WO2008142319A2

WO2008142319A2 - Peptide analogues of melanocortin receptors

Info

Publication number: WO2008142319A2
Application number: PCT/FR2008/050588
Authority: WO
Inventors: Christel Gros; Roger Lahana
Original assignee: Neorphys
Priority date: 2007-04-04
Filing date: 2008-04-03
Publication date: 2008-11-27
Also published as: WO2008142319A3; FR2914646A1

Abstract

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

Description

Peptide analogs of melanocortin receptors

Field of the invention

The present application relates to novel cyclic peptides 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 of the context of the invention

Melanocortins are a family of structurally close peptides, obtained from the same precursor, pro-opiomelanocortin (POMC). The melanocortin family includes ACTH (adrenocorticotropic hormone), α-MSH (α-melanocyte stimulating hormone), β-MSH and γ-MSH (Eipper, 1980, 1, 27, Endoc Rev). These peptides bind to the 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 35-65% sequence homology (Cone et al, Recog Hormone Res., 1996, 51, 287-318), are expressed in different tissues such as brain (MC3-R, MC4-R, MC5-R), exocrine (MC5-R), adrenal (MC2-R) and skin (MC1-R) glands.

The melanocortin system is involved in many physiological functions, including skin pigmentation (MC-IR), 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 strong affinity for α-MSH and β-MSH, is widely expressed centrally and has multiple effects.

The compounds, agonists or antagonists of the melanocortin receptors may be used to treat disease states or metabolic diseases, Dermatological or nervous system, in which one or more receptors of melanocortins are involved: inflammatory conditions, 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, substance abuse, skin diseases ( such as dermatoses, skin cancers, melanomas, acne).

Considerable studies of structure - activity relationships have been carried out on melanocortins, in particular on α - 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. A shortened analogue of α-MSH, NDP-α-MSH or [Nie ⁴ , D-Phe ⁷ ] -α-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-Nle ⁴ -c [Asp ⁵ , D-Phe ⁷ , Lys ^lo ] -α- MSH (4-1O) -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 various 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. It exists therefore always 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 exhibiting agonist / antagonist activity of melanocortin receptors, particularly at type 4 (MC4-R) receptors.

The present invention relates to a compound of general formula (II):

(il) in which:

R ¹ represents a hydrogen, a linear or branched, saturated or unsaturated C 1 -C ₅ alkyl radical, or a CO-alkyl group;

R ² represents a hydrogen, a halogen (F, Cl, Br, I), an alkyl group, an alkoxy group or a nitro group; R ³ represents a hydrogen, a hydroxyl, an alkyl group or a nitro group;

R ⁴ represents a 3-indolyl, 5-hydroxy-3-indolyl or 2-naphthyl group; m and n are, independently of each other, an integer of 0 to 9; p and q are, independently of one another, 0 or 1; R ⁵ and R ⁶ are, independently of one another, a hydrogen or an alkyl radical selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 1-ethylpropyl and neopentyl;

L is -YZ- or -V- (CH ₂ VZ'- with Y and Z together form -CH = CH- or -C≡C-; or Y is O, S, NH or CH ₂ and Z is S, CH ₂ or C = B where B is S or O with the proviso that Y and Z are not both CH ₂ ;

Y 'and Z' are, independently of each other, O, S or NR ⁷ , with R ⁷ being hydrogen, unsubstituted linear or branched saturated C 1 -C ₅ alkyl or an acetyl group represented by CH _3- CO, preferably a saturated, unsubstituted, branched C ₁ -C ₂ or C ₃ -C ₅ alkyl or an acetyl CH ₃ -CO group; and r is an integer between 0 and 5, m + n + r being less than or equal to 9; Si represents a glycinyl or a β-alanyl or is absent; S ₂ represents a glycinyl or a β-alanyl or is absent; and the ring comprises 19 to 25 atoms; the phenylalanyl residue or the derivative thereof is of D configuration; as well as one of its pharmaceutically acceptable salts.

In a first particular embodiment, p and / or q are 1 and L is -Y-Z-.

In this particular embodiment, the compound preferably has at least one of the following characteristics: a) R ⁵ is an alkyl radical selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, sec-butyl, tert-butyl isopentyl, 1-ethylpropyl and neo-pentyl; and / or b) R ⁶ is hydrogen; and / or c) If is absent; and / or d) S ₂ is absent or represents a glycinyl or a β-alanyl, preferably a glycinyl; and / or e) p is 1; and / or f) q is 1; and / or g) m is 0 or 1; and / or h) n is an integer from 0 to 5; and / or i) if m + p is 2 and n + q is 1, then Si or S ₂ is present; and / or j) Y is NH or S; and / or k) Z is CO or S; and / or 1) R ⁵ is hydrogen; and / or m) m + p is 2; and or n) the ring comprises 20 to 25 atoms.

In a second particular embodiment, p and q are 0 and L is -Y'- (CH ₂ VZ'- 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

- If is absent, and / or

- S ₂ is absent.

The compound according to the present invention may be selected from the group consisting of Cyclo (Aeh-His-DPhe-Arg-Trp-εAhx), Cyclo (Aeh-His-DPhe-Arg-Trp-Ahp), Cyclo (Aeh-His-DPhe-Arg-Trp- His-DPhe-Arg-Trp-βAla), Cyclo (3Mp-His-DPhe-Arg-Trp-Gly-GIy), Cyclo (3MP-Gly-His-DPhe-Arg-Trp-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-Trp-Gly-βAla), as well as one of its pharmaceutically acceptable salts.

The present invention also relates to a pharmaceutical composition comprising, as active principle, a compound according to the present invention in combination with a pharmaceutically acceptable carrier. The composition may be in a parenteral, oral, inhaled, intranasal, topical, rectal, vaginal, perlingual or ophthalmic form. In particular, it may be in the form of an injectable preparation parenterally, subcutaneously, intravenously or intramuscularly. The composition may further comprise another active ingredient.

The present invention further relates to a compound according to the invention as a medicament. In particular, the present invention relates to a compound according to the invention for the treatment of disorders of male or female sexual activity, diabetes, weight disorders such as obesity and anorexia, mental disorders associated with the anxiety and depression and / or pain 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.

Brief description of the figures

Figure 1: Graph showing the chemical stability of the products tested at a concentration of 1 mg / ml for 30 days at 60 ⁰ C and pH 5.

Figure 2: Chart showing the chemical stability of the products tested at a concentration of 1 mg / ml for 30 days at 60 ⁰ C and pH 7.

Figure 3: Graph showing the chemical stability of the products tested at a concentration of 1 mg / ml for 30 days at 60 ⁰ C and pH 9.2.

Detailed description of the invention

The main subject of the present invention is novel cyclic peptide analogues exhibiting agonist / antagonist activity of melanocortin receptors, particularly at type 4 (MC4-R) receptors. The analogs according to the invention are free of extracyclic 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. In addition, 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

(I):

(I) 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 linkage, an ether, a thioether and a thioester, no carbon the carbon chain being substituted with a functional group;

R ² represents a hydrogen, a halogen (F, Cl, Br, I), an alkyl group, an alkoxy group or a nitro group;

R ³ represents a hydrogen, a hydroxyl, an alkyl group or a nitro group; R ⁴ represents a 3-indolyl, 5-hydroxy-3-indolyl or 2-naphthyl group;

₁ represents a glycinyl or a β-alanyl or is absent;

₂ represents glycinyl or β-alanyl or is absent; and the ring comprises 19 to 25 atoms; as well as one of its pharmaceutically acceptable salts. The phenylalanyl residue or a derivative thereof present in all the compounds of the present invention is still of configuration D.

By "functional group" is meant a group containing a heteroatom or a multiple bond, especially double or triple bond. This term does not include an unsubstituted linear or branched saturated alkyl. By "amine" is meant a group -NR- where R can be hydrogen or unsubstituted linear or branched saturated C 1 -C ₅ alkyl or an acetyl group represented by CH ₃ -CO. By "amide" is meant a -CO-NH- group. By "disulfide" is meant a group -SS-. By "dithioester" is meant -S- (C = S) -. By "ethylenic" is meant -CH = CH- or -C≡C-, preferably -CH = CH-. By "ether" is understood -O-. By "thioether" is meant a group -S-. By "thioester" is meant a group -S- (C = O) -. By "alkoxy" is meant a group -O-R where R is a saturated linear or branched unsubstituted C1-C5 alkyl. By "nitro" is meant - NO ₂ . Preferably, R is saturated, unsubstituted, C ₁ -C ₂ or branched C ₃ -C ₅ alkyl.

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, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl , pentyl, isopentyl, neo-pentyl and 1-ethylpropyl. In a particular embodiment, an alkyl is a Ci -C ₅ linear or branched, saturated and unsubstituted. In a more particular embodiment, an alkyl is a saturated and unsubstituted C ₁ -C ₂ or branched C ₃ -C ₅ alkyl, that is to say selected from the following list a methyl, an ethyl or an isopropyl isobutyl, sec-butyl, tert-butyl, isopentyl, 1-ethylpropyl and neo-pentyl.

In a preferred embodiment, Cy is - (CH ₂ ) _m - [CHR ⁵ ] _p -L- (CH ₂ ) _n - [CHR ⁶ ] _q - with m and n are, independently of one another an integer between 0 and 9; p and q are, independently of one another, 0 or 1;

R ⁵ and R ⁶ are, independently of one another, a hydrogen or a saturated linear or branched C ₁ -C ₅ alkyl radical, preferably an unsubstituted, saturated, C ₁ , C ₂ alkyl radical or Branched C3-C5;

L is -YZ- or -V- (CH ₂ VZ'- with

Y represents O, S, NH or CH ₂ , preferably O, S, NH, or CH ₂ ; Z represents S, CH ₂ or a group C = B where B is S or O, preferably S, CH ₂ , or a group C = B; or Y and Z together form -CH = CH- or -C≡C-;

Y 'and Z' are, independently of one another, O, S or NR ⁷ , with R ⁷ being hydrogen, unsubstituted linear or branched saturated C ₁ -C ₅ alkyl or acetyl group represented by CH ₃ -CO, preferably a saturated, unsubstituted, branched C ₁ -C ₂ or C ₃ -C ₅ alkyl or an acetyl group CH ₃ -CO, preferably O, S, or NH; and r is an integer between 0 and 5; m + n + r being less than or equal to 9.

Preferably, R ⁵ and R ⁶ are, independently of one another, a hydrogen or an alkyl radical selected from the group consisting of a methyl, ethyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 1-ethylpropyl and neopentyl. In a preferred embodiment, R ⁶ is hydrogen and R ⁵ is hydrogen or an alkyl radical selected from the group consisting of methyl, ethyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, and the like. -ethylpropyl and neo-pentyl. In a particular embodiment, R ⁶ is hydrogen and R ⁵ is hydrogen or an alkyl radical selected from the group consisting of methyl and 1-ethylpropyl. When L is -YZ-, Y and Z together form -CH = CH- or -C≡C- or Y represents O, S, NH or C; and Z is S, CH ₂ or a group C = B where B is S or O; provided that Y and Z are not both CH ₂ . For example, L can be selected from the group consisting of -NH-CO-, -SS-, -S-CO-, -S-CS-, -CH = CH- and -C≡C-, preferably from NH-CO-, -SS- and -S-CO-. Thus, the compound may have the following formula (II):

(II) m, p, L, n, q, R ⁵ and R ⁶ being as defined above. The phenylalanyl residue or a derivative thereof present in all the compounds of formula (II) is always of configuration D. In a particular embodiment, the ring comprises 20 to 25 atoms or 21 to 24 atoms. In a first particular embodiment, p and / or q are 1 and L is -YZ-. Thus, Cy represents - (CH ₂ ) _m - [CHR ⁵ ] _p -YZ- (CH ₂ ) _n - [CHR ⁶ ] _q -.

In a preferred embodiment, the compound of formula (II) preferably has at least one of the following characteristics: a) R ⁵ is an alkyl radical selected from the group consisting of a methyl, ethyl, isopropyl, isobutyl, sec-butyl tert-butyl, isopentyl, 1-ethylpropyl and neo-pentyl; and / or b) R ⁶ is hydrogen; and / or c) If is absent; and / or d) S ₂ is absent or represents a glycinyl or a β-alanyl, preferably a glycinyl; and / or e) p is 1; and / or f) q is 1; and / or g) m is 0 or 1; and / or h) n is an integer from 0 to 5; and / or i) if m + p is 2 and n + q is 1, then Si or S ₂ is present; and / or j) Y is NH or S; and / or k) Z is CO or S; and or

1) R ⁵ is hydrogen; and / or m) m + p is 2; and / or n) the ring comprises 20 to 25 atoms.

In particular, the following combinations are preferred:

- e) and g); or

- e), f) and g); or - e), f), g) and h); or

- e), f), g), h), and i); or

- e), f), g), h), and m); or

e), f), g), h), i) and m); or - b), d), e), f), g), h), j) k) and n); or b), e), f), g), h), j), k), 1), m), n) and optionally i); or

- j) and k); or

- a), e) and g); or

- a), e), g), j) and k); or - e), g) and i); or

- a), b), c), d), e), f), g), h), j), k), 1) and n); or - b), h) i), j), k), m) and n).

Thus, the compound may have the following formula (III):

("I ⁾ m, p, Y, Z, n, q, R ⁵ and R ⁶ 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, preferably a branched C ₁ -C ₂ alkyl or C 3 -C ₅ alkyl radical; and or

- R ⁶ represents a 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

- If is absent; and / or - S ₂ represents a glycinyl or a β-alanyl.

Preferably, the compound of formula (III) preferably has at least one of the following characteristics: a) R ⁵ is an alkyl radical selected from the group consisting of a methyl, ethyl, isopropyl, isobutyl, sec-butyl, te / t butyl, isopentyl, 1-ethylpropyl and neo-pentyl; and / or b) R ⁶ is hydrogen; and / or c) If is absent; and / or d) S ₂ is absent or represents a glycinyl or a β-alanyl, preferably a glycinyl; and / or e) p is 1; and / or f) q is 1; and / or g) m is 0 or 1; and / or h) n is an integer from 0 to 5; and / or i) if m + p is 2 and n + q is 1, then Si or S ₂ is present; and / or j) Y is NH or S; and or k) Z is CO or S; and / or 1) R ⁵ is hydrogen; and / or m) m + p is 2; and / or n) the ring comprises 20 to 25 atoms. In particular, the following combinations are preferred:

- e) and g); or

- e), f) and g); or

- e), f), g) and h); or

- e), f), g), h), and i); or - e), f), g), h), and m); or

e), f), g), h), i) and m); or - b), d), e), f), g), h), j) k) and n); or

b), e), f), g), h), j), k), 1), m), n) and optionally i); or

- j) and k); or - (a), (e) and (g); or

- a), e), g), j) and k); or

- e), g) and i); or

- a), b), c), d), e), f), g), h), j), k), 1) and n); or - b), h) i), j), k), m) and n). In a second particular embodiment, p and q are O and L is -Y'-

(CH ₂ ) _r -Z'-. Thus, Cy represents - (CH ₂ ) _m - Y '- (CH ₂ ) _r --Z' - (CH ₂ ) _n -. Thus the compound may have the formula (IV): f ^(CH ₂₎ _m Y '(CH ₂₎ _r T (CH ₂₎ _n - ^

O = C NH

^ S ¹ His DPhe Arg Trp S ² ^

(IV) m, Y ', Z', n and r being as defined above. In this particular embodiment, the compound of formula (IV) 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

- If is absent. Nonlimiting examples of compounds according to the invention are the following: Cyclo (Aeh-His-DPhe-Arg-Trp-εAhx), Cyclo (Aeh-His-DPhe-Arg-Trp-Ahp), Cyclo ( Aeh-His-DPhe-Arg-Trp-βAla), Cyclo (3Mp-His-DPhe-Arg-Trp-Gly-Gly), Cyclo (3MP-Gly-His-DPhe-Arg-Trp-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-Trp-Gly-βAla ), as well as one of its pharmaceutically acceptable salts. Aeh represents 3-amino-4-ethylhexanoic acid; εAhx represents ε-aminohexanoic acid; Ahp represents 3-amino-4-ethylheptanoic acid; represents MP represents mercaptopropionic acid; more specifically, 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 meant 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 the Kd or 1ΕD50.

The invention also relates to a pharmaceutical composition containing as active principle one of the compounds described above or one of its salts. pharmaceutically acceptable, such as for example and without limitation, an acetate, a sulfate 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 meant, in particular, an improvement or disappearance of the symptoms, a slowing of the progression of the disease, an arrest 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.

Although the effective dose may vary from one individual to another and according to the applications, the skilled person knows how to determine this effective dose. By way of example, the compounds according to the present invention can be used at a dosage of between 0.1 ng / kg and 1 g / kg of body weight per day.

Disorders of sexual activity include conditions that prevent or hinder 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 reducing food intake or to increase weight gain in a subject comprising the administration of a pharmaceutically effective dose of a compound or a pharmaceutical composition according to the invention, in particular a compound exhibiting antagonist activity

MC4-R.

By "patient" or "subject" is meant a mammal, preferably a human.

The examples, synthesis 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-Hιs-DPhe-Arg-Trp-εAhx) Cyclo (3-Mp-H₂-DPhe-Arg-Trp-Gly-Gly)

CyCiO (NH-CH ₂ -CH ₂ -SS-CH ₂ -CH ₂ -CO-HiS-DPhB-ArQ-TrP-GIy) Cyclo (2-Mp-Hι-DPhe-Arg-Trp-Gly-Gly)

Cyclo (Hιs-DPhe-Arg-Trp-NH-CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CO) Cyclo (Aeh-Hι-DPhe-Arg-Trp-Gly-βAla)

Cyclo (Aeh-His-DPhe-Arg-Trp-βAla) Cyclo (Aeh-His-DPhe-Arg-Trp-Ahp)

Cyclo (3MP-Gly-His-DPhe-Arg-Trp-Gly)

The various synthesis methods used to obtain the products in question are detailed below.

The amino acids used for this invention are commercially available. Natural amino acids and their derivatives are represented by the three-letter code according to the IUPAC nomenclature.

Materials and methods

A. Peptide synthesis in solid phase

The peptide syntheses of the present invention were carried out stepwise from the C-terminus to the N-terminus according to the "Fmoc" (fluorenylmethoxycarbonyl) strategy, a temporary protective group of amino 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; For example, the Trt (trityl) group for histidine, the Pbf (2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl) group for arginine, the Boc (t-butoxycarbonyl) group are exemplified. ) for tryptophan.

The resins used (Novabiochem) were weighed, placed in the reactor in the presence of DMF (Λ /, Λ-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) (Λ /, Λf'-diisopropyl-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 Et ₂ O 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

The homogeneous phase reactions were monitored by reversed-phase analytical high pressure liquid chromatography (HPLC) (Beckman System GoId) using a SymetrieShield RP 18.5 μm column (100 Å, 250 x 4.6 mm). The elution is carried out using a linear gradient of acetonitrile at 0.08% TFA (solvent B) and FtO 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 C 18 column (5 μm, 100 μm, 250 μm). 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-NO resin VASYN-TGT (Novabiochem):

This resin is represented for all the synthesis schemes by

HO NOVASYN -TGT

- the resin HMPB-MBHA (4-hydroxymethyl-3-methoxyphenoxy butyric acid)

This is a representative of all synthesis schemes by

HO HMPB-MBHA These two resins allow the synthesis of peptides with C-terminal acid function.

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.

Fmoc-AA, ^ _^ , O - [HMPB-MBHA

FmOC-AA ₁ -OH, DIPCDI, "" ^ Y

HO [HMPB-MBHA ^{• *} o

IQ DMAP, CH ₂ CI ₂

5 equivalents of Fmoc-amino acid to be coupled to the resin are solubilized in CH ₂ Cl ₂ (10 ml / g). 2.5 equivalents of DIPCDI (N, N'-diisopropylcarbodiimide) (previously solubilized in 1 to 2 ml / g of CH ₂ Cl ₂ ) 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-termini 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 equiv) 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. Thioester cycling

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 mole), PyBOP (1.6 equivalents), DIEA (4 equivalents) in DMF (1O ^"M). The reaction is left with magnetic stirring and at ambient temperature for 6 to 24 hours The cyclization reaction is monitored by analytical HPLC and by 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 by the Fmoc group

Fmoc-OSu, NaHCO ₃

H ₂ O / acetone

In a reactor, 1 molar equivalent of 3-amino-4-ethylhexanoic acid is dissolved in H ₂ O at 100 g / l. 0.98 equivalent of Fmoc-OSu (N- (9-fluorenylmethoxycarbonyloxy) succinimide), previously solubilized in acetone (100 g / l), is added dropwise to the reaction mixture and 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 MgSO ₄ , filtered and concentrated to dryness under reduced pressure to give a white solid. 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-CH ₁ -CH ₁ -SS-CH ₁ -CH ₁ -COOH svnthon

/ \ _/ S-S / FmocOSu, Na ₂ CO ₃ s- S ^ / ^ TCEP, DMF / \ / SH

HCLH ₂ N ^ - ^/ NH ₂ .HCl • - Fπroc-HN ^{x /} ^ / NH-Fmoc → ~ Fmoc-NH ^

H ₂ O / acetone

PySSPy, T = ⁰ ° C DMF

DMF 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 ₂ CO 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 MgSO ₄ , 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 -SS-CH 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-CH ₂ -CH ₂ -SS-CH ₂ -CH ₂ -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-CH ₂ -CH ₂ -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 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. and 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 melting. the ice bath. The reaction crude is then purified by preparative HPLC. The product of the reaction is obtained: FmOC-NH-CH ₂ -CH ₂ -SS-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-εAhx)

The resin used was a HMPB-MBHA resin (140 mg, 0.115 mmol). The summary scheme is shown below:

Fmoc-εAhx-OH, DIPCDI,

H-Aeh-His (Trt) -DPhe-Arg (Pbf) -Trp (Boc) -NH- (CH ₂₎ ₅ - ¹ O ^ "^ HMPB-MBHA

O

TFA / TIS / H ₂ O (95 / 2.5 / 2.5)

H-Aeh-His-DPhe-Arg-Trp-NH- (CH ₂ ) ₅ -CθθH

PyBOP, NaHCO ₃ , DMF

The grafting of the first residue was carried out according to the procedure described in paragraph D using Fmoc-εAhx-OH (200 mg, 0.575 mmol), DIPCDI (45 μl, 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 μl, 0.93 mmol) and:

Amino acid Mass No. of moles No. eq. Coupling

Fmoc-Trp (Boc) -OH 182 mg 0.345 mmol 3 1

Fmoc-Arg (Pbf) -OH 224 mg 0.345 mmol 3 1

Fmoc-DPhe-OH 134 mg 0.345 mmol 3 1

Fmoc-His (Trt) -OH 214 mg 0.345 mmol 3 1

Fmoc-Aeh-OH 132 mg 0.345 mmol 3 1

At the end of synthesis and after deprotection of the N-terminal function, the resin was washed according to the protocol described in paragraph A.

300 mg of peptidyl resin were placed in the presence of 4 ml of TFA, 105 μl of TIS and 105 μl of FtO 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 Et2θ. 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 NaHCO ₃ (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-εAhx) 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)

The resin used was Fmoc-Gly-NO resin VASYN-TGT (500 mg, 0.12 mmol). The summary scheme is shown below:

Fmoc-Gly-O NOVASYN -TGT

Trt-S-CH, -CH, -CO-His (Trt) -DPhe-Arg (Pbf) -Trp (Boc) -Gly-Gly-O NOVASYN -TGT

TFA / TIS / H ₂ O (95 / 2.5 / 2.5)

HS-CH ₂ -CH ₂ -C O-His-DPhe-Arg-Trp-G Iy-GIy-OH DMF

The peptide elongation stepwise was carried out in DMF for 40 minutes in the presence of PyBOP (188 mg, 0.36 mmol), DIEA (130 μl, 0.72 mmol) and: Amino acid Mass No. of moles No. eq. Coupling

Fmoc-G Iy-OH 107 mg 0.36 mmol 3 T

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 μl of TIS and 158 μl of FtO 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 Et2θ. 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 μl, 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 -SS-CH ₂ -CH ₂ -CO-His-DPhe-Arg-Trp-Gly)

Fmoc-Gly-0 - NOVASYN -TGT

SPPS

H ₂ N-CH ₂ -CH ₂ -SS-CH ₂ -CH ₂ -CO-His (Trt) -DPhe-Arg (Pbf) -Trp (Boc) -Gly-0 - NOVASYN -TGT

TFA / TIS / H ₂ O (95 / 2.5 / 2.5)

H ₂ N-CH ₂ -CH ₂ -SS-CH ₂ -CH ₂ -CO-His-DPhe-Arg-Trp-Gly-OH

I

PyBOP, NaHCO ₃ , DMF

The peptide elongation stepwise was then carried out in DMF for 40 minutes in the presence of PyBOP (188 mg, 0.36 mmol), DIEA (125 μl, 0.72 mmol) and:

Amino acid Mass No. of moles No. eq. VS

Fmoc-frp (Boc 1 -H 190 mg? 36 mmol 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

FmOC-NH-CH ₂ -CH ₂ -SS-CH ₂ -CH ₂ -COOH 145 mg 0.36 mmol 3 1

At the end of the synthesis, the resin was washed according to the protocol described in paragraph A. 575 mg of peptidyl resin were placed in the presence of 6 ml of TFA, 158 μl of

TIS and 158 μl of FtO 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 (1'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 NaHCO ₃ (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 linear gradient from O to

60% of solvent B in 60 minutes. Fractions of interest were recovered and lyophilized. 15 mg of CyCiO (NH-CH ₂ -CH ₂ -SS-CH ₂ -CH ₂ -CO-HiS-DPlIe-Ar ₈ -TrP-GIy) 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 ⁺ ].

EXAMPLE 4 Synthesis of Cyclo (Aeh-His-DPhe-Arg-Trp-Ahp)

The resin used was a HMPB-MBHA resin (500 mg, 0.41 mmol). The summary scheme is shown below:

Fmoc-Ahp-OH, DIPCDI,

HMPB-MBHA

H-Aeh-His (Trt) -Phe-Arg (Pbf) -Trp (Boc) -NH- (CH ₂ ) ₍

TFA / TIS / H ₂ O (95 / 2.5 / 2.5) H-Aeh-His-DPhe-Arg-Trp-NH- (CH ₂ ) ₆ -COOH

PyBOP, NaHCO ₃ , DMF

The grafting of the first residue was carried out according to the procedure described in paragraph D using Fmoc-Ahp-OH (750 mg, 2.05 mmol), DIPCDI (205 μl, 1.025 mmol) and DMAP (50 mg, 0.41 mmol).

The peptide elongation stepwise was then carried out in DMF for 40 minutes in the presence of PyBOP (640 mg, 1.23 mmol), DIEA (460 μl, 2.46 mmol) and:

Amino acid Mass No. of moles No. eq. Coupling

__

Fmoc- FRP (Bocj? H-648 ^~~ ^{^"~} 3 mg I.23 mmol

Fmoc-Arg (Pbf) -OH 800 mg 1.23 mmol 3

Fmoc-DPhe-OH 477 mg 1.23 mmol 3

Fmoc-His (Trt) -OH 762 mg 1.23 mmol 3 1

Fmoc-Aeh-OH 468 mg 1.23 mmol 3 1 At the end of synthesis and after deprotection of the N-terminal function, the resin was washed according to the protocol described in paragraph A.

800 mg of peptidyl resin were placed in the presence of 15 ml of TFA, 395 μl of TIS and 395 μl of FtO 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 Et2θ. The crude peptide was purified by preparative HPLC before being cyclized.

165 mg (0.13 mmol) of linear peptide were solubilized in 12 ml of DMF in the presence of PyBOP (82 mg, 0.156 mmol) and NaHCO ₃ (55 mg, 0.65 mmol) at room temperature. Cyclization was monitored by analytical HPLC. The cyclized peptide was then purified by preparative HPLC using a linear gradient from 0 to

60% of solvent B in 60 minutes. Fractions of interest were recovered and lyophilized. 69 mg of cyclo (Aeh-His-DPhe-Arg-Trp-Ahp) were obtained, ie a yield of 48%: HPLC: tr = 8.45 min; purity>95%; calculated mass: 894 - MALDI MS m / z [DHB] 895.8 [M + H ⁺ ].

Example 5 Synthesis of cyclo-Ala-Aeh-His-DPhe-Arg-Trp-Gly

The resin used was Fmoc-Gly-NO resin VASYN-TGT (500 mg, 0.12 mmol). The summary scheme is shown below: Fmoc-Gly-0 NOVASYN -TGT

H ₂ N-CH ₂ -CH, -CO-Aeh-His (Trt) -DPhe-Arg (Pbf) -Trp (Boc) -Gly-O NOVASYN -TGT

TFA / TIS / H ₂ O (95 / 2.5 / 2.5)

H N-CH-CH-CO-AeH-His-DPhe-Arg-Trp-G Iy-OH

PyBOP, NaHCO ₃ , DMF

Amino acid Mass No. of moles No. eq. Coupling

Fmoc-Trp (Boc) -OH 190 mg 0.36 mmol 3

Fmoc-Arg (Pbf) -OH 234 mg 0.36 mmol 3

Fmoc-DPhe-OH 140 mg 0.36 mmol 3

Fmoc-His (Trt) -OH 223 mg 0.36 mmol 3

Fmoc-Aeh-OH 137 mg 0.36 mmol 3

Fmoc-βAla-OH 112 mg 0.36 mmol 3

At the end of the synthesis, the resin was washed according to the protocol described in section A. 575 mg of peptidyl resin were placed in the presence of 6 ml of TFA, 158 μl of TIS and 158 μl of FtO at room temperature 180 minutes. The resin then was filtered and washed with 4 ml of TFA. The crude peptide was then precipitated and washed with two volumes of Et2θ. The crude peptide was purified by preparative HPLC before being cyclized.

50 mg (0.04 mmol) of linear peptide were solubilized in 4 ml of DMF in the presence of PyBOP (25 mg, 0.048 mmol) and NaHCO ₃ (17 mg, 0.2 mmol) at room temperature. Cyclization was monitored by analytical HPLC. The cyclized peptide was then purified by preparative HPLC using a linear gradient from O to

60% of solvent B in 60 minutes. Fractions of interest were recovered and lyophilized. 36 mg of cyclo (βAla-Aeh-His-DPhe-Arg-Trp-Gly) were obtained, ie a yield of 80%: HPLC: tr = 8.1 min; purity> 95%; calculated mass: 895 -

MALDI MS m / z [DHB] 895.7 [M + H ⁺ ].

Example 6 Synthesis of Cyclo (Aeh-His-DPhe-Arg-Trp- $ Ala)

H-Aeh-His (Trt) -Phe-Arg (Pbf) -Trp (Boc) -NH- (CH ₂ ) ₂ - ~ _π - ' ^{• (} O- - HMPB-MBHA

O

TFA / TIS / H ₂ O (95 / 2.5 / 2.5)

H-Aeh-His-DPhe-Arg-Trp-NH- (CH ₂ ) 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-βAla-OH (640 mg, 2.05 mmol), DIPCDI (160 μl, 1.025 mmol) and DMAP (50 mg, 0.41 mmol).

The peptide elongation stepwise was then carried out in DMF for 40 minutes in the presence of PyBOP (640 mg, 1.23 mmol), DIEA (430 μl, 2.46 mmol) and:

Amino acid Mass No. of moles No. eq. Coupling

Fmoc-Trp (Boc) -OH 648 mg 1.23 mmol 3 1

Fmoc-Arg (Pbf) -OH 800 mg 1.23 mmol 3

Fmoc-DPhe-OH 477 mg 1.23 mmol 3

Fmoc-His (Trt) -OH 762 mg 1.23 mmol 3 1

900 mg of peptidyl resin were placed in the presence of 15 ml of TFA, 395 μl of TIS and 395 μl of FtO 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 Et2θ. The crude peptide was purified by preparative HPLC before being cyclized.

225 mg (0.19 mmol) of linear peptide were solubilized in 14 ml of DMF in the presence of PyBOP (119 mg, 0.23 mmol) and NaHCO ₃ (80 mg, 0.95 mmol) at room temperature. Cyclization was monitored by analytical HPLC. The cyclized peptide was then purified by preparative HPLC using a linear gradient from 0 to

60% of solvent B in 60 minutes. Fractions of interest were recovered and lyophilized. 100 mg of cyclo (Aeh-His-DPhe-Arg-Trp-βAla) were obtained, ie a yield of 50%: HPLC: tr = 8.28 min; purity>95%; calculated mass: 838 - MALDI MS m / z [DHB] 839.2 [M + H ⁺ ].

Example 7 Synthesis of Cyclo (3MP-Gfy-His-DPhe-Arg-Trp-Gfy)

The resin used was Fmoc-Gly-NO resin VASYN-TGT (500 mg, 0.12 mmol). The summary scheme is shown below: Fmoc-Gly-O NOVASYN -TGT

SPPS

Trt-S-CH ₂ -CH ₂ -CO-Gly-His (Trt) -DPhe-Arg (Pbf) -Trp (Boc) -Gly-O - NOVASYN -TGT

TFA / TIS / H ₂ O (95 / 2.5 / 2.5)

HS-CH ₂ -CH ₂ -CO-Gly-His-DPhe-Arg-Trp-Gly-OH

PyBOP, NaHCO ₃ , DMF

Amino acid Mass No. of moles No. eq. VS

Fmoc-frp (Boc) -6Hmg 0.36 mmol

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

Fmoc-Gly-OH 107 mg 0.36 mmol 3 1

TrtS-CH ₂ -CH ₂ -COOH 126 mg 0.36 mmol 3 1

At the end of the synthesis, the resin was washed according to the protocol described in paragraph A. 680 mg of peptidyl resin were placed in the presence of 2 ml of TFA, 53 μl of TIS and 53 μl (1'H ₂ O 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 Et 2 O. The crude peptide was purified by preparative HPLC before being cyclized.

50 mg (0.045 mmol) of linear peptide were solubilized in 4 ml of DMF in the presence of PyBOP (28 mg, 0.054 mmol) and NaHCO ₃ (19 mg, 0.225 mmol) at room temperature. Cyclization was monitored by analytical HPLC. The cyclized peptide was then purified by preparative HPLC using a linear gradient of 0 to 60% solvent B in 60 minutes. Fractions of interest were recovered and lyophilized. 15 mg of cyclo (S-CH ₂ -CH ₂ -CO-Gly-His-DPhe-Arg-Trp-Gly) were obtained, ie a yield of 32%: HPLC: tr = 7.6 min; purity>95%; calculated mass: 828 - MALDI MS m / z [DHB] 829.1 [M + H ⁺ ].

Example 8 Synthesis of Cyclo (His-DPhe-Arg-Trp-NH-CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CO)

The resin used was a HMPB-MBHA resin (350 mg, 0.3 mmol). The summary scheme is shown below:

H-His (Trt) -DPhe-Arg (Pbf) -Trp (Boc) - N 'HMPB-MBHA

H nr ^

TFA / TIS / H ₂ O (95 / 2.5 / 2.5)

H-Aeh-His-DPhe-Arg-Trp-NH- (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -O-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-PEG-OH (500 mg, 1.3 mmol), DIPCDI (101 μl, 0.65 mmol) and DMAP (37 mg, 0.3 mmol).

The peptide elongation stepwise was then carried out in DMF for 40 minutes in the presence of PyBOP (470 mg, 0.9 mmol), DIEA (325 μl, 1.8 mmol) and:

Amino acid Mass No. of moles No. eq. Coupling

Fmoc-Trp (Boc) -OH 474 mg 0.9 mmol 3 1

Fmoc-Arg (Pbf) -OH 584 mg 0.9 mmol 3 1

Fmoc-DPhe-OH 350 mg 0.9 mmol 3 1

Fmoc-His (Trt) -OH 558 mg 0.9 mmol 3 1 At the end of synthesis and after deprotection of the N-terminal function, the resin was washed according to the protocol described in paragraph A.

820 mg of peptidyl resin were placed in the presence of 12 ml of TFA, 315 μl of TIS and 315 μl of FtO at room temperature for 180 minutes. The resin was then filtered and washed with 3 ml of TFA. The crude peptide was then precipitated and washed with two volumes of Et2θ. The crude peptide was purified by preparative HPLC before being cyclized.

70 mg (0.062 mmol) of linear peptide were solubilized in 14 ml of DMF in the presence of PyBOP (39 mg, 0.075 mmol) and NaHCO ₃ (26 mg, 0.31 mmol) at room temperature. Cyclization was monitored by analytical HPLC. The cyclized peptide was then purified by preparative HPLC using a linear gradient from 0 to

60% of solvent B in 60 minutes. Fractions of interest were recovered and lyophilized. 29 mg of cyclo (His-DPhe-Arg-Trp-NH-CH ₂ -CH ₂ -O-CH ₂ -CH ₂ -O-CH ₂ -CO) were obtained, ie a yield of 47%: HPLC: tr = 7.3 min; purity>95%; calculated mass: 771 - MALDI MS m / z [DHB] 771.9 [M + H ⁺ ].

Pharmacological study

The compounds of the present invention are subject to pharmacological tests to determine the agonist activity of the melanocortin receptors, and more particularly the agonist effect on the hMC-4 receptor.

Study of the affinity of the compounds for the MC4 melanocortin receptors The affinity of the compounds of the invention is determined after measuring the displacement with increasing doses of each product, the binding of ^[125 I] - [Nle ⁴ , D-Phe ⁷ ] -α-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 LVmL penicillin and 100 μg / 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 [ ¹²⁵ I] - [NIe ⁴ , D-Phe ⁷ ] -α-MSH binding on human MC4 receptors is carried out in triplicates. The [Nie ⁴ , D-Phe ⁷ ] -α-MSH used was labeled with iodine-125 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 ^[125 I] - [Nle ^4, D-Phe ^7] -CC-MSH is fixed and corresponds to a final value of 3.10 ^"10 M in the well concentrations of each unlabeled compound varies from ^{10." 10} M to 10 ^"6 M.

The cell binding is carried out for 90 minutes at room temperature. She is stopped by placing the cells on ice. The free [ ¹²⁵ I] - [NIe ⁴ , D-Phe ⁷ ] -α-MSH, thus 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 γ counter.

The specific binding is obtained by subtracting the total binding (cell-bound cpm radioactivity counted for each well) from the radioactivity obtained for the cells placed in the presence of 10 ^-6 M non-radioactive compound (non-specific binding). analyzed and the values of 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 cyclic AMP production via MC4 receptors HEK-293 cells expressing human MC4 receptors of melanocortins are grown 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. 135: 541-546).

Results

The affinity results for the human MC4 receptor are reported in the table below:

Compounds Kd (nM) ED ₅₀ (nM)

_____

Meinotan II

Cyclo (Aeh-His-DPhe-Arg-Trp-εAhx) 417 46

Cyclo (3MP-His-DPhe-Arg-Trp-Gly-Gly) 500 40

Cyclo (Aeh-His-DPhe-Arg-Trp-Ahp) 400 N.D.

Cyclo (βAla-Aeh-His-DPhe-Arg-Trp-Gly) 370 40

Cyclo (Aeh-His-DPhe-Arg-Trp-βAla) 550 99

Cyclo (3Mp-Gly-His-DPhe-Arg-Trp-Gly) 140 27

Cyclo (His-DPhe-Arg-Trp-NH- (CH ₂ -CH ₂ -O) ₂ -CH ₂ -CO) 420 100

NDP-α-MSH N.D. 2.9

N.D. = 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 agonists of the melanocortin type 4 receptor.

Stability study

The compounds of the present invention were the subject of a chemical stability study for 30 days under different experimental conditions at 60 ^° C. at a concentration of 1 mg / ml. The monitoring was performed in analytical HPLC by measuring the percentage of product remaining at each time. Three pH conditions were used to demonstrate the difference in stability between the compounds of the present invention and Melanotan II, used as the reference molecule.

The stability results are reported in Figures 1-3. The compounds of the present invention described above are more stable than the reference molecule at pH 5, pH 7 and pH 9.2. In particular, whatever the pH (5, 7 or 9.2), the compounds of the present invention have a very high stability compared to that obtained for Melanotan IL

Claims

claims

1- Compound of general formula (II):

(") in which :

R ³ represents a hydrogen, a hydroxyl, an alkyl group or a nitro group;

R ⁴ represents a 3-indolyl, 5-hydroxy-3-indolyl or 2-naphthyl group; m and n are, independently of each other, an integer of 0 to 9; p and q are, independently of one another, 0 or 1;

R ⁵ and R ⁶ are, independently of one another, a hydrogen or an alkyl radical selected from the group consisting of a methyl, an ethyl, an isopropyl, an isobutyl, a sec-butyl, a tert-butyl, isopentyl, 1-ethylpropyl and neopentyl; L is -YZ- or -Y '- (CH ₂ ) _r -Z'- with

Y and Z together form -CH = CH- or -C≡C-; or Y is O, S, NH or CH ₂ and Z is S, CH ₂ or C = B where B is S or O with the proviso that Y and Z are not both CH ₂ ; Y 'and Z' are, independently of each other, O, S or NR ⁷ , with R ⁷ being hydrogen, unsubstituted linear or branched saturated C 1 -C ₅ alkyl or an acetyl group represented by CH _3- CO; and r is an integer between 0 and 5, m + n + r being less than or equal to 9; Si represents a glycinyl or a β-alanyl or is absent;

S ₂ represents a glycinyl or a β-alanyl or is absent; and the ring comprises 19 to 25 atoms; the phenylalanyl residue or the derivative thereof is of D configuration; as well as one of its pharmaceutically acceptable salts.

The compound of claim 1, wherein p and / or q are 1 and L is -Y-Z.

The compound of claim 1, wherein p and q are O and L is -Y'- (CH ₂ ) _r -Z'-.

4- compound according to claim 1 or 2, characterized in that it has at least one of the following characteristics: a) R ⁵ is an alkyl radical selected from the group consisting of a methyl, ethyl, isopropyl, isobutyl, sec-butyl tert-butyl, isopentyl, 1-ethylpropyl and neo-pentyl; and / or b) R ⁶ is hydrogen; and / or c) If is absent; and / or d) S ₂ is absent or represents a glycinyl or a β-alanyl, preferably a glycinyl; and / or e) p is 1; and / or f) q is 1; and / or g) m is 0 or 1; and / or h) n is an integer from 0 to 5; and / or i) if m + p is 2 and n + q is 1, then Si or S ₂ is present; and / or j) Y is NH or S; and / or k) Z is CO or S; and / or 1) R ⁵ is hydrogen; and / or m) m + p is 2; and or n) the ring comprises 20 to 25 atoms.

5- compound according to claim 1 or 3, 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

- If is absent; and or

- S ₂ is absent.

6. A compound according to any one of claims 1-5, selected from the group consisting of Cyclo (Aeh-His-DPhe-Arg-Trp-εAhx), Cyclo (Aeh-His-DPhe-Arg-Trp-Ahp) , Cyclo (Aeh-His-DPhe-Arg-Trp-βAla), Cyclo (3Mp-His-DPhe-Arg-Trp-Gly-Gly), Cyclo (3MP-Gly-His-DPhe-Arg-Trp- Gly), Cyclo (NH-CH ₂ -CH ₂ -S-S-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) -Trp-Gly-βAla), as well as one of its pharmaceutically acceptable salts.

Pharmaceutical composition comprising, as active principle, a compound according to any one of claims 1 to 6, in association with a pharmaceutically acceptable carrier.

8- Pharmaceutical composition according to claim 7, characterized in that it is in a parenterally administrable form, oral, inhaled, intranasal, topical, rectal, vaginal, perlingual or ophthalmic.

9- Pharmaceutical composition according to claim 7 or 8, characterized in that it is in the form of injectable preparation parenterally, subcutaneously, intravenously or intramuscularly.

10- Pharmaceutical composition according to any one of claims 7 to 9, further comprising another active ingredient. 11. A compound according to any one of claims 1 to 6 as a medicament.

12- Use of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to any one of claims 7 to 10 for the preparation of a medicament for the treatment of disorders of sexual activity feminine or masculine.

13- Use of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to any one of claims 7 to 10 for the preparation of a medicament for the treatment of diabetes, weight disorders such as than obesity and anorexia.

14- Use of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to any one of claims 7 to 10 for the preparation of a medicament for the treatment of mental disorders associated with the anxiety and depression.

15- Use of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to any one of claims 7 to 10 for the preparation of a medicament for the treatment of pain.

16- A compound according to any one of claims 1 to 6 for the treatment of disorders of male or female sexual activity, diabetes, weight disorders such as obesity and anorexia, mental disorders associated with the anxiety and depression and / or pain.