Title of Invention

"LH-RH PEPTIDE ANALOGUES"

Abstract A LH-RH peptide analogue of the formula (SEQ ID N°: 1): in which: - A1 is pGlu; D-pGlu; Sar; AcSar; Pro; AcPro; ForPro; OH-Pro; Ac-OH-Pro; dehydro-Pro; Ac-dehydro-Pro; Ser; D-Ser; Ac-D-Ser; Thr; D-Thr; Ac-D-Thr; or an aromatic D-amino acid which may be acylated; - A2 is a direct bond; His ; or an aromatic D-amino acid; - A3 is an aromatic L- or D-amino acid; - A4 is Ala, Ser, D-Ser, MeSer, Ser(OBut), Ser(OBzl) or Thr; - A5 is an aromatic L-amino acid or a basic L- or D-amino acid.
Full Text LH-RH peptide analogues, their uses and pharmaceutical compositions containing them
This invention relates to LH-RH peptide analogues, to their use and to pharmaceutical compositions m which they are present
LH-RH, or luteinizing hormone-releasing hormone, is a neurohumoral hormone produced in the hypothalamus which stimulates the secretion of the gonadotrophins, LH (luteinizing hormone) and FSH (follicle-stimulating hormone), which in turn regulate the endocnne and exocnne functions of the ovary in the female, and of the testis in the male. It has the following structural formula :
1234567 89 10 pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2
Histoncally (Karten and Rivier, Endocr. Rev., 1986, 7(1), 44-66), synthetic improvement of LH-RH activity has been achieved first, by replacement of the C-terminal glycmamide by an ethylamide directly bound to Pro', and then, by introduction of D-Ala in position 6. Both independent breakthroughs yielded analogs each about 5 times more active than LH-RH. All therapeutically useful agonists result from further major improvement in position 6 with the introduction of hydrophobic aliphatic or aromatic D-ammo acids instead of D-Ala, with or without the combined Pro'-N-ethylamide modification. On this C-termmal end, only slight improvements have been obtained with fluorinated amides or with azaglycinamide. Replacement of Trp in position 3 by INal has been reported (Karten and Rivier, 1986, cf above) to give an agonist twice as potent as LH-RH, without further synthetic or therapeutic developments.
The only other individual ammo acid modification noticed to increase the biological activity of some agonists was found in position 7. Thus, N-methylation of Leu7 in LH-RH itself did not increase its potency, but enhanced the activity of some already potent synthetic agonists with certain D-amino acids in position 6 such as D-Trp (Karten and Rivier, 1986, cf above) , fijrthermore, charged and bulkier L-amino acids than leucine (Ser(OBut), Asp(O-But), Glu(O-But), BocLys) somewhat improved the activity of [des-Gly'"; Pro'-N-ethylamide]-LH-RH but reduced the potency of 6-modified agonists (Karten and Rivier, 1986, cf above).
As far as antagonists are concerned, numerous modifications in all positions but Pro9, and a wide variety of combinations among them, have been tned with unequal success to achieve inhibition of endogenous LH-RH activity (Dutta, Drugs of the Future,
1988,11(8), 761-787 ; Karten and Rivier, Endoc. Rev., 1986, 7(1), 44-66). For example, antide, a standard potent LH-RH antagonist, results from amino acid changes in positions 1, 2, 3, 5, 6, 8 and 10. N-methylation of Leu7 brought about a decrease in potency, and the only changes m this position reported to increase it (2-fold maximum)
were the replacement of Leu by Trp7 or Phe7.
It has now been found that the replacement of Leu7 by highly hydrophobic amino acids, increases the activity of LH-RH itself or of known highly active analogues (agonists or antagonists) of LH-RH.
Especially, it has been found that the replacement of Leu7 by adamantylalanine (Ada) or neopentylglycine (Npg) increases the activity of LH-RH itself and makes it possible to obtain analogs with a high affinity for the LH-RH receptors. More specifically, the [Npg']-LH-RH analogues of this invention are potent LH-RH agonists / antagonists in vivo.
Thus, according to one aspect of the present invention, LH-RH peptide analogues with high affinity for the LH-RH receptors are provided, m which a non-aromatic hydrophobic ammo acid having from 7 to 20 carbon atoms, such as for example Ada' or, preferably, Npg' is substituted for Leu7. Preferably these peptide analogues are of the formula (SEQ ID N° : 1).
A1 -A2-A3-A4-A5-A6-HAA-A7-Pro-Z (I)
in which:
- Al is pGlu ; D-pGlu ; Sar ; AcSar ; Pro or a derivative thereof such as AcPro, ForPro, OH-Pro, Ac-OH-Pro, dehydro-Pro or Ac-dehydro-Pro ; Ser; D-Ser; Ac-D-Ser ; Thr ; D-Thr ; Ac-D-Thr ; or an aromatic D-amino acid which may be acylated, such as D-Phe, D-HPhe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-Bal, D-Pal, D-4Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups;
- A2 is a direct bond ; His ; or an aromatic D-amino acid such as D-Phe, D-HPhe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-Bal, D-Pal, D-4Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups.
- A3 is an aromatic L- or D-amino acid such as Phe, HPhe, Tyr, Trp, Nal, INal, diphenyl-Ala, Bal, Pal, 4Pal or Qal, where Phe and Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups;
- A4 is Ala, Ser, D-Ser, MeSer, Ser(OBut), Ser(OBzl) or Thr;
- A5 is an aromatic L-amino acid such as Phe, HPhe, Tyr, Trp, Nal, INal, diphenyl-Ala, Bal, Pal, 4Pal or Qal, where Phe and Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C]-C4)alkoxy, nitro or tnfluoromethyl groups ; or a basic L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg and HArg may be N-substituted by a (C1-C6)alkyl or a (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a nicotinoyl, isonicotmoyl, 6-methyl-nicotinoyl, glycyl-mcotinoyl, nicotinyl-azaglycyl, furyl, glycyl-furyl, furyl-azaglycyl, pyrazinyl, pyrazinyl-carbonyl, picolmoyl, 6-methyl-picolinoyl, shikimyl, shikimyl-glycyl, Fmoc or Boc group;
- A6 is Gly ; D-Pro ; D-Ser ; D-Thr ; D-Cys , D-Met; D-Pen ; D-(S-Me)Pen ; D-(S-Et)Pen ; D-Ser(OBut) ; D-Asp(OBut) ; D-Glu(OBut) ; D-Thr(OBut) ; D-Cys(OBut) , D-Ser(ORi) where Ri is a sugar moiety ; an aza-amino acid such as azaGly or azaAla , D-His which may be substituted on the imidazole nng by a (C1-C6)alkyl or by a (C2-C7)acyl group ; an aliphatic D-amino acid with a (C1-C8)alkyl or a (C3-C6)cycloalkyl side chain such as D-Ala, D-Abu, D-Aib, D-3Aib, D-Val, D-Nva, D-Leu, D-IIe, D-Tle, D-Nle, D-Hol, D-Npg, D-CPa, D-Cpa, D-Cba or D-Cha ; an aromatic D-amino acid such as D-Phe, D-HPhe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-anthryl-Ala, D-phenanthryl-Ala, D-benzhydryl-Ala, D-fluorenyl-Ala, D-Bal, D-Pal, D-4Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups , D-cyclohexadienyl-Gly ; D-perhydronaphthyl-Ala ; D-perhydrodiphenyl-Ala , or a basic L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg and HArg may be N-substituted by a (C1-C6)alkyl or a (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a nicotmoyl, isonicotinoyl, 6-methyl-nicotinoyl, glycyl-nicotmoyl, nicotinyl-azaglycyl, fiiryl, glycyl-furyl, furyl-azaglycyl.
pyrazinyl, pyrazinyl-carbonyl, picohnoyl, 6-methyl-picolinoyl, shikimyl, shikimyl-glycyl, Fmoc or Boc group,
- HAA is a non-aromatic hydrophobic amino acid of from 7 to 20 carbon atoms;
- A7 is a basic L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg or HArg may be N-substituted by a (C1-C6)alkyl or a (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe or ACha may be N-substituted by one or two (C|-C6)alkyl or (C3-C6)cycloalkyl groups, or by a nicotinoyl, isomcotinoyl, 6-methyl-nicotinoyl, glycyl-mcotinoyl, nicotinyl-azaglycyl, furyl, glycyl-furyl, furyl-azaglycyl, pyrazinyl, pyrazinyl-carbonyl, picohnoyl, 6-methyI-picoIinoyI, shikimyl, shikimyl-glycyl, Fmoc or Boc group;
- Z is GlyNH2 ; D-AlaNHi , azaGlyNHi ; or a group -NHR2 where R2 is a (C1-C4)alkyl which may be substituted by an hydroxy or one or several fluorine atoms, a (C3-C6)cycloalkyl, or a heterocyclic radical selected from morpholinyl, pyrrohdinyl and pipendyl,
as well as their pharmaceutically acceptable salts
hi these peptide analogues, HAA is preferably Ada or Npg which may be N-alpha-substituted by a (C1-C4)-alkyl group optionally substituted by one or several fluonne atoms, Npg being especially preferred.
A preferred group of peptide analogues (I) comprises the peptides of the formula (SEQ ID N° • 6)
A1 -A2-A3-A4-A5-A6-Npg-A7-Pro-Z (!')
in which .
- Al is pGlu ; D-pGlu ; Sar , AcSar , Pro or a derivative thereof such as AcPro, ForPro, OH-Pro, Ac-OH-Pro, dehydro-Pro or Ac-dehydro-Pro ; Ser ; D-Ser , Ac-D-Ser , Thr , D-Thr ; Ac-D-Thr , or an aromatic D-ammo acid which may be acylated, such as D-Phe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-Bal, D-Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups;
- A2 is a direct bond , His ; or an aromatic D-amino acid such as D-Phe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-Bal, D-Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups;
- A3 is an aromatic L-'or D-amino acid such as Phe, Tyr, Trp, Nal, INal, diphenyl-Ala, Bal, Pal or Qal, where Phe and Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C)-C4)alkoxy, nitro or trifluoromethyl groups,
- A4 is Ala, Ser, D-Ser, MeSer, Ser(OBut), Ser(OBzl) or Thr;
- A5 is an aromatic L-amino acid such as Phe, Tyr, Trp, Nal, INal, diphenyl-Ala, Bal, Pal or Qal, where Phe and Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups ; or a basic L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg and HArg may be N-substituted by a (C1-C6)alkyl or a (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a nicotinoyl, isonicotinoyl, 6-methyl-nicotinoyl, glycyl-nicotinoyl, mcotinyl-azaglycyl, furyl, glycyl-furyl, fiiryl-azaglycyl, pyrazinyl, pyrazinyl-carbonyl, picolmoyl, 6-methyl-picolinoyl, shikimyl, shikimyl-glycyl, Fmoc or Boc group;
- A6 is Gly ; D-Pro , D-Ser ; D-Thr ; D-Cys , D-Met; D-Pen ; D-(S-Me)Pen ; D-(S-Et)Pen ; D-Ser(OBut) ; D-Asp(OBut) ; D-Glu(OBut) ; D-Thr(OBut) ; D-Cys(OBut) ; D-Ser(ORi) where Ri is a sugar moiety ; an aza-amino acid such as azaGly or azaAla ; D-His which may be substituted on the imidazole ring by a (C1-C6)alkyl or by a (C2-C7)acyl group ; an aliphatic D-amino acid with a (C1-Cg)alkyl or a (C3-C6)cycloalkyl side chain such as D-Ala, D-Abu, D-Aib, D-3Aib, D-Val, D-Nva, D-Leu, D-Ile, D-Tle, D-Nle, D-Hol, D-Npg, D-CPa, D-Cpa, D-Cba or D-Cha ; an aromatic D-amino acid such as D-Phe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-anthryl-Ala, D-phenanthryl-Ala, D-benzhydryl-Ala, D-fluorenyl-Ala, D-Bal, D-Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups ; D-cyclohexadienyl-Gly , D-perhydronaphthyl-Ala , D-perhydrodiphenyl-Ala , or a basic L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg and HArg may be N-substituted by a (C1-C6)alkyl or a (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a nicotinoyl, isonicotinoyl, 6-methyl-nicotinoyl, glycyl-nicotmoyl, mcotinyl-azaglycyl, furyl, glycyl-furyl, furyl-azaglycyl.
pyrazmyl, pyrazmyl-carbonyl, picolmoyl, 6-methyl-picolinoyl, shikimyl, shikimyl-glycyl, Fmoc or Boo group,
- Npg may be N-alpha-substituted by a (C1-C4)alkyl group which may be
substituted by one or several fluorine atoms;
- A7 is a basic L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg or HArg may be N-substituted by a (C1-C6)alkyl or a (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe or ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a nicotinoyl, isonicotmoyl, 6-methyl-nicotinoyl, glycyl-nicotmoyl, nicotinyl-azaglycyl, ftiryl, glycyl-iuryl, furyl-azaglycyl, pyrazinyl, pyrazinyl-carbonyl, picolinoyl, 6-methyl-picolinoyl, shikimyl, shikimyl-glycyl, Fmoc or Boc group;
- Z is GlyNH2 , D-AlaNH2 ; azaGlyNHi ; or a group -NHR2 where R2 is a (C1-C4)alkyl which may be substituted by an hydroxy or one or several fluonne atoms, a (C3-C6)cycloalkyl, or a heterocyclic radical selected from morpholmyl, pyrrolidmyl and piperidyl;
as well as their pharmaceutically acceptable salts.
hi the present descnption the term "(C1-C4)alkyr' denotes methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl groups
The term "(C1-Cgjalkyl" denotes methyl, ethyl, n-propyl, i-propyl, n-butyl, 1-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl and hexyl groups.
The term "(C1-C8)alkyl" denotes methyl, ethyl, n-propyl, i-propyl, n-butyl, 1-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, s-pentyl, t-pentyl, hexyl, heptyl and octyl groups;
The term "(C1-C4)alkoxy" denotes a group -OR where R is a (C1-C4)alkyl.
The term "(C2-C7)acyr' denotes a group -COR where R is a (C1-C6)alkyl.
The term "(C3-C6)cycloalkyl" denotes cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups.
The term "sugar moiety" denotes D- or L-pentoses or hexoses and their amino-derivatives.
The term "LH-RH analogues" denotes peptides in which at least one amino acid has been modified in the sequence of LH-RH
The term "non-aromatic hydrophobic amino acid" denotes a hnear, branched or cyclic amino acid with a side chain of from 5 to 18, more preferably 5 to 11 carbon atoms (beginning at the ß-carbon included) ; the hydrophobic nature of a suitable ammo-acid can be defined by a positive difference of at least 0.5 when compared with leucine, in either log P (P partition coefficient in the n-octanol/water system) or the Hansch hydrophobicity constant n.
In the present descnption and in the claims, the following abbreviations are used
Abu : 2-aminobutyric acid Ac acetyl
ACha . ammocyclohexylalanine Aib 2-aminoisobutync acid
3Aib • 3-aminoisobutync acid Ala : alanine
AlaNH2: alaninamide APhe p-ammophenylalanine
Arg : argmine Asp . aspartic acid
azaAla aza-alanme azaGly aza-glycine
azaGlyNH2 . azaglycmamide Bal benzothienylalanine
Boc . tert-butoxycarbonyl Cba : cyclobutylalanme
Cha cyclohexylalanme Cit citruUine
CPa : cyclopropylalanme Cpa cylopentylalanine
Fmoc : fluorenylmethoxycarbonyl For : formyl
Glu glutamic acid Gly glycine
GlyNH2 : glycmamide HArg . homoarginine
HCit homocitrulline His : histidine
HLys : homolysine Hoi homoleucine
He : isoleucme IprLys : NE-isopropyllysine
Leu leucine Lys lysine
MeSer • N-methylsenne Met. methionine
Nal 3-(2-naphtyl)alanine INal 3-(l-naphtyl)alamne
NEt. N-ethylamide NicLys NE-mcotinoyllysine
Nle norleucine Npg neopentylglycine
Nva norvaline OBu' tert-hutoxy
OBzl. benzyl ester Om ornithine
Pal 3-(3-pyndyl)alanine pClPhe : 3-(4-chlorophenyl)alanine
Pen penicillamine pGlu : pyroglutamic acid
Phe phenylalanine Pro proline
Qal 3-(3-quinolyl)alamne Sar sarcosine
Ser : senne (S-Me)Pen : S-methyl-peniciUamine
(S-Et)Pen . S-ethyl-peniciUamme Thr threonine
Tie : tert-leucine Trp . tryptophan
Tyr : tyrosine Val. valine
Ada : adamantylalanine HPhe . homophenylalanine
MeNpg : N-methylneopentylglycine 4Pal 3-(4-pyridyl)alanine
A preferred group of peptide analogues according to the invention, having LH-RH agonist activity, compnses the peptides of the formula (SEQ ID N° : 2).
Al-A2-A3-A4-A5-A6-HAA-A7-Pro-Z (IIa)
m which:
- Al is pGlu, Sar or AcSar;
- A2 is His,
- A3 is an aromatic L-amino acid such as Phe, HPhe, Tyr, Trp, Nal, INal,
diphenyl-Ala, Bal, Pal, 4Pal or Qal, where Phe and Trp may be substituted by one or
more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups;
- A4 is Ala, Ser, D-Ser, MeSer, Ser(OBut), Ser(OBzl) or Thr;
- A5 is an aromatic L-ammo acid such as Phe, HPhe, Tyr, Trp, Nal, INal,
diphenyl-Ala, Bal, Pal, 4Pal or Qal, where Phe and Trp may be substituted by one or
more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups;
- A6 is Gly ; D-Pro , D-Ser , D-Thr ; D-Cys ; D-Met; D-Pen ; D-(S-Me)Pen ; D-
(S-Et)Pen , D-Ser(OBut) ; D-Asp(OBut) , D-Glu(OBut) ; D-Thr(OBut) ; D-Cys(OBut) ;
D-Ser(ORi) where Ri is a sugar moiety ; an aza-amino acid such as azaGly or azaAla ;
D-His which may be substituted on the imidazole ring by a (C1-C6)alkyl or by a (C2-
C7)acyl group ; an aliphatic D-amino acid with a (C1-C8)alkyl or a (C3-C6)cycloalkyl
side chain such as D-Ala, D-Abu, D-Aib, D-3Aib, D-Val, D-Nva, D-Leu, D-Ile, D-Tle,
D-Nle, D-Hol, D-Npg, D-CPa, D-Cpa, D-Cba or D-Cha ; an aromatic D-amino acid
such as D-Phe, D-HPhe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-anthryl-Ala,
D-phenanthryl-Ala, D-benzhydryl-Ala, D-fluorenyl-Ala, D-Bal, D-Pal, D-4Pal or D-Qal,
where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups ; D-cyclohexadienyl-Gly , D-perhydronaphthyl-Ala ; D-perhydrodiphenyl-Ala ; or a basic D-amino acid such as D-Arg, D-HArg, D-Om, D-Lys, D-HLys, D-Cit, D-HCit, D-APhe or D-ACha, where D-Arg and D-HArg may be N-substituted by a (C1-C6)alkyl or a (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where D-Om, D-Lys, D-HLys, D-APhe and D-ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a Fmoc or Boc group;
- HAA is as defined for (I);
- A7 is a basic L-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha;
- Z is GlyNHi; azaGlyNHi; or a group -NHR2 where R2 is a (C1-C4)alkyl which may be substituted by an hydroxy or one or several fluorine atoms, a (C3-C6)cycloalkyl or a heterocyclic radical selected from morpholinyl, pyrrolidinyl and pipendyl,
as well as their pharmaceutically acceptable salts.
In these peptide analogues, HAA is preferably Ada or Npg which may be N-alpha-substituted by a (C1-C4)alkyl group optionally substituted by one or several fluorine atoms, Npg being especially preferred.
A preferred group of peptide analogues (IIa) comprises the peptides of the formula (SEQ ID N° 7) •
Al -A2-A3-A4-A5-A6-Npg-A7-Pro-Z (Il'a)
in which :
Al is pGlu, Sar or AcSar;
- A2 is His,
- A3 is an aromatic L-ammo acid such as Phe, Tyr, Trp, Nal, INal, diphenyl-Ala, Bal, Pal or Qal, where Phe and Trp may be substituted by one or more halogens, (d-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups;
- A4 is Ala, Ser, D-Ser, MeSer, Ser(OBut), Ser(OBzl) or Thr;
- A5 is an aromatic L-amino acid such as Phe, Tyr, Trp, Nal, INal, diphenyl-Ala, Bal, Pal or Qal, where Phe and Trp may be substituted by one or more halogens, (Cj-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups;
- A6 is Gly ; D-Pro ; D-Ser ; D-Thr ; D-Cys , D-Met; D-Pen ; D-(S-Me)Pen ; D-
(S-Et)Pen ; D-Ser(OBut) ; D-Asp(OBut) ; D-Glu(OBut) ; D-Thr(OBut) ; D-Cys(OBut) ,
D-Ser(ORi) where R1 is a sugar moiety , an aza-ammo acid such as azaGly or azaAla ;
D-His which may be substituted on the imidazole ring by a (C1-C6)alkyl or by a (C2-
C7)acyl group ; an aliphatic D-ammo acid with a (C1-Cg)alkyl or a (C3-C6)cycloalkyl
side chain such as D-Ala, D-Abu, D-Aib, D-3Aib, D-Val, D-Nva, D-Leu, D-Ile, D-Tle,
D-Nle, D-Hol, D-Npg, D-CPa, D-Cpa, D-Cba or D-Cha ; an aromatic D-amino acid
such as D-Phe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-anthryl-AIa, D-
phenanthryl-AIa, D-benzhydryl-Ala, D-fluorenyl-Ala, D-Bal, D-Pal or D-Qal, where D-
Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-
C4)alkoxy, nitro or tnfluoromethyl groups , D-cyclohexadienyl-Gly ; D-
perhydronaphthyl-Ala ; D-perhydrodiphenyl-Ala ; or a basic D-ammo acid such as D-
Arg, D-HArg, D-Om, D-Lys, D-HLys, D-Cit, D-HCit, D-APhe or D-ACha, where D-
Arg and D-HArg may be N-substituted by a {C1-C6)alkyl or a (C3-C6)cycloalkyl group
on one or both nitrogen atoms, and where D-Om, D-Lys, D-HLys, D-APhe and D-ACha
may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a
Fmoc or Boc group;
- Npg may be N-alpha-substituted by a (C1-C4)alkyl group which may be
substituted by one or several fluorine atoms,
- A7 is a basic L-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha;
- Z is GlyNH2 ; azaGlyNH2 ; or a group -NHR2 where R2 is a (C1-C4)alkyl which may be substituted by an hydroxy or one or several fluorine atoms, a (C3-C6)cycloalkyl or a heterocyclic radical selected from morpholinyl, pyrrolidinyl and pipendyl,
as well as their pharmaceutically acceptable salts.
Another preferred group of peptide analogues according to the invention, having
LH-RH antagonistic activity, comprises the peptides of the formula (SEQ ID N° . 3):
Al-A2-A3-A4-A5-A6-HAA-A7-Pro-Z (IIb)
in which:
- Al is pGlu ; D-pGlu ; Sar , AcSar ; Pro or a denvative thereof such as AcPro,
ForPro, OH-Pro, Ac-OH-Pro, dehydro-Pro or Ac-dehydro-Pro ; Ser; D-Ser ; Ac-D-Ser ;
Thr , D-Thr , Ac-D-Thr , or an aromatic D-amino acid which may be acylated such as
D-Phe, D-HPhe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-Bal, D-Pal, D-4Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (Cp C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups,
- A2 is a direct bond or an aromatic D-amino acid such as D-Phe, D-HPhe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-Bal, D-Pal, D-4Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C]-C4)alkoxy, nitro or trifluoromethyl groups;
- A3 is an aromatic L- or D-ammo acid such as Phe, HPhe, Tyr, Trp, Nal, INal, diphenyl-Ala, Bal, Pal, 4Pal or Qal, where Phe and Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups;
- A4 is Ala, Ser, D-Ser, MeSer, Ser(OBut), Ser(OBzl) or Thr;
- A5 is an aromatic L-amino acid such as Phe, HPhe, Tyr, Trp, Nal, INal,
diphenyl-Ala, Bal, Pal, 4Pal or Qal, where Phe and Trp may be substituted by one or
more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups ; or a basic
L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha,
where Arg and HArg may be N-substituted by a (C1-C6)alkyl or (C3-C6)cycloalkyl group
on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-
substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a nicotinoyl,
isonicotinoyl, 6-methyl-nicotinoyl, glycyl-nicotinoyl, nicotmyl-azaglycyl, furyl, glycyl-
furyl, furyl-azaglycyl, pyrazinyl, pyrazinyl-carbonyl, picolinoyl, 6-methyl-picolinoyl,
shikimyl, shikimyl-glycyl, Fmoc or Boc group,
- A6 is Gly ; D-Pro , D-Ser ; D-Thr ; D-Cys , D-Met; D-Pen ; D-(S-Me)Pen ; D-
(S-Et)Pen ; D-Ser(OBut) , D-Asp(OBut) ; D-Glu(O-But) ; D-Thr(O-But) ; D-Cys(O-But)
, D-Ser(O-Ri) where Ri is a sugar moiety , an aliphatic D-amino acid with a (Cj-
C8)alkyl or a (C3-C6)cycloalkyl side chain such as D-Ala, D-Abu, D-Aib, D-3Aib, D-
Val, D-Nva, D-Leu, D-Ile, D-Tle, D-Nle, D-Hol, D-Npg, D-CPa, D-Cpa, D-Cba or D-
Cha ; an aromatic D-amino acid such as D-Phe, D-HPhe, D-Tyr, D-Trp, D-Nal, D-INal,
D-diphenyl-Ala, D-anthryl-Ala, D-phenanthryl-Ala, D-benzhydryl-Ala, D-fluorenyl-Ala,
D-Bal, D-Pal, D-4Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or
more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups ; D-
cyclohexadienyl-Gly ; D-perhydronaphthyl-Ala ; D-perhydrodiphenyl-Ala ; or a basic L-
or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where
Arg and HArg may be N-substituted by a (C1-C6)alkyl or (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a mcotinoyl, isonicotinoyl, 6-methyl-nicotinoyl, glycyl-nicotinoyl, mcotinyl-azaglycyl, furyl, glycyl-furyl, furyl-azaglycyl, pyrazinyl, pyrazmyl-carbonyl, picohnoyl, 6-methyl-picolinoyl, shikimyl, shikimyl-glycyl, Fmoc or Boc group,
- HAA is as defined for (I),
- A7 is a basic L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg and HArg may be N-substituted by a (C1-C6)alkyl or (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a mcotinoyl, isonicotinoyl, 6-methyl-nicotinoyl, glycyl-nicotinoyl, nicotinyl-azaglycyl, fiiryl, glycyl-fiiryl, fiiryl-azaglycyl, pyrazinyl, pyrazmyl-carbonyl, picohnoyl, 6-methyl-picolinoyl, shikimyl, shikimyl-glycyl, Fmoc or Boc group;
- Z is GlyNH2 or D-AlaNHa;
as well as their pharmaceutically acceptable salts
In these peptide analogues, HAA is preferably Ada or Npg which may be N-alpha-substituted by a (C1-C4)alkyl group optionally substituted by one or several fluorine atoms, Npg being especially preferred.
A preferred group of peptide analogues (IIb) composes the peptides of the formula (SEQ ID N° . 8):
Al-A2-A3-A4-A5-A6-Npg-A7-Pro-Z (IIb)
in which .
- Al is pGlu ; D-pGlu , Sar , AcSar ; Pro or a derivative thereof such as AcPro, ForPro, OH-Pro, Ac-OH-Pro, dehydro-Pro or Ac-dehydro-Pro ; Ser ; D-Ser ; Ac-D-Ser ; Thr ; D-Thr ; Ac-D-Thr , or an aromatic D-amino acid which may be acylated such as D-Phe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-Bal, D-Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups;
- A2 is a direct bond or an aromatic D-amino acid such as D-Phe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-Bal, D-Pal or D-Qal, where D-Phe and D-Trp may
be substituted by one or more halogens, (C1-C4)alkyl, (C|-C4)alkoxy, nitro or tnfluoromethyl groups;
- A3 is an aromatic L- or D-amino acid such as Phe, Tyr, Trp, Nal, INai, diphenyl-Ala, Bal, Pal or Qal, where Phe and Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups;
- A4 is Ala, Ser, D-Ser, MeSer, Ser(OBu*), Ser(OBzl) or Thr;
- A5 is an aromatic L-amino acid such as Phe, Tyr, Trp, Nal, INal, diphenyl-Ala, Bal, Pal or Qal, where Phe and Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or trifluoromethyl groups ; or a basic L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg and HArg may be N-substituted by a (C1-C6)alkyl or (C3-C6)cycloaIkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a nicotmoyl, isonicotinoyl, 6-methyl-nicotinoyl, glycyl-nicotmoyl, mcotinyl-azaglycyl, furyl, glycyl-furyl, fiiryl-azaglycyl, pyrazinyl, pyrazinyl-carbonyl, picolinoyl, 6-methyl-picolmoyl, shikimyl, shikimyl-glycyl, Fmoc or Boc group;
- A6 is Gly , D-Pro ; D-Ser ; D-Thr ; D-Cys , D-Met, D-Pen ; D-(S-Me)Pen , D-(S-Et)Pen ; D-Ser(OBut) ; D-Asp(OBut) ; D-Glu(O-But) ; D-Thr(O-But) ; D-Cys(O-Bu*) , D-Ser(O-Ri) where Ri is a sugar moiety ; an aliphatic D-amino acid with a (C1-C8)alkyl or a (C3-C6)cycloalkyl side chain such as D-Ala, D-Abu, D-Aib, D-3Aib, D-Val, D-Nva, D-Leu, D-Ile, D-Tle, D-Nle, D-Hol, D-Npg, D-CPa, D-Cpa, D-Cba or D-Cha ; an aromatic D-amino acid such as D-Phe, D-Tyr, D-Trp, D-Nal, D-lNal, D-diphenyl-Ala, D-anthryl-Ala, D-phenanthryl-Ala, D-benzhydryl-Ala, D-fluorenyl-Ala, D-Bal, D-Pal or D-Qal, where D-Phe and D-Trp may be substituted by one or more halogens, (C1-C4)alkyl, (C1-C4)alkoxy, nitro or tnfluoromethyl groups , D-cyclohexadienyl-Gly ; D-perhydronaphthyl-Ala ; D-perhydrodiphenyl-Ala ; or a basic L-or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg and HArg may be N-substituted by a (C1-C6)alkyl or (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a nicotinoyl, isonicotmoyl, 6-methyl-nicotmoyl, glycyl-nicotinoyl, mcotmyl-azaglycyl, fixryl, glycyl-
furyl, furyl-azaglycyl, pyrazmyl, pyrazmyl-carbonyl, picohnoyl, 6-methyl-picohnoyl, shikimyl, shikimyl-glycyl, Fmoc or Boc group,
- Npg may be N-alpha-substituted by a (C1-C4)alkyl group which may be
substituted by one or several fluorine atoms;
- A7 is a basic L- or D-amino acid such as Arg, HArg, Om, Lys, HLys, Cit, HCit, APhe or ACha, where Arg and HArg may be N-substituted by a (C1-C6)alkyl or (C3-C6)cycloalkyl group on one or both nitrogen atoms, and where Om, Lys, HLys, APhe and ACha may be N-substituted by one or two (C1-C6)alkyl or (C3-C6)cycloalkyl groups, or by a mcotinoyl, isonicotinoyl, 6-methyl-nicotinoyl, glycyl-nicotmoyl, mcotinyl-azaglycyl, furyl, glycyl-fiiryl, furyl-azaglycyl, pyrazmyl, pyrazmyl-carbonyl, picohnoyl, 6-methyl-picolinoyl, shikimyl, shikimyl-glycyl, Fmoc or Boc group;
- Z is GlyNH2 or D-AlaNHi;
as well as their pharmaceutically acceptable salts
Among the peptide analogues of formula (IIa), those of the formula (SEQ ID N° . 4)
pGlu-His-A3-Ser-Tyr-A6-HAA-Arg-Pro-Z (Ilia)
in which .
- A3 and HAA are as defined for (IIa),
- A6 is Gly ; an aliphatic D-amino acid with a (C1-C8)alkyl side chain ; or an aromatic D-amino acid ;
- Z is GlyNHi or a group -NHC2H5; and their pharmaceutically acceptable salts, are especially preferred.
Among the peptide analogues of formula (IIIa), those where A3 is Trp are preferred , among the latter, those where HAA is Npg which may be N-alpha-methylated, are especially preferred.
Among the peptides of formula (IIb), those of the formula (SEQ ID N° 5).
Ac-D-Nal-D-pClPhe-D-Pal-Ser-A5-A6-HAA-A7-Pro-D-AlaNH2 (IIIb)
in which .
- A5 and A7 are as defined above for (IIb);
- A6 is Gly or a basic L- or D-amino acid ,
- HAA is as defined for (IIb),
and their pharmaceutically acceptable salts, are especially preferred.
Among the peptide analogues of formula (Illb), those where HAA is Npg which may be N-alpha-methylated, are preferred.
Examples of the salts with pharmaceutically acceptable acids are those with mineral acids, such as for example the hydrochlonde, hydrobromide, sulfate, phosphate, borate, hydrogensulfate, dihydrogenphosphate or nitrate, and those with organic acids, such as for example the acetate, oxalate, tartrate, succinate, maleate, fumarate, gluconate, citrate, pamoate, malate, ascorbate, benzoate, p-toluenesulfonate or naphtalenesulfonate
Examples of the salts with pharmaceutically acceptable bases are those with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, and those with organic bases such as amines, trometamol, N-methylglutamine, and the like.
The peptides according to the present invention can be prepared by the well-known techniques of peptide chemistry such as for example peptide synthesis in solution or solid phase peptide synthesis, hi general, these techniques involve the stepwise addition of one or more ammo acids -which may be suitably protected- to a forming peptide chain.
Preferably, the peptides according to the invention are synthesized using stepwise solid phase sjmthesis (1,2) with N-a-Fmoc protection. For example, the peptides are assembled on a 4-methylbenzylhydrylamine resin (Peninsula Laboratones, UK) or on an aminomethyl resin (Peninsula Laboratories, UK). The C-terminal proline is introduced as 4-(Boc-Prolyloxymethyl)phenyl acetic acid Subsequent removal of the Boc protecting group is achieved with trifluoroacetic acid followed by dichloromethane and dimethylformamide (DMF) washing as well as diisopropylethylamine neutralization. It is also possible to use a "Rink" resin (4-(2',4'-dimethoxyphenyl)-Fmoc-aminomethylphenoxy resm) using Fmoc strategy of synthesis (2).
The synthesis comprises assembling, cleavage and purification steps, as described below
I Assembling
For all the peptides the following deprotection/couphng procedure is used .
1 - DMF washing (3 times - 1 min.)
2 - Pipendine 25 % in DMF (1 mm.)
3 - Pipendine 25 % in DMF (twice - 15 mm)
4 - DMF washing (7 times - 1 mm.)
For each step 15 ml of solvent per gram of peptide resm are used.
Coupling of all ammo acid (three fold excess) is performed in DMF in the presence of BOP, Hobt and DIEA (3). Each coupling step is controlled for completion by the ninhydnne test (4) and double coupling is performed if necessary. If, after the second coupling the test still remains positive, the resm is acetylated (acetic acid anhydride, 10 fold excess and DIEA)
Generally, a tnfluoroacetic acid (TFA) treatment is performed pnor to the deprotection/cleavage step
II. Cleavage
The peptides are cleaved from the resm and fully deprotected by a treatment with either liquid hydrogen fluoride (HF) or TFA. 10 ml of HF or TFA per gram of peptide resm are used classically at 0° C for 45 mm, or 2.5 hours, respectively, in the presence of p-cresol and ethanedithiol (for tryptophan-containing peptides) as scavengers.
After evaporation of the HF, the crude reaction mixture is washed with diethyl ether, dissolved m TFA, precipitated with diethyl ether and dned under reduced pressure
If need be, pnor to HF deprotection the peptide is cleaved from the resin and subsequently amidated by a treatment with ethylamme (5 ml ethylamine per gram of petpide resin, - 78°C, 20 hours).
When a benzyl group is present in the final product, TFA is used (10 ml per gram of peptide resin, 0° C, 2,5 hours) for the final cleavage/deprotection.
The composition of the TFA cleavage mixture in v % is the following .
TFA • 83.3 %
Ethanedithiol :2.1%
Thioanisol : 4 2 %
Water 4.2 %
Phenol : 6.2 %
After filtration of the resin, the peptide is precipitated fi-om the reaction mixture by addition of a large amount of diethylether. After several washings with diethylether the crude peptide is dried under reduced pressure.
Ill Punfication
All the peptides are punfied by reverse phase liquid chromatography.
The general procedure of punfication is identical for each peptide ; however the gradient of organic solvent is adjusted depending on the initial retention time of the peptide.
General conditions of purification :
Equipment : KRONWALD SPERATIONSTECHNIK, Medium
Pressure liquid chromatography system (Germany) equipped with Glass column.
Stationnary phase silica Bondapack C18 (Waters) 15-25 \xm, 100 A
Size of column 40 x 340 mm
Elution conditions. Mobile phase: Eluant A . 0.1 % TFA in water
Eluant B • CH3CN/A 60/40 (volume)
Temperature • Room
Flow rate . 40 ml
Detection UV210nm
Fractionnmg : 5 ml per fraction
All fractions containing the target compound are individually analyzed by analytical HPLC. The fractions with a purity higher than 95 % are pooled and freeze-dried. In case the requested punty is not reached after the first purification step, a second purification step and, if need be, a third purification step are performed. The conditions of punfication for the second and third steps are similar as those described above except that the slope of the gradient is modified in order to increase the resolution.
After lyophilisation, all punfied peptides are present as their trifluoroacetate salt The final powder corresponding to each peptide is controlled by analytical HPLC. The structure of each compound is also assessed by mass spectral analysis and the net peptide content is determinated by UV absorption.
The peptides according to the present invention have a potent affinity for the LH-RH receptors.
This affinity has been determined according to the following method :
Pituitanes from female Sprague Dawley rats were removed and homogenized with a Potter homogenizer in a 25 mM HEPES buffer (pH 7.4) containing 0.32 M sucrose, 100 µg/1 PMSF (phenylmethylsulfonylfluonde), 5.6 U/1 aprotinin and 10 000 U/1 bacitracin. The homogenates were centnfuged at 700 g for 10 minutes and the supematants were further centnfuged at 12,500 g for 30 minutes. The pellets were homogenized and centnfuged as described above, but m the same buffer without sucrose.
All homogenization, centnfugation and subsequent incubation steps were earned out at 4° C.
Aliquots of membrane fractions were incubated for 2 hours in duplicate with increasing concentrations of test compounds in the presence of 20 to 70 pM of [l^Sjj. buserehn (between 1000 and 2000 Ci/mmol depending on ligand batches). The assay was terminated by filtration under suction (Brandel 96-well harvester) through Whatman GF/B glass fiber filters. After repeated washes, filters were placed m counting vials with scintillation cocktail to measure the radioactivity of 125I For each expenment, curve-fitting of residual specific binding against concentrations of test compound gave the 50 % inhibitory concentration (IC50). Each compound was tested m at least 4 experiments
This LH-RH receptor assay was charactenzed by 4 saturation expenments using increasing concentration of [125I].buserelin in the absence or presence of 1 µM unlabelled buserehn for non specific binding determination Specific binding data were analysed according to Scatchard's method At equihbnum (2 hours of incubation), the dissociation constant (Kd) and the number of binding sites for [125I]-byserelin were respectively equal to 88 ± 6 pM and 15 6 ± 2 9 pM
For each test compound, the inhibitory constant (Ki) was calculated from its IC50 according to the Cheng and Prussofs equation K1 = IC50/(l + [radioligand]/Kd). K1 were then transformed into pKi (= - log K1) for final expression of affinity scales
The natural ligand, LH-RH itself, displays a strong affinity with experimental IC50 in the 10 nM range, i.e , a pKi equal to about 8

So-called superagonists like buserelm, leuprorelin, tryptorelm, histrelm or deslorelin and antagonists like antide show an even stronger binding to LH-RH receptors with IC50 m the subnanomolar range, i.e. pKi > 9.
The affinity of test peptides of the invention for the LH-RH receptors is given in Table 1 below
Table 1 . Affinity for LH-RH receptors

(Table Removed)

(n). number of determinations
The peptides according to the general formula (IIa) exert an agonist activity upon the LH-RH receptors in vivo, resulting m the stimulation of LH secretion by the pimitary, which, m males, stimulates the secretion of testosterone by the testis.
Adult male Sprague-Dawley rats received a subcutaneous injection of various doses of LH-RH, tryptorelm or leuprorelin, or their respective counterpart with Npg replacing Leu7 . example 1 ([Npg7]-LH-RH), example 6 ([Npg7]-leuprorelin) or example 11 ([Npg7]-tryptorelin), dissolved in phosphate-buffered saline (PBS) Two hours later, blood samples were drawn for total plasma testosterone determination by
direct radioimmunoassay (Immunotech). Example 1 was a little more than twice as active as LH-RH itself, and the other compounds behaved like so-called « superagonists » by inducing a stronger stimulation of testosterone secretion at much lower doses (loganthmic x axis) than LH-RH (Figure 1; 8 animals per point). At 20 ng/kg, the secretion of testosterone was equally maximally stimulated by the four superagonists (exponential y axis scale), but at 10 ng/kg, examples 6 and 11 were a little more than twice as active as leuprorelin and tryptorelin, respectively.
At this intermediate dose of 10 ng/kg, several examples with Npg7 or Ada7 in place of Leu7 according to the invention were screened for agonistic activity (Tables 2 and 3). When available from Bachem (France) or Sigma (France), the corresponding standard agonist with Leu7 was tested for companson In all the six instances, both Npg'- and Ada7- modified examples were more active, by a slight or wide margin depending on the Leu7 counterpart compound (Table 2). In two cases, the Ada7 replacement was more favorable than Npg' (examples 27 and 28). Conversely, Npg7 led to more potent agonists than Ada7 in two other structures (examples 6 and 11).
Those findings illustrate that increasing the overall hydrophobicity of the ammo-acid m position 7 of LH-RH analogues is a general mean to achieve a greater potency in vivo Depending on the rest of the molecule, other characteristics of the side chain m position 7, such as stenc hindrance, modulate the resulting gam in activity.
An increase in hydrophobicity, as exemplified with Npg', was compatible with several changes in positions 3 and 6 to give several agonists in a range of potency similar to that of leuprorelin or tryptorelin (examples 5, 9, 13, 20, 21, 22 or 25 ; Tables 2 and 3) N-methylation of Npg' was also compatible with a very strong agonistic activity (example 23 ; Table 3).
Table 2 . Stimulation of testosterone secretion


(Table Removed)
TABLE 3 : Stimulation of testosterone secretion

(Table Removed)
n : number of animals
In conclusion, examples 6 and 23 on the one hand, and example 28 on the other hand, are the best examples to date of more potent LH-RH agonists than current therapeutic LH-RH analogues, obtained by increasing the hydrophobicity of the amino acid in position 7, respectively with Npg or Ada
The peptides according to the general formula (IIb) exert an antagonistic activity upon the LH-RH receptors in vivo, resulting in the inhibition of ovulation in the female.
Adult female Wistar rats are first monitored for normal estrous cyclicity by daily vaginal smears. After at least 2 regular 4-day cycles, they received by subcutaneous injection either the vehicle alone (0.5 ml of a mixture of propylene-glycol and water . 20/80 vol/vol), or the LH-RH antagonist according to the formula (IIb) dissolved in this vehicle, around 2:00 PM on the day of proestrus All but one vehicle-treated animals ovulated spontaneously as demonstrated by the recovery of numerous ovocytes in the oviducts the following morning.
When effective, LH-RH antagonists totally block ovulation. Antide, a commercially available standard LH-RH antagonist (from Bachem, France) showed a dose-related inhibition of ovulation (Table 4) Semi-loganthmic regression analysis gave a 50 % inhibitory dose (ID50) equal to 0 99 |ig/rat When Leu7 was replaced by
Npg7 in the structure of antide (example 15), the inhibitory potency was markedly increased at 0.5 and 0.25 )ig/rat, resulting in an ID50 of 0.26 µg/rat. Variations m basic ammo acids in positions 6 and 8 were compatible with Npg7 to give antagonists more potent than antide, as seen with the maximal or sub-maximal activity of examples 16, 17, 18 or 19 at the dose of 1 µg/rat (Table 4). Example 17 was especially active, but its effects were not related to the dose in the dose range studied. Therefore, introduction of an hydrophobic amino acid in position 7 is favorable to achieve stronger LH-RH antagonistic properties, as best examplified to date by the 4-fold increase in the antiovulatory potency of antide by replacement of Leu7' by Npg (example 15).
Table 4 : inhibition of ovulation

(Table Removed)
this conclusion of both agonistic and antagonistic studies in vivo, it has been shown that replacement of Leu by a more hydrophobic non aromatic amino acid, such as Npg or Ada, systematically increased the potency of existing analogues. Furthermore, closely related analogues having an hydrophobic amino acid in position 7 without direct Leu7 counterpart for comparison, often displayed interesting levels of activity per se.
Therefore, the use of Npg, Ada or any other hydrophobic ammo acid in position 7 of an LH-RH analogue sequence corresponding to the definition of general formula (I) is claimed as a general feature to obtain new LH-RH agonists or antagonists with high or enhanced potency in vivo.
No sign of toxicity is observed with the peptides of the invention at pharmaceutically active doses
Thus, the peptides of the invention and their pharmaceutically acceptable salts may be used m the treatment or prevention of various complaints or diseases wherein a LH-RH agonist or antagonist activity is required
The main target of LH-RH analogues is the pituitary gland, but direct actions have been reported on the gonads themselves (testis and ovary), on the thymus and some lymphoid cell lines, on mast cells and on breast, prostate or pancreatic tumors.
LH-RH agonists according to formula (IIa) exert on any LH-RH sensitive target, either a stimulatory activity by short-term acute or pulsatile administrations, or an inhibitory effect by repeated or continuous administrations that induce the desensitization and the down-regulation of LH-RH receptors, hi the case of the hypothalamo-pituitary-gonadal axis, prolonged administration results in a so-called "chemical" castration.
LH-RH antagonists according to formula (IIb) exert pnmanly an inhibitory effect on any LH-RH-sensitive target, but are also useful in obtaining or planning a rebound stimulatory release of LH and FSH when treatment is discontinued
Due to this ambivalent potential of both LH-RH agonists and antagonists, all analogues according to formula (I) can find an appropriate therapeutic use in humans as well as in animals, depending on doses, treatment regimens and routes of administration, m reproductive endocnnology and in the treatment or prevention of sex hormone-dependent benign or malignant tumors, alone or m combination with other
hormonal or antitumoral agents. LH-RH sensitive sex hormone-mdependent benign or malignant tumors can also regress upon treatment with LH-RH analogues according to formula (I), alone or in combination with antitumoral agents. Immune mechanisms can also be modified by LH-RH analogues according to formula (I), alone or in combination with immuno-modulatmg or -suppresive agents such as glucocorticoids, cyclosponn, rapamycin, tacrolimus, their derivatives, and the like. The LH-RH analogues according to the invention are therefore very valuable in the treatment and prevention of autoimmune diseases, graft rejection or atopic diseases, and in the treatment of benign or malignant lymphoproliferative disorders.
LH-RH analogues according to formula (I) are especially usefiil, alone or in combination with sex steroids or gonadotrophins, m the inhibition, planning and triggenng of ovulation in in vitro fertilization programs, and in the treatment of male and female infertility or hypogonadic states. Conversely, they can also be used in male or female contraception or treatment of hypergonadic states, alone or in combination with sex steroids or gonadotrophins. This applies to men and women, but also to wild or domestic animals in uses such as improvement or control of reproductive performance, or as a tool to optimize breeding strategies.
LH-RH analogues according to formula (I) are also especially usefiil in men to treat advanced prostate cancer, but can also be used as a first line therapy in this indication and in benign prostatic hypertrophy, alone or in combination with inhibitors of androgen action, i.e. antiandrogens such as cyproterone acetate, osaterone acetate, chlormadinone acetate, flutamide, nilutamide or bicalutamide and the like, or 5a-reductase inhibitors such as finastende, epnsteride or turostende and the like, or C17-20 lyase inhibitors such as abiraterone and the like
LH-RH analogues according to formula (I) are also especially usefiil in the treatment or prevention of breast cancer in women and in men, especially estrogen receptor positive tumors, alone or in combination with antiestrogens such as tamoxifen, raloxifen or droloxifen and the like, or with aromatase inhibitors such as atamestane, formestane, letrozole, anastrozole and the like, or with C 17.20 lyase inhibitors such as abiraterone and the like, but also of certain estrogen receptor negative tumors that respond to the direct effects of LH-RH analogues or indirectly to their gonadal suppressive activity
Other gynecological conditions, such as endometnal hyperplasia, leiomyoma, adenomyoma, endometriosis, polycystic ovary syndrome, hirsutism and benign breast disease (pam, cysts or fibrosis), can also be prevented by or benefit from treatment with the LH-RH analogues according to formula (I), alone or in combination with antiestrogens (cited above), progestins such as cyproterone acetate, osaterone acetate, chlormadmone acetate, nomegestrol acetate, promegestone, demegestone, tnmegestone and the like, or their contraceptive or post-menopausal replacement combination formulations with estrogens such as estradiol or ethynylestradiol. The peptides of the invention can also interfere with gestation by inducing abortion or by triggering labor, alone or m combination with estrogens (cited above), antiprogestins such as mifepnstone or prostaglandin analogs such as sulprostone
Similar indications can be encountered in vetennary medicine for male or female domestic or wild animals that may require the use of LH-RH analogues according to formula (I)
Another aspect of the present invention is therefore pharmaceutical compositions containing an effective amount of at least one peptide of formula (I) or a pharmaceutically acceptable salt thereof, alone or mixed with suitable pharmaceutical excipients.
A further aspect of the invention relates to a method of treating and/or preventing the above diseases which compnses administering to patients or animals in need thereof a therapeutically effective amount of a peptide of formula (I) or a pharmaceutically acceptable salt thereof
A further aspect of the invention relates to the use of the peptides of formula (IIa), or of their pharmaceutically acceptable salts, for the preparation of a medicament having LH-RH agonist activity. Also within the scope of the invention is the use of the peptides of formula (IIb), or of their pharmaceutically acceptable salts, for the preparation of a medicament having LH-RH antagonist activity
The peptides of the invention are preferentially administered by parenteral administration, although oral formulations are also effective provided that the dosage is appropriately increased
Preferred delivery systems for LH-RH agonists of formula (IIa) in long term pituitary-gonadal suppressive indications are slow-release implantable devices, or
injectable biodegradable polymenc micro- or nano-particles or -capsules, or micro- or nano-emulsions, with unit doses of the peptides or of their appropnate salts ranging from 1 mg to 100 mg per human patient for a duration of action ranging from 1 month to 1 year. Long term administration of LH-RH antagonists of formula (IIb) will generally require higher dosages m the same slow-release formulations, ranging from 10 mg to 1 g for 1 week to 1 year of activity. Animal doses will be adapted on a body weight basis depending on the wild or domestic species to be treated either by LH-RH agonists or antagonists according to formula (I).
All other means of parenteral administration are suited for immediate, delayed or planned delivery of the peptides of the invention : subcutaneous, intramuscular, intravenous, intragonadal or mtratumoral needle bolus injections, or prolonged continuous, pulsatile or planned perfusions or micromfusions using the appropriate pump technology ; gas-propelled subcutaneous microinjection ; vaginal creams, gels or pessaries ; rectal enemas or suppositones ; transdermal creams, gels, lotions, solutions, patches or lontophoretic devices ; nasal spray or dry powder inhalation device ; ophtalmic solutions, gels, creams or contact lenses ; pulmonary inhalation of micro- or nano-particles or droplets generated manually or with an appropriate pulverization or nebulization device.
The unit dose of these parenteral administrations will range in humans from 0.001 mg to 10 mg/day for LH-RH agonists of formula (IIa) and from 0.01 to 100 mg/day for LH-RH antagonists of formula (IIb), one to 16 times per day (in the case of pulsatile administration).
Oral administration of peptides according to the invention is preferentially effected using gastro-resistant and delayed enteric or colonic release formulations which can be coated pills or tablets containing two or more components, hardened gelatin capules, special polymeric macro-, micro- or nano-beads containing them, or any device designed to protect them from gastrointestinal degradation and to release them when needed All other formulations to be taken orally such as solutions, suspensions, syrups, gels and the like, or lingual, sublingual or chewable formulations are suited provided that the dosage is increased.
Overall, effective oral treatment may be achieved with any of the above formulations with unit doses of peptides of formula (I) ranging from 1 mg to 1 g per human patient, from one to 16 times per day (in the case of pulsatile administration).
All the above-mentioned oral or parenteral formulations of the peptides according to the invention and their pharmaceutical acceptable salts may contain one or several pharmaceutically appropnate excipients, one or several inhibitors of proteases, and one or several absorption enhancers as needed by the specific route of administration.
Raw powder of pure peptides according to the invention or their pharmaceutically acceptable salts can also be used, especially m the lyophilized form for fast sublingual application.
The invention will now be descnbed with reference to the following examples, which are not intended to limit the invention in any respect. In these examples, the starting materials used were either commercially available or synthetized, as mentioned below
- Fmoc-Glu-OH, Fmoc-Tyr(OBut)-OH, Fmoc-Trp-OH and Fmoc-His(Trt) were purchased from Propeptide (France).
- Fmoc-p-Nal-OH and Fmoc-pClPhe were synthesized as racemates. These ammo acids and their corresponding acetyl ethylesters were enz>'mat]cally resolved using subtihsin (5);
- Other Fmoc protected amino-acids were purchased from Bachem (Switzerland), Novabiochem (Switzerland), American Peptide C° (USA) or Neosystem (France)
- Adamantylalanine was synthesized as described by Kim Quang Do et al (6).
EXAMPLE 1 pGlu-His-Trp-Ser-Tyr-Gly-Npg-Arg-Pro-Gly-NH2
Example 1 was synthesized on a Rink resin using a Fmoc strategy as mentioned above in the general synthesis of the invention peptides. Cleavage was carried out with TFA in the presence of scavengers.
Punfication was carried out using a linear gradient of from 10 to 40 % of eluent B (CH3CN/O.I % TFA 60/40 v/v) over 30 min
68 mg (approximate yield 24 %) of purified material were obtained.
Mass spectral analysis - ES+ mode •
expected- 1195.3
found: 1195.7

net peptide content 73.9 % ; punty 97.2 % ; retention time 16.4 mm.
EXAMPLE 2 • pGlu-His-Trp-Ser-Tyr-Gly-Npg-Arg-Pro-NEt
The synthesis was carried out on Boc-Pro-PAM resin The second amino acid, arginme, was also incorporated via a Boc strategy. The subsequent ammo acids were incorporated via a Fmoc strategy. After coupling of the N-terminal amino acid, the peptide was cleaved from the resin and converted into ethylamide by ammolysis using ethylamine (5 ml of ethylamme per gram of peptide resin for 20 hours, -78°C).
After cleavage the protected peptide was extracted with methanol, dried and deprotected with HF as described.
Punfication was carried out using a linear gradient of fi-om 10 to 60 % of eluent B over 30 mm. 15 mg (approximate yield 8 %) of purified material were obtained.
Mass spectral analysis - ES"*" mode
expected- 1166.3
found: 1166.8.
net peptide content 72.7 % ; punty 95.0 % , retention time 15.1 min.
EXAMPLE 3 : pGlu-His-Trp-Ser-Tyr-D-Ala-Npg-Arg-Pro-Gly-NH2
Assembling and cleavage of the peptide were carried out as descnbed for Example 1
Punfication was earned out using a linear gradient of from 10 to 50 % of eluent B over 30 mm
66 mg (approximate yield 27 %) of purified material were obtained.
Mass spectral analysis - ES+ mode :
expected 1209 4
found 1209.5.
net peptide content 72.6 % ; purity 95 2 % ; retention time 14 5 min.
EXAMPLE 4 : pGlu-His-Trp-Ser-Tyr-D-Ala-Npg-Arg-Pro-NEt
Assembling and cleavage of the peptide were carried out as described for Example 2.
Purification was carried out using a linear gradient of from 10 to 60 % of eluent B over 30 min
8 mg (approximate yield 7 %) of purified material were obtained.
Mass spectral analysis - ES+ mode
expected- 1180.3
found. 1181.0
net peptide content 69 5 % ; punty 96.9 % , retention time 17.7 min
EXAMPLE 5 : pGlu-His-Trp-Ser-Tyr-D-Leu-Npg-Arg-Pro-Gly-NH2
Assembling and cleavage of the peptide were carried out as described for Example 1
Punfication was earned out using a linear gradient of fi-om 15 to 50 % of eluent B over 30 mm.
123 mg (approximate yield 36 %) of purified material were obtained.
Mass spectral analysis - ES+ mode
expected 1251.4
found- 1251.9
net peptide content 71.7 % ; punty 95.7 % , retention time 13.9 mm.
EXAMPLE 6 : pGlu-His-Trp-Ser-Tyr-D-Leu-Npg-Arg-Pro-NEt
Assembling and cleavage of the peptide were carried out as described for Example 2
Punfication was earned out in two steps, the first one using a linear gradient of from 15 to 50 % of eluent B over 30 mm , and the second one using a linear gradient of from 15 to 40 % of eluent B over 30 mm.
49 mg (approximate yield 20 %) of punfied material were obtained.
Mass spectral analysis - ES+ mode .
expected. 1222.4
found . 1223 6 (MH+)
net peptide content 73.6 % ; punty 95 3 % , retention time 14.6 min.
EXAMPLE 7 pGlu-His-Trp-Ser-Tyr-D-Npg-Npg-Arg-Pro-Gly-NH2
Assembling and cleavage of the peptide were carried out as descnbed for Example 1
Punfication was earned out in two steps, the first one using a linear gradient of from 30 to 60 % of eluent B over 30 mm., and the second one using a linear gradient of from 25 to 60 % of eluent B over 30 mm.
13 mg (approximate yield 4 %) of purified material were obtained.
Mass spectral analysis - ES+ mode

expected: 1265.5
found 1266.0
net peptide content 71.1 % ; punty 97.8 % , retention time 15.1 min.
EXAMPLE 8 : pGlu-His-Trp-Ser-Tyr-D-Npg-Npg-Arg-Pro-NEt
Assembling and cleavage of the peptide were earned out as descnbed for Example 2
Punfication was carried out using a linear gradient of from 20 to 80 % of eluent B over 30 min
13 mg (approximate yield 4 %) of punfied matenal were obtained.
Mass spectral analysis - ES+ mode :
expected. 1236.4
found : 1237.5 (MH+)
net peptide content 68.5 % ; punty 96.2 % ; retention time 13.9 mm.
EXAMPLE 9 : pGlu-His-Trp-Ser-Tyr-D-Phe-Npg-Arg-Pro-Gly-NH2
The synthesis was earned out as described for example 1.
Punfication was carried out using a linear gradient of from 25 to 80 % of eluent B over 30 min.
61 mg (approximate yield 16 %) of purified material were obtained.
Mass spectral analysis - ES+ mode
expected : 1285 5
found . 1286.2 (MH+)
net peptide content 71.8 % ; punty 96 8 % ; retention time 14.9 mm.
EXAMPLE 10 : pGlu-His-Trp-Ser-Tyr-D-Phe-Npg-Arg-Pro-NEt
Assembling and cleavage of the peptide were earned out as descnbed for example 2
Purification was carried out using a linear gradient of from 20 to 80 % of eluent B over 30 mm.
6 mg (approximate yield 4 %) of purified matenal were obtained.
Mass spectral analysis - ES+ mode :
expected. 1256.4
found 1257 4 (MH+)

net peptide content 63.2 % , punty 96.9 % , retention time 13.9 mm.
EXAMPLE 11 : pGlu-His-Trp-Ser-Tyr-D-Trp-Npg-Arg-Pro-Gly-NH2
Assembling and cleavage of the peptide were carried out as descnbed for example 1
Punfication was earned out using a linear grandient of from 20 to 80 % of eluent B over 30 min.
22 mg (approximate yield 7 %) of punfied matenal were obtained.
Mass spectral analysis - ES+ mode
expected: 1324.5
found : 1325.5 (MH+)
net peptide content 71.6 % ; purity 97.1 % , retention time 13.1 min.
EXAMPLE 12 : pGlu-His-Trp-Ser-Tyr-D-Trp-Npg-Arg-Pro-NEt
Assembling and cleavage of the peptide were earned out as described for example 2
Punfication was carried out using a linear gradient of from 20 to 80 % of eluent B over 30 min.
10 mg (approximate yield 5 %) of punfied material were obtained.
Mass spectral analysis - ES+ mode
expected . 1295 4
found • 1296.3 (MH+)
net peptide content 713%, punty 98.4 % , retention time 13.8 mm.
EXAMPLE 13 : pGlu-His-Trp-Ser-Tyr-D-Nal-Npg-Arg-Pro-Gly-NH2
Assembling and cleavage of the peptide were carried out as descnbed for example 1
Purification was earned out using a linear gradient of from 15 to 75 % of eluent B over 30 min
205 mg (approximate yield 50 %) of purified material were obtained
Mass spectral analysis - ES+ mode
expected 1335.6
found 1336.2 (MH+)
net peptide content 74 8 % ; purity 95 6 % , retention time 14.9 min.
EXAMPLE 14 . pGlu-His-Trp-Ser-Tyr-D-Nal-Npg-Arg-Pro-NEt

Assembhng and cleavage were carried out as descnbed for example 2 Purification was carried out using a linear gradient of from 25 to 50 % of eluent B over 30 mm.
82 mg (approximate yield 22 %) of punfied material were obtained.
Mass spectral analysis - ES+ mode :
expected. 1306.5
found : 1307.2 (MH+)
net peptide content 76 0 % ; punty 97.4 % , retention time 15.8 mm.
EXAMPLE 15 AcD-Nal-D-pClPhe-D-Pal-Ser-NicLys-D-NicLys-Npg-IprLys-
Pro-D-Ala-NH2
The synthesis was carried out on a 4-methylbenzhydrylamine resin
D-alanine and proline were introduced using a Boc strategy as described above for the general synthesis of the invention peptides. The other amino acids were incorporated via a Fmoc strategy as descnbed above.
The synthesis was started with Boc-D-Ala-OH
The peptides were deprotected and cleaved from the resm using HF as descnbed above
Purification was carried out using a hnear gradient of from 15 to 70 % of eluent B over 30 mm.
49 mg (approximate yield 31 %) of punfied material were obtained.
Mass spectral analysis - ES+ mode :
expected • 1605.3
found: 1605.5
net peptide content 67 6 % ; punty 98 3 % ; retention time 15.5 mm.
EXAMPLE 16 AcD-Nal-D-pClPhe-D-Pal-Ser-Tyr-D-Cit-Npg-Arg-Pro-D-Ala-NH.
Assembling and cleavage of the peptide were earned out as described for example 15, arginine being introduced using a Boc strategy.
Punfication was carried out using a linear gradient of from 30 to 60 % of eluent B over 30 min.
16 mg (approximate yield 9 %) of purified material were obtained.
Mass spectral analysis - ES+ mode :

expected: 1444.9
found 1444.6
net peptide content 67 1 % ; punty 97.0 % , retention time 16.8 mm.
EXAMPLE 17 AcD-Nal-D-pClPhe-D-Pal-Ser-Tyr-D-Cit-Npg-IprLys-Pro-D-Ala-NH.
Assembling and cleavage of the peptide were earned out as described for Example 15
Purification was earned out using a linear gradient of from 10 to 60 % of eluent B over 30 mm.
55 mg (approximate yield 29 %) of purified material were obtained.
Mass spectral analysis - ES"*" mode :
expected value : 1459.9
found: 1459.3
net peptide content 69.8 % ; purity : 96.4 % ; retention time 11.2 mm.
EXAMPLE 18 : AcD-Nal-D-pClPhe-D-Pal-Ser-Tyr-D-HCit-Npg-IprLys-Pro-D-Ala-NH2
Assembling and cleavage of the peptide were earned out as described for example 15
Punfication was carried out using a linear gradient of from 30 to 50 % of eluent B over 30 mm.
40 mg (approximate yield 17 %) of purified material were obtained.
Mass spectral analysis - ES+ mode .
expected 1473.2
found. 1473.2
net peptide content 69.8 % ; purity 95.7 % , retention time 15.9 mm.
EXAMPLE 19 AcD-Nal-D-pClPhe-D-Pal-Ser-Tvr-D-HCit-Npg-Arg-Pro-D-Ala-NH2
Assembling and cleavage of the peptide were carried out described for example 16
Purification was carried out using a linear gradient of from 30 to 60 % of eluent B over 30 min
55 mg (approximate yield 21 %) of purified material were obtained.

Mass spectral analysis - ES+ mode :
expected; 1459.1
found 1459.2
net peptide content 68.2 % ; punty 96.6 % , retention time 15.7 mm.
EXAMPLE 20 • pGlu-His-Trp-Ser-Tyr-D-Pal-Npg-Arg-Pro-Gly-NHj
Assembling and cleavage of the peptide were earned out as described for Example
1
Purification was earned out using a linear gradient of from 5 to 50 % of eluent B over 30 mm.
74 mg (approximate yield 29 %) of punfied material were obtained.
Mass spectral analysis - ES+ mode :
expected: 1287.3
found. 1287.3
net peptide content 72.1 % ; punty 98.6 % , retention time 12.5 mm
EXAMPLE 21 : pGlu-His-Trp-Ser-Tyr-D-4Pal-Npg-Arg-Pro-Gly-NH2
Assembling and cleavage of the peptide were earned out as described for Example 1.
Purification was earned out using a linear gradient of from 10 to 30 % of eluent B over 30 mm
7 mg of purified material were obtained
Mass spectral analysis - ES+ mode
expected: 1287.3
found • 1287.2
net peptide content 64.3 % ; punty 98.4 % , retention time 12.2 min.
EXAMPLE 22 pGlu-His-Trp-Ser-Tyr-D-HPhe-Npg-Arg-Pro-Gly-NH2
Assembling and cleavage of the peptide were earned out as described for Example 1.
Punfication was carried out using a linear gradient of from 15 to 70 % of eluent B over 30 min
94 mg (approximative yield 36 %) of punfied material were obtained.
Mass spectral analysis - ES+ mode .
expected. 1300.3

found. 1300.2
net peptide content 74 2 % ; punty 97 5 % , retention time 15.5 min.
EXAMPLE 23 . pGlu-His-Trp-Ser-Tyr-D-Leu-MeNpg-Arg-Pro-NEt
Assembling and cleavage of the peptide were earned out as described for Example 2
Punfication was earned out using a linear gradient of from 20 to 80 % of eluent B over 30 mm.
50 mg (approximate yield 17 %) of purified material were obtained.
Mass spectral analysis - ES+ mode .
expected: 1237.5
found: 1237.4
net peptide content 73.7 % ; punty 95.0 % , retention time 16.2 mm.
EXAMPLE 24 : pGlu-His-lNal-Ser-Tyr-D-Leu-Npg-Arg-Pro-NEt
Assembling and cleavage of the peptide were carried out as descnbed for Example 2
Purification was carried out using a linear gradient of from 10 to 70 % of eluent B over 30 mm.
68 mg (approximate yield 7 %) of purified matenal were obtained.
Mass spectral analysis - ES+ mode :
expected. 1234.5
found 1234.2
net peptide content 73 3 % ; purity 98.5 % , retention time 15.5 mm.
EXAMPLE 25 pGlu-His-2Nal-Ser-Tyr-D-Leu-Npg-Arg-Pro-NEt
Assembling and cleavage of the peptide were earned out described for example 2
Punfication was carried out using a linear gradient of from 10 to 65 % of eluent B over 30 min.
17 mg (approximate yield 7 %) of punfied material were obtained.
Mass spectral analysis - ES+ mode :
expected : 1234 5
found: 1234.2
net peptide content 71.5 % ; purity 98.0 % , retention time 14.0 mm
EXAMPLE 26 . pGIu-His-Bal-Ser-Tyr-D-Leu-Npg-Arg-Pro-NEt

Assembling and cleavage of the peptide were carried out as descnbed for Example 2.
Punfication was carried out using a linear gradient of from 20 to 70 % of eluent B over 30 min
41 mg (approximate yield 16 %) of punfied material were obtained.
Mass spectral analysis - ES"^ mode :
expected: 1240.5
found: 1240.4
net peptide content 89.0 % ; punty 97.4 % ; retention time 15.6 min.
EXAMPLE 27 : pGlu-His-Trp-Ser-Tyr-D-Phe-Ada-Arg-Pro-NEt
Assembling and cleavage of the peptide were earned out as described for Example 2.
Punfication was earned out using a linear gradient of from 15 to 50 % of Eluent B over 30 min.
90 mg (approximate yield 14 %) of purified material were obtained.
Mass spectral analysis - ES* mode •
expected • 1335.6
found: 1335.5
net peptide content 76.3 % ; punty 97 8 % ; retention time 17.0 min.
EXAMPLE 28 : pGlu-His-Trp-Ser-Tyr-D-Ala-Ada-Arg-Pro-NEt
Assembling and cleavage of the peptide were carried out as described for Example 2.
Punfication was carried out using a hnear gradient of from 15 to 50 % of eluent B over 30 min.
150 mg (approximate yield 24 %) of purified material were obtained.
Mass spectral analysis - ES"^ mode .
expected. 1259.5
found: 1259.0
net peptide content 72.9 % ; punty 97.4 % , retention time 14.1 mm.
EXAMPLE 29 • pGlu-His-Trp-Ser-Tyr-D-Leu-Ada-Arg-Pro-NEt
Assembling and cleavage of the peptide were carried out as descnbed for Example

Punfication was earned out using a linear gradient of from 15 to 70 % of eluent B over 30 mm
100 mg (approximate yield 15 %) of punfied material were obtained.
Mass spectral analysis - ES+ mode :
expected: 1301.6
found: 1301.5
net peptide content 72.7 % ; punty 97.3 % , retention time 17 7 mm.
EXAMPLE 30 : pGlu-His-Trp-Ser-Tyr-D-Trp-Ada-Arg-Pro-Gly-NH2
Assembling and cleavage of the peptide were carried out as described for Example 1
Punfication was earned out using a linear gradient of from 15 to 70 % of eluent B over 30 min.
30 mg (approximate yield 11 %) of punfied material were obtained.
Mass spectral analysis - ES+ mode :
expected: 1403.6
found: 1403.2
net peptide content 82.9 % ; purity 95.0 % , retention time 16.0 min.

REFERENCES
(1) G. BARANY and R.B. MERRIFIELD (1979)
The Peptides, Analysis, Synthesis, Biology, Vol. 2, Chapter 1.
(2) E. ATHERTON and R.C SHEPPARD (1989) Solid phase peptide synthesis, IRL Press, OXFORD
(3) D. Le NGUEN, A. HEITZ and B CASTRO (1987) J Chem. Soc. Perkm Trans. 1,1915
(4) E. KAisER, R.L. COLESCOTT, CD. BOSSINGER and P.I. COOK (1970) Anal. Biochem., 34, 595
(5) P.N. RAO, J.E. BURDETT Jr, J.W. CESSAD, CM. DI NUNNO, D.M. PETERSON and H.K. KIM (1987)
Int. J. Pept. Protein Res., 29, 118
(6) KIM QUANG DO, P. THANEI, M. CAVIEZEL and R. SCHWYZER (1979)
Helvetica Chimica Acta, 62, 956-964









We claim:
1. A LH-RH peptide analogue of the formula (SEQ ID N°: 1):
(Formula Removed)
in which:
- Al is pGlu ; D-pGlu ; Sar ; AcSar ; Pro ; AcPro ; ForPro ; OH-Pro ; Ac-OH-Pro ; dehydro-Pro ; Ac-dehydro-Pro ; Ser ; D-Ser ; Ac-D-Ser ; Thr ; D-Thr ; Ac-D-Thr ; or an aromatic D-amino acid which may be acylated;
- A2 is a direct bond; His ; or an aromatic D-amino acid;
- A3 is an aromatic L- or D-amino acid;
- A4 is Ala, Ser, D-Ser, MeSer, Ser(OBut), Ser(OBzl) or Thr;
- A5 is an aromatic L-amino acid or a basic L- or D-amino acid;
- A6 is Gly ; D-Pro ; D-Ser ; D-Thr ; D-Cys ; D-Met; D-Pen ; D-(S-Me)Pen ; D-(S-Et)Pen ; D-Ser(OBut) ; D-Asp(OBut) ; D-Glu(OBut); D-Thr(OBut) ; D-Cys(OBut) ; D-Ser(OR1) where R1 is a sugar moiety ; an aza-amino acid; D-His which may be substituted on the imidazole ring by a (C1-C6)alkyl or by a (C2-C7)acyl group ; an aliphatic D-amino acid with a (C1-C8)alkyl or a (C3-C6)cycloalkyl side chain ; an aromatic D-amino acid ; D-cyclohexadienyl-Gly ; D-perhydronaphthyl-Ala ; D-perhydrodiphenyl-Ala ; or a basic L-or D-amino acid;
- HAA is Ada or Npg, wherein Npg7 is unsubstituted or N-alpha substituted by a (C1-C4) alkyl group optionally substituted by one or several fluor atoms.
- A7 is a basic L- or D-amino acid;
- Z is GlyNH2 ; D-AlaNH2 ; azaGlyNH2 ; or a group -NHR2 where R2 is a (C1-C4)alkyl which may be substituted by an hydroxy or one or several fluorine atoms, a (C3-C6)cycloalkyl or a heterocyclic radical selected from morpholinyl, pyrrolidinyl and piperidyl; and its pharmaceutically acceptable salts.
2. A peptide analogue as claimed in claim 1, of the formula (SEQ ID N°: 2):
(Formula Removed)
wherein:
- Al is pGlu, Sar or AcSar;
- A2 is His;
- A3 and A4 are as defined for (I) in claim 1;
- A5 is an aromatic L-amino acid;
- A6 is Gly ; D-Pro ; D-Ser ; D-Thr ; D-Cys ; D-Met; D-Pen ; D-(S-Me)Pen ; D-(S-Et)Pen ; D-Ser(OBut) ; D-Asp(OBut) ; D-Glu(OBut) ; D-ThrCOBut) ; D-Cys(OBut) ; D-Ser(OR1) where R1 is a sugar moiety ; an aza-amino acid; D-His which may be substituted on the imidazole ring by a (C1-C6)alkyl or by a (C2-C7)acyl group ; an aliphatic D-amino acid with a (C1-C8)alkyl or a (C3-C6)cycloalkyl side chain ; an aromatic D-amino acid ; D-cyclohexadienyl-Gly ; D-perhydronaphthyl-Ala ; D-perhydrodiphenyl-Ala ; or a basic D-amino acid;
HAA is as defined for (I) in claim 1;
- A7 is a basic L-amino acid;
- Z is GlyNH2, azaGlyNH2 or a group -NHR2 where R2 is as defined for (I) in claim 1; and its pharmaceutically acceptable salts.
3. A peptide analogue according to claim 2, of the formula (SEQ ID N°: 4):
(Formula Removed)
wherein:
- A3 and HAA are as defined for (IIa) in claim 2;
- A6 is Gly; and aliphatic D-amino acid with a (C1-C8)alkyl side chain ; or an aromatic D-amino acid;
- Z is Gly or a group -NHC2H5;
and its pharmaceutically acceptable salts.
4. A peptide analogue as claimed in claim 3 wherein A3 is Trp; and its pharmaceutically acceptable salts.
5. A peptide analogue as claimed in claims 2 to 4 wherein HAA is Npg where Npg may be N-alpha substituted by a (C1-C4)alkyl group which may be substituted by one or several fluorine atoms; and its pharmaceutically acceptable salts.
6. A peptide analogue as claimed in claim 5, wherein HAA is Npg which may be N-alpha-methylated;
and its pharmaceutically acceptable salts.
7. A peptide analogue as claimed in claim 1, of the formula (SEQ ID N°: 3):
(Formula Removed)
wherein:
- Al as defined for (I) in claim 1;
- A2 is a direct bond or an aromatic D-amino acid;
- A3, A4 and A5 are as defined for (I) in claim 1;
- A6 is Gly; D-Pro; D-Ser; D-Thr; D-Cys; D-Met; D-Pen; D-(S-Me)Pen; D-(S-Et)Pen; D-Ser(OBul); D-AspCOBut); D-Glu(O-But); D-Thr(O-But); D-Cys(O-But); D-Ser(O-R1) where R1 is a sugar moiety; an aliphatic D-amino acid with a (C1-C8)alkyl or a (C3-C6)cycloalkyl side chain; an aromatic D-amino acid; D-cyclohexadienyl-Gly; D-perhydronaphthyl-Ala; D-perhydrodiphenyl-Ala; or a basic L- or D-amino acid;
- HAA and A7 are as defined for (I) in claim 1;
- Z is GlyNH2 or D-AlaNH2;
and its pharmaceutically acceptable salts.
8. A peptide analogue as claimed in claim 7, of the formula (SEQ ID N°: 5):
(Formula Removed)
wherein:
- A5 is as defined for (IIb) in claim 7;
- A6 is Gly or a basic L- or D- amino acid;
- HAA and A7 are as defined for (IIb) in claim 7; and its pharmaceutically acceptable salts.

9. A peptide analogue as claimed in 7 or 8 wherein HAA is Npg where Npg may be N-alpha substituted by a (C1-C4)alkyl group which may be substituted by one or several fluorine atoms ; and its pharmaceutically acceptable salts.
10. A peptide analogue as claimed in claim 9, wherein HAA is Npg which may be N-alpha-methylated; and its pharmaceutically acceptable salts.
11. A peptide analogue as claimed in claim 2, which is pGlu-His-Trp-Ser-Tyr-D-Ala-Npg-Arg-Pro-NEt.
12. A peptide analogue as claimed in 2, which is pGlu-His-Trp-Ser-Tyr-D-Leu-Npg-Arg-Pro-NEt.

13. A peptide analogue as claimed in claim 2, which is pGlu-His-Trp-Ser-Tyr-D-Ala-Ada-
Arg-Pro-NEt.
14. A peptide analogue as claimed in claim 7, which is selected from the group consisting of:
AcD-Nal-D-pClPhe-D-Pal-Ser-NicLys-DNicLys-Npg-IprLys-Pro-D-AlaNH2
AcD-Nal-D-pClPhe-D-Pal-Ser-Tyr-D-Cit-Npg-Arg-Pro-D-AlaNH2
AcD-Nal-D-pClPhe-D-Pal-Ser-Tyr-D-Cit-Npg-IprLys-Pro-D-AlaNH2
AcD-Nal-D-pClPhe-D-Pal-Ser-Tyr-D-HCit-Npg-IprLys-Pro-D-AlaNH2, and
AcD-Nal-D-pClPhe-D-Pal-Ser-Tyr-D-HCit-Npg-Arg-Pro-D-AlaNH2.
15. A LH-RH peptide analogue as claimed in claim 1, which is pGlu-His-Trp-Ser-Tyr-D-
Leu-MeNpg-Arg-Pro-NEt.
16. A peptide agonist or antagonist of LH-RH as claimed in claim 1 of formula :
(Formula Removed)
in which Leu is replaced by Ada or Npg , wherein Npg is unsubstituted or N-alpha substituted by a (C1-C4) alkyl group optionally substituted by one or several fluor atoms.

Documents:

1263-DEL-1998-Abstract-(23-02-2011).pdf

1263-DEL-1998-Abstract-(29-04-2011).pdf

1263-del-1998-abstract.pdf

1263-DEL-1998-Claims-(23-02-2011).pdf

1263-DEL-1998-Claims-(29-04-2011).pdf

1263-del-1998-claims.pdf

1263-DEL-1998-Correspondence Others-(29-04-2011).pdf

1263-DEL-1998-Correspondence-Others-(09-03-2011).pdf

1263-DEL-1998-Correspondence-Others-(23-02-2011).pdf

1263-del-1998-correspondence-others.pdf

1263-DEL-1998-Description (Complete).pdf

1263-del-1998-drawings.pdf

1263-DEL-1998-Form-1-(29-04-2011).pdf

1263-del-1998-form-1.pdf

1263-del-1998-form-19.pdf

1263-DEL-1998-Form-2-(29-04-2011).pdf

1263-del-1998-form-2.pdf

1263-DEL-1998-Form-3-(23-02-2011).pdf

1263-del-1998-form-3.pdf

1263-DEL-1998-GPA-(23-02-2011).pdf

1263-del-1998-gpa.pdf

1263-DEL-1998-Petition 137-(23-02-2011).pdf


Patent Number 248355
Indian Patent Application Number 1263/DEL/1998
PG Journal Number 27/2011
Publication Date 08-Jul-2011
Grant Date 06-Jul-2011
Date of Filing 13-May-1998
Name of Patentee LABORATOIRE THERMEX
Applicant Address 6, AVENUE DU PRINCE HEREDITAIRE ALBERT, 98000 MONACO, (MC), FRANCE.
Inventors:
# Inventor's Name Inventor's Address
1 REMI DELANSORNE 7 TER, AVENUE EDITH CAVELL, 06000 NICE, FRANCE.
2 JACQUES PARIS 31 AVENUE CAP DE CROIX, 06100 NICE, FRANCE.
PCT International Classification Number C07K14/47
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA