FR2914646A1 - PEPTIDE ANALOGUES OF MELANOCORTIN RECEPTORS - Google Patents

Abstract

The present application relates to novel cyclic peptide analogues which exhibit good affinity for certain melanocortin receptor subtypes, specifically MC-4 receptors. The invention also relates to pharmaceutical compositions containing said products and their use for the treatment of disease states and diseases in which one or more melanocortin receptors are involved.

Description

Peptide analogs of melanocortin receptors

  The present invention relates to novel cyclic peptides that exhibit good affinity for certain melanocortin receptor subtypes, specifically MC-4 receptors. The invention also relates to pharmaceutical compositions containing said products and their use for the treatment of disease states and diseases in which one or more melanocortin receptors are involved.

  Description of the Background of the Invention Melanocortins are a family of structurally close peptides obtained from the same precursor, pro-opiomelanocortin (POMC). The family of melanocortins includes ACTH (adrenocorticotropic hormone), α-MSH (melanocyte stimulating hormone cc), R-MSH and γ-MSH (Eipper, 1980, 1, 27, Endoc Rev). These peptides bind to melanocortin receptors, seven G-protein-coupled transmembrane receptors, of which there are five subtypes called MC1-R to MC5-R. These five receptor subtypes, which have 35 to 65% sequence homology (Cone et al., Rec., Prog Hormone Res., 1996, 51, 287-318), are expressed in different tissues such as as the brain (MC3-R, MC4-R, MC5-R), the exocrine glands (MC5-R), the adrenal glands (MC2-R) and the skin (MC1-R). The melanocortin system is involved in many physiological functions, including skin pigmentation (MC-1R), inflammation (MC1-R, MC-3R, MC-5R), synthesis and secretion of adrenal glucocorticoids (MC). - 2R), energy homeostasis (MC-3R and MC-4R), feeding behavior (MC-4R), sexual dysfunction (MC-3R, MC-4R) and exocrine secretions (MC-5R). Stimulation of melanocortin receptors activates adenylate cyclase with production of cAMP. The MC-4R receptor, which has a high affinity for α-MSH and 13-MSH, is widely expressed centrally and has multiple effects. The compounds, agonists or antagonists of melanocortin receptors can be used to treat pathological conditions or metabolic, dermatological or nervous system diseases, in which one or more melanocortin receptors are involved: inflammatory states, disorders of energy homeostasis, food intake, weight disorders (such as weight gain, cachexia, anorexia), disorders of sexual activity (such as erectile dysfunction, desire disorders), pain, diabetes , mental disorders associated with anxiety and depression, addictions, skin diseases (such as skin diseases, skin cancers, melanomas, acne). Considerable studies of structure-activity relationships have been carried out on melanocortins, in particular on a-MSH, and have led to the production of potent non-selective peptide agonists of the different receptor subtypes, most of which have a common sequence. of amino acids, His-Phe-Arg-Trp, essential for receptor recognition and activation of adenylate cyclase. An abbreviated analogue of? -MSH, NDP-a-MSH or [N1e4, D-Phe '] -a-MSH, also called Melanotan I (MT I) (Sawyer TK, Sanfilippo PJ, Hruby VJ, Engel MH , Heward CB, Burnett JB, Hadley ME, PNAS USA, 1980, 77, 5754-5758) and the corresponding cyclic analog, Ac-N1e4-c [Asps, D-Phe ', Lys1O] -a-MSH (4- 10) -NH 2 or Melanotan II (Al-Obeidi F, Lauro Castrucci AM, Hadley ME, Hruby VJ, J. Med Chem, 1989, 32, 2555-2561, Al-Obeidi F, Hadley ME, Pettitt BM, Hruby VJ, J. Am Chem Soc., 1989, 111, 3413-3416) have been identified as potent agonists of the different melanocortin receptor subtypes hMC1-R, hMC3-R, hMC4-R and hMC5-R. . Thus, many linear and cyclic peptide analogues for targeting melanocortin receptors have been described in the literature, of which the following is a non-exhaustive list: US 4,457,864; US 5,674,839; US 5,731,408; WO 98/27113; US 5,714,576; WO 99/54351; WO 00/35952; US 6,051,555; WO 01/00224; WO 01/52880; US 6,284,735; WO 01/74844; WO 01/05401; US 2001/0056179; US 2002/0143141; WO 02/18437; US 6,613,874; WO 03/006604; US 6,579,968; WO 03/095474; WO 03/006620; US 2004/0171793; US 2004/0138136; WO 2004/099246; US 6,794,489; WO 2005/000339; US 2005/0250215; US 6,960,646; US 6,951, 916; WO 2005/014617; US 2005/0037951; WO 2006/073772; and US 7,045,591. Structure - activity relationship studies clearly demonstrate the difficulty of obtaining selective analogues of each receptor subtype. There is therefore still a need for selective agonists of each receptor subtype. In addition, increased stability of these analogs is also sought. SUMMARY OF THE INVENTION The main subject of the present invention is novel cyclic peptide analogues having melanocortin receptor agonist / antagonist activity, particularly at type 4 (MC4-R) receptors.

  The present invention relates to a compound of general formula (I): ## STR2 ## wherein: ## STR2 ##

  ~ R4 N / vN RR1 R3-H NH (I) in which:

  Cy represents a carbon chain interrupted by at least one group selected from the group consisting of an amine, an amide, a disulfide, a dithioester, an ethylenic group, an ether, a thioether, and a thioester, no carbon of the carbon chain. being substituted by a functional group at its side chain;

  RI represents a hydrogen, a linear or branched, saturated or unsaturated C1-C5 alkyl radical, or a CO-alkyl group;

  R2 represents a hydrogen, a halogen (F, Cl, Br, I), an alkyl group, an alkoxy group or a nitro group; R3 represents a hydrogen, a hydroxyl, an alkyl group or a nitro group;

  R4 represents a 3-indolyl, 5-hydroxy-3-indolyl or 2-naphthyl group;

  S1 represents a glycinyl or a (3-alanyl or is absent, S2 represents a glycinyl or a 13-alanyl or is absent, and the ring comprises 19 to 25 atoms; HN as well as a pharmaceutically acceptable salt thereof. , Cy is - (CH2) m [CHR5] pL- (CH2) ri [CHR6] q with m and n are, independently of each other, an integer of 0 to 9; p and q are independently 0 or 1; R5 and R6 are, independently of one another, hydrogen or a linear or branched saturated C105 alkyl radical; L is ùY-Z- or ùY '- ( CH2), Z'- where Y is O, S, NH, or CH2, Z is S, CH2, or a group C = B where B is S or O; Y 'and Z' are, independently of one another, other, O, S, or NH; and r is an integer between 0 and 5, m + n + r being less than or equal to 9.

  In a first particular embodiment, p and / or q are 1 and L is ùY-Z-. In this particular embodiment, the compound preferably has at least one of the following characteristics: R 5 represents a linear or branched saturated C 1 -C 5 alkyl radical; and / or - R6 represents hydrogen; and / or - m and n, independently of each other, are an integer of 0 to 5; and / or - S 1 is absent; and / or - S2 represents a glycinyl or a (3-alanyl) In a second particular embodiment, p and q are 0 and L is ùY'- (CH2), -Z'- In this particular embodiment, the compound preferably has at least one of the following characteristics: - m, n and r, independently of each other, are an integer between 1 and 4, and / or - S1 is absent.

  The compound according to the present invention may be selected from the group consisting of Cyclo (Aeh-His-DPhe-Arg-Trp-eAhx), Cyclo (3Mp-His-DPhe-Arg-Trp-Gly-Gly), Cyclo ( NH-CH 2 -CH 2 -SS-CH 2 -CH 2 -CO-His-DPhe-Arg-Trp-Gly), Cyclo (2Mp-His-DPhe-Arg-Trp-Gly-Gly), Cyclo (His-DPhe- Arg-Trp-NH-CH 2 -CH 2 -O-CH 2 -CH 2 -O-CH 2 -CO), and Cyclo (Aeh-His-DPhe-Arg-TrpGly- (3Ala), as well as one of its pharmaceutically The present invention also relates to a pharmaceutical composition comprising, as active ingredient, a compound according to the present invention in association with a pharmaceutically acceptable carrier, the composition may be in a form that can be administered parenterally, orally, or inhaled. intranasal, topical, rectal, vaginal, perlingual or ophthalmic, in particular it may be in the form of an injectable preparation parenterally, subcutaneously, intravenously or intramuscularly. besides another active ingredient. The present invention further relates to a compound according to the invention as a medicament. The present invention finally relates to the use of a compound or a pharmaceutical composition according to the invention for the preparation of a medicament, in particular for the treatment of disorders of female or male sexual activity; the treatment of diabetes, weight disorders such as obesity and anorexia; mental disorders associated with anxiety and depression, or the treatment of pain.

  DETAILED DESCRIPTION OF THE INVENTION The main subject of the present invention is novel cyclic peptide analogues having melanocortin receptor agonist / antagonist activity, particularly at type 4 (MC4-R) receptors. The analogs according to the invention are free of extra-cyclic functional groups except those of the side chain of the four reference amino acids. In particular, the analogs according to the invention are free of amino or carboxy-terminal extra-cyclic ends, modified or otherwise. The cycle length of these analogs was chosen to promote selectivity for the melanocortin type 4 receptor. Thus, the cyclic analogues according to the invention have between 19 and 25 atoms in the ring. The chemical modifications performed on the compounds of the present invention are intended to limit enzymatic degradations, to increase the stability and therefore to prolong the biological activity in the bloodstream and other biological fluids. Furthermore, these modifications are also intended to increase the selectivity of analogs for type 4 receptors. Thus, the compounds according to the present invention have a general formula R 3 -H NH (1) in which: Cy represents a carbon chain interrupted by at least one group selected from the group consisting of an amine, an amide, a disulfide, a dithioester, an ethylenic group, an ether, a thioether, and a thioester, no carbon of the carbon chain being substituted by a functional group; RI represents a hydrogen, a linear or branched, saturated or unsaturated C1-C5 alkyl radical, or a CO-alkyl group; R2 represents a hydrogen, a halogen (F, Cl, Br, I), an alkyl group, an alkoxy group or a nitro group; R3 represents a hydrogen, a hydroxyl, an alkyl group or a nitro group; R4 represents a 3-indolyl, 5-hydroxy-3-indolyl or 2-naphthyl group; S1 represents a glycinyl or a (3-alanyl or is absent, S2 represents a glycinyl or a 13-alanyl or is absent, and the ring comprises from 19 to 25 atoms, and a pharmaceutically acceptable salt thereof. is meant a heteroatom or double bond-containing group.This term does not include an unsubstituted linear or branched saturated alkyl.On amine is understood to be a group -NR- where R may be a hydrogen or a linear saturated C 1 -C 5 alkyl or unsubstituted branched chain or an acetyl group represented by CH 3 -C CO. Amide means a group -CO-NH-. By O = C II SN cy 1 NH NHI II NI II s, HN 7 disulfide is understood a group ûS-S The term "dithioester" is understood to mean a group of CH 2 -CH 2 -. A thioester is understood to mean a group ûSCH 2 -CH 2 -. (C = O) - By alkoxy is understood a group ûO-R o R is a linear or branched unsubstituted C1-C5 alkyl. By nitro is meant ûNO2. An alkyl radical is a saturated or unsaturated hydrocarbon radical, linear or branched, substituted or unsubstituted. C1-C5 alkyl is understood to mean a radical as defined above having 1 to 5 carbon atoms, preferably a group selected from methyl, ethyl, propyl, ispropyl, butyl, isobutyl, sec-butyl, tert-butyl , pentyl, isopentyl, neo-pentyl, isoamyl. In a particular embodiment, an alkyl is a linear or branched C1-C6 alkyl, saturated and unsubstituted. In a preferred embodiment, Cy is - (CH2) m [CHR5] p-L- (CH2) r1 [CHR6] q with m and n are, independently of each other, an integer from 0 to 9; p and q are, independently of one another, 0 or 1; R5 and R6 are, independently of one another, hydrogen or a linear or branched saturated C1-C5 alkyl radical; L is ûY-Z- or ûY '- (CH2) r Z'- where Y is O, S, NH, or CH2; Z represents S, CH2, or a group C = B where B is S or O; Y 'and Z' are, independently of each other, O, S, or NH; and r is an integer between 0 and 5; m + n + r being less than or equal to 9.

  Thus, the compound can have the following formula (II): ## STR2 ## ## STR2 ## ## STR1 ## R5 and R6 being as defined above.

  In a first particular embodiment, p and / or q are 1 and L is ûY-Z-. Thus, Cy represents - (CH2) m [CHR5] p-Y-Z- (CH2) r1 [CHR6] q.

  Thus, the compound may have the following formula (III): ## STR2 ## where [CHR6] q -1 = C NH ù SI ù His ù DPhe ù Arg ùTrp S2 -1 (III) m, p, Y, Z, n, q, R5 and R6 being as defined above. In this particular embodiment, the compound of formula (III) preferably has at least one of the following characteristics: R 5 represents a linear or branched saturated C 1 -C 5 alkyl radical; and / or -R6 represents hydrogen; and / or - m and n, independently of one another, are an integer between 0 and 5, preferably between 0 and 3, even more preferably between 0 and 2; and / or - S 1 is absent; and / or - S2 represents a glycinyl or a (3-alanyl) In a second particular embodiment, p and q are 0 and L is - Y '- (CH2) r Z'- Thus Cy represents - (CH2) For example, the compound may have the following formula (IV): ## STR2 ## ## STR2 ## ## STR2 ## ## STR3 ## where ## STR1 ## where ## STR1 ## where ## STR2 ## where R is as defined above, in this particular embodiment, the compound of formula ) preferably has at least one of the following characteristics: - m, n and r, independently of each other, are an integer between 1 and 4, preferably between 1 and 2, and / or - S1 is absent. Non-limiting compounds according to the invention are the following: Cyclo (Aeh-His-DPhe-Arg-Trp-eAhx), Cyclo (3Mp-His-DPhe-Arg-Trp-Gly-Gly), Cyclo 9 (NH-CH2-CH2-SS-CH2-CH2-CO-His-DPhe-Arg-Trp-Gly), Cyclo (2Mp-His-DPhe-Arg-Trp-Gly-Gly), Cyclo (His-DPhe -Arg-Trp-NH-CHz-CHz-O-CHz-CHz-OCH2-CO), and Cyclo (Aeh-His-DPhe-Arg-TrpGly- (3Ala), as well as one of its pharmaceutically acceptable salts. Aeh represents 3-amino-4-ethylhexanoic acid; $ Ahx represents e-aminohexanoic acid; MP represents mercaptopropionic acid; more precisely, 2-MP represents 2-mercaptopropionic acid and 3-MP represents 3-mercaptopropionic acid. The natural amino acids used are of the L series (with the exception of glycine). When no indication is given in the three-letter code, it is the L-isomer. For example, His represents L-histidine; Arg which represents L-arginine or Trp which represents L-tryptophan. The presence of isomers of the D series is indicated by the use of the letter D in front of the residue concerned. An example is D-Phe, which represents D-phenylalanine. The compounds according to the invention may comprise asymmetric carbons apart from those of the amino acids His, Phe, Arg and Trp. They can therefore exist in the form of enantiomers and diastereoisomers. These enantiomers, diastereoisomers, as well as their mixtures, including the racemic mixtures, form part of the invention. The invention therefore relates to a compound according to the invention having an agonist or antagonist activity of at least one melanocortin receptor. In a preferred embodiment, the compound is an agonist. In an alternative embodiment, it is an antagonist. Preferably, the compound is selective for one or more melanocortin receptor subtypes. In a preferred embodiment, it is selective for the MC4-R receptor. By selective is understood that its agonist / antagonist activity towards the receptor is 5, 10, 25, 50, 75, 100, 250, 500, or 1000 times more effective than for another melanocortin receptor. Its effectiveness can be evaluated by determining, for example, Kd or ED50. The invention also relates to a pharmaceutical composition containing, as active principle, one of the compounds described above or one of its pharmaceutically acceptable salts, such as, for example and without limitation, an acetate, a sulphate or a hydrochloride. The pharmaceutical composition according to the invention may be in a form suitable for administration: parenterally, as for example in the form of injectable preparations subcutaneously, intravenously or intramuscularly; orally, for example in the form of tablets, capsules, capsules, powders, syrups, oral gels, granules, suspensions or oral solutions, either with immediate release or with prolonged or delayed release; topically, particularly transdermally, such as in the form of patch, ointment or gel; intranasally, for example in the form of aerosols and sprays; rectally, such as in the form of suppositories; vaginally, for example in the form of ovules or vaginal tablets; perlinguale; by inhalation, for example in the form of powders, aerosols or sprays; ophthalmic. The pharmaceutically acceptable vehicle may be selected from vehicles conventionally used in each of the modes of administration. The pharmaceutical compositions may contain excipients, diluents, stabilizing agents, solubilizing agents, preservatives, buffers, etc.

  The present invention further relates to a compound according to the invention as a medicament. In one embodiment, the composition may further comprise another active ingredient. The present invention finally relates to the use of a compound or a pharmaceutical composition according to the invention for the preparation of a medicament, in particular for the treatment of disorders of female or male sexual activity; to the treatment of diabetes; the treatment of weight disorders such as obesity and anorexia; the treatment of mental disorders associated with anxiety and depression; or the treatment of pain.

  The present invention relates to a method for treating disorders of female or male sexual activity, diabetes, weight disorders such as obesity and anorexia, mental disorders associated with anxiety and depression, or pain in a subject comprising administering a therapeutically effective dose of a compound or pharmaceutical composition of the invention. By treatment is understood in particular an improvement or disappearance of the symptoms, a slowing of the progression of the disease, a stop of the evolution of the disease or a disappearance of the disease. By effective amount is meant an amount sufficient to have an agonist or antagonist effect on the targeted receptor, or to treat the targeted disease or disorder. Disorders of sexual activity include conditions that prevent or interfere with normal sexual functions. They include erectile dysfunctions and desire disorders. The invention also relates to a method for stimulating sexual activity in a subject comprising administering a pharmaceutically effective dose of a compound or a pharmaceutical composition according to the invention. The invention also relates to a method for decreasing food intake or to increase weight gain in a subject comprising administering a pharmaceutically effective dose of a compound or a pharmaceutical composition according to the invention, in particular a compound exhibiting MC4-R antagonistic activity. By patient or subject is meant a mammal, preferably a human.

  The examples, synthetic strategies, choice of resins for the supported synthesis, procedures and methods of analysis and purification cited in this document are presented to illustrate the procedures used but should in no way be considered as a limit to the scope of the invention.

  Examples The invention is illustrated in a nonlimiting manner by the examples below. Cyclo (Aeh-H is-DPhe-Arg-Trp-sAhx) Y NH NH H = -N, NH N

  NH 3 NH 2 Cyclo (3-Mp-His-DPhe-Arg-Trp-Gly-Gly) O-O NH NH Oy HL ~ ••• NH NH 2 O NH NH 2 NH 2 NH 3 Cyclo (NH-CH 2 -CH 2 -S-CH 2 CH 2 CO-His-DPhe-Arg-Trp-Gly) ONH 0 0,, NH HN YNH NH 2 Cyclo (2-Mp-His-DPhe-Arg-Trp-Gly-Gly ) NH HN YNH NH 2 Cyclo (His-DPhe-Arg-Trp-NH-CH 2 CH 2 O-CH 2 CH 2 O-CH 2 CO) N HNYNH NH 2 Cyclo (Aeh-H is-D Phe-Arg-Trp-Gly- (3Ala) The various synthetic methods used to obtain the products in question are detailed below: The amino acids used for this invention are commercially available The natural amino acids and their derivatives are represented by the three-letter code according to the nomenclature of the invention. Materials and Methods A. Solid Phase Peptide Synthesis The peptide syntheses of the present invention were run stepwise from the C-terminus to the N-terminus according to the Fmoc (fluorenylmethoxycarbonyl) strategy, grouping. temporary protector of amine functions used during peptide elongation. This group is cleavable under basic conditions. The permanent protective groups for protecting the side chains of the amino acids used are acid-labile; Examples are the Trt (trityl) group for histidine, the Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) group for arginine, the Boc (t-butoxycarbonyl) group for tryptophan. The resins used (Novabiochem) were weighed, placed in the reactor in the presence of DMF (N, N-dimethylformamide) and then washed twice with DMF.

  Deprotection was carried out using a solution of 20% piperidine in DMF (1 x 1 min then 2 x 3 min and then 1 x 10 min) and followed by several washes with DMF (4 x 1 min) . The coupling of natural or exotic Fmoc-amino acids (3 eq) was carried out in DMF in the presence of PyBOP (3 eq) (1H-benzotriazol-1-yl-1-oxy) tripyrrolidino phosphonium hexafluorophosphate) and DIEA ( 6eq) (N, N'-DiisopropylN-ethylamine). The coupling times for each residue are between 30 and 60 minutes. After each coupling, the resin is washed successively with DMF (2 × 1 min), with CH 2 Cl 2 (dichloromethane) (2 × 1 min) and with DMF (2 × 1 min). Each end of coupling was checked using the Kaiser test.

  At the end of the synthesis, the resin is washed with DMF (3 x 1 min), with CH 2 Cl 2 (3 x 1 min), with absolute EtOH (2 x 1 min) and finally with Et 2 O (ethyl ether) (4 x 1 min). The deprotection and / or cleavage steps are carried out in the presence of a solution of TFA (trifluoroacetic acid), TIS (triisopropylsilane) and H 2 O in the proportions 95, 2.5, 2.5. The resin is then stirred for 180 minutes at room temperature before being filtered. The filtrate is recovered, the crude peptide is precipitated with Et2O and then centrifuged. The peptide thus recovered is washed several times with ether. The reaction crude is solubilized and then injected into preparative HPLC.

  B. Purifications and Analyzes Homogeneous phase reactions were monitored by reverse-phase analytical high-pressure liquid chromatography (HPLC) (Beckman System Gold) using a SymetrieShield RP 18.5 m column (100 À, 250 x 4.6 mm). The elution is carried out using a linear gradient of acetonitrile at 0.08% TFA (solvent B) and H20 at 0.1% TFA (solvent A). The elution conditions vary from 5 to 95% of B in 15 minutes with a flow rate of 1.5 ml / min. The detection is carried out at 215 nm and 280 nm. Preparative HPLCs (Waters) were made with the same elution system at a flow rate of 20 ml / min on a Chromasil C18 column (5 m, 100 Å, 250 x 20 mm). The fractions of interest are recovered and lyophilized. The mass spectra were carried out according to the MALDI-TOF mode on a Voyager DE Elite mass spectrometer (PerSeptive Biosystems). For each analysis, the sample was mixed volume to volume with a template (2,5-dihydrobenzoic acid) with which it co-crystallizes. At the end of the synthesis, each compound is characterized by its retention time in analytical HPLC and its molecular peak obtained in mass spectrometry.

  C. Choice of Resins Two resins were used for the syntheses: the Fmoc-Gly-NOVASYN-TGT resin (Novabiochem): Fmoc-HN O This resin is represented for all the synthesis schemes by: HO NOVASYN -TGT 25 - the resin HMPB-MBHA (4-hydroxymethyl-3-methoxyphenoxy butyric acid): This resin is represented for all synthesis schemes by: HO-HMPB-MBHA These two resins allow the synthesis of peptides to function C-terminal acid.

  D. Grafting of the First Fmoc-Amino Acid on the HMPB-MBHA Resin The grafting of the first amino residue is carried out by forming an ester linkage between the carboxylic acid of the amino acid of the hydroxyl function carried by the resin. This grafting is carried out in two steps: activation of the amino acid in the form of symmetrical anhydride followed by coupling on the resin of the first amino acid via an ester bond. HMPB-MBHA Fmoc-AA1-OH, DIPCDI, Fmoc-AA, O-HMPB-MBHA O HO-DMAP, CHZCl 2

  5 equivalents of Fmoc-amino acid to be coupled to the resin are solubilized in CH2Cl2 (10 ml / g). 2.5 equivalents of DIPCDI (N, N'-diisopropylcarbodiimide) (previously solubilized in 1 to 2 ml / g of CH2Cl2) are added dropwise.

  The reaction mixture is stirred at room temperature for 30 minutes and then evaporated to dryness. The symmetrical anhydride formed is recovered as a white solid. The latter is solubilized in DMF (10 ml / g) and added to the resin in the presence of 1 equivalent of DMAP (4-dimethylaminopyridine). The reactor is stirred slowly at room temperature for 3 hours. The resin is then filtered, washed successively with DMF (3 x 1 min), CH 2 Cl 2 (3 x 1 min) and then with Et 2 O (3 x 1 min). The resin is placed in the desiccator to establish the graft yield and its rate of substitution.

  E. Lactam Cyclization The cyclization reaction between the N- and C-terminal ends is carried out in solution in the presence of the peptide to be cyclized (n moles), PyBOP (1.2 equivalents), NaHCO 3 (sodium bicarbonate) (5 equivalents) in DMF (10.2 M). The reaction is left stirring magnetically and at room temperature between 6 and 24 hours. The cyclization reaction is monitored by analytical HPLC and mass spectrometry.

  At the end of the reaction, the cyclized peptide is recovered after purification and lyophilization.

  F. Cyclization thioester The cyclization reaction between the S- and C-terminal ends is carried out in solution in the presence of the peptide to be cyclized (n moles), PyBOP (1.6 equivalents), DIEA (4 equivalents) in DMF (10.2 M). The reaction is left stirring magnetically and at room temperature between 6 and 24 hours. The cyclization reaction is monitored by analytical HPLC and mass spectrometry. At the end of the reaction, the cyclized peptide is recovered after purification and lyophilization.

  G. Protection of 3-amino-4-ethylhexanoic acid with the Fmoc o Fmoc-OSu group, NaHCO3 O Fmoc-NH "OH H2N OH H2O / acetone In a reactor, 1 molar equivalent of 3-amino-4-acid -ethylhexanoic acid is dissolved in H 2 O at 100 g / l 0.98 equivalents of Fmoc-OSu (N- (9-fluorenylmethoxycarbonyloxy) succinimide) previously solubilized in acetone (100 g / l) is added dropwise to the reaction mixture then the mixture is stirred at room temperature for 3 hours.Once the reaction is complete, the acetone is evaporated to dryness and the crude reaction product is taken up in ethyl acetate (AcOEt) .The organic phase is washed twice successively. with a solution of KHSO4 (potassium acid sulphate) and then with a saturated solution of NaCl The organic phase is then dried with MgSO4, filtered and concentrated to dryness under reduced pressure to give a solid white, the latter will be used as it is without further purification This synthon is introduced as a classical amino acid during peptide elongation.

  H. Synthesis of the Fmoc-NH-CH2-CH2-SS-CH2-CH2-COOH Synthon Fmoc-NH2H2N NH2.HCl Fmoc-HN TCEP, DMF NH-Fmoc Fmoc-NH Fmoc.OSu, Na2CO3 H20 / Acetone PySSPy, T = In a reactor, 1 molar equivalent of cystamine dihydrochloride is dissolved in water at 75 g / l in the presence of 1.96 molar equivalents of Na.sub.2 CO.sub.3 (sodium carbonate). 1.96 equivalents of Fmoc-OSu (N- (9-fluorenylmethoxycarbonyloxy) succinimide) previously solubilized in acetone (75 g / l) are introduced dropwise into the reaction mixture and the mixture is then stirred at room temperature for 3 hours. Once the reaction is complete, the acetone is evaporated to dryness and the reaction product is extracted with CH 2 Cl 2. The organic phases are combined and washed with a saturated solution of NaCl. The organic phase is then dried with MgSO4, filtered and concentrated to dryness under reduced pressure to give a white solid which is purified by preparative HPLC. The product of the reaction is obtained: Fmoc-NH-CH 2 -CH 2 -S-SCH 2 -CH 2 -NH-Fmoc: purity> 95%; yield = 82%; calculated mass: 596 - MALDI MS m / z [DHB] 597.7 [M + H +]; 620.0 [M + Na +]; 636.0 [M + K +]. In a reactor, 1 molar equivalent of Fmoc-NH-CH2-CH2-S-S-CH2-CH2-NH-Fmoc is dissolved in DMF (200 g / l). 2. 5 molar equivalents of TCEP (tris (2-carboxyethyl) phosphine) are added to the reaction mixture. After 1 hour of reaction, the product is purified by preparative HPLC. The product of the reaction is obtained: Fmoc-NH-CH 2 -CH 2 -SH: purity> 95%; yield = 72%; calculated mass: 299 MALDI MS m / z [DHB] 322.5 [M + Na +]; 338.5 [M + K +]. In a reactor, 1 molar equivalent of Fmoc-NH-CH2-CH2-SH is dissolved in DMF at 145 g / l and placed in an ice-water bath (0 C). 3 equivalents of PySSPy (dithiodipyridine) solubilized in DMF (0.2 g / ml) and placed in an ice-cold water bath at 0 ° C. are introduced dropwise into the reaction mixture. The reaction mixture is stirred for 3 hours, gradually melting the ice bath and the mixture is stirred at room temperature for one hour. The reaction mixture is then placed again at 0 ° C., then 4 molar equivalents of 3-mercaptopropionic acid (3-MP), previously placed at 0 ° C., are added dropwise. The reaction mixture is stirred for 24 hours, gradually allowing the bath to melt. of ice. The reaction crude is then purified by preparative HPLC. The product of the reaction is obtained: Fmoc-NH-CH 2 -CH 2 -S-S-CH 2 -CH 2 -COOH: purity> 95%; yield = 62%; calculated mass: 402 MALDI MS m / z [DHB] 426.8 [M + Na +]; 442.8 [M + K +].

  EXAMPLE 1 Synthesis of Cyclo (Aeh-His-DPhe Arg-Trp-eAhx) The resin used was a HMPB-MBHA resin (140 mg, 0.155 mmol). The synthetic scheme is shown below: HMPB-MBHA Fmoc-EAhx-OH, DIPCDI, DMAP, CH, Cl, Fmoc-NH HMPB-MBHA HO-SPPS H-Aeh-H is (Trt) -DPhe-Arg (Pbf) -Trp (Boc) -NH- (CH2) n (HMPB-MBHA O / O TFA / TIS / H2O (95 / 2.5 / 2.5) H-Aeh-His (Trt) -DPhe-Arg (Pbf) - Trp (Boc) -NH- (CH 2) COOH PyBOP, NaHCO 3 DMF The grafting of the first residue was carried out according to the procedure described in paragraph D using Fmoc-eAhx-OH (200 mg, 0. 575 mmol), DIPCDI ( 45 L, 0.28 mmol) and DMAP (14 mg, 0.115 mmol) The peptide elongation stepwise was then carried out in DMF for 40 minutes in the presence of PyBOP (242 mg, 0.645 mmol), DIEA (168 l). 0.93 mmol) and: Amino acid Mass No. of moles No. of equivalents Fmoc-Trp (Boc) -OH coupling 245 mg 0.465 mmol 3 1 Fmoc-Arg (Pbf) -OH 302 mg 0.465 mmol 3 1 Fmoc-DPhe- OH 180 mg 0.465 mmol 3 1 Fmoc-His (Trt) -OH 288 mg 0.465 mmol 3 1 Fmoc-Aeh-OH 177 mg 0.465 mmol 3 1 At the end of synthesis and after deprotection of the N-terminal function, the resin was the According to the protocol described in section A, 300 mg of peptidyl resin were placed in the presence of 4 ml of TFA, 105 l of TIS and 105 l of H 2 O at room temperature for 180 minutes. The resin was then filtered and washed with 2 ml of TFA. The crude peptide was then precipitated and washed with two volumes of Et2O. The crude peptide was purified by preparative HPLC before being cyclized. 65 mg (0.0525 mmol) of linear peptide were solubilized in 5.2 ml of DMF in the presence of PyBOP (33 mg, 0.063 mmol) and NaHCO3 (22 mg, 0.26 mmol) at room temperature. Cyclization was monitored by analytical HPLC. The cyclized peptide was then purified by preparative HPLC using a 0 to 60% linear gradient of Solvent B over 60 minutes. Fractions of interest were recovered and lyophilized. 29.5 mg of cyclo (Aeh-His-DPhe-Arg-Trp-eAhx) were obtained, ie a yield of 51%: HPLC: tr = 7.85 min; : purity> 95%; calculated mass: 880 - MALDI MS m / z [DHB] 881.4 [M + H +].

  EXAMPLE 2 Synthesis of Cyclo (3Mp-His-DPhe Arg-Trp-Gly-Gly) ## STR1 ## The resin used was the Fmoc-Gly-NOVASYN-TGT resin (500 mg, 0.12 mmol). ). The synthesis scheme is shown below: Fmoc-Gly-O NOVASYN -TGT SPPS

  Trt-S-CH 2 CH 2 -CO-His (Trt) -DPhe-Arg (Pbf) -Trp (Boc) -Gly-GIy-O NOVASYN -TGT TFA / TIS / H2O HS-CH2-CH2-CO-H is DPhe-Arg-Trp-G ly-GIy-OH CYCLISATION HN NH NH2 Peptide elongation stepwise was carried out in DMF for 40 minutes in the presence of PyBOP (188 mg, 0.36 mmol), DIEA (130 l, 0.72 g). mmol) and of: HN \ LN o ~ Amino acid Mass No. of moles No. eq. Coupling Fmoc-Gly-OH 107 mg 0.36 mmol 3 1 Fmoc-Trp (Boc) -OH 190 mg 0.36 mmol 3 1 Fmoc-Arg (Pbf) -OH 234 mg 0.36 mmol 3 1 Fmoc-DPhe-OH 140 mg 0.36 mmol 3 1 Fmoc-His (Trt) -OH 223 mg 0.36 mmol 3 1 Trt-S- (CH 2) 2 -COOH 125 mg 0.36 mmol 3 1 At the end of the synthesis, the resin was washed according to the protocol described in paragraph A. 600 mg of peptidyl resin were placed in the presence of 6 ml of TFA, 158 l of TIS and 158 l of H2O at room temperature for 180 minutes. The resin was then filtered and washed with 4 ml of TFA. The crude peptide was then precipitated and washed with two volumes of Et20. The crude peptide was purified by preparative HPLC before being cyclized. 70 mg (0.065 mmol) of linear peptide were solubilized in 6.5 ml of DMF in the presence of PyBOP (52 mg, 0.1 mmol) and DIEA (47 l, 0.26 mmol) at room temperature. Cyclization was monitored by analytical HPLC. The cyclized peptide was then purified by preparative HPLC using a 0 to 60% linear gradient of Solvent B over 60 minutes. Fractions of interest were recovered and lyophilized. 40 mg of cyclo (3Mp-His-DPhe-Arg-Trp-Gly-Gly) were obtained, ie a yield of 58%: HPLC: tr = 6.66 min; : purity> 95%; calculated mass: 828 MALDI MS m / z [DHB] 829.3 [M + H +]. EXAMPLE 3 Synthesis of Cyclo (NH-CH 2 -CH 2 -SH-CH 2 -CH 2 -CO-His-DPhe-Arg-Trp-Gly) ## STR2 ## was Fmoc-Gly-NOVASYN-TGT resin (500 mg, 0.12 mmol). The synthetic scheme is shown below: Fmoc-Gly-O NOVASYN -TGT sPPs NOVASYN -TGT NH HNNH NH2 Peptide elongation stepwise was then carried out in DMF for 40 minutes in the presence of PyBOP (188 mg , 0.36 mmol), DIEA (125 l, 0.72 mmol) and: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Amino acid Mass Number of moles Number of eq. Coupling Fmoc-Trp (Boc) -OH 190 mg 0.36 mmol 3 1 Fmoc-Arg (Pbf) -OH 234 mg 0.36 mmol 3 1 Fmoc-DPhe-OH 140 mg 0.36 mmol 3 1 Fmoc-His (Trt) -OH 223 mg 0.36 mmol 3 Fmoc-NH-CH 2 -CH 2 -SH-CH 2 -CH 2 -145 mg 0.36 mmol 3 1 COOH At the end of the synthesis, the resin was washed according to the protocol described in paragraph 10 A. 575 mg of peptidyl resin were placed in the presence of 6 ml of TFA, 158 tl of TIS and 158 l of H2O at room temperature for 180 minutes. The resin was then filtered and washed with 4 ml of TFA. The crude peptide was then precipitated and washed with Fmoc-NH-CH2 CH2-SS-CHZCH2-CO-His (Trt) -DPhe-Arg (Pbf) -Trp (Boc) -GIy-O-TFA / TIS / H2O H2N- CH 2 -CH 2 S-CH 2 -CH 2 -CO-His-DPhe-Arg-Trp-GIy-OH 1 CYCLING SS NH HL NH NH 2 HnO N 2 volumes of Et 2 O. The crude peptide was purified by preparative HPLC before being cyclized. 50 mg (0.042 mmol) of linear peptide were solubilized in 4 ml of DMF in the presence of PyBOP (26 mg, 0.05 mmol) and NaHCO3 (18 mg, 0.21 mmol) at room temperature. Cyclization was monitored by analytical HPLC. The cyclized peptide was then purified by preparative HPLC using a 0 to 60% linear gradient of Solvent B over 60 minutes. Fractions of interest were recovered and lyophilized. 15 mg of cyclo (NH-CH 2 -CH 2 -S-S-CH 2 -CH 2 -CO-His-DPhe-Arg-Trp-Gly) were obtained, ie a yield of 33%: HPLC: tr = 7.3 min; : purity> 95%; calculated mass: 846 - MALDI MS m / z [DHB] 847.5 [M + H +]. Pharmacological study The compounds of the present invention are the subject of pharmacological tests for determining the agonist activity of melanocortin receptors, and more particularly the agonist effect on the hMC-4 receptor.

  Study of the affinity of the compounds for the MC4 receptors of the melanocortins The affinity of the compounds of the invention is determined after measurement of the displacement by increasing doses of each of the products, the binding of [125 I] -20 [N1e4, D-Phe '- a-MSH to HEK-293 cells (Human Embryonic Kidney) stably expressing the human MC4 receptor. These cells are cultured in F12-DME medium containing 7.5% fetal calf serum, 0.5 mM glutamine, 100 U / mL penicillin and 100 g / mL streptomycin. The cells are placed the day before the binding measurement in 12-well dishes coated with poly-D-lysine. The measurement of the competitive inhibition of [? 25I] - [N1e4, D-Phe '] - a-MSH binding on human MC4 receptors is carried out in triplicates. The [N1e4, D-Phe '] - α-MSH used was 125 I-labeled on lysine at position 11 (Amersham, Bolton and Hunter labeling, 2000 Ci / mmol). Two hours before binding, the cell medium is replaced with serum-free medium. The binding medium is F12-DME medium with 0.5% BSA (bovine serum albumin) and 0.1% bacitracin. The concentration of ['25I] - [N1e4, D-Phe'] - a-MSH is fixed and corresponds to a final value of 3.10-10 M in the well. The concentrations of each unlabeled compound ranged from 10-10 M to 10-6 M. The cell binding was for 90 minutes at room temperature. She is stopped by placing the cells on ice. The free [125I] - [N1e4, D-Phe '] - α-MSH, therefore not bound to the cells, is aspirated then the cells are washed once with NaCl 0.9% containing 1% BSA and then 3 times with NaCl 0.9%. The cells are then solubilized in 0.4% sodium deoxycholate containing 0.5M NaOH and then counted on the counter. The specific binding is obtained by subtracting the total binding (cell-bound cpm radioactivity counted for each well) of the radioactivity obtained for the cells placed in the presence of 10 -6 M non-radioactive compound (non-specific binding). The data are analyzed and the values of the dissociation constants (Kd) are determined. The MC4 receptor agonist or antagonist activity of the compounds of the present invention was determined by measuring cAMP production by HEK-293 cells stably transfected with the human MC4 receptor.

  Measurement of intracellular claval AMP production via MC4 receptors HEK-293 cells expressing human MC4 receptors of melanocortins are cultured in 12-well plates coated with poly-D-lysine at 200,000 cells per well. The cells are placed just before the assay in a simple F12-DME culture medium. To measure the agonist effect of a compound, the cells are incubated for 5 minutes at 37 ° C. in the presence of 1 mM of IBMX (isobutylxanthine), and stimulation of cyclic AMP production is obtained by incubating the cells. in the presence of a given compound at concentrations ranging from 10-10 M to 10-6 M in triplicates for 20 minutes at 37 C. The reaction medium is then removed and replaced with 60% alcohol. The lysed cells are then recovered in 1.5 mL tubes. These are placed overnight at -20 C before being centrifuged for 10 minutes. The supernatant is recovered and evaporated and then taken up in a 50 mM sodium acetate buffer at pH 5.6. The level of intracellular cyclic AMP is measured by a competition test with 125 I-labeled cyclic AMP in the presence of a specific antibody (radioimmunoassay) (Blondet et al., J. Biochem. 2004, 135: 541-546).

  Results The affinity results for the human MC4 receptor are shown in the table below: Compounds Kd (nM) ED50 (nM) Melanotan II 8.45 12 Cyclo (Aeh-His-DPhe-Arg-Trp-EAhx) 417 46 Cyclo ( 3Mp-His-DPhe-Arg-Trp-Gly-Gly) 500 NDP-αμMSH ND 2.9 ND = not determined

  The agonist activity of the compounds mentioned above was measured as described in the previous chapter: the compounds according to the invention behave as melanocortin type 4 receptor agonists.

Claims (16)

claims   1. R3-H NH (1) wherein: Cy represents a carbon chain interrupted by at least one group selected from the group consisting of an amine, an amide, a disulfide, a dithioester, an ethylenic group, an ether, a thioether , and a thioester, no carbon in the carbon chain being substituted by a functional group; RI represents a hydrogen, a linear or branched, saturated or unsaturated C1-C5 alkyl radical, or a CO-alkyl group; R2 represents a hydrogen, a halogen (F, Cl, Br, I), an alkyl group, an alkoxy group or a nitro group; R3 represents a hydrogen, a hydroxyl, an alkyl group or a nitro group; R4 represents a 3-indolyl, 5-hydroxy-3-indolyl or   2-naphthyl; S1 represents a glycinyl or a (3-alanyl or is absent, S2 represents a glycinyl or a 13-alanyl or is absent, and the ring comprises 19 to 25 atoms, and a pharmaceutically acceptable salt thereof. according to claim 1, wherein Cy is - (CH2) m [CHR5] pL- (CH2) r1 [CHR6] q with 1 0 = C II SN 1- Compound of the general formula (I): cy HN 26 1 NHm and n are, independently of one another, an integer from 0 to 9; p and q are, independently of one another, 0 or 1; R5 and R6 are, independently of one another, other, a hydrogen or a linear or branched, saturated C 1 -C 5 alkyl radical, L is Y-Z- or Y '- (CH 2) r Z' - with Y is O, S, NH, or CH 2, Z is S, CH 2 , or a group C = B where B is S or O; Y 'and Z' are, independently of each other, O, S, or NH; and r is an integer between 0 and 5 m + n + r being less than or equal to 9.   3. The compound of claim 2 wherein p and / or q are 1 and L is Y-Z-.   4. A compound according to claim 2 wherein p and q are 0 and L is Y '- (CH 2) r Z' -.   5- compound according to claim 2 or 3, characterized in that it has at least one of the following characteristics - R5 represents a linear or branched saturated C1-C5 alkyl radical; and / or - R6 represents hydrogen; and / or - m and n, independently of each other, are an integer of 0 to 5; and / or - S 1 is absent; and / or - S2 represents a glycinyl or a (3-alanyl.   6. Compound according to claim 2 or 4, characterized in that it has at least one of the following characteristics - m, n and r, independently of each other, are an integer between 1 and 4; and / or - S1 is absent.   7. A compound according to claim 1 or 2 selected from the group consisting of Cyclo (Aeh-His-DPhe-Arg-Trp-eAhx), Cyclo (3Mp-His-DPhe-Arg-Trp-Gly-Gly), Cyclo (NH-CH 2 -CH 2 -SS-CH 2 -CH 2 -CO-His-DPhe-Arg-Trp-Gly), Cyclo (2Mp-His-DPhe-Arg-Trp-Gly-Gly), Cyclo (His-DPhe -Arg-Trp-NH-CH2-CH2-O-CH2-CH2-O-CH2-CO), and Cyc10 (Aeh-His-DPhe-Arg-TrpG1y- (3Ala), as well as one of its salts pharmaceutically acceptable.   8. A pharmaceutical composition comprising, as active principle, a compound according to any one of claims 1 to 7, in association with a pharmaceutically acceptable carrier. 10   9- Pharmaceutical composition according to claim 8, characterized in that it is in a form administrable parenterally, orally, inhaled, intranasal, topical, rectal, vaginal, perlingual or ophthalmic.   10- Pharmaceutical composition according to claim 8 or 9, characterized in that it is in the form of injectable preparation parenterally, subcutaneously, intravenously or intramuscularly.   11. Pharmaceutical composition according to any one of claims 8 to 10, further comprising another active ingredient.   12. A compound according to any one of claims 1 to 7 as a medicament.   13- Use of a compound according to any one of claims 1 to 7 or of a pharmaceutical composition according to any one of claims 8 to 11 for the preparation of a medicament for the treatment of disorders of the activity sexual feminine or masculine.   14- Use of a compound according to any one of claims 1 to 7 or 30 of a pharmaceutical composition according to any one of claims 8 to 11 for the preparation of a medicament for the treatment of diabetes, disorders of weight such as obesity and anorexia. 20 5   15- Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 8 to 11 for the preparation of a medicament for the treatment of mental disorders associated with the anxiety and depression.   16- Use of a compound according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 8 to 1 1 for the preparation of a medicament for the treatment of pain.