, X, Y и Z являются H, то R₃ и R₄ каждый не является триптофаном;
и Q является амидной связью или подобием вставленной амидной связи.The following formula (1) represents the compounds of the present invention:

and their derivatives and analogues, wherein X is selected from the group consisting of H and C _1-6 alkyl;
Y and Z are independently selected from the group consisting of H, cyclic aralkyl, and C _1-6 alkyl;
R ₁ is a tyrosyl residue, 2 ', 6'-dimethyltyrosyl residue, or an analog or derivative thereof;
R ₃ is an aromatic amino acid;
R ₄ is an aromatic amino acid residue;
R ₂ is an amino acid having an R configuration, provided that when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are H, and R ₃ is phenylalanine, then R _{4 is} not unsubstituted phenylalanine or phenylalanine substituted with 4NO ₂ or 4N ₃ ;
when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are H, and R ₄ is phenylalanine, then R _{3 is} not unsubstituted phenylalanine or phenylalanine substituted with 4NO ₂ ;
when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are H, and R ₄ is 1'-naphthylalanine, then R _{3 is} not 1'-naphthylalanine or 2'-naphthylalanine; and
when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are H, then R ₃ and R _{4 are} each not tryptophan;
and Q is an amide bond or the like of an inserted amide bond.

 The invention also provides pharmaceutically acceptable compositions comprising these peptides for use in pain relief.

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

The invention also provides the use of peptides of the formula H-Tyr-D-Ala-Phe-Phe-NH ₂ for the production of peripheral analgesics for analgesia.

Tables and Shapes
Table 1 lists the in vivo and in vitro activities of hydrophobic tetrapeptides related to dermorphin.

 Figure 1 shows the time variation of the analgesic effect of morphine (10 mg / kg) (Fig. 1A) and typical test compounds (Fig. B: BCH2463; C: BCH2462; D: BCH2687).

In FIG. Figure 2 shows a dose-response diagram for BCH2463 in experiments on the study of convulsions induced by phenylquinone administered subcutaneously to mice, ED ₅₀ = 0.5 mg / kg 20 min after administration.

 In FIG. Figure 3 shows a comparative change in time of the analgesic effect of BCH1774 and BCH2463 in experiments on the study of convulsions caused by phenylquinone, which is administered to mice subcutaneously.

Description of the invention
In the description and claims, the following general notations are used:
Abu - aminobutyric acid; Ala is alanine; Arg is arginine; Cle is cycloleucine; Gln is glutamine; Gly glycine; His is histidine; Hph - homophenylalanine; Aib is aminoisomalic acid; Chl is cyclohomoleucine; Cis (Bzl) - cysteine (benzyl); Dmt - 2 ', 6'-dimethyl tyrosyl; Glu - glutamic acid; GPI — Guinea pig ileum; Ile - isoleucine; Leu - leucine; Nle - norleucine; Phe - phenylalanine; Phg is phenylglycine; Ser is serine; Trp - tryptophan; Met is methionine; MVD - seminal duct of the mouse; Nva - norvaline; Pro is proline; Thr is threonine; Tyr is tyrosine; Nal is 1'- or 2'-naphthylalanine; PBQ is phenyl para-benzoquinoline; Tic - tetrahydroisoquinoline-3-carboxylic acid; TAPP - H-Tyr-D-Ala-Phe-Phe-NH ₂ ; TSPP - H-Tyr-D-Ser-Phe-Phe-NH ₂ .

The terms “amino acid” and “aromatic amino acid” as used herein include naturally occurring amino acids, as well as non-naturally occurring amino acids, their derivatives and analogs commonly used by those skilled in the art of chemical synthesis and peptide chemistry. Also included are TAPP analogues, where phenylalanine is para-substituted at position 4 with a nitro or azido residue. A list of non-naturally occurring amino acids can be found in "The Peptide", Vol. 5, 1983, Academic Press, Chapter 6, DC Roberts and F. Vellaccio, this inclusion monograph is here by reference. Examples of aromatic amino acids include tyrosine, tryptophan, phenylglycine, histidine, naphthylalanine, tetrahydroisoquiniline-3-carboxylic acid, and benzylcysteine. Other examples of aromatic amino acids include phenylalanine substituted on the aromatic ring, for example, with CH ₃ , C ₂ H ₅ , F, Cl, Br, NO ₂ , OH, SH, CF ₃ , CN, COOH and CH ₂ COOH or substituted at β -carbon lower alkyl radicals, OH, SH or benzene group. The aromatic ring may have several substituents. Aromatic amino acids may also include aromatic carbocycles of the phenylglycine type, where the aromatic phenylglycine ring is substituted with CH ₃ , C ₂ H ₅ , F, Cl, Br, NO ₂ , OH, SH, CF ₃ , CN, COOH and CH ₂ COOH. These examples are provided only to illustrate the invention, but in no way limit it.

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

 The term “similarity of an inserted amide bond” means a bond in which the carbonyl group and the NH group of the amide bond are interchanged.

The term "K _i " refers to the binding inhibition constant.

The term "K $\genfrac{}{}{0ex}{}{\text{δ}}{\text{i}}$ / K $\genfrac{}{}{0ex}{}{\text{μ}}{\text{i}}$ "denotes a value that can be used to measure selectivity. This ratio represents the ratio of the ability of opioid peptides to bind to μ and δ receptors.

 The term "R-configuration" refers to the three-dimensional arrangement of substituents around a chiral element. The basic system for designating an absolute configuration is based on a priority system that is well known to those skilled in the art and is briefly described below. Each group attached to the chiral center is indicated by a number according to priorities. A configuration is defined as “R” if, when moving from the highest priority group to the lower priority eye groups, it follows the clockwise direction.

 The term “residue” as applied to an amino acid means a radical derived from the corresponding amino acid when the hydroxyl of the carboxyl group and one hydrogen of the amino group are removed.

и их производные и аналоги, где X выбирают из группы, состоящей из H и C_1-6-алкила;
Y и Z независимо выбирают из группы, состоящей из H, циклического аралкила и C_1-6-алкила;
R₁ является тирозильным остатком, 2',6'-диметилтирозильным остатком или его аналогом или производным;
R₃ является аминокислотным остатком, выбранным из группы, состоящей из ароматических аминокислот;
R₄ является остатком ароматической аминокислоты;
R₂ является аминокислотой, имеющей R-конфигурацию при условии, что когда R₁ является тирозильным остатком, R₂ является D-аланином, X, Y и Z являются H, и R₃ является фенилаланином, то R₄ не является незамещенным фенилаланином или фенилаланином, замещенным 4NO₂ или 4N₃;
когда R₁ является тирозильным остатком, R₂ является D-аланином, X, Y и Z являются H, и R₄ является фенилаланином, то R₃ не является незамещенным фенилаланином или фенилаланином, замещенным 4NO₂;
когда R₁ является тирозильным остатком, R₂ является D-аланином, X, Y и Z являются H, и R₄ является 1'-нафтилаланином, то R₃ не является 1'-нафтилаланином или 2'-нафтилаланином; и
когда R₂ является D-аланином, R₁ является тирозильным остатком и X, Y и Z являются H, то R₃ и R₄ каждый не является триптофаном; и
Q является амидной связью или подобием вставленной амидной связи.Compounds of the present invention are compounds represented by formula (1):

and their derivatives and analogues, wherein X is selected from the group consisting of H and C _1-6 alkyl;
Y and Z are independently selected from the group consisting of H, cyclic aralkyl, and C _1-6 alkyl;
R ₁ is a tyrosyl residue, a 2 ′, 6′-dimethyltyrosyl residue or an analogue or derivative thereof;
R ₃ is an amino acid residue selected from the group consisting of aromatic amino acids;
R ₄ is an aromatic amino acid residue;
R ₂ is an amino acid having an R configuration, provided that when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are H, and R ₃ is phenylalanine, then R _{4 is} not unsubstituted phenylalanine or phenylalanine substituted with 4NO ₂ or 4N ₃ ;
when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are H, and R ₄ is phenylalanine, then R _{3 is} not unsubstituted phenylalanine or phenylalanine substituted with 4NO ₂ ;
when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are H, and R ₄ is 1'-naphthylalanine, then R _{3 is} not 1'-naphthylalanine or 2'-naphthylalanine; and
when R ₂ is D-alanine, R ₁ is a tyrosyl residue, and X, Y and Z are H, then R ₃ and R _{4 are} each not tryptophan; and
Q is an amide bond or the like of an inserted amide bond.

 Preferred are the compounds represented by formula (1) and derivatives and analogs thereof, wherein X is H.

Other preferred are compounds represented by formula (1), and their derivatives and analogues, where
R ₂ is an amino acid residue having an R configuration, provided that when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are H, then R ₃ and R _{4 are} different and selected from the group consisting of from phenylalanine and tryptophan.

Other preferred are compounds represented by formula (1), and their derivatives and analogues, where
Q is an amide bond or a similarity of an inserted amide bond of the formula Q ₁ -Q ₂ , where Q _{1 is} selected from the group consisting of CH ₂ , CHOH, C = O, C = S and CH =, and Q _{2 is} selected from the group consisting of CH ₂ , NH, S, SO, SO ₂ , O and CH =, provided that when Q ₁ is CH =, then Q ₂ is CH =.

In addition, preferred are the compounds represented by formula (1), and their derivatives and analogues, where
Y and Z are H;
R ₃ and R ₄ are independently aromatic amino acids, and
R ₂ is an amino acid having an R configuration, provided that when R ₁ is a tyrosyl residue and R ₂ is D-alanine, then R ₃ and R _{4 are} different and selected from the group consisting of phenylalanine and tryptophan.

In addition, preferred are the compounds represented by formula (1), and their derivatives and analogues, where
R ₂ is an amino acid having an R configuration, provided that R _{2 is} not D-alanine and R ₃ and R ₄ are phenylalanine residues.

Still more preferred are the compounds represented by formula (1) and derivatives and analogues thereof, wherein R ₁ is a tyrosyl residue, R _{2 is} selected from the group consisting of D-norvaline, D-serine and D-arginine;
R ₃ and R ₄ are phenylalanine residues; and
Q is a peptide bond.

More preferred are the compounds represented by formula (1), and their derivatives and analogues, where
X is H,
Y and Z are independently selected from the group consisting of H, aralkyl, and C _1-6 alkyl;
R ₁ is a tyrosyl residue, a 2 ′, 6′-dimethyltyrosyl residue or an analogue or derivative thereof;
R ₃ is aromatic acid,
R ₄ independently selected from the group consisting of aromatic and aliphatic amino acids; and
R ₂ is an amino acid residue having an R configuration, provided that when R ₂ is D-alanine, R ₁ is a tyrosyl residue, and Y and Z are H, then R ₃ and R _{4 are} independently selected from the group consisting of phenylalanine and tryptophan, but they are not the same, Q is an amide bond or the like of an inserted amide bond of the formula Q ₁ -Q ₂ , where Q _{1 is} selected from the group consisting of CH ₂ , CHOH, C = O, C = S and CH =, and Q _{2 is} selected from the group consisting of CH ₂ , NH, S, SO, SO ₂ , O and CH =, provided that when Q ₁ is CH =, then Q ₂ is CH =.

More preferred are the compounds represented by formula (1) and derivatives and analogs thereof, wherein X is H, Y and Z are H;
R ₁ is a tyrosyl residue, a 2 ′, 6′-dimethyltyrosyl residue or an analogue or derivative thereof;
R ₃ and R ₄ are independently aromatic amino acids;
R ₂ is an amino acid having an R configuration, provided that when R ₂ is D-alanine and R ₁ is a tyrosyl residue, R ₃ and R _{4 are} independently selected from the group consisting of phenylalanine and tryptophan, but they are not the same, Q is an amide bond or a similarity of an inserted amide bond of the formula Q ₁ -Q ₂ , where Q _{1 is} selected from the group consisting of CH ₂ , CHOH, C = O, C = S and CH =, and Q _{2 is} selected from the group consisting of CH ₂ , NH, S, SO, SO ₂ , O and CH =, provided that when Q ₁ is CH =, then Q ₂ is CH =.

More preferred are the compounds represented by formula (1) and derivatives and analogs thereof, wherein X is H, Y and Z are H,
R ₁ is a tyrosyl residue, a 2 ′, 6′-dimethyltyrosyl residue or an analogue or derivative thereof;
R ₂ is an amino acid having an R configuration, provided that R _{2 is} not alanine,
R ₃ and R ₄ are phenylalanine residues;
Q is an amide bond or a similarity of an inserted amide bond of the formula Q ₁ -Q ₂ , where Q _{1 is} selected from the group consisting of CH ₂ , CHOH, C = O, C = S and CH =, and Q _{2 is} selected from the group consisting of CH ₂ , NH, S, SO, SO ₂ , O and CH =, provided that when Q ₁ is CH =, then Q ₂ is CH =.

Most preferred are compounds represented by formula (1) and derivatives and analogs thereof, wherein X is H; Y and Z are H;
R ₁ is a tyrosyl residue; R _{2 is} selected from the group consisting of D-norvaline, D-serine and D-arginine; R ₃ and R ₄ are phenylalanine residues; and
Q is a peptide bond.

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

The currently best known method of implementing the present invention is the use of the compound H-Tyr-D-Arg-Phe-Phe-NH ₂ .

The invention also includes the use of a TAPP compound H-Tyr-D-Ala-Phe-Phe-NH ₂ as a peripheral analgesic.

 As ligands of opioid receptors and analgesic agents of general action, a large number of tetrapeptides based on the general formula (1) have been obtained and studied. These compounds are listed in table 1 together with the corresponding binding inhibition constants and receptor selectivity ratios.

2 ', 6'-dimethyl tyrosine (Dmt) can be replaced with tyrosine in the opioid peptide compounds. Experiments show that replacing Dmt with tyrosine at position R ₁ in general formula 1 (the first amino acid residue) increases the potential of the opioid peptide relative to the μ receptor by two orders of magnitude. Selectivity for μ receptors increases when the compound includes Dmt at position R ₁ . This substitution causes a corresponding shift in the ratio of binding inhibition constants, which reflects increased selectivity for μ receptors.

Many of the compounds listed in Table 1 exhibit good binding to μ receptors, but exhibit weak analgesic effects in the study of mouse writhing assay. This anomaly can be observed due to rapid proteolysis, rapid cleansing, or both. For example, when the lipophilic peptide Demorphin TAPP (BCH1774) of the prototype is exposed at the border of the renal membranes, rapid degradation is observed in 15-30 minutes. Among the peptides listed in Table 1, three preferred compounds, in addition to TAPP itself, exhibit an increased in vivo analgesic effect. These three compounds are H-Tyr-Nva-Phe-Phe-NH ₂ (BCH2462), H-Tyr-D-Ser-Phe-Phe-NH ₂ (BCH2463) and H-Tyr-D-Arg-Phe-Phe-NH ₂ (BCH2687). BCH2462, BCH2463, and BCH2687 were shown to exhibit peripheral analgesic effects. When using these peptides, no central analgesic effect was observed, even at doses of 100 mg / kg in the test on mice with a hot plate.

As shown in table 1, the value of the ED ₅₀ for TAPP (BCH1774) is 1.4. The corresponding values for H-Tyr-D-Nva-Phe-Phe-NH ₂ (BCH2462), H-Tyr-D-Ser-Phe-Phe-NH ₂ (BCH2463) and H-Tyr-D-Arg-Phe-Phe -NH ₂ (BCH2687) are 2.7, 0.5, and 0.5, respectively. The ED ₅₀ values for the remaining compounds in table 1 are greater than these values. Although the ED ₅₀ value for BCH2813 is only 0.15, it was found that this compound acts centrally at doses of about 40 mg / kg in the hot plate test.

 The results show that compounds BCH1774, BCH2462, and BCH2463 still undergo proteolysis, but have a longer half-life and, therefore, are more effective analgesics. In FIG. 3 compares the duration of in vivo analgesia caused by BCH1774 (TAPP) and BCH2463 (TSPP). When using 30 mg / kg BCH2463 (subcutaneously) and 20 mg / kg BCH1774 (subcutaneous) in FIG. 3 shows that the analgesic effect of BCH1774 lasts longer than BCH2463, indicating probably a slightly accelerated proteolysis of BCH2463 compared to BCH1774 in vivo.

 In FIG. 1A-D show the effects of morphine, BCH2463 (TSPP), BCH2462 (TNPP) and BCH2687 on mice when evaluating the response of mice in a hot plate test. As shown in FIG. 1A, the reaction time of a mouse treated with 10 mg / kg of morphine is approximately 17 s. The reaction time of a mouse treated with 100 mg / kg BCH2463 (FIG. 1B) is about 9 s compared to a control value of about 7 s. These results indicate that while morphine inhibits a thermal pain stimulus, BCH2463 does not; but BCH2463 is a potential analgesic agent, as shown by inhibition of chemically induced convulsions (Fig. 2). The reaction time of the mouse treated with BCH2462 and BCH2687 (FIG. 1C, 1D) is approximately 8 s, which indicates a similar result as for BCH2463.

 The effects of inhibiting the proteolytic metabolism of BCH2463 by a DL-thiophane inhibitor, as well as the metabolic breakdown of BCH2463 mediated by brush border kindney membranes, were studied. The findings indicate that the kidneys may be the main cleansing and metabolism site for compound BCH2463. In figure 2 it is clear that the enzyme endopeptidase EC24-11, which is inhibited by DL-thiophan, is a preliminary mediator of proteolysis of BCH2463 by means of brush border kidney extract.

 Both BCH1774 (TAPP) and BCH2462 (TNPP) have a lethal effect on mice when a dose of 1-5 mg / kg is administered intravenously. In contrast, BCH2463 (TSPP), surprisingly, has no lethal effect at doses up to 20 mg / kg intravenously. In addition, peptides are safe when administered subcutaneously (s. C.) At doses greater than 100 mg / kg. Therefore, a subcutaneous administration is a desirable route of administration of these compounds. Thus, the structural example presented by BCH1774 can be modified while maintaining exclusion from the central nervous system even at doses higher than 100 mg / kg subcutaneously, and harmful toxicity by intravenous administration can be minimized. Thus, BCH2463 is not lethal for mice at doses above at least 20 mg / kg (intravenously).

 Pharmaceutically acceptable salts of the peptides of the present invention can be obtained in the usual way by adding acids such as hydrochloric, hydrobromic, phosphoric, acetic, fumaric, salicylic, citric, lactic, almond, tartaric, oxalic, methanesulfonic and other suitable acids known to those skilled in the art.

 The present invention also provides pharmaceutical compositions. Suitable compositions contain a pharmaceutically effective amount of the peptide of the present invention or its pharmaceutically acceptable salts and a pharmaceutically acceptable carrier or diluent.

 The present invention also provides a method of analgesia in animals such as mammals, including humans. The method includes the steps of administering to the patient a pharmaceutically effective amount of a peptide of formula 1 or a pharmaceutically acceptable salt thereof. The pharmaceutical compositions described above may also be used.

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

Examples
The opioid activity of the peptides was evaluated in vitro using the guinea pig ileum longitudinal muscle (GPI) preparation, and their analgesic activity was determined on an in vivo rodent on a model of convulsions induced by PBQ (peripheral activity) and in a hot plate test (central activity). Antagonism of analgesia by a peripheral opioid antagonist N-methylnalorfine and comparison of activity in convulsions and in tests with a hot plate show that the analgesic effect is predominantly mediated at the periphery. Peripheral anesthesia is indicated by a high potential in the observation of convulsions and a low potential in a hot plate test.

 Mouse-induced PBQ (phenyl-para-benzoquinone) convulsions of mice serve to evaluate central and peripheral analgesia. For experimental conditions see Sigmund et al., Proc. Soc. Exp. Biol. Med., 95, p. 729 (1957), which is incorporated herein by reference. Central anesthesia is determined by inhibiting the response of mice to a hot plate. For experimental conditions see G. Woolf and A. Macdonald, J. Pharmacol. Exp. Ther., 80, p. 300 (1944), which is incorporated herein by reference. Studies determining the binding of opioid receptors (for both receptors), as well as GPI and MVD scores, were made under the experimental conditions described by Schiller et al., Biophis. Res. Commun., 85, p. 1322 (1975), which is incorporated herein by reference.

 The compounds of the present invention are prepared using solid-phase synthesis, described generally below and generally known to those skilled in the art.

Example 1. Solid-phase synthesis of opioid peptides
Synthetic peptides are prepared using the Rink ^{* 1} resin [4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxy resin] (Novabiochem of Advanced Chemtech) and the corresponding C-terminal N-Fmoc-L-amino acid residue of each synthesized peptide (* - trademark).

 All L- and D-amino acids (Novabiochem of Advanced Chemtech) have their Fmoc-protected (9-fluorenylmethyloxycarbonyl) alpha groups and the following side protecting groups: tert-butyl ether (tBu) for serine, threonine and tyrosine; tert-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 and trityl (trt) for cysteine.

 Dimethylamine-free dimethylformamide (DMF) (Anachemia) is treated with 4A activated molecular sieves. Piperidine (Advanced Chemtech) is used without further purification. DCC (dicyclohexylcarbodiimide) and HOBt (hydroxybenzotriazole) are obtained from Fluka and Advanced Chemtech, respectively.

Solid-phase synthesis of peptides is carried out manually on Rink ^{* 2} resin (* - trademark). The charge is about 0.6 mmol / g. Peptide condensation is carried out using:
1) the interaction of 2 equivalents of each Fmoc amino acid, HOBt and DCC in DMF for 1-4 hours at room temperature;
2) re-interaction of 1 equivalent of each Fmoc amino acid, HOBt and DCC;
3) acetylation of 20% (v / v) (CH ₂ CO) ₂ O / DMC for 1 h at room temperature;
4) removal of N-Fmoc protection: 20% (v / v) piperidine in DMF for 25 min.

The removal of side protective groups (tBu, Boc, Trt, Pmc) and cleavage of the peptide from the resin is carried out when exposed to a cocktail containing TFA:
TFA / Anisole / DCM = 55/5/40 (v / v) for 90 min at room temperature under N ₂ . The peptide is precipitated from diethyl ether, filtered and dried. The crude peptide is purified and analyzed by reverse phase column high performance liquid chromatography (HPLC) using a gradient elution column using 0.06% TFA / H ₂ O and 0.06% TFA / acetonitrile.

Example 2. Study with a hot plate
Determination of analgesic activity
For the test, male CD # 1 mice weighing from 20 to 25 g are used. Mice are weighed, labeled and divided into groups of 10 animals.

 Typically, mice are given a subcutaneous injection of a compound (either standard or medium)), the injection volume is 0.1 ml per 10 g of mouse weight (10 ml / kg). If an antagonist is used, such as Nalaxone or N-methyl-levallorphan (N-methyl-Levallorphan), it is administered intraperitoneally 20 minutes before administration of the compound (or standard or medium). The injected volume is also 0.1 ml per 10 g of mouse weight. The dose of the antagonist is 10 ml / kg.

For each mouse, the reaction time on the hot plate is evaluated. The temperature of the hot plate (Sorel, model DS37) is set to 55 ^° C. The mouse is observed, noting signs of discomfort, such as licking its paws or shaking, attempting to escape (jumping off the plate) or trembling. The reaction time (in s) is detected when one of these signs appears. Each mouse was observed for a maximum of 30 s, so as to prevent damage to the paw tissue. Mice can be observed after various time intervals after administration of the test compound (or medium or standard). The time intervals may be 30, 60 or 120 minutes (or others).

 For each time indicator, the average reaction time of the control group is multiplied by 1.5. The reaction time of each treated mouse is compared with an “average control • 1.5”. If the reaction time is less than the "average control • 1.5", consider that the mouse did not receive analgesia. If the reaction time is higher than the “average control • 1.5", then it is believed that an analgesic effect was exerted on the mouse. The number of mice that experienced analgesia in the group determines the percentage of analgesia with this compound for this indicator. If the percentage of analgesia is less than 30%, the compound is considered inactive.

Example 3. The study of convulsions
Determination of the number of dislocations
For the test, male CD # 1 mice weighing 18 to 22 g are used. Mice are weighed and labeled. Mice were injected intraperitoneally with a 0.02% phenylquinone solution, 0.3 ml / 20 g weight. Count the number of dislocations for a period of 15 minutes after injection. Phenylquinone is administered subcutaneously at intervals of 5, 20, or 60 minutes after administration of the test compound (or medium or standard). Phenylquinone is administered orally with an interval of 60 min after administration of the test compound (or medium or standard).

 A 0.02% phenylquinone solution - (2-phenyl-1,4-benzoquinone (Sigma) - is prepared as follows. 20 mg of phenylquinone is dissolved in 5 ml of 90% ethanol (Sigma, reagent, alcohol). Dissolved phenylquinone, continuously shaking and preheating ( not to boil), slowly add to 95 ml of distilled water.The phenylquinone solution is constantly protected from light and a new solution is prepared every day for testing. Before using the phenylquinone solution, it is recommended to wait 2 hours.

 Testing can be carried out on 5 mice at a time. Each group usually includes 10 mice. If an antagonist is used, such as naloxone (naloxone), it is administered intraperitoneally 20 minutes before the test compound (or medium, or standard).

Claims (19)

1. Opioid peptides of the general formula I

and their derivatives and analogues with a peripheral analgesic effect, where X = hydrogen; Y and Z = hydrogen; R ₁ is a tyrosyl residue, a 2 ′, 6′-dimethyltyrosyl residue or an analogue or derivative thereof; R ₃ is the residue of an aromatic amino acid; R ₄ is the residue of an aromatic amino acid; R ₂ is an amino acid residue having an R - configuration, or is selected from the group consisting of aminoisobutyric acid, cyclopropylalanine, cyclohomoleucine, cycloleucine, provided that when R ₁ is a tyrosyl residue, R ₂ is - alanine, X, Y and Z are hydrogen and R ₃ is phenylalanine, then R _{4 is} not unsubstituted phenylalanine or phenylalanine substituted with 4NO ₂ or 4N ₃ ; when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are hydrogen and R ₄ is phenylalanine, then R _{3 is} not unsubstituted phenylalanine or phenylalanine substituted with 4NO ₂ ; when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are hydrogen and R ₄ is 1'-naphthylalanine, then R _{3 is} not 1'-naphthylalanine or 2'-naphthylalanine; and when R ₁ is a tyrosyl residue, R ₂ is D-alanine, X, Y and Z are H, then R ₃ and R ₄ are not tryptophan; and Q is an amide bond, and are excluded
H-Tyr-D-Phe-Phe-Phe-NH ₂
H-Tyr-D-NMePhe-Phe-Phe-NH ₂
H-Tyr-D-Tic-Phe-Phe-NH ₂
2. The compound of formula I according to claim 1, characterized in that R ₂ is an amino acid residue having an R configuration, provided that when R ₁ is a tyrosyl residue, R ₂ is D-alanine, Y and Z are H, then R ₃ and R _{4 are} different and selected from the group consisting of phenylalanine and tryptophan.  3. The compound according to claim 2 and its pharmaceutical derivatives in order to produce peripheral pain relievers. 4. The compound of formula I according to claim 1, characterized in that Y and Z are H; R ₃ and R ₄ are independently aromatic amino acids, and R ₂ is an amino acid having an R configuration, provided that when R ₁ is a tyrosyl residue, R ₂ is D-alanine, then R ₃ and R _{4 are} different and selected from the group consisting of phenylalanine and tryptophan.  5. The compound according to claim 4 and its pharmaceutical derivatives in order to produce peripheral pain relievers. 6. The compound of formula I according to claim 4, characterized in that R ₂ is an amino acid having an R-configuration, provided that R _{2 is} not D-alanine, and R ₃ and R ₄ are phenylalanine residues. 7. The compound according to claim 4, characterized in that R ₁ is a tyrosyl residue, R _{2 is} selected from the group consisting of D-norvaline, D-serine and D-arginine; R ₃ and R ₄ are phenylalanyl residues; and Q is an amide bond. 8. The compound according to claim 1, characterized in that it is selected from the group consisting of
H-Tyr-Aib-Phe-Phe-NH ₂
H-Tyr-D-Nle-Phe-Phe-NH ₂
H-Tyr-D-Ala-Phe-2'-Nal-NH ₂
H-Tyr-D-Ala-D-Phe-Phe-NH ₂
H-Tyr-D-Ala-Phe (4NO ₂ ) -Phe (4NO ₂ ) -NH ₂
H-Tyr-D-Ala-Phe-Tic-NH ₂
H-Tyr-D-Ala-Phe-1'-Nal-NH ₂
H-Tyr-D-Ala-Trp-Phe-NH ₂
H-Tyr-D-Ala-Phe-Trp-NH ₂
H-Tyr-D-Nle-Phe-Trp-NH ₂
H-Tyr-D-Nle-Phe-2'-Nal-NH ₂
H-Tyr-D-Nle-Trp-Phe-NH ₂
H-Tyr-D-Ala-Trp-2'-Nal-NH ₂
H-Tyr-D-Nle-Trp-2'-Nal-NH ₂
H-Tyr-D-Nle-Trp-Trp-NH ₂
H-Tyr-D-Nva-Phe-Phe-Phe-NH ₂
H-Tyr-D-Ser-Phe-Phe-NH ₂
H-Tyr-D-Val-Phe-Phe-NH ₂
H-Tyr-D-Leu-Phe-Phe-NH ₂
H-Tyr-D-Ile-Phe-Phe-NH ₂
H-Tyr-D-Abu-Phe-Phe-NH ₂
H-Tyr-Chl-Phe-Phe-NH ₂
H-Tyr-Cle-Phe-Phe-NH ₂
H-Tyr-D-Arg-Phe-Phe-NH ₂
H-Tyr-D-Cys-Phe-Phe-NH ₂
H-Tyr-D-Thr-Phe-Phe-NH ₂
H-DMT-D-Ser-Phe-Phe-NH ₂
H-Tyr-D-Ala-Phe-Phg-NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Arg-Phe-Hgh-NH ₂ - salt of bis-trifluoroacetic acid
H-DMT-D-Ala-Phe-Phe-NH ₂ - trifluoroacetic acid
HD-DMT-D-Ala-Phe-Phe-NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Ala-Phe-Hph-NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Ala-Phe-Cys (Bzl) -NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Arg-Phg-Phe-NH ₂ - salt of bis-trifluoroacetic acid
H-Tyr-D-Arg-Hph-Phe-NH ₂ - salt of bis-trifluoroacetic acid
H-Tyr-D-Ala-Phe-Phe (pf) -NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Ala-Phe-D-Phe (pf) -NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Ala-Hph-Phe-NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Met-Phe-Phe-NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Arg-Phe-D-Phe-NH ₂ - salt of bis-trifluoroacetic acid
H-Tyr-D-Ala-Phg-Phe-NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Ala-D-Phg-Phe-NH ₂ - trifluoroacetic acid salt
H-Tyr-D-Arg-Phe-Phe (pf) -NH ₂ - salt of bis-trifluoroacetic acid
H-Tyr-D-Arg-Phe-D-Phe (pf) -NH ₂ - Ditrifluoroacetic Acid Salt
9. The compound according to claim 1, characterized in that said compound is H-Tyr-D-Nva-Phe-Phe-NH ₂ . 10. The compound according to claim 1, characterized in that said compound is H-Tyr-D-Ser-Phe-Phe-NH ₂ . 11. The compound according to claim 1, characterized in that said compound is H-Tyr-D-Arg-Phe-Phe-NH ₂ .  12. The compound of claims 8 to 11 and their pharmaceutical derivatives for the production of peripheral pain relievers.  13. The compound of formula I according to claim 1 for the production of peripheral pain relievers. 14. The compound according to item 13, where the aforementioned compound H-Tyr-D-Ala-Phe-Phe-NH ₂ or its analogues or pharmaceutical derivatives intended for the production of peripheral analgesics, to relieve pain. 15. The compound of claim 14, wherein said analogue is selected from 4-Tyr-D-Ala-Phe-Phe (4-NO ₂ ) NH ₂ and H-Tyr-D-Ala-Phe-Phe (4- N ₃ ) -NH ₂ .  16. A pharmaceutical composition having analgesic activity, comprising an effective amount of at least one compound of formula I according to claim 1, in admixture with a pharmaceutically acceptable carrier.  17. The pharmaceutical composition according to clause 16, comprising an effective amount of at least one compound according to claims 8 to 11 in a mixture with a pharmaceutically acceptable carrier.  18. The pharmaceutical composition according to clause 16, having peripheral analgesic activity, comprising an effective amount of at least one compound according to claim 2 in a mixture with a pharmaceutically acceptable carrier.  19. The pharmaceutical composition according to clause 16, characterized in that, in addition, includes an effective amount of at least one compound according to claim 4 in a mixture with a pharmaceutically acceptable carrier.  20. The pharmaceutical composition according to 17, further comprising at least another therapeutically active agent.  21. The pharmaceutical composition according to claims 18 and 19, further comprising an effective amount of at least one therapeutically active agent.

Images