FR3128958A1 - KISS1R RECEPTOR AGONIST CYCLIC PEPTIDE COMPOUNDS AND THEIR THERAPEUTIC USES - Google Patents

Abstract

The invention relates to a peptide compound agonist of the KISS1R receptor, chosen from the compounds of formula (I) and their analogues capable of binding the KISS1R receptor, or one of their salts: R1-Xaa1-Xaa2-Xaa3-Asn-Xaa4-Phe -Xaa5-Xaa6-Xaa7-Xaa8-NH2 (I), in which Xaa5 and one of the residues Xaa1, Xaa2 or Xaa3, both simultaneously represent either a cysteine residue or a D-cysteine residue, the sulfur atoms of these residues cysteine or these D-cysteine residues being covalently linked to each other by a disulphide, methylene, cis-but-2-enylene, m-xylylene, p-xylylene or 2,5-dichloro-p -xylylene. The peptide compound according to the invention has a particularly long duration of action in the body, and proves to be particularly effective in improving fertility in mammals.

Description

KISS1R RECEPTOR AGONIST CYCLIC PEPTIDE COMPOUNDS AND THEIR THERAPEUTIC USES

The present invention falls within the field of improving fertility in mammals.

More particularly, the present invention relates to a peptide compound agonist of the KISS1R receptor capable of improving this fertility, as well as the use of such a compound as a medicament, in particular for improving fertility in mammals, and a veterinary composition and/or or pharmaceutical containing it.

The peptide compound according to the invention finds particular application both in the field of animal husbandry and in that of human therapy.

In the present description, the term "improvement of fertility" means both the treatment of sterility, in particular in humans, and the stimulation and improvement of the control of reproduction, in particular the induction and synchronization of ovulation in female mammals, particularly for the resumption of postpartum cyclicity, and the stimulation of testosterone secretion in male mammals.

In the field of breeding, the control of reproduction is an important issue, in order to optimize the production of products, such as meat, milk or its derivatives, throughout the year. For example, sheep and goat farming is subject to strong seasonal variations in productivity, due to the seasonal nature of reproduction and the course of lactation. The methods currently used to induce ovulation at any time during the off-season and/or to synchronize ovulation within herds rely on the use of steroid hormones, such as progesterone or estradiol, prostaglandin F2α (PGF2α) and PMSG (for English Pregnant Mare Serum Gonadotropin, equine chorionic gonadotropin). However, these methods do not allow complete control of ovulation. In addition, estrogen-like hormones implemented are pollutants that are not easily degraded, accumulate in land and waters, and pose a threat to human health. The treatment of animals with such hormones also presents significant constraints for breeders, who are obliged to comply with very strict specifications. For example, the methods currently used to induce ovulation in sheep provide for the implantation for 12 to 14 days of a vaginal sponge containing progesterone, combined with co-treatment by an injection intramuscular PMSG at the time of sponge removal. Similarly, in cattle, an injection of PGF2α must be performed 6 days after the vaginal implantation of a device containing progesterone, which must be followed the day after the removal of this vaginal device. Such processing is cumbersome to implement.

In human clinical practice, currently available treatments for assisted reproduction have certain undesirable side effects, such as ovarian hyperstimulation. These treatments also use hormones to induce ovulation, and therefore act either at the level of the pituitary gland or at the level of the gonads. The same is true for treatments intended to treat hypothalamic amenorrhea or delayed puberty.

Alternative treatments have thus been sought by the prior art, which implement non-steroidal molecules capable of triggering / synchronizing ovulation both in ruminants, so as to be able to program their reproduction and their milk production throughout the year, improve yields of artificial insemination, optimize herd profitability, reduce infertility problems in herds, etc., than in humans, to induce the release of gonadotropins in a more natural way than hormonal treatments current ones, thus reducing the risks of ovarian hyperstimulation syndrome.

The neurotransmission system formed by a receptor, the KISS1R, also called KISS1 receptor, or GPR54 (GenBank accession number, for the human receptor: NM_032551.4, GI:189163516), and its endogenous ligands, called kisspeptides (or kisspeptins), derived from the cleavage of a precursor peptide, KISS1 (GenBank accession number, for the human peptide: NM_002256.3, GI:116829963; NP_002247.3, GI:116829964; for the murine peptide: AB666166. 1, GI:384367966; BAM11250.1, GI:384367967; for the ovine peptide: AFW03832.1, GI:411100741), which are neurotransmitters with the ability to stimulate the KISS1R receptor (Kotani et al., 2011, J. Biol. Chem. 276(37): 34631-34636) has in particular formed the basis of a line of research aimed at achieving these objectives.

In the present description, the abbreviation KISS1R will be used to designate both the human receptor and the receptor of other species (indicated by the abbreviation Kiss1r in the official nomenclature). A complete list of synonyms used to designate the KISS1R is notably available on the IUPHAR website.

The kisspeptide (also called kisspeptin) primarily responsible for the biological stimulatory activity of KISS1R is the decapeptide called kisspeptin-10, or KP10. We know in particular the sequences of kisspeptin-10 derived from the mouse (mKP10, Mus musculus , of sequence:
Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 1)
in which the C-terminus is modified by amidation) and human-derived kisspeptin-10 (hKP10, Homo sapiens , of sequence:
Tyr-Asn-Trp-Asn-Ser-Phe-Gly-Leu-Arg-Phe-NH ₂ (SEQ ID No: 2)
in which the C-terminus is also modified by amidation). Ewe-derived kisspeptin-10 is notably identical to mKP10.

The activation of KISS1R by these ligands, and more particularly by KP10, induces a very powerful stimulatory effect on the release of LH and FSH hormones in mammals, this effect resulting from an increase in the secretion of GnRH (for l 'English Gonadotropin Releasing Hormone, pituitary gonadotropin releasing hormone) (Caraty and Franceschini, 2008, Reprod. Dom. Anim. (Suppl. 2) : 172-178). In particular, it has been shown that KP10, administered to ewes intravenously at the end of the follicular phase, is capable of inducing a peak in luteinizing hormone (LH) which is followed, 21 hours later, by synchronized ovulations at hour (Caraty et al., 2007, Endocrin. 148(11): 5258-5267). It has also been shown that during the period of sexual rest (seasonal anestrus), prolonged infusion of KP10 is able to reactivate the gonadotropic axis and induce ovulation (Sébert et al., 2010, Domest. Anim. Endocrinol 38 (4): 289-298). These results thus demonstrated the feasibility of controlling ovulation in farm animals by stimulating the KISS1R/KP10 system. The human KISS1R and that of the sheep also show a very high degree of homology, with more than 60% identity, and the kisspeptides (or kisspeptins) show similar in vivo activity in primates and sheep (Seminara et al. al., 2006, Endocrinology 147(5): 2122-2126; Caraty et al., 2007, cited above), which makes it possible to envisage an application in human therapy.

Compared to steroid hormones, KP10 has the particular advantages of being very quickly eliminated from the body and easily destroyed in the natural environment, leaving only amino acids as residues. In addition, it has a mechanism of action, resulting in the triggering of the secretion of the LH hormone necessary for the induction of ovulation, which is completely different from that of current treatments, and which allows a more localized action. and fine avoiding unwanted side effects.

However, since KP10 is rapidly degraded and excreted by the body, it has a limited duration of action.

It has therefore been sought by the prior art to develop compounds derived from KP10 which have a prolonged and controlled duration of action in the body, while retaining properties of degradability in the environment similar to those of KP10. .

Document WO 2014/118318 in particular describes peptide compounds agonists of the KISS1R receptor derived from KP10, exhibiting a strong capacity for inducing / synchronizing ovulation in mammals, in which a peptide bond within the KP10 peptide is replaced by a 1,2,3-triazole-1,4-disubstituted group.

The present invention aims to provide non-steroidal compounds which have further improved properties compared to these compounds proposed by the prior art, and which, in particular, combine the advantages of a strong effect of improving fertility, and in particular a strong ability to induce / synchronize ovulation in female mammals, and a prolonged duration of action in the body.

Additional objects of the invention are that these compounds are easy to synthesize and at low cost, and that they exhibit low toxicity for living organisms and for the environment.

In order to achieve these objectives, the present inventors are also interested in derivatives of kisspeptins. They discovered that cyclic derivatives of kisspeptin-10 responding to a particular structure exhibit, surprisingly, a very good activation efficiency of the KISS1R receptor as well as, compared to the linear derivatives of this kisspeptin proposed by the prior art , a prolonged effect, more gradual over time.

Thus, according to a first aspect, the present invention provides a peptide compound agonist of the KISS1R receptor, chosen from:
- a compound of formula (I):
R₁-Xaa₁-Xaa₂-Xaa₃-Asn-Xaa₄-Phe-Xaa₅-Xaa₆-Xaa₇-Xaa₈-NH₂(I)
in which
the C-terminal end is modified by amidation,
Xaa₅and one, preferably only one, of the Xaa residues₁, Xaa₂or Xaa₃, both simultaneously represent either a cysteine residue or a D-cysteine residue, the sulfur atoms of said cysteine residues or of said D-cysteine residues, i.e. of Xaa₅and one of Xaa₁, Xaa₂and Xaa₃, being linked to each other covalently by a disulphide bridge, methylene,cis-but-2-enylene, m-xylylene, p-xylylene or 2,5-dichloro-p-xylylene,
when they do not represent the said cysteine or D-cysteine residue: Xaa₁represents a tyrosine, D-tyrosine or lysine residue, Xaa₂is zero or represents an asparagine or lysine residue, and Xaa₃represents a tryptophan, lysine, proline or hydroxyproline residue,
Xaa₄represents a serine or threonine residue,
Xaa₆represents a leucine residue or an analogous aliphatic α-amino acyl residue, such as Ile, Val, Ala(cPr), Nle or Nval,
Xaa₇represents an arginine residue, whose –NH function₂is optionally substituted by a C1-C3 alkyl radical, or a similar positively charged α-amino acyl residue, such as Arg(asymMe₂), Lys, where appropriate substituted, or Orn, where appropriate substituted,
Xaa₈represents a tyrosine, phenylalanine, tryptophan residue or an α-amino acyl residue of an analogous aryl alanine type, such as 1-, 2- or 3-naphthylalanine, 2- or 3-thienylalanine, 2- or 3-furylalanine, 2-, 3- or 4-fluorophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or 4-bromorophenylalanine, 2-, 3- or 4-cyanophenylalanine, 2-, 3- or 4-iodophenylalanine, 2-, 3- or 4-methylphenylalanine or 2-, 3- or 4-trifluoromethylphenylalanine,
Asn represents an asparagine residue,
Phe represents a phenylalanine residue,
R₁represents a hydrogen atom, an acetyl group, an alkanoyl group, a benzoyl group or a group of general formula (II):

(II)

in which Y represents a covalent bond or a spacer arm and Z represents a unit capable of binding to serum albumin,
- an analog of the compound of formula (I) capable of binding the KISS1R receptor,
- or any of their salts, in particular any of their pharmaceutically acceptable salts.

In the present description, the term “pharmaceutically acceptable salt”, in a conventional manner per se, means any salt of the compound of formula (I) or of its analogue comprising, as counterion, a substance which does not produce any adverse, allergic or otherwise undesirable reaction when administered to a subject, particularly a mammal. Any conventional pharmaceutically acceptable salt of the compound of formula (I) or its analogue can be used according to the invention. Examples include chlorides, bromides, formates, acetates, etc.

In the present description, the positions of the amino acids in the peptides are defined in the conventional way in itself, the numbering starting at the level of the N-terminal end of the peptide, appearing on the left in all the sequences, whereas the C-terminal end is shown on the right.

The peptide compound according to the invention is thus such that two residues located in specific positions within the KP10 peptide are replaced simultaneously by cysteine residues or by D-cysteine residues, these cysteine or D-cysteine residues being linked, at the level of their respective sulfur atoms, covalently via an arm, called a "clip", so as to carry out a macrocyclization of the peptide compound, this clip having a particular chemical formula, chosen from:
- a disulphide bridge,
- a methylene bridge, of formula:

- a cis -but-2-enylene bridge, of formula:

- an m-xylylene bridge, of formula:

- a p-xylylene bridge, of formula:

- and a 2,5-dichloro-p-xylylene bridge, of formula:

the reasons in these formulas representing the bonds to the sulfur atoms of the cysteines or D-cysteines residues.

The peptide compound according to the invention advantageously has a very powerful stimulatory effect on the release of the luteinizing hormone LH in a female mammal, in particular much stronger than cyclized peptide compounds of similar structure but not falling within the formula ( I), in particular by the structure of the clip, the position of the amino acid residues substituted by the cysteine or D-cysteine residues, and even in which one amino acid residue is substituted by a cysteine and the other by a D-cysteine.

In addition, the peptide compound according to the invention has a prolonged duration of action in vivo . Without prejudging the mechanisms underlying this extension of the effect of the peptide compound according to the invention over time, it can be assumed that the particular combined choice of the anchoring positions of the clip on the peptide compound and of the chemical structure of this staple, forces the peptide compound into a particular three-dimensional configuration which, on the one hand, promotes its interaction with the KISS1R receptor, and on the other hand, disfavors its interaction with the active sites of proteases, so that it is more resistant to proteolysis phenomena in vivo .

In particular, it has been demonstrated by the present inventors, by a calcium mobilization test carried out in vitro on cells of a cell line derived from human embryonic kidney (HEK) stably transfected with the human kisspeptin receptor, this test making it possible to evaluate the activation of this receptor by measuring the intracellular increase in calcium ions, that the peptide compound according to the invention activates the KISS1R receptor in a dose-dependent manner, with median concentrations of activation of the receptor Very low EC ₅₀ , in the range of 1.1 to 45 nM. Depending on its specific formula, the peptide compound according to the invention may even have a greater capacity for activating the KISS1R receptor than kisspeptin itself.

A test carried out in vivo on ovariectomized female mice implanted subcutaneously with a device containing estradiol which is released gradually (which makes it possible to eliminate the ovarian cycle while maintaining a low and stable level of gonadotropin secretion ), the peptide compound being injected intraperitoneally at three different doses ranging from 1.3 to 10 nmol, also shows in particular a strong effect on the secretion of luteinizing hormone (LH), this effect increasing as a function of the increase of the dose administered and being durable over time. These results demonstrate in particular that the peptide compound according to the invention has a strong ability to stimulate the hypothalamic-pituitary axis over a long period.

The peptide compound in accordance with the invention thus advantageously exhibits, compared to KP10, an extended lifespan in the organism, and all the more so in the blood serum, when it is coupled to a unit capable of binding the serum albumin, while maintaining KISS1R stimulation efficiency as great as that of KP10, or even better.

By analogue of the compound of formula (I) capable of binding the KISS1R, is meant in the present description any peptide compound retaining the ability of this compound to bind the KISS1R receptor and whose sequence is different from the sequence of the peptide compound of formula (I), in particular by:
- one or more substitutions of a natural amino acid by its D enantiomer or by another natural or unnatural amino acid, in particular one or more conservative substitutions, i.e. of an amino acid residue by another having chemical and/or physical properties, for example of size, polarity, charge, etc., which are similar, for example by substitution of a basic residue such as arginine by another basic residue such as a residue lysine, of an acid residue such as aspartate by another acid residue such as glutamate, of a polar residue such as serine by another polar residue such as threonine, of an aliphatic residue such as leucine by another residue aliphatic such as isoleucine, etc. ;
- and/or by a post-translational modification or a chemical modification of the N-terminal end and/or of the C-terminal end and/or of any functional group carried by the side chain of an acid residue amine of the sequence, for example glycosylation, amidation, esterification, acylation, acetylation, methylation, etc.,
- and/or by the replacement of one or more peptide bonds by a non-peptide isosteric bond,
- and/or by one or more additions of amino acids to the N-terminal end of the sequence of the peptide compound of formula (I), between the R ₁ group and the Xaa ₁ residue.
The ability of peptide compounds to bind KISS1R can be assessed in different ways. It can in particular be evaluated by an in vivo test, by measuring the LH concentration in blood samples from a female mammal, for example from mice, into which the compound to be tested has been previously injected, for example according to the protocol described below. later in this description. An increase in this concentration, compared to an untreated control, demonstrates an ability of the analog to bind KISS1R.

Furthermore, this ability can be checked by in vitro test, by measuring the amount of intracellular calcium in a cell line expressing the KISS1R receptor after incubation with the molecule to be tested. Indeed, stimulation of the KISS1R receptor leads to the activation of two distinct intracellular pathways, which induce an increase in the intracellular concentration of calcium ions: by release of intracellular reserves, following the production of IP3, and by entry , following the opening of the ion channels (for example TRPC) of the plasmatic membrane of the cell, of calcium ions present in the extracellular medium. An example of such a test, called a calcium mobilization test, is described in detail below in this description. An increase in the amount of intracellular calcium, relative to an untreated control, or relative to the same cell line not expressing the KISS1R receptor, then demonstrates the ability of the analog to bind the KISS1R receptor.

By unit capable of binding to serum albumin, is meant any unit exhibiting a capacity for binding to serum albumin, preferably with a dissociation constant Kd of the order of 10 ⁻³ M or less.

Such patterns are well known to those skilled in the art, who can in particular, to identify them, refer to numerous publications in the field, for example to the publications of Angelini 2012, J. Med. Chem. , 55:10187-10197, by Dennis et al., 2002, J. Biol. Chem. , 277: 35035−35043, by Dumelin et al., 2008, Angew. Chem., Int. Ed. , 47: 3196-3201, by Knudsen et al., 2000, J. Med. Chem . 43: 1664-1669, from Zarandi et al., 2006, Proc. Natl. Acad. Science. USA , 103(12): 4610-4615, or in the reviews by Pollaro and Heinis, 2010, Med. Chem. Commmon. 1, 319-324 or Zorzi et al., 2019, Med. Chem. Commmon. 10, 1068-1081.

The person skilled in the art can otherwise proceed empirically, by determining the dissociation constant Kd of a given unit by a test for studying the classic binding in itself, for example by one of the tests described in the publication of Phizicky et al., Microbiological Reviews, 1995, 59, 94-123, in particular by the so-called saturation method, or by the so-called displacement method, or by affinity chromatography, the serum albumin being immobilized on the column, etc Examples of protocols for the implementation of such binding tests, which are all well known to those skilled in the art and which can easily be implemented, are for example described in the publication by Kurtzhals et al., Biochem. J., 1995, 3212, 725-731. For the interpretation of the results, a comparison may for example be made with the results obtained, by the same technique, with one of the compounds described in the literature as capable of binding to serum albumin, or with one of the examples of such units described below in the present description, in particular a hexadecanoyl unit, in particular included in a γ-(N-hexadecanoyl-Glu-OH) group.

The presence in the peptide compound according to the invention of a unit capable of binding to serum albumin increases in particular even more, and very significantly, the duration of action of the peptide compound in the body, by an increase of its half-life in the bloodstream, its renal excretion being in particular delayed.

Any pattern capable of binding to serum albumin can be implemented according to the invention. Particularly preferred units are lipid chains, and in particular fatty acids, in particular hexadecanoic acid. Thus, preferably, in the general formula (II), Z represents a hexadecanoyl group (also called palmitoyl). Z may otherwise represent, for example, an ω-carboxylate fatty acid, as described in the aforementioned publication by Zarandi et al., 2006, the Albu-tag as described in the aforementioned Dumelin et al., 2008 publication, or a cyclopeptide such as described in the publications by Dennis et al., 2002 or Angelini et al., 2012 cited above, etc.

In the peptide compound according to the invention, a unit capable of binding to serum albumin is preferably attached, directly or indirectly via a spacer arm, to an amino acid located in the N-terminal region of the peptide compound, preferably at the N-terminal end of the latter within the R ₁ group of general formula (II) above.

A unit capable of binding to serum albumin can also, or otherwise, be attached, directly or indirectly via a spacer arm, to the side chain of an amino acid residue located in position 1, in position 2 and/or in position 3 of the peptide compound, that is to say of Xaa ₁ , Xaa ₂ and/or Xaa ₃ , or residues which are analogous thereto, for those of these residues which do not represent a cysteine or D-cysteine residue. When the unit capable of binding to serum albumin is attached to the side chain of an amino acid residue located in position 1, 2 or 3 of the peptide compound, the residue concerned is preferably a lysine residue.

Thus, in particular embodiments of the invention, Xaa ₁ , Xaa ₂ and/or Xaa ₃ , if it does not represent a cysteine or D-cysteine residue, can be substituted, on its side chain, by a group of general formula (II), comprising a unit capable of binding to serum albumin, as defined in the present description, in particular when, in formula (I), R ₁ does not represent such a group of general formula (II).

The peptide compound according to the invention may also meet one or more of the characteristics described below, implemented in isolation or in each of their technically effective combinations.

In general formula (II), Y represents a covalent bond or a spacer arm, preferably a pharmacologically inactive spacer arm. This spacer arm is also preferably of the hydrophilic type.

Y preferably represents a linear, branched and/or cyclic, saturated or unsaturated, optionally substituted hydrocarbon radical, which may be interrupted by one or more heteroatoms, preferably by at least, or only, one or more oxygen atoms, and/ or one or more groups comprising at least one heteroatom, preferably at least, or only, one or more primary amine -NH ₂ , secondary amine -NH- and/or carbonyl -CO- groups.

Y may in particular represent such a hydrocarbon radical comprising one or more amino acid residues, in particular a D-lysine or L-lysine residue, or a sequence of several D- and/or L-lysine residues, in particular six D-residues - and/or L-lysine, any sequence of D-lysine and L-lysine residues falling within the scope of the invention. Such a characteristic notably advantageously increases the solubility of the peptide compound in water.

In particularly preferred embodiments of the invention, Y comprises, or consists of, a γ-glutamyl group. The presence of such a group between the amino acid residue of the peptide compound and the unit capable of binding to serum albumin, in particular advantageously increases the affinity of this unit for serum albumin.

In formula (I), R ₁ can thus comprise, or consist of, a γ-( N -hexadecanoyl-L-glutamyl) group, also called γ-( N -hexadecanoyl-Glu-OH).

R ₁ may otherwise comprise, or consist of, a 2-(succinamido)-6-(4-(4-iodophenyl)butanamido)hexanoate moiety.

In particular embodiments of the invention, Y comprises one or more, in particular from 1 to 24, ethylene glycol groups. Such a characteristic promotes in particular the binding to serum albumin of the unit capable of binding to serum albumin Z, in particular thanks to a greater conformational freedom then procured for the group R ₁ .

Y may in particular comprise, or consist of, a group having the general formula (IV):

(IV)

in which
m ₁ is equal to 1 or 2,
m ₂ is equal to 0 or 1,
m ₃ is equal to 1 or 2,
n is an integer between 1 and 24,
and Y ₁ represents an oxygen atom or an amide group.

Preferably, the unit capable of binding to serum albumin Z is coupled to this group of formula (IV) on the side of the terminal secondary amine function of the latter, and the amino acid residue of the peptide compound is coupled to it at level of its opposite terminal carbonyl function.

In particular embodiments of the invention, Y comprises, or consists of, a group having the general formula (IV'), comprising at least one D- and/or L-lysine residue:

(VI')

in which
m ₁ is equal to 1 or 2,
m ₂ is equal to 0 or 1,
m ₃ is equal to 1 or 2,
n is an integer between 1 and 24,
Y ₁ represents an oxygen atom or an amide group,
p is an integer between 1 and 12, for example equal to 6,
m ₄ is equal to 1 or 2,
m ₅ is equal to 0 or 1,
m ₆ is equal to 1 or 2,
n ₁ is an integer between 1 and 24,
and Y ₂ represents an oxygen atom or an amide group.

Specific examples of groups of formula (IV) or (IV') which can be used according to the invention correspond to the respective formulas (IVa), (IVb), (IVc), (IVd) and (IV'a):

(IVa)

(IVb)

(IVc)

(IVd)

(IV'a)

in which
n represents an integer between 1 and 24,
n ₁ represents an integer between 1 and 24,
and p represents an integer between 1 and 12, for example equal to 6.

In the general formula (IVd), n can for example be equal to 3.

Preferably, in general formula (II), Y represents a group of formula (III):

(III)

in which
represents binding to the motif capable of binding to serum albumin Z,
and R ₃ represents a covalent bond to the amino acid residue of said peptide compound, in particular to the terminal amine group of this peptide compound (R ₃ then representing a motif ),
or R ₃ represents a group of formula (IV) or (IV') as defined above, for example a group of formula (IVa), (IVb), (IVc), (IVd) or (IV'a) , this group being coupled, directly or indirectly, preferably via its terminal carbonyl function, to the amino acid residue of the peptide compound according to the invention.

The group Y can for example be chosen from the groups of formulas (IIIa), (IIIb), (IIIc), (IIId), (IIIe) and (III'a):

(IIIa)

(IIIb)

(IIIc)

(IIId)

(III)

(III'a)

in which n represents an integer between 1 and 24, n ₁ represents an integer between 1 and 24 and p represents an integer between 1 and 12, for example equal to 6.

Preferably, for all of these groups, the unit capable of binding to serum albumin Z, in particular the hexadecanoyl unit, is covalently linked to the terminal secondary amine function of said group, and the amino acid residue of the peptide compound, in particular at the N-terminal end of the latter, is covalently linked to the opposite terminal carbonyl function of said Y group.

R ₁ can otherwise represent a hydrogen atom, an acetyl group, an alkanoyl group, in particular chosen from linear alkanoyls, preferably C1-C6, for example C2, or a benzoyl group.

In particular embodiments of the invention, the peptide compound is an analog of the compound of formula (I), in which a peptide bond is replaced by a non-peptide isostere bond, for example by a 1,2,3- triazole-1,4-disubstituted. Several of the peptide bonds of the compound of formula (I) can thus be replaced by non-peptide isosteric bonds, which are identical or different from each other.

In formula (I), when Xaa ₇ represents an arginine residue, the –NH ₂ primary amine function of this residue may be substituted by a C1-C3 alkyl radical, in particular by a methyl radical. Such a substitution advantageously avoids degradation of the C-terminal part of the compound by trypsin-type proteases.

A particularly preferred combination in the context of the invention is that in which Xaa ₆ represents Leu and Xaa ₇ represents Arg, the –NH ₂ function of which is optionally substituted by a methyl group.

The peptide compound according to the invention derived from KP10 may have the amino acid sequence:
R ₁ -Tyr-Asn-Xaa ₃ -Asn-Ser-Phe-Xaa ₅ -Leu-Arg(R ₂ )-Tyr-NH ₂ (SEQ ID No: 3)
in which
Xaa ₃ and Xaa ₅ each represent a cysteine residue or Xaa ₃ and Xaa ₅ each represent a D-cysteine residue,
R ₁ is as defined above (N-terminal modification),
R ₂ represents a hydrogen atom or a C1-C3 alkyl radical, for example a methyl radical (optional substitution of the NH ₂ group of the arginine residue by a C1-C3 alkyl radical),
and the sulfur atoms of Xaa ₃ and Xaa ₅ are covalently bonded to each other by a disulfide, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2,5 -dichloro-p-xylylene,
or consist of one of its analogues capable of binding the KISS1R receptor, or of one of its salts or one of the salts of said analogue, this salt preferably being pharmaceutically acceptable.

In particular, the peptide compound according to the invention derived from KP10 may have the amino acid sequence:
R ₁ -Tyr-Asn-Cys-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 4)
in which
R ₁ represents an acetyl group,
and the sulfur atoms of the two cysteines at positions 3 and 7 are covalently bonded to each other by a disulfide, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2 ,5-dichloro-p-xylylene,
or consist of one of its analogues capable of binding the KISS1R receptor, or of one of its salts or one of the salts of said analogue, this salt preferably being pharmaceutically acceptable.

The peptide compounds of sequence SEQ ID No: 4 in which the clip connecting the sulfur atoms of the cysteine residues is a p-xylylene, m-xylylene, methylene, cis -but-2-enylene or 2,5-dichloro- p-xylylene, are particularly preferred within the scope of the invention.

Otherwise, the peptide compound according to the invention derived from KP10 may have the amino acid sequence:
R ₁ -Xaa ₁ -Asn-Trp-Asn-Ser-Phe-Xaa ₅ -Leu-Arg(R ₂ )-Tyr-NH ₂ (SEQ ID No: 5)
in which
Xaa ₁ and Xaa ₅ each represent a cysteine residue or Xaa ₁ and Xaa ₅ each represent a D-cysteine residue,
R ₁ is as defined above (N-terminal modification),
R ₂ represents a hydrogen atom or a C1-C3 alkyl radical, for example a methyl radical (optional substitution of the NH ₂ group of the arginine residue by a C1-C3 alkyl radical),
and the sulfur atoms of Xaa ₁ and Xaa ₅ are covalently bonded to each other by a disulfide, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2,5 -dichloro-p-xylylene,
or consist of one of its analogues capable of binding the KISS1R receptor, or of one of its salts or one of the salts of said analogue, this salt preferably being pharmaceutically acceptable.

In particular, the peptide compound according to the invention derived from KP10 may have the amino acid sequence:
R ₁ -Cys-Asn-Trp-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 6)
in which
R ₁ represents an acetyl group,
and the sulfur atoms of the two cysteines at positions 1 and 7 are covalently bonded to each other by a disulfide, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2 ,5-dichloro-p-xylylene,
or consist of one of its analogues capable of binding the KISS1R receptor, or of one of its salts or one of the salts of said analogue, this salt preferably being pharmaceutically acceptable.

The peptide compound of sequence SEQ ID No: 6 in which the clip connecting the sulfur atoms of the cysteine residues is a disulphide bridge is particularly preferred within the scope of the invention.

The peptide compound according to the invention derived from KP10 may otherwise have the amino acid sequence:
R ₁ -D-Cys-Asn-Trp-Asn-Ser-Phe-D-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 7)
in which
R ₁ represents an acetyl group,
and the sulfur atoms of the two cysteines at positions 1 and 7 are covalently bonded to each other by a disulfide, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2 ,5-dichloro-p-xylylene,
or consist of one of its analogues capable of binding the KISS1R receptor, or of one of its salts or one of the salts of said analogue, this salt preferably being pharmaceutically acceptable.

The peptide compounds of the amino acid sequence SEQ ID No: 7 in which the clip connecting the sulfur atoms of the D-cysteine residues is a disulphide bridge or a methylene bridge are particularly preferred in the context of the invention.

The peptide compound according to the invention derived from KP10 may otherwise have the amino acid sequence:
R ₁ -Tyr-Xaa ₂ -Trp-Asn-Ser-Phe-Xaa ₅ -Leu-Arg(R ₂ )-Tyr-NH ₂ (SEQ ID No: 8)
in which
Xaa ₂ and Xaa ₅ each represent a cysteine residue or Xaa ₂ and Xaa ₅ each represent a D-cysteine residue,
R ₁ is as defined above (N-terminal modification),
R ₂ represents a hydrogen atom or a C1-C3 alkyl radical, for example a methyl radical (optional substitution of the NH ₂ group of the arginine residue by a C1-C3 alkyl radical),
and the sulfur atoms of Xaa ₂ and Xaa ₅ are covalently bonded to each other by a disulfide, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2,5 -dichloro-p-xylylene,
or consist of one of its analogues capable of binding the KISS1R receptor, or of one of its salts or one of the salts of said analogue, this salt preferably being pharmaceutically acceptable.

In particular, the peptide compound according to the invention derived from KP10 may have the amino acid sequence:
R ₁ -Tyr-Cys-Trp-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 9)
in which
R ₁ represents an acetyl group,
and the sulfur atoms of the two cysteines at positions 2 and 7 are covalently bonded to each other by a disulfide, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2 ,5-dichloro-p-xylylene,
or consist of one of its analogues capable of binding the KISS1R receptor, or of one of its salts or one of the salts of said analogue, this salt preferably being pharmaceutically acceptable.

The peptide compound of sequence SEQ ID No: 9 in which the staple connecting the sulfur atoms of the cysteine residues is a p-xylylene bridge is particularly preferred in the context of the invention.

The peptide compound according to the invention derived from KP10 may in particular have the amino acid sequence:
R ₁ -Tyr-Asn-Cys-Asn-Ser-Phe-Cys-Leu-Arg(R ₂ )-Tyr-NH ₂ (SEQ ID No: 10)
in which
R ₁ represents a group of general formula (II) as defined above (N-terminal modification),
R ₂ represents a methyl group,
and the sulfur atoms of the two cysteine residues at positions 3 and 7 are covalently linked to each other by a disulfide bridge, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2 ,5-dichloro-p-xylylene, the p-xylylene bridge being particularly preferred,
or consist of one of its analogues capable of binding the KISS1R receptor, or of one of its salts or one of the salts of said analogue, this salt preferably being pharmaceutically acceptable.

Preferably, in such embodiments, R ₁ is then chosen from the groups of general formula (II) in which:
- Z represents a hexadecanoyl group,
- and/or Y represents a group of formula (IIIe) above or a group of formula (IIIa) above or a group of formula (III'a) above, n being an integer between 1 and 24, preferably comprised between 1 and 12, preferentially comprised between 1 and 6, preferentially still comprised between 2 and 4, in particular equal to 2; n ₁ being an integer comprised between 1 and 24, preferably comprised between 1 and 12, preferentially comprised between 1 and 6, preferentially still comprised between 2 and 4, in particular equal to 2; and p being an integer between 1 and 12, preferably between 3 and 8, in particular equal to 6.

R ₁ can in particular be chosen from the groups of formulas (Va), (Ve) and (V'a):

(Goes)

(F)

(Goes)

The peptide compound according to the invention can be prepared by any conventional method per se, in particular by chemical synthesis on a solid support, according to the conventional techniques of peptide synthesis, for example on solid phase in Fmoc/tBu strategy. Preferably, the chemical modification(s) of the amino acid residues of the peptide compound, in particular to form the clip between the cystein residues or between the D-cystein residues, to substitute the side chains of acid residues amines, and/or coupling the R ₁ group when the latter is not a hydrogen atom, are then produced on this same solid support. Such a characteristic advantageously simplifies the synthesis steps as well as the subsequent steps of purification of the peptide compound.

The process for preparing the peptide compound according to the invention may also comprise one or more stages of purification of the peptide compound at the end of the synthesis stages, this or these purification stage(s) possibly being carried out in any conventional manner by itself for those skilled in the art, for example by high performance liquid chromatography (HPLC).

The peptide compound according to the invention finds in particular applications in the veterinary field, within livestock, for the control of reproduction, in particular, but not limited to, with a view to programming reproduction throughout the year, by example in small ruminants, such as sheep and goats, for the resumption of postpartum cyclicity or the improvement of fertility in cattle, for the synchronization of gilt flocks, to advance puberty in young bulls, to stimulate testosterone secretion in males, especially in small ruminants such as sheep and goats, etc.

It can also be used for fertility enhancement, including ovulation induction, in endangered wildlife species kept in captivity for possible reintroduction into the wild.

Furthermore, it also finds applications in the field of human therapy, in particular to reduce infertility problems, and in particular in human clinics for the treatment of reproductive pathologies, in particular within the framework of the implementation medically assisted procreation techniques.

Thus, another aspect of the present invention relates to the use of a peptide compound according to the invention as a medicament, in particular for improving fertility in a mammal, in particular for stimulating reproduction, in particular inducing and/or synchronizing ovulation in a female mammal, or to treat infertility. This mammal can in particular be a livestock animal such as a sheep, a goat, a bovine, a pig, etc., a pet, such as a dog or a cat, a horse, etc., or even , for example a wild animal, such as one encounters in zoos and animal parks, etc. ; otherwise, this mammal can be a human.

More generally, the invention relates to the use of a peptide compound according to the invention to stimulate the KISS1R receptor, with a view to increasing the secretion of GnRH, and consequently to stimulate the release of LH hormones and/or FSH in a mammal.

The peptide compound according to the invention can in particular be used in the context of the treatment of pathological states resulting from low circulating levels of LH and FSH, for example pathological states resulting from insufficient pituitary stimulation. More generally, it can be used for the treatment of pathologies linked to a deficit activity of the reproductive axis, more particularly to a reduction in the activity of the hypothalamic-pituitary-gonad axis, such as amenorrhea of origin hypothalamic or delayed puberty and any other pathologies which require an increase in the secretion of GnRH and gonadotropins.

In the present description, the term "treatment" means obtaining a desired pharmacological and physiological effect. The term "treatment" includes the prevention or partial prevention of one or more of the symptoms of a pathology and/or the partial or total cure of a pathology and/or the total or partial disappearance of one or more of his symptoms.

Other applications of the peptide compound according to the invention are in particular the treatment of certain forms of cancer, sensitive to steroid hormones, the delay of aging by stimulation of the secretion of GnRH, or even the treatment of any other pathology associated with the functions of the kisspeptin system, for example the treatment of cystic follicular syndrome in female mammals, in particular non-humans, the treatment of polycystic ovary syndrome in women, the restoration of the libido in the case of a reduction in this ci or the treatment of certain metabolic disorders that impact reproduction, such as obesity induced by diabetes, etc.

The peptide compound according to the invention can also be used, for example, to remedy the inhibition of testosterone secretion in males after chronic treatment, in particular of hormone-dependent tumors such as encountered in prostate cancers.

The peptide compound according to the invention can be administered to any subject in need thereof. This subject may in particular be a mammal, such as a livestock or pet animal, or a human.

The administration of the peptide compound according to the invention to the subject can be carried out by any conventional route in itself, in particular by the parenteral route, for example by the subcutaneous, subdural, intravenous, intramuscular, intrathecal, intraperitoneal, intracerebral route. , intra-arterial or intra-lesional; intranasally; rectally; by the pulmonary route, for example by aerosol or inhalation; or even topically. It is preferably carried out systemically, in particular parenterally, in particular by injection, in particular intraperitoneal, intramuscular, intravenous, subcutaneous or intradermal, or orally.

The treatment can for example consist of a single injection of the compound into the subject to be treated.

The peptide compound according to the invention is preferably administered to said subject in a therapeutically effective amount.

By "therapeutically effective amount" is meant that amount of the peptide compound which, when administered to a subject, is sufficient to provide the desired treatment effect.

The therapeutically effective amount of the peptide compound according to the invention depends on several factors, such as the disease and its severity, the age, weight, etc., of the subject to be treated, the particular compound used, the route and the form of administration, etc. The therapeutically effective amount of the peptide compound according to the invention will be determined by the doctor or the veterinarian for each individual case.

By way of example, the dose of the peptide compound according to the invention administered to the treated subject can be between 1 μg and 1 mg, in particular between 1 μg and 250 μg, between 10 and 250 μg or even between 50 and 250 μg, depending on the mammal and the molecular mass of the compound. For example, for a sheep, the dose administered may be around 60 µg.

The invention is also expressed in terms of a method for improving fertility in a mammal, in particular for inducing ovulation in a female mammal, for the therapeutic treatment of pathologies linked to a reduction in the activity of the hypothalamic-pituitary-gonad axis, for treating cancers sensitive to steroid hormones and/or for delaying aging, in a subject in need thereof, this method comprising a step of administering to said subject in need thereof an amount therapeutically effective of a peptide compound according to the invention. This method can meet one or more of the characteristics described above with reference to the use of the peptide compound according to the invention as a drug.

The invention also relates to the use of a peptide compound according to the invention for the manufacture of a medicament, in particular a medicament for improving fertility in a mammal, in particular for inducing ovulation in a female mammal.

According to another aspect, the present invention relates to a veterinary or pharmaceutical composition, in particular for improving fertility in a mammal, for example inducing and/or synchronizing estrus and/or ovulation in a female mammal. This veterinary or pharmaceutical composition contains, as active principle, a peptide compound according to the invention, corresponding to one or more of the above characteristics, in a pharmaceutically acceptable vehicle.

The term "pharmaceutically acceptable" means any vehicle useful for the preparation of a pharmaceutical or veterinary composition and which is generally safe, non-toxic and neither biologically nor otherwise undesirable for the subject to be treated, in particular for mammals and in particular livestock or humans.

The vehicle of the pharmaceutical composition according to the invention can equally well be solid, semi-solid or liquid. It may be a diluent, an adjuvant or any other conventional vehicle in itself for the constitution of pharmaceutical or veterinary compositions.

This pharmaceutical or veterinary composition may be in any galenic form, in particular in a form suitable for parenteral, oral, intranasal, rectal, pulmonary or topical administration. Preferably, it is in a form suitable for parenteral administration, in particular by injection by the intraperitoneal, intramuscular, subcutaneous, intravenous or intradermal route, or in a form that can be administered orally.

The pharmaceutical or veterinary composition according to the invention may contain one or more excipients/additives conventional in themselves for the constitution of pharmaceutical or veterinary compositions, as well as, optionally, one or more other active principles, this or these principle(s) ) active ingredient(s) which may or may not act synergistically with the peptide compound according to the invention.

The pharmaceutical or veterinary composition according to the invention can be used for the therapeutic applications mentioned above, in particular for improving fertility in a mammal, in particular a livestock or companion animal or a human.

The characteristics and advantages of the invention will appear more clearly in the light of the examples of implementation below, provided purely by way of illustration and in no way limiting of the invention, with the support of figures 1 to 3, in which:

There represents general reaction schemes for the cyclization step of a process for preparing peptide compounds according to the invention, this step being carried out on a solid support (route A) or in solution (routes B and C).

There depicts a graph showing the concentration of LH measured by enzyme-linked immunosorbent (ELISA) in blood samples taken from mice pretreated to secrete low and constant levels of LH, as a function of time after injection of a compliant peptide compound to the invention C1 at different doses (1, 3 or 10 nmol) or murine peptide KP10 at a dose of 5 nmol.

There depicts a graph showing the concentration of LH measured by enzyme-linked immunosorbent (ELISA) in blood samples taken from mice pretreated to secrete low and constant levels of LH, as a function of time after injection of a compliant peptide compound to the invention C2 at different doses (1, 3 or 10 nmol) or murine peptide KP10 at a dose of 5 nmol.

Example 1 - Synthesis of Peptide Compounds

1.1/ Operating mode

General operating mode

The peptide syntheses are carried out on a solid phase using the Fmoc/ t Bu strategy, on a scale of 0.025 mmol. The solid support used is a ChemMatrix® resin functionalized by a “Rink amide” arm.

Automated peptide elongation is performed using Protein Technologies' Prelude® synthesizer. Couplings are made using 10 equivalents of protected amino acid, 9.5 equivalents of HCTU (2-(6-chloro- 1H -benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate ) and 20 equivalents of di-isopropylethylamine in N -methyl-2-pyrrolidone (NMP) for 30 min. Each coupling step is followed by an acetylation step carried out using 60 equivalents of acetic anhydride, 15.5 equivalents of di-isopropylethylamine and 1.8 equivalents of HOBt hydrate (1-hydroxybenzotriaole) for 7 min . The deprotection of the Fmoc group is carried out by three successive treatments using a 20% solution of piperidine in NMP for 3 min. The side chain protecting groups used are Arg(Pbf), Arg(Me,Pbf), Asn(Trt), Cys(Acm) or Cys(Trt), D-Cys(Acm), Ser( t Bu), Trp( Boc), Tyr( t Bu). The N-terminal acetyl groups are incorporated by coupling acetic acid following a protocol identical to that used for the protected amino acids.

The macrocyclization of the peptides by means of the clip according to the invention is carried out on a solid support (route A) or in solution (routes B and C), according to the general diagrams shown on the .

The crude macrocyclic peptide is finally purified by reverse phase high performance liquid chromatography (RP-HPLC) (Nucleosil® C18 300 Å column, 5 µm, 10x250 mm, 3mL/min, eluent A = H ₂ O + 0.1% TFA , eluent B = CH ₃ CN + 0.1% TFA). The pure macrocyclic peptide is analyzed by HPLC (Chromolith® HighResolution RP-18 column, 4.6×100 mm, 3 mL/min) and mass spectrometry (instrument: Agilent 6120, ESI+ mode). The theoretical and experimental values given for m / z correspond to the monoisotopic ion in the case of the [M+H] ⁺ peaks, and to an average of the isotopic mass in the case of the [M+2H] ²⁺ peaks.

Deprotection of groups S -acetamidomethyl (Acm)

Depending on the general synthesis mode implemented, AgBF₄(195 mg, 1 mmol, 40 eq.) are dissolved in 10 ml of an NMP/water mixture (9:1) under an argon atmosphere. The solution is then drawn into a syringe fitted with a polypropylene frit, and containing the resin bearing the peptide whose cysteine or D-cysteine residues are protected by Acm groups (0.025 mmol). The reaction medium is stirred for 2 h at room temperature. The resin is then washed successively with NMP (5 x 5 mL), pyridine (5 x 3 mL), 0.5 M sodium diethyldithiocarbamate solution in NMP (5 x 5 mL), 1 M pyridinium chloride in CH₂Cl₂/MeOH 95:5 (5 x 5 mL) then CH₂Cl₂(5x5mL).

Macrocyclization on solid support (route A)

According to the general method of synthesis implemented, in the case of staples of the bis-thioether type, 2 equivalents of a di-halogenated derivative of the staple and 2.5 equivalents of di-isopropylethylamine are dissolved in 1.5 mL of DMF under an argon atmosphere. The solution is then drawn into a syringe fitted with a polypropylene frit, and containing the resin carrying the peptide whose cysteine or D-cysteine residues are deprotected (0.005 mmol). The reaction medium is stirred for 5 h at room temperature. The resin is then washed successively with NMP (5 x 3 mL) then CH₂Cl₂(5x3mL). In the case of the methylene bridge, the dihalo derivative of the clip is diiodomethane; in the case of the cis-but-2-enylene bridge the dihalo derivative of the clip is cis-1,4-dibromo-but-2-ene and in the case of the m-xylylene, p-xylylene and 2,5-dichloro-p-xylylene, the di-halo derivative is the corresponding α,α'-dibromo-xylene compound.

The crude macrocyclic peptide is finally released from the resin with a solution of TFA/H ₂ O/ i Pr ₃ SiH/phenol, 87.5/5/2.5/5 for 2 h. The peptide is precipitated in cold Et ₂ O, centrifuged and then washed 3 times with Et ₂ O.

Macrocyclization by formation of a disulphide bridge in solution (path B)

According to the general synthesis mode implemented, in the case of disulfide bridge type staples, the peptide whose cysteine or D-cysteine residues are deprotected (0.005 mmol) is released from the resin with a solution of TFA/H ₂ O / i Pr ₃ SiH/phenol, 87.5/5/2.5/5 for 2 h. The peptide is precipitated in cold Et ₂ O, centrifuged and then washed 3 times with Et ₂ O. The crude peptide is dissolved in 5 mL of a 1:3 DMSO/water mixture and stirred at room temperature for 18 h then freeze-dried.

Macrocyclization by formation of a p-xylylene staple in solution (lane C)

Depending on the general synthesis mode implemented, in the case of staples of the p-xylylene type, the reaction can alternatively be carried out in solution. The peptide whose cysteine residues are protected by S-trityl groups (0.005 mmol) is released from the resin with a solution of TFA/H ₂ O/ i Pr ₃ SiH/phenol, 87.5/5/2.5/ 5 for 2 hours. The peptide is precipitated in cold Et ₂ O, centrifuged then washed 3 times with Et ₂ O. The crude peptide is dissolved in 2.4 mL of NMP then a solution of 1.58 mg (0.006 mmol, 1.2 equivalents) of α,α-dibromo-p-xylene and 26 μL (0.15 mmol, 30 equivalents) of di-isopropylethylamine in 100 μL of NMP are added. The reaction is stirred for 30 min at ambient temperature, then 2.87 mg (0.01 mmol, 2 equivalents) of tris -carboxyethylphosphine hydrochloride (TCEP.HCl) in 100 μl of water are added. After 30 min of additional stirring, the medium is diluted with 25 mL of an H ₂ O/MeCN/CF ₃ COOH 7:3:0.01 mixture.

1.2/ Peptide compounds according to the invention

1.2.1/ Compounds 10c , 10f, 10g, 10i, 10j

The peptide compounds of this family meet the amino acid sequence:
Ac-Tyr-Asn-Cys-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 4)
the two cysteine residues being linked to one another by a clip according to the invention. In this sequence, as in all the following ones, Ac denotes an acetyl group (N-terminal modification).

Compound 10g

The peptide compound 10g, of chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 13.5 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.39 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: m/z observed = 1425.5 ([M+H] ⁺ calculated for C _{6 6} H _{8 9} N _{1 6} O ₁₆ S ₂ = 1425.6).

Compound 10f

The peptide compound 10f, with the chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 12.8 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.37 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: m/z observed = 713.3 ([M+2H] ^{2 +} calculated for C ₆₆ H ₈₉ N ₁₆ O ₁₆ S ₂ = 713.8).

Compound 10c

The peptide compound 10c, of chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 17.0 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.48 min (gradient: 5-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: m/z observed = 668.6 ([M+2H] ²⁺ calculated for C ₅₉ H _{8 4} N ₁₆ O ₁₆ S ₂ = 668.8).

Compound 10i

The peptide compound 10i, of chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 15.9 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.73 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: m/z observed = 1492.6 ([M+H] ⁺ calculated for C ₆₆ H _{8 7} Cl ₂ N ₁₆ O ₁₆ S ₂ = 1493.5).

Compound 10d

The peptide compound 10j, with the chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 15.1 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.77 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1375.5 ([M+H] ⁺ calculated for C _{6 2} H _{8 7} N ₁₆ O ₁₆ S ₂ = 1375.5).

1.2.2/ Compound 4b

This peptide compound meets the amino acid sequence:
Ac-Cys-Asn-Trp-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 6)
the two cysteine residues being linked to each other by a clip in accordance with the invention, and has the chemical formula:

It is prepared according to route B described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 15.2 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.95 min (gradient: 5-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1344.6 ([M+H] ⁺ calculated for C ₆₀ H ₈₂ N ₁₇ O ₁₅ S ₂ = 1344.6).

1.2.3/Compounds 7b, 7c

The peptide compounds of this family meet the amino acid sequence:
Ac-D-Cys-Asn-Trp-Asn-Ser-Phe-D-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 7)
the two D-cysteine residues being linked to one another by a staple in accordance with the invention.

Compound 7c

The peptide compound 7c, of chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 15.7 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 4.18 min (gradient: 5-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1358.4 ([M+H] ⁺ calculated for C ₆₁ H ₈₄ N ₁₇ O ₁₅ S ₂ = 1358.6).

Compound 7b

The peptide compound 7b, of chemical formula:

is prepared according to route B described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 15.5 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.80 min (gradient: 5-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1344.4 ([M+H] ⁺ calculated for C ₆₀ H ₈₂ N ₁₇ O ₁₅ S ₂ = 1344.6).

1.2.4/ Compound 8g

This peptide compound responds to the amino acid sequence:
Ac-Tyr-Cys-Trp-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 9)
the two cysteine residues being linked to each other by a clip in accordance with the invention, and with the chemical formula:

It is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 12.8 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.12 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1497.5 ([M+H] ⁺ calculated for C ₇₃ H ₉₃ N ₁₆ O ₁₅ S ₂ = 1497.6).

1.3/ Comparative peptide compounds not in accordance with the invention

1.3.1/ Compounds 10k , 10l, 10m

The KP10-derived peptide compounds of this family meet the amino acid sequence:
Ac-Tyr-Asn-Cys-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 11)
the two cysteine residues being linked to one another by a clip not in accordance with the invention (2,5-dimethoxy-p-xylylene, ethylene or 2,5-dimethyl-p-xylylene).

Compound 10k

The 10k peptide compound, whose staple is a 2,5-dimethoxy-p-xylylene group, with the chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 16.2 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.39 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1484.8 ([M+H] ⁺ calculated for C ₆₈ H ₉₃ N ₁₆ O ₁₈ S ₂ = 1485.6).

Compound 10l

The peptide compound 10l, whose staple is an ethylene group, with the chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 10.2 min (gradient: 30-80% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 2.49 min (gradient: 30-60% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1349.4 ([M+H] ⁺ calculated for C ₆₀ H ₈₅ N ₁₆ O ₁₆ S ₂ = 1349.6).

Compound 10m

The peptide compound 10m, whose staple is a 2,5-dimethyl-p-xylylene group, with the chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 15.2 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.50 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1452.8 ([M+H] ⁺ calculated for C ₆₈ H ₉₃ N ₁₆ O ₁₆ S ₂ = 1453.6).

1.3.2/Compound 4th

This peptide compound derived from KP10 meets the amino acid sequence:
Ac-Cys-Asn-Trp-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 12)
the two cysteine residues being linked to each other by an o-xylylene clip not in accordance with the invention, and with the chemical formula:

is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 13.6 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.19 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1448.5 ([M+H] ⁺ calculated for C ₆₈ H ₉₀ N ₁₇ O ₁₅ S ₂ = 1448.5).

1.3.3/Compound 5b

This peptide compound derived from KP10 responds to the amino acid sequence:
Ac-D-Cys-Asn-Trp-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 13)
the cysteine and D-cysteine residues being linked to each other by a disulphide bridge, and to the chemical formula:

It is prepared according to route B described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 16.2 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 4.08 min (gradient: 5-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1344.5 ([M+H] ⁺ calculated for C ₆₀ H ₈₂ N ₁₇ O ₁₅ S ₂ = 1344.6).

1.3.4/Compound 6b

This peptide compound derived from KP10 meets the amino acid sequence:
Ac-Cys-Asn-Trp-Asn-Ser-Phe-D-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 14)
the cysteine and D-cysteine residues being linked to each other by a disulphide bridge, and to the chemical formula:

It is prepared according to route B described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 16.2 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.96 min (gradient: 5-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1344.6 ([M+H] ⁺ calculated for C ₆₀ H ₈₂ N ₁₇ O ₁₅ S ₂ = 1344.5).

1.3.5/Compound 9g

This peptide compound derived from KP10 responds to the amino acid sequence:
Ac-Tyr-D-Cys-Trp-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 15)
the cysteine and D-cysteine residues being linked to each other by a p-xylylene bridge, and to the chemical formula:

It is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 11.6 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 2.99 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1497.5 ([M+H] ⁺ calculated for C ₇₃ H ₉₃ N ₁₆ O ₁₅ S ₂ = 1497.6).

1.3.6/Compound 11g

This peptide compound derived from KP10 responds to the amino acid sequence:
Ac-Tyr-Asn-D-Cys-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 16)
the cysteine and D-cysteine residues being linked to each other by a p-xylylene bridge, and to the chemical formula:

It is prepared according to route A described above, following the general procedure. Purification by semi-preparative HPLC: t _R = 13.15 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.22 min (gradient: 20-50% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1425.4 ([M+H] ⁺ calculated for C ₆₆ H ₈₉ N ₁₆ O ₁₆ S ₂ = 1425.6).

Example 2 - In vitro test of mobilization of extracellular calcium

The peptide compounds prepared in example 1 are subjected to an in vitro test of mobilization of extracellular calcium, according to the following protocol.

The HEK293A cell line (ATCC, American Type Culture Collection – CRL-1573) was stably transfected with the human KISS1R receptor (KISS1 receptor (KISS1R) mRNA, Homo sapiens , GenBank accession number: NM_032551.5). For transfection, the pcDNA3.1 vector (Invitrogen) was used, in which the human KISS1R sequence was inserted with a hemagglutinin (HA) tag fused to its 5' end. Transfection and clone selection were performed as described in the literature (Mancini et al. 2009, British Journal of Pharmacology , 158(1): 382-391).

The KISS1R receptor is coupled to Gq proteins and its activation produces an increase in the intracellular concentration of calcium. To verify the agonist activity and measure the median KISS1R receptor activation concentration (EC ₅₀ ) of the peptide compounds tested, the following protocol was implemented.

The HEK293A cells expressing the human KISS1R receptor were seeded in a 96-well plate (μclear® black plate) at a concentration of 40,000 cells/well, and placed in an incubator at 37°C. After 48 h, the medium (DMEM with GlutaMAX® and without pyruvate, 10% fetal calf serum, 1% penicillin, 1% streptomycin, 200 μg/mL Geneticin, and HEPES buffer pH 7.4 (25 mM)) was changed and the cells were incubated with the fluorescent dye Fluo-4NW, according to the manufacturer's instructions (Molecular Probes).

To avoid adhesion of the peptide compound to the plastic, the compounds to be tested were prediluted in a so-called “non-binding” plate (Corning), at a concentration 20 times greater than the desired final concentration (20X solution).

After measuring the basal fluorescence, 5 μl of the 20X solution containing the compound to be tested were added to each well (containing 95 μl) so as to obtain the desired concentration. Fluorescence variations were recorded every 7 s for 5-7 min with a Polarstar® Optima plate reader (BMG Labtech). Activity-concentration curves were generated using GraphPad Prism 5 software, and the EC ₅₀ of each peptide compound was calculated by fitting the curve to a sigmoid.

For comparison, the murine KP10 peptide was also tested.

The results obtained are shown in Table 1 below.

Compound according to the invention _EC50 (nM) Comparative compound _EC50 (nM) 10g 1.1 10l 52 10f 12 10k 66.1 10c 33 4th >100 10i 10 5b >100 10d 27 6b >100 4b 4.7 9g >100 7c 17 11g >100 7b 24 KP10 4.1 8g 36 - -

All of the compounds in accordance with the invention have EC _50s that are much lower than those of the comparative compounds not in accordance with the invention, which clearly demonstrates that they have a particularly strong capacity for activating the KISS1R receptor. In particular, compound 10g shows an activity even more potent than that of KP10.

Example 3 - Synthesis of peptide compounds in accordance with the invention with a motif capable of binding serum albumin

The following two peptide compounds in accordance with the invention are prepared.

Compound C1

Peptide compound C1 derived from KP10 has the amino acid sequence:
R ₁ -Tyr-Asn-Cys-Asn-Ser-Phe-Cys-Leu-Arg(Me)-Tyr-NH ₂ (SEQ ID No: 17)
in which the sulfur atoms of the two cysteine residues are linked by a p-xylylene bridge, Me represents, in a manner conventional per se, a methyl group substituting the -NH ₂ group of the side chain of the arginine residue, and R ₁ represents a group comprising a hexadecanoyl unit, capable of binding serum albumin, coupled via a γ-glutamyl spacer arm to the N-terminal end of the peptide compound, R ₁ thus exhibiting a gamma-( N -hexadecanoyl) group -Glu-OH) (N-terminal modification), of formula (Ve):

(F)

Compound C1 thus has the chemical formula:

This peptide compound is prepared according to route A described in Example 1, following the general procedure. Prior to the deprotection of the Acm groups and the macrocyclization on a solid support using α,α'-dibromo-m-xylene to form the m-xylylene bridge, the γ-glutamyl residue is introduced by coupling of the protected amino acid Fmoc-Glu-O t Bu according to the general procedure, followed by the coupling of palmitic acid according to a slightly modified protocol for reasons of solubility: this reaction is carried out in a 1:3 mixture of NMP and dichloromethane rather than in NMP, for 2 h rather than 30 min. Purification by semi-preparative HPLC: t _R = 14.6 min (gradient: 50-90% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.94 min (gradient: 50-90% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: observed m/z =1764.8 ([M+H] ⁺ calculated for C ₈₆ H ₁₂₆ N ₁₇ O ₁₉ S ₂ = 1764.9.).

This compound, tested according to the protocol described in Example 2, has an EC ₅₀ of 45 nM.

Compound C2

The C2 peptide compound derived from KP10 has the amino acid sequence:
R₁-Tyr-Asn-Cys-Asn-Ser-Phe-Cys-Leu-Arg(Me)-Tyr-NH₂ (SEQ ID No: 18)
wherein the sulfur atoms of the two cysteine residues are linked by a p-xylylene bridge and R₁represents a hexadecanoyl unit, capable of binding serum albumin, coupled via a spacer arm with a γ-glutamyl group and ethylene glycol groups at the N-terminal end of the peptide compound (N-terminal modification), R₁having the formula (Va):

(Goes)

Compound C2 thus has the chemical formula:

This peptide compound is prepared according to route A described in Example 1, following the general procedure. Prior to the deprotection of the Acm groups and the macrocyclization on a solid support using α,α'-dibromo-m-xylene to form the m-xylylene bridge, an Ebes spacer arm, then a γ-glutamyl residue are introduced by the successive couplings of the Fmoc-Ebes-OH derivative ( N -[8-(9-fluorenylmethyloxycarbonyl)amino-3,6-dioxaoctyl]succinamic acid) then of the protected amino acid Fmoc-Glu-O t Bu according to the general procedure , followed by the coupling of palmitic acid according to a slightly modified protocol to ensure its solubility: this reaction is carried out in a 1:3 mixture of NMP and dichloromethane rather than in NMP, for 2 h rather than 30 min. Purification by semi-preparative HPLC: t _R = 11.6 min (gradient: 50-90% MeCN/H ₂ O + 0.1% TFA in 20 min); Analytical HPLC: t _R = 3.71 min (gradient: 50-90% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: m/z observed = 998.7 ([M+2H] ²⁺ calculated for C ₉₆ H ₁₄₅ N ₁₉ O ₂₃ S ₂ = 998.4.).

This compound, tested according to the protocol described in Example 2, has an EC ₅₀ of 15 nM.

Example 4 - Tests in mice

All experiments were performed on 9-week-old female mice of the C57BL/6 strain in accordance with European (directive 2010/63/EU) and French (decree no. 2013–118) legislation on the use of animals for research.

Mice were housed 3-4 per cage in a light-controlled environment (12 h light and 12 h dark) and with ad libitum access to food and water.

Mice were ovariectomized following a standard procedure and implanted with a capsule containing sufficient estradiol to induce a plasma level of 4-7 pg/mL necessary to maintain negative feedback on luteinizing hormone LH secretion ( Quennell et al., 2011, Endocrinology 152(4):1541-50). This maintains constant low levels of LH making it easy to see stimulation of its secretion.

The surgery was performed under anesthesia induced by a solution of ketamine (80 mg/kg) and medetomidine (1 mg/kg) diluted in sterile physiological solution (0.25 mL/100 g of weight).

At the beginning of the surgery 0.05 mg/mL of buprenorphine (1 µL/gram of weight) was injected subcutaneously and at the end of the surgery the mice were injected with atipamezole (Antisedan® 0.015 mL /100 grams of weight).

The mice were injected intraperitoneally with the peptide compounds in accordance with the invention C1 and C2 at three different doses: 1, 3 or 10 nmol (n=3 per dose), as well as with the murine peptide KP10 at the dose of 5 nmol for comparison, and blood samples (4 μL) were collected from the tip of the tail every 20 min from 40 min before the injection of the compounds and until 10 h after. Collected samples were immediately mixed with phosphate buffered saline and frozen on dry ice.

The concentration of LH, used as a biomarker of the activation of the gonadotropic axis, was carried out by adapting an immuno-enzymatic ELISA method as described in the publication by Steyn et al., 2013, Endocrinology 154(12) :4939-45. A monoclonal antibody against bovine LH (LHβ, 518 B7, 1:1,000, from the University of California Davis) was used to capture LH and a polyclonal rabbit against LH (AFP240580Rb, AF Parlow) was used to capture LH. was used for detection. The standard was the LH mouse NIDDK (AF Parlow AFP5306A mouse RIA kit). The detection limit was 0.2 ng/mL and the coefficient of variation averaged 11%.

The 96-well plates were incubated with the LHβ 518 B7 antibody overnight at 4°C, the antibody was removed and 200 µL of blocking buffer (5% skimmed milk powder) was added to each well for 2 hours at room temperature. Then the samples were deposited in each well in duplicate, left at room temperature for 2 h, then the AFP240580Rb antibody was added to each well and left to incubate for 90 min. A polyclonal secondary antibody coupled to horseradish peroxidase was then added, the mixture was incubated for 90 min, before development with the substrate OPD (o-phenylenediamine, 100 µL) diluted in citrate buffer containing H ₂ O ₂ (6µL). After 30 min of incubation the reaction was quenched with 3 M HCl (50 µL) and the plate read at 490 and 650 nm. LH concentration time course curves were generated using GraphPad Prism 5 software.

These curves are shown on the for compound C1, at the 3 doses tested, and on the for compound C2, also at the 3 doses tested. It is clearly observed in these figures, for each of the two compounds, a strong effect on the secretion of LH, this effect increasing as a function of the increase in the dose of compound. At concentrations of 3 and 10 nmol, the increase in LH induced by either of the compounds according to the invention is still present 10 hours after the injection. These results clearly show the ability of compounds C1 and C2 in accordance with the invention to stimulate the hypothalamic-pituitary axis over a long period. In comparison, the action of the murine peptide KP10 is exerted over a much shorter duration.

Example 5 - Peptide compound in accordance with the invention with a motif capable of binding serum albumin

The C3 peptide compound derived from KP10 has the amino acid sequence:
R₁-Tyr-Asn-Cys-Asn-Ser-Phe-Cys-Leu-Arg(Me)-Tyr-NH₂ (SEQ ID No: 19)
wherein the sulfur atoms of the two cysteine residues are linked by a p-xylylene bridge and R₁represents a hexadecanoyl unit, capable of binding serum albumin, coupled via a spacer arm with a γ-glutamyl group, lysine residues and ethylene glycol groups at the N-terminal end of the peptide compound (N-terminal modification ), R₁having the formula (V’a):

(Goes)

The compound C3 in accordance with the invention thus has the chemical formula:

This peptide compound is prepared according to route C described above, following the general procedure, according to which the cysteines are introduced protected by a trityl group, and the macrocyclization is carried out in solution after release of the peptide from the resin and deprotection, by reaction with α,α-dibromo-p-xylene. Prior to the resin release step, an Ebes spacer arm, 6 lysine residues, a second Ebes spacer arm, then a γ-glutamyl residue are introduced by successive couplings of the derivative Fmoc-Ebes-OH then Fmoc-Lys( Boc)-OH (repeated six times), then the Fmoc derivative Ebes-OH, then the protected amino acid Fmoc-Glu-O t Bu, according to the general procedure, followed by the coupling of palmitic acid according to a slightly modified to ensure its solubility: this reaction is carried out in a 1:3 mixture of NMP and dichloromethane rather than in NMP, for 2 h rather than 30 min.

Purification by semi-preparative HPLC: t _R = 18.6 min (gradient: 40-60% MeCN/H ₂ O + 0.1% TFA in 30 min); Analytical HPLC: t _R = 2.52 min (gradient: 40-60% MeCN/H ₂ O + 0.1% TFA in 5 min); UV detection (λ= 214 and 280 nm); MS: m/z observed = 1498.0 ([M+2H] ²⁺ calculated for C ₁₄₂ H _{23 5} N ₃₃ O _{3 3} S ₂ = 1498.4).

This compound, tested according to the protocol described in Example 2 above, has an EC ₅₀ of 1.2 nM.

It has improved solubility in water compared to compounds C1 and C2 (it is in particular soluble in water at a concentration as high as 10 mg/ml, i.e. 2.64 nM), and good performance in terms of ability to stimulate the hypothalamic-pituitary axis over a long period.

Claims (15)

Peptide compound agonist of the KISS1R receptor, characterized in that it is chosen from:
- a compound of formula (I):
R₁-Xaa₁-Xaa₂-Xaa₃-Asn-Xaa₄-Phe-Xaa₅-Xaa₆-Xaa₇-Xaa₈-NH₂(I)
in which
Xaa₅and one of the residues Xaa₁, Xaa₂or Xaa₃, both simultaneously represent either a cysteine residue or a D-cysteine residue, the sulfur atoms of said cysteine residues or of said D-cysteine residues being covalently linked to each other by a disulphide, methylene,cis-but-2-enylene, m-xylylene, p-xylylene or 2,5-dichloro-p-xylylene,
when they do not represent the said cysteine or D-cysteine residue: Xaa₁represents a tyrosine, D-tyrosine or lysine residue, Xaa₂is zero or represents an asparagine or lysine residue and Xaa₃represents a tryptophan, lysine, proline or hydroxyproline residue,
Xaa₄represents a serine or threonine residue,
Xaa₆represents a leucine residue or a similar aliphatic α-amino acyl residue,
Xaa₇represents an arginine residue, whose –NH function₂is optionally substituted by a C1-C3 alkyl radical, or a similar positively charged α-amino acyl residue,
Xaa₈represents a tyrosine, phenylalanine, tryptophan residue or an α-amino acyl residue of the analogous aryl alanine type,
Asn represents an asparagine residue,
Phe represents a phenylalanine residue,
R₁represents a hydrogen atom, an acetyl group, an alkanoyl group, a benzoyl group or a group of general formula (II):
[Chem 1]
(II)
in which Y represents a covalent bond or a spacer arm and Z represents a unit capable of binding to serum albumin,
- an analog of said compound of formula (I) capable of binding the KISS1R receptor,
- or one of their salts. Peptide compound according to Claim 1, in which, in the general formula (II), Y represents a linear, branched and/or cyclic, saturated or unsaturated, optionally substituted hydrocarbon-based radical interrupted by one or more heteroatoms and/or one or more groups comprising at least one heteroatom. Peptide compound according to Claim 1 or 2, in which Y comprises a γ-glutamyl group. Peptide compound according to any one of Claims 1 to 3, in which, in the general formula (II), Y contains one or more ethylene glycol groups. Peptide compound according to any one of Claims 1 to 4, in which, in the general formula (II), Y represents a group of formula (III):
[Chem 14]
(III)
in which R ₃ represents a covalent bond to the amino acid residue of said peptide compound or a group of formula (IV):
[Chem 7]
(IV)
in which
m ₁ is equal to 1 or 2,
m ₂ is equal to 0 or 1,
m ₃ is equal to 1 or 2,
n is an integer between 1 and 24
and Y ₁ represents an oxygen atom or an amide group. Peptide compound according to any one of Claims 1 to 5, in which, in the general formula (II), Z represents a hexadecanoyl group. Peptide compound according to any one of Claims 1 to 6, in which, in formula (I), when R ₁ does not represent a group of general formula (II), Xaa ₁ , Xaa ₂ and/or Xaa ₃ , s it does not represent a cysteine or D-cysteine residue, is substituted on its side chain by a group of general formula (II) as defined in one of claims 1 to 6. Peptide compound according to any one of Claims 1 to 7, of amino acid sequence:
R ₁ -Tyr-Asn-Xaa ₃ -Asn-Ser-Phe-Xaa ₅ -Leu-Arg(R ₂ )-Tyr-NH ₂ (SEQ ID No: 3)
in which
Xaa ₃ and Xaa ₅ each represent a cysteine residue or Xaa ₃ and Xaa ₅ each represent a D-cysteine residue,
R ₁ is as defined in one of claims 1 to 6,
R ₂ represents a hydrogen atom or a C1-C3 alkyl radical,
and the sulfur atoms of Xaa ₃ and Xaa ₅ are covalently bonded to each other by a disulfide, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2,5 -dichloro-p-xylylene. Peptide compound according to any one of Claims 1 to 8, of amino acid sequence:
R ₁ -Tyr-Asn-Cys-Asn-Ser-Phe-Cys-Leu-Arg-Tyr-NH ₂ (SEQ ID No: 4)
in which
R ₁ represents an acetyl group,
and the sulfur atoms of the two cysteine residues are covalently linked to each other by a disulfide bridge, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2,5-dichloro- p-xylylene. Peptide compound according to any one of Claims 1 to 8, of amino acid sequence:
R ₁ -Tyr-Asn-Cys-Asn-Ser-Phe-Cys-Leu-Arg(R ₂ )-Tyr-NH ₂ (SEQ ID No: 10)
in which
R ₁ represents a group of general formula (II) as defined in one of claims 1 to 6,
R ₂ represents a methyl group,
and the sulfur atoms of the two cysteine residues are covalently linked to each other by a disulfide bridge, methylene, cis -but-2-enylene, m-xylylene, p-xylylene or 2,5-dichloro- p-xylylene. Peptide compound according to any one of Claims 1 to 10, for its use as a medicament. Peptide compound for its use according to claim 11, for the improvement of fertility in a mammal. Peptide compound for its use according to claim 11, for the treatment of pathologies linked to a reduction in the activity of the hypothalamic-pituitary-gonad axis. Peptide compound for its use according to claim 11, for the treatment of cancers sensitive to steroid hormones or for delaying ageing. Pharmaceutical or veterinary composition, in particular for improving fertility in a mammal, characterized in that it contains a peptide compound according to any one of Claims 1 to 10 in a pharmaceutically acceptable vehicle.