HU211932A9 - Lhrh antagonists - Google Patents

Description

This invention is described in N.C.I. (NIH) Government grants 40,003 and 40,004. The Government of the United States of America has certain rights in this application.

This application is in part a continuation of filing pending application 07/404,667, filed September 7, 1989, which is in part a continuation of application filed October 21, 1988, and is now withdrawn.

The present invention relates to novel peptides which inhibit the release of gonadotropins from the pituitary gland in mammals, including humans, and have an effect on the growth of cancerous tumors in humans. In particular, the present invention is a luteinizing hormone releasing hormone (LHRH), which has the formula:

_{pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2,} refers antagonist analogs, salts and pharmaceutical compositions and methods of using these analogs.

The luteinising hormone releasing hormone (LHRH) from the hypothalamus regulates the synthesis and secretion of gonadotropins (LH and FSH) in the pituitary gland, which are essential for the regulation of sex hormone synthesis in the gonads.

Discovery and Structure of LHRH [A. V. Schally et al., Fertil. Steril., 22, 703721 (1971)], more than 2,500 new synthetic LHRH analogues (agonist and antagonist analogues) have been reported for medical use [M. J. Karten and J.E. Rivier, Endocrine Rev. 7, 44-66 (1986); A. Dutta, Drugs of the Future, 13,761-787 (1988)]. LHRH antagonists compete with endogenous LHRH at the pituitary receptors and directly inhibit the secretion of gonadotropins. The pharmacologically significant advantage of agonists over antagonists is that they inhibit gonadotropin secretion almost immediately, without initially increasing the amount of gonadotropins typical of LHRH agonists. LHRH antagonists are used in endocrinology and gynecology to control fertility and treat premature puberty by blocking ovulation in women and suppressing the production of semen in men. The use of antagonists in the treatment of hormone-sensitive tumors in oncology is very recent but the most promising [A. V. Schally et al., GnRH Analogues in Cancer and Human Reproduction. Basic Aspects, edited by H. Vickery and V. Lunenfeld, eds., Kluwer Academic Publishers, Dordrecht / Boston / London, Vol. 1, 5-31, 1989].

The most interesting antagonists so far are compounds whose structure is a modified version of the structure of LHRH. A systematic change in the structure of the molecule has shown how each amino acid and its side chains contribute to the biological effect. The former most potent antagonists often contained a hydrophobic D-amino acid residue at the N-terminus and a highly basic hydrophilic D-amino acid at the 6- and / or 8-positions [D. H. Coy et al., Endocrinology 100: 1445-1447 (1982); A. Horváth et al., Peptides, 3, 969971 (1982); J. Rivier et al., J. Med. Chem.

29: 1848-1851 (1986)]. However, these potential hydrophilic antagonists caused transient systemic edema on the face and limbs and inflammation at the injection site when the compounds were injected subcutaneously in rats at 1.25 or -1.5 mg / kg body weight. These analogs, which increase white blood cell secretion, release histamine and when administered intravenously at a dose of 1.25 mg / kg to the rat can also cause cyanosis and respiratory depression, leading to cell death [Smith et al., Contraception, 29, 283- 289 (1984); Morgan et al., Int. Arch. Allergy Appl. Immunology 80: 70-75 (1986)]. In order to maintain the potent ovarian inhibitory effect of the antagonists, but to avoid these side effects, studies have been directed to derivatives in which

In the range of 5-8 amino acids, the basicity of the side chains varies. Hocart et al., J. Med. Med. Chem.

30, 1910-1914 (1987)] found that substitution of the alkylated Lys at the 6-position did not cause any significant change in the histamine release effect of the analogs, whereas similar substitution at the 8-position reduced the release of histamine to one tenth. Detirelix [Ac-D-Na (2) ', D-Ph (4Cl) ² , D-Trp ³ , D-hArg (Et) ₂ ⁶ , D-Ala'] was shown to be a potent antagonist [LA Adams et al. J. Clin. Endocrinol. Metab. 62, 58 (1986)] and showed antihypertensive and bradycardic side effects [CJ Lee et al. Life Sci., 45, 67 (1989)]. Nal-Glu-GnRHant antagonists retain their anti-ovarian inhibitory activity and exhibit significantly less in vitro histamine release activity (JE Rivier et al., J. Med. Chem., 29, 1846-1851 (1986)), but some of their local allergic responses in human subjects cannot be ignored. N ^{E is} nicotinoyl lysine

The introduction of ^this 5.6- and N-isopropyl-lysine at position 8 led to a compound having a high releasing effect of inhibiting ovulation and negligible histamine is [Ljungqist et al, Proc. Natl. Acad. Sci. USA 85: 8236-8240 (1988). Modification by Bajusz et al., Int. J. Peptides. Prot. Sl., 32, 425-435 (1988)], i.e., incorporation of D-citrulline and D-homocitrulline at the 6-position resulted in peptides that did not cause edema and anaphylactoid side effects and had high inhibitory activity, such as Ac-D Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DCit-Leu-Arg-Pro-D-Ala-NH ₂ . Some of these compounds have been found to inhibit the growth of various animal tumors and inhibit the growth of various human cancer cell lines in in vivo models (AV Schally, General Gynecology, Vol. 6, Parthenon Press Camforth, England, 1989, 1- P. 20, 1990, 1990, J. Natl. Cancer Inst., 82, 513-517; 1990, Cancer Research, 50, 3716-3721; E. Korkut et al., Proc. Natl. Acad. Sci.

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884-848 (1991)] and thus are potential drugs for the treatment of various cancers (prostate, breast, uterus, ovary, and pancreas).

Many human tumors are hormone dependent or hormone responsive and contain hormone receptors; for example, breast carcinomas contain estrogen, progesterone, glucocorticoid, LHRH, EGF, IGF-I and somatotatin receptors. Peptide hormone receptors have also been found in acute leukemia, prostate, breast, pancreas, uterine, ovarian, colon, and brain tumors [Μ. N. Pollak et al., Cancer Lett., 38, 223-230 (1987); F. Pekonen et al., Cancer Res., 48, 1343-1347 (1988); M. Fekete et al., J. Clin. Foot. Anal., 3, 137-147 (1989); G. Emons et al., Eur. J. Cancer Oncol., 25, 215-221 (1989). Our recent results [M. Black et al., 1989, Endocrinology 124: 946-955; M. Black et al., Pancreas, 4: 521-528 (1989)] show that LHRH agonist and antagonist analogues bind to human breast cancer cell membranes and to pancreatic cancer cell membranes, although the latter tumor is thought to be non-hormone dependent. Biologically active peptides such as melanotropin (MSH), epidermal growth factor, insulin and agonist and antagonist analogues of LHRH have been shown [L. Jennes et al., Peptides. 5: 215-220 (1984)] endocytosis to their target cells.

The present invention relates to a novel antagonist decapeptide of LHRH from the hypothalamus, which inhibits high ovulation and abnormal tissue formation, and is free of anaphylactoid side effects and is believed to cause no edema formation.

The compounds of the present invention are represented by the following general formula (I):

XR ¹ -R ² -R ³ -Ser-R ⁵ -R ⁶ (AY ₂ ) -Leu-Arg-Pro-D-AlaNH ₂ in which

R ^{1 is} D-Phe, D-Phe (4Cl), D-Nal (1) or D-Nal (2),

R ^{2 is} D-Phe or D-Ph (4Hl),

R ^{3 is} D-Trp, D-Phe, D-Phe (4Hl), D-Nal (1), D-Nal (2) or D-Pal (3),

R ^{5 is} Tyr or Arg,

R ^{6 is} D-Lys or D-Orn,

Hl is fluorine, chlorine or bromine,

X is a C 2 -C 5 alkanoyl group,

H ₂ N-CH ₂ - (CH ₂ ) _m -CH (NH ₂ ) - (CH ₂ ) _n -CO- (II) diaminoacyl wherein m is 0 or 1 and n is 0 or 1

Y means Y ^! or Y ² where

Y ^{1 is an} acyl group derived from straight or branched aliphatic or alicyclic carboxylic acids having from 3 to 12 carbon atoms or from aromatic carboxylic acids containing 6 or 10 ring carbon atoms,

Y ^{2 is} carbamoyl or alkyl-substituted carbamoyl of formula H- (CH ₂ ) _n -NH-CO- (III) wherein n is 0-3.

Pharmaceutically acceptable salts of the compounds of formula (I) are also included within the scope of the present invention.

The peptides of formula (I) may be synthesized by classical in-solution synthesis, or preferably by solid phase technique, using methyl benzhydrylamine (MBHA), benzhydrylamine (BAH) resin or

2-methoxy-4-alkoxybenzyl alcohol (Sasrin) with a resin having a suitable amido linker.

Such procedures

X ¹ -R ¹ -R ² -R ³ -Ser (X ⁴ ) -R ⁵ (X ⁵ ) -R ⁶ (X ⁶ ) -Leu-Arg (X ⁸ )

^{-Pro-D-Ala-NH-X10} (IV) The peptides and / or intermediate of the formula peptide-resins are made available, wherein

R ^1, R ^2, R ^3, R ⁵ and R ⁶ are as defined above,

X ^{1 is C} 2 -C 5 alkanoyl,

X ⁴ is hydrogen or a protecting group of the hydroxyl group of Ser,

^X5 is hydrogen or a phenolic hydroxyl group of Tyr or Arg guanidino protecting group,

X ^{6 is} hydrogen or the protecting group of Lys or Om, X ^{8 is} hydrogen or the guanidino protecting group of Arg,

X is ¹⁰ hydrogen atoms or a spacer in the resin.

In order to carry out the reaction selectively on the diaminoalkanoyl side chain of R ^{6 to} obtain the peptide of formula (I),

X * -R * R ² -R ³ -Ser (X ⁴ ) -R ⁵ X ⁵ -R ⁶ [A (X ⁶ ) 2] -Leu-ArgX ⁸ Pro-D-Ala-NHX ¹⁰ (V) to form intermediate peptides were synthesized by solid-phase synthesis, these peptides differ from the general formulas from (I) that R ⁶ (X ⁶⁾ in accordance with the 6-position protected ^R6 [A ^(X6) 2] contain, in the formula

X ¹ , R ¹ , R ² , R ³ , R ⁵ , R ⁶ and A are as defined above, X ¹ , X ⁴ , X ⁵ and X ⁸ are as defined above but are not hydrogen,

X ^{6 is} hydrogen or the protecting group of the diamino side chain,

X ^{10 is} a resin switch group.

For the preparation of compounds of formula I wherein QA (Y *) ₂ or A (Y ² ) _{2 is} used, four different reaction sequences are used:

a) (IV) peptide (in which R ^1, R ^2, of the formula

R ³ , R ⁵ , R ⁶ and X ¹ are as defined above, X ⁴ , X ⁵ , X ⁶ , X ⁸ and

X ¹⁰ H) preformed A (Y ') ₂ or

A compound of formula (Y ² ) ₂ wherein A, Y ¹ and Y ² are as defined above.

b) Alternatively, a compound of Formula V (wherein R ¹ , R ² , R ³ , R ⁵ , R ⁶ and X ¹ is as defined above)

X ⁴ , X ⁵ side chain protecting group, X ⁶ , hydrogen atom and

X ^{10 is} the linking group of the resin) as an intermediate peptide. Compounds of formula (I) in which YY ¹ is prepared by direct acylation of a compound of formula (V) with an acyl halide or anhydride followed by cleavage of the peptide from the resin and deprotection of the compound step.

c) According to another method, the R ⁶ [A (Y 2)] molecule is prepared by reaction of a suitably protected R ⁶ and A, then Y or the already prepared A (Y) 2

And then incorporated into the peptide during solid phase peptide synthesis.

d) The intermediate peptide of formula (V)

R ¹ , R ² , R ³ , R ⁵ , R ⁶ , A and X ¹ are as defined above, X ⁴ , X ⁵ ,

X ⁶ , X ⁸ and X ¹⁰ H) The two free amino groups of the moiety are acylated with acylimidazole.

The invention also provides methods for removing one or more protecting groups and / or cleavage of the peptides from the resin, purification of the peptides prepared by synthesis, and, if the salt retains the desired biological activity of the parent compound, non-toxic pharmaceutically acceptable salts.

The peptides of the present invention inhibit ovulation in female rats at a dose of 0.15-1.0 micrograms / kg body weight administered subcutaneously on the day of the pre-estrus. These peptides sustainably suppress LH, FSH and testosterone levels. when injected into castrated male rats at a dose of 0.5 to 2.0 micrograms / kg body weight. Peptides 7 and 8 cause significant reductions in LH levels for more than 24 hours (p, 01). At 48 hours after injection, both antagonists show significant inhibition (p, 05) at a dose of 5 micrograms. Peptide 8 was effective even at a dose of 1.25 micrograms (p, 05) 48 hours after administration. Most of the compounds of formula I have high affinity for the rat pituitary and human breast cancer membrane receptors. In cytotoxicity assays in culturing human breast and prostate cancer cell lines, some analogs potently inhibit the incorporation of ³ H-thymidine.

Inhibition of growth of Dunning R3327H prostate cancer was detected after treatment of rats with 8 peptides. The time required for the tumor to double in size increased from 12 days in the control group to 42 days. Body weight did not change during treatment, although testicular, seminal vesicle, and ventral prostate weight was significantly reduced in the group receiving 8 peptides. The results show that peptide 8, when released from sustained release systems, effectively suppresses the growth of prostate cancer.

The pharmaceutical compositions are prepared as follows. mixing the compound of formula (I) with a pharmaceutically acceptable carrier, including microcapsules (microspheres) or microgranules (microparticles) for extended release from poly (DL-lactide-co-glycolide).

The usual abbreviations for amino acids, peptides and derivatives thereof, which are generally accepted in the field of peptide chemistry and are recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (Europen J. Biochem., 138, 9-37 (1984)), are used herein.

Abbreviations for each amino acid residue are based on trivial names of amino acids such as pGlu pyroglutamic acid, His histidine, Trp tryptophan, Ser serine, Tyr tyrosine, Lys lysine, Orn ornithine, Leu leucine, Arg arginine, Pro proline, Gly glycine, Ala alanine Phe phenyl analine. Where an amino acid residue exists in an isomeric form, the abbreviation means the L-form of the amino acid, unless otherwise specified.

Abbreviations used in the present application include: A ₂ pr: 2,3-diaminopropionic acid, A ₂ bu: 2,4-diaminobutyric acid, Nal (2): 3- (2-naphthyl / alanine, D-Pal). (3): 3- (3-Pyridyl) -alanine, Phe (4Cl):

4-chloro-phenylalanine.

The peptide sequence is written according to the convention, i.e., the N-terminal amino acid is on the left and the C-terminal amino acid is on the right.

Further abbreviations:

AcOH acetic acid Ac ₂ O acetic anhydride Boc tert-butoxycarbonyl group bz benzoyl Bzl benzyl tsar carbamoyl CHC ciklohexanoilcsoport DCB 2,6-dichloro-benzyl DCC N.N '-dicyclohexylcarbodiimide DCM dichloromethane DIC N.N '-diisopropylcarbodiimide DMF dimethylformamide EtCar ethylcarbamoyl group FMOC fluorenyl-methyloxy-carbonyl group HOBt 1-hydroxybenzotriazole HOPCP Pentachlorophenol HPLC high performance liquid chromatography iPrOH isopropyl alcohol LAU lauryl MeCN acetonitrile MeOH Methanol OSu N-hydroxy-succinimide ester PRL propionyl TEA triethylamine TFA trifluoroacetic acid Tos 4-toluene sulfonyl Z (2-C1) 2-chloro-benzyloxy carbonyl z benzyloxy carbonyl.

Particularly preferred compounds of formula (I) according to the present invention are those wherein R ^{1 is} D-Nal (2).

R ² D-Phe (4Cl),

R ^{3 is} D-Trp or D-Pal (3),

R ^{5 is} Tyr or Arg

R ^{6 is} D-Lys or D-orn,

X is acetyl,

AA ₂ pr or DL-A ₂ bu,

YY ¹ or Y ² , in this

Y ^{1 is} formyl, acetyl, propionyl, butyryl, protobutyryl, cyclohexanoyl or benzoyl,

Y ^{2 is} carbamoyl, N-methylcarbamoyl or N-ethylcarbamoyl.

The most preferred compounds are:

1. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-DLys- [A ₂ pr (Car) ₂ ] -Leu-Arg-Pro-D-Ala -NH ₂ ,

2. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-DLys [A ₂ pr) Ac) ₂ ] -Leu-Arg-Pro-D-Ala- NH ₂ ,

3. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-DLys [A ₂ pr (For) ₂ ] -Leu-Arg-Pro-D-Ala- NH ₂ ,

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4. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-DLys [A ₂ pr (EtCar) ₂ ] -Leu-Arg-Pro-D-Ala- NH ₂ ,

5. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-DLys [A ₂ pr (CHC) ₂ ] -Leu-Arg-Pro-D-Ala- NH ₂ ,

6. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-DLys [A ₂ pr (Bz) ₂ ] -Leu-Arg-Pro-D-Ala- NH ₂ ,

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A ₂ pr (Car) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ .

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A ₂ pr (Ac) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ .

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A ₂ pr (EtCar) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ .

10. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A ₂ pr (For) ₂ ] -Leu-Arg-Pro-D-Ala- NH ₂ ,

11. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A ₂ pr (PRL) ₂ ] -Leu-Arg-Pro-D-Ala- NH ₂ ,

12. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A ₂ pr (CHC) ₂ ] -Leu-Arg-Pro-D-Ala- NH ₂ ,

13. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A ₂ pr (Bz) ₂ ] -Leu-Arg-Pro-D-Ala- NH ₂ ,

14. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu (Ac) ₂ ] -Leu-Arg-Pro-D- Lower NH ₂ ,

15. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu (For) ₂ ] -Leu-Arg-Pro-D- Lower NH ₂ ,

16. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu (Car) ₂ ] -Leu-Arg-Pro-D- Lower NH ₂ ,

17. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys (DL-A ₂ bu (EtCar) ₂ ] -Leu-Arg-Pro-D- Lower NH ₂ ,

18. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu (PRL) ₂ ] -Leu-Arg-Pro-D- Lower NH ₂ ,

19. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu (LAU) ₂ ] -Leu-Arg-Pro-D- Lower NH ₂ ,

20. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu (Bz) ₂ ] -Leu-Arg-Pro-D- Lower NH ₂ ,

21. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu (CHC) ₂ ] -Leu-Arg-Pro-D- Lower NH ₂ ,

22. Ac-D-Nap (2) -D-Phe (4Cl) -D-Trp-Ser-Arg-DLys [A ₂ pr (Car) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ ,

23. Ac-D-Nal (2) -D-Phe (4Cl) -D-Trp-Ser-Arg-DLys [A ₂ pr (Ac) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ ,

24. Ac-D-Nal (2) -D-Phe (4Cl) -D-Trp-Ser-Arg-DLys [A ₂ pr (For) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ ,

25. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A ₂ pr] -Leu-Arg-Pro-D-Ala-NH ₂ ,

26. Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu] -Leu-Arg-Pro-D-Ala-NH ₂ .

The LHRH antagonizing properties of the compounds of the present invention make them suitable for use in human and veterinary medicine. For example, the compounds of formula (I) may be used to alleviate complications caused by undesired physiological availability of pituitary gonadotropins in mammals. Such complications include premature puberty; hormone-dependent tumors such as benign and malignant prostate tumors such as secondary amenorrhea; endometriosis and cystic ovarian and breast diseases in humans and animals. The compounds of formula (I) regulate ovulation, and can therefore be used, for example, as a pre- or post-partum contraceptive for fertility control, and are useful in livestock estrogen synchronization and for the development of a "rhythmic" method. The compounds may also be used to regulate human menopausal gonadotropin, follicle stimulating hormone (FSH) and luteinising hormone (LH) in women with menopausal or peri-periodic conditions. Because they suppress semen production and testosterone levels in men, they can potentially serve as a contraceptive for men.

The peptides of the invention are often administered in the form of pharmaceutically acceptable, non-toxic salts such as acid addition salts. Examples of such salts are hydrochloride, hydrobromide, sulfate, phosphate, fumarate, gluconate, tannate, maleate, acetate, citrate, benzoate, succinate, alginate, pamoate, malate, ascorbate and tartrate.

Microcapsules or microparticles of these peptides made of poly (DL-lactide-co-glycolide) may be the preferred sustained release systems. Intravenous administration in isotonic saline, phosphate buffer solutions or the like can also be used.

The pharmaceutical compositions will generally contain the peptide together with a conventional pharmaceutically acceptable carrier. The dose will usually be in the range of approx. It ranges from 1 to 100 micrograms peptide / kg body weight for intravenous administration. In general, individuals are treated with these peptides in the same way as with other LHRH agonists or antagonists.

The peptides of the invention may be administered to mammals intravenously, subcutaneously, intramuscularly, intranasally to achieve LHRH antagonizing and antitumor activity. The effective dose will depend on the dosage form and the species of mammal being treated. A typical dosage form is a physiological saline solution containing the peptide, which is ca. It is administered in a daily dose of from 0.01 to 0.05 mg / kg body weight.

Although the invention will be described with reference to preferred embodiments, it will be appreciated that changes and modifications will be apparent to those skilled in the art without departing from the scope of the invention as defined in the claims. In the practice of the present invention, substitutions known in the art that do not significantly reduce efficacy may be used.

The compounds of the present invention have a potent effect on the release of gonadotropin from the pituitary gland, binding to tumor cell membranes and inhibiting the incorporation of [ ³ H] thymidine into cellular cultures.

(a) LHRH inhibitory effect

The effect of the compounds on LH release was measured in vitro using a superfused rat pituitary cell system (S. Vígh and A. V. Schally, Peptides, 5 Suppl., 1984, 241-247; V. Csemus and A. V. Schally, in Neuroendocrine Research Methods, Ed. B. Greenstein, Harwood Academic Publishers, London (1990)].

The LHRH inhibitory activity of your peptide was measured as follows: Each peptide was perfused through the cells (3 ml of perfusate) for 9 minutes at a concentration of 1 nM.

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A mixture of peptide and 3 nM LHRH was then added directly at the same concentration for 3 minutes. Subsequently, four 3 nM infusions of LHRH were administered for 3 minutes (1 ml perfusate) at 30 minute intervals (30.60.90 and 120 minutes). The LH content of the collected 1 ml fraction was determined by radioimmunoassay (RIA).

b) The ovarian inhibitory activity of the peptides was determined in vivo in 4-day cycling rats according to A. Corbin and C. W. Beattie [Endocr. Gap. Commun., 2, 1-23 (1975)].

c) Receptor binding

The affinity of peptides for cellular membranes of rat pituitary and human breast cancer was determined using labeled LHRH and [D-Trp ⁶ ] LHRH. Measurements were made by T. Kadar et al., Proc. Natl. Acad. Sci. USA 85: 890-894 (1988) and by M. Black et al. (1989) Endocrinology 124: 946-955.

d) The effect on LH and FSH levels in vivo was measured by the method of L. Bokser et al., Proc. Natl. Acad. Sci. USA, 87, 7100-7104 (1990). Castrated male rats (350410 g) were anesthetized with urethane and subcutaneously injected with 1.25 and 5.0 micrograms of peptides 7 and 8. Blood samples were taken from the jugular vein before, 1, 2, 3, 4, 6, 24 and 48 hours after injection of the peptide. Control animals received saline only. LH and FSH levels were determined by specific RIA methods.

(e) Cytotoxicity test

To determine the ability of peptides of formula (I) to inhibit the incorporation of [ ³ H] thymidine in the monolayer culture of the MCF-7 human breast tumor cell line, the procedures described in VK Sondak et al. Cancer Research, 44, 1725-1728 (1984); F. Holzer et al., J. Cancer Slit. Clin. Oncol. 109: 217-226 (1985); M. Albert et al., J. Cancer Slit. Clin. Oncol., 109, 210-216 (1985).

f) In vivo antitumor activity

In rats, inhibition of the growth of cancerous tumors by compounds of formula I is described by Sende et al., J. Med. Natl. Cancer Inst., 82, 513-517 (1990); Sende et al., Cancer Research, 50, 3716-3721 (1990)], A. V. Schally and T. Redding, Proc. Natl. Acad. Sci., U.S.A. 84, 7279-7282 (1987)] and by E. Korkut et al., (Proc. Natl. Acad. Sci. U.S.A. 88, 844-848, 1991). The δ peptide was dissolved in 45% aqueous propylene glycol and administered at a dose of 25 micrograms / day in an ALZET mini syringe in androgen-dependent, well-differentiated male R3327 Dunning rat prostate adenocarcinoma. Tumors were measured weekly with a micrometer, and tumor volumes were calculated. The duration of treatment was 8 weeks and the mini-syringes were replaced at the end of week 4.

The peptides of the present invention may be prepared by any method known to those skilled in the art in the field of peptides. A summary of available methods can be found in M. Bodanszky's J. Principles of Peptide Synthesis (Springer-Verlag, Heidelberg, 1984). Classical solution synthesis is described in detail in Vol. 15 of the "Methoden dér Organische Chemie" (Houben-Weyl) monograph (Synthese von Peptiden, Parts I and II, Georg Thieme Verlag, Stuttgart, 1974). Solid-State Synthesis Only J. M. Stewart and J. D. Young (Solid Phase Peptide Synthesis, Pierca Chem Co., Rockford, IL, 1984, 2nd ed.) And G. Barany et al., Int. J. Peptide Protein Res., 30, 705-739 (1987).

The basic peptides of the invention were prepared by the solid phase method, but in some cases the 6-position side chain was incorporated by the "classic" method. In solid phase synthesis, the appropriate protected amino acids (sometimes protected peptides) are linked stepwise in the C-N direction after the C-terminal amino acid is attached to an inert solid support (resin). After completion of the coupling step, the N-terminal protecting group of the newly added amino acid residue is removed and the next appropriately protected amino acid is coupled. This is continued until all the desired amino acids are linked in the appropriate order, at which time the peptide is cleaved from the support and any protecting groups still present are removed under the least damaging conditions of the sequence groups. After all, a thorough cleaning must be followed to ensure that the product obtained has the desired structure.

A particularly preferred method of preparing the compounds of formula (I) of the present invention is solid phase synthesis, but may also be prepared by combining solid phase and classical (in solution) methods. According to this particularly preferred method, the alpha-amino group of the amino acid is protected by an acid or base sensitive moiety. Such protecting groups must be stable under the conditions of peptide bond formation and can be readily removed without damage to the growing peptide chain or racemization of any chiral center in the peptide.

Preferably, the peptides of formula (I) are X'-R ^, -D-Phe (4 HI) -R ³ -Ser (X ⁴ ) -R ⁵ (X ⁵ ) -R ⁶ (X ⁶ ) -LeuArg (X ⁸ ) - prepared ^{Pro-D-Ala-NH-X10} (IV) the peptides of formula, wherein

R ¹ , R ³ , R ⁵ , R ⁶ , H ¹ and X ¹ are as defined above,

X ^{4 is} a protecting group for serine hydroxyl groups such as benzyl (Bzl) or 2,6-dichlorobenzyl (DCB); the preferred protecting group is Bzl;

X ^{5 is} benzyl, 2-bromobenzyloxycarbonyl or

2,6-dichlorobenzyl (the latter being preferred) is the

R ^{5 for} protecting the phenolic hydroxy group of Tyr; or a tosyl group (preferred), a nitro or methyl (tert-butylbenzene) sulfonyl group to protect the guanidino group when R ^{5 is} Arg;

X ⁶ is an amino protecting group on the side chain of Lys or Orn, such as Z, Z (2-C 1) (preferred) or FMOC;

X ⁸ is a protecting group for Arg, which may be nitro, methyl 6

A9 (tert-butylbenzene) sulfonyl or tosyl group (preferred);

After X ¹⁰ = amido to protect a benzhydryl or methylbenzhydryl group incorporated in the resin support; a commercially available benzhydrylaminopolystyrene-2% divinylbenzene copolymer is preferred for the preparation of peptide amides.

Solid-phase synthesis of peptides of formula (IV) is accomplished by coupling a Boc-protected D-Ala amino acid to a benzhydrylamine resin in methylene chloride. DIC or DIC / HOBt reagents were used for coupling at ambient temperature. After removal of the Boc protecting group, sequential coupling of the protected amino acids (3 molar excess) is carried out, depending on the solubility of the Bocamino acids in methylene chloride or in dimethylformamide / methylene chloride. The success of the coupling after each step is preferably verified by a ninhydrin reaction by Kaiser et al., Anal. Biochem., 34, 595 (1970)]. When coupling is not complete, the coupling procedure is repeated before removing the alpha amino protecting group prior to reaction with the next amino acid.

After the desired amino acid sequence of the intermediate peptide of formula IV is prepared, the N-terminal is acylated with Ac ₂ O / TEA reagents and the peptide resin is then treated with liquid hydrogen fluoride in anisole to give a compound of formula IV wherein X ⁴ , X ⁵ , X ⁶ , X ⁸ and X ¹⁰ are each hydrogen.

These peptides are converted to the peptides of formula (I) wherein Y is Y ¹ , by a carbodiimide coupling method and a

2,3-bis-benzene-diaminopropionic acid, 2,3-bis-cyclohexanoyl-diaminopropionic acid, 2,3-bis-lauroyl-diaminopropionic acid, 2,4-bis-benzyl-1-diaminobutyric acid,

2.4-bis-cyclohexanoyl-diaminobutyric acid and 2.4-bis-lauroyl-diaminobutyric acid are used.

For the preparation of compounds of the formula I in which Y ^{1 is a} lower alkanoyl group and Y ^{2 is} a lower alkyl carbamoyl group, the second synthetic process is the preferred substituent on the ^6- side chain of lysine, such as 2,3-bisformyl diaminopropionic acid. due to its hydrophilicity. These peptides

X ¹ -R ¹ -D-Phe (4H 1) -R ³ -Ser (X ⁴ ) -R ⁵ (X ⁵ ) -R ⁶ [A (X ⁶ ) ₂ ] Leu-Arg (X ⁸ ) -Pro-D was prepared from compound ¹⁰ -Ala-NH-X (V) wherein

R ^! After ^{R3, R5, R6,} HI and X @ ¹ = letters are as defined above, X ⁴ is serine hydroxyl group protection, such as benzyl (Bzl) or 2,6-dichloro-benzyl (DCB); the preferred protecting group is Bzl;

X ^{5 is} benzyl, 2-bromobenzyloxycarbonyl or

The 2,6-dichlorobenzyl group (the latter being preferred) is the

R ^{5 for} protecting the phenolic hydroxy group of Tyr; or tosyl (preferred), nitro or methyl (tert-butylbenzene) sulfonyl to protect the guanidino group when R ^{5 is} Arg;

X ⁶ 'is an amino protecting group on the diaminoacyl side chain of Lys, such as Z, Z (2-C 1) or FMOC;

X ⁸ is a protecting group for Arg, which may be nitro, methyl (tert-butylbenzene) sulfonyl or tosyl (preferred);

X ^{10 is an} amido group for protecting a benzhydryl or methyl benzhydryl group incorporated in the resin support; a commercially available benzhydrylaminopolystyrene-2% divinylbenzene copolymer is preferred for the preparation of peptide amides.

Each of the protected peptides of formula (V) is prepared by solid-phase peptide synthesis as described for peptides of formula (IV), however, at position ⁶ instead of Boc-R ⁶ X ⁶ , a suitably protected R ⁶ (A) group is preferred. A BocR ⁶ [A (FMOC) ₂ ] group was introduced. The protecting group A is selected so that it can be selectively removed, i.e. the other protecting groups remain intact when the two X ⁶ 'groups are removed. For example, this can be accomplished by cleaving the FMOC protecting group with piperidine when

X ¹ -R ¹ -R ² -R ³ -Ser (X ⁴ ) -R ⁵ (X ⁵ ) -R ⁶ A-Leu-Arg (X ⁸ ) Pro-D-Ala-NH-X ¹⁰ (Va) General a peptide of the formula X ',

R ¹ , R ² , R ³ , R ⁵ , R ⁶ and A are as defined above and X ⁴ , X ⁵ , X ⁸ and X ^{10 are} not hydrogen.

The free amino groups at the 6-position are then acylated with a mixture of formic acetic anhydride, acetic acid halides or anhydride, propionic acid or pivalic acid to give compounds of formula I in which Y is Y ¹ after deprotection.

Deprotection and cleavage of the peptide from the resin is accomplished after the R ⁶ side chain is formed.

In another process, it is completely free of protecting groups

Obtained compounds ^X'-R, -R ² -R ³ -Ser-R ⁵ -R ⁶ (A) -Leu-Arg-Pro-D-Ala-NH ₂ (Vb) of the formula wherein X ^1, R ¹ , R ² , R ³ , R ⁵ , R ⁶ and A are as defined above such that Boc-R ⁶ [A (FMOC) 2] is preferably Boc-R ⁶ [A (Z) 2] at the 6-position. is deprotected from the intermediate peptide of formula (V).

The synthesis of peptides of formula (I) containing the side chain A (Y ² ) ₂ can be accomplished by the reaction of a peptide of formula (Vb) with a suitable cyanate atom, suitably metal cyanates such as potassium cyanate or N-alkyl isocyanate, e.g. with ethyl isocyanate.

Compounds of formula (I) wherein Y is Y ¹ are readily acylated by direct acylation of the 6-position diamino group of peptides of formula (Vb) using an equivalent amount of acyl halide, acetic anhydride, formyl or propionyl imidazole. The reaction yields a single compound7

A9 that the ⁴ hydroxy group of serine is present in free form.

Alternatively, the peptides of formula (I) may be prepared by incorporating, instead of protected R ⁶ , a suitably protected disubstituted diaminoacyl R ⁶ moiety. The procedure above was carried out exactly as mentioned above with the exception that instead of BocR ^{6 (X6)} of the synthesis step 5 BocR ⁶ (Y ₂₎ was reacted.

The crude synthetic products (over 500 mg) were purified on a BECKMAN Prep-350 preparative HPLC system equipped with a DYNAMAX MACRO column (41.4 x 250 mm) and column loading: C18 silica gel (spherical, pore size: 300A, particle size: 12) micrometer) (RAININ Inc. Co. Woburn, MA) (column A). Smaller amounts of peptides (Ν250 mg) were purified on a BECKMAN HPLC system (Model 142) on a DYNAMAC MACRO (21.2 x 260 mm) column (column B) filled with the above adsorbent. For purification of peptide less than 50 mg, reverse phase 10 x 250 mm VYDAC Protein & Peptide C, 8 columns (pore size: 300A, particle size 5 micrometers) (ALTECH, Deerfield. IL) (column C) or 10 x 250 mm W-POREX C, 8 columns (pore size: 300A, particle size: micrometer) (Phenomenex, Rancho Paulos Verdes, CA) (column D) were used. The columns were eluted with (i) a solvent system consisting of (A) 0.1% aqueous TFA and (B) 0.1% TFA in 70% aqueous acetonitrile, generally in a gradient manner. The column was monitored for elution progress with a UV detector at 230 or 280 nm. Chromatography was carried out at ambient temperature.

For analysis of crude and purified peptides, a HewlettPackard Model 1090 liquid chromatograph with a diode array detector at 220 and 280 nm and a reversed phase 4.6 x 25 O W-POREX C] 8 column (pore size: 300A, 5 micrometer) was used. column). The flow rate was 1.2 ml / min; (i) or (ii) the solvent system, the latter components being (A) 0.05 M ammonium acetate pH 7.0 and (B) 0.05 M ammonium acetate in 65% aqueous acetonitrile. The separations were carried out at room temperature.

Peptide samples were hydrolysed at 110 ° C for 20 hours in evacuated solder tubes containing 4 M methanesulfonic acid. Beckman 6300 amino acid analyzer was used for analysis.

Preparation 1

Bis-benzoyl-2,4-diaminopropionic acid (Ia), Bis-benzoyl-2,4-DL-diaminobutyric acid (1b)

To a solution of 140 mg (1 mmol) of ₂ pr in 2 ml of 10% sodium hydroxide was added dropwise 1.5 ml of 25% solution of benzoyl chloride in dioxane at 4 ° C. After stirring for 24 hours at 4 ° C, the title compound was extracted with ethyl acetate and purified by recrystallization from chloroform / hexane. (Bz) ₂ -DL-A ₂ bu is similarly prepared, but DL-A ₂ bu is used instead of A ₂ pr. The silica retention factors were 0.60 (la) and 0.69 (lb) on a thin layer of ethyl acetate: pyridine: acetic acid: water = 60: 20: 6: 11.

II. production

Bis-cyclohexanoyl-2,3-diaminopropionic acid (Ha), bis-cyclohexanoyl-2,3-DL-diaminobutyric acid (IIb)

To a solution of ₂ pr.HCl in 2 mL of 10% sodium hydroxide (140 mg, 1 mmol) was added dropwise a solution of cyclohexanecarbonyl chloride (1.6 mL, 3 mmol) in dioxane (1.6 mL, 3 mmol) and stirred at room temperature for 24 h. . The title compound was purified by solvent extraction followed by crystallization from a mixture of benzene and hexane.

Compound (IIb) was prepared in a similar manner, but using DL-2,4-diaminobutyric acid dihydrochloride (191 mg, 1 mmol) instead of 2,3-diaminopropionic acid. The retention factors on a silica gel thin film solvent system of Preparation I were 0.69 (Ha) and 0.79 (IIb).

Hl

Boc-D-Lys (A ₂ pr) -OH (IIIa), Boc-D-Lys (DL-A ₂ bu) OH (Illb)

To a mixed anhydride solution of 0.72 g of Z ₂ -A ₂ pr and 0.2 ml of ethyl chloroformate in 4 ml of dimethylformamide and 0.28 ml of triethylamine, 0.5 g of N ^alpha- Boc- A solution of D-Lys in dimethylformamide (4 ml) and triethylamine (0.3 ml) was added with stirring at 0 ° C. After 2 hours, the reaction mixture was concentrated under reduced pressure, the residual oil dissolved in water and ethyl acetate and acidified with 1M KHSO ₄ . The organic layer was washed with water, dried over anhydrous sodium sulfate and concentrated in vacuo. 0.5 g of this protected dipeptide are dissolved in 25 ml of 50% aqueous acetic acid and hydrogenated at room temperature in the presence of 0.1 g of 10% carbon-palladium catalyst for 2 hours. The reaction mixture was filtered and the filtrate was evaporated to dryness. The resulting white product was triturated with diethyl ether, then filtered and dried.

The (IIIb) obtained according to a similar manner, however, the acylation is carried out _Z2 -DL-A ₂ bu compound instead of Z ₂ -A ₂ pr.

ARC. Preparation (FMOC) ₂ A ₂ pr-OH

Dissolve 0.5 g (3.55 mmol) of 2,3-diaminopropionic acid in 7.1 ml of N sodium hydroxide and add 2.75 g (15% excess) of a solution of FMOC-OSu in 25 ml of acetone while stirring. at room temperature. After stirring for a further 4 hours, 3.55 ml of N sulfuric acid was added, which was then filtered, the filtrate was washed with water (3 x 10 ml) and air-dried on the filter. The product was recrystallized from ethyl acetate / petroleum ether. The purity of the white precipitate (1.9 g) was checked by TLC on a silica gel plate with ethyl acetate: pyridine: acetic acid: water = 120: 20: 6: 11. _Rf value: 0.62 to 0.66.

V. Production

Boc-D-Lys [(FMOC) ₂ A ₂ pr] -OH

0.75 g (3 mmol) N ^alpha- Boc-D-Lys-OH 0.42 ml (3 mmol) triethylamine in 3 ml 50% aqueous dimethylformamide

Suspend in a solution of A9 with amide. thereafter

1.8 g (3.2 mmol) of (FMOC) ₂ A ₂ pr (Preparation VIII) and 0.37 g of HOBt are dissolved in 3 ml of dimethylformamide and mixed with 0.5 ml of diisopropylcarbodiimide at 0 ° C. C. After 10 minutes, this solution is added to the above suspension. The reaction mixture, which is stirred at room temperature, clears within 1 hour. The mixture was poured into water (30 mL) to give a yellow voluminous precipitate which crystallized on trituration with diethyl ether. The crystalline material was recrystallized from a mixture of methanol, dichloromethane and hexane. 1.2 g of product, the silica gel, ethyl acetate: pyridine: acetic acid: water 960: 11 oldószerrrendszerrel made homogeneous by TLC, _Rf value of from 0.68 to 0.72: 20: 6th

Virus. production

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-D-LysLeu-Arg-Pro-D-Ala-NH ₂ (Via),

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-D-LysLeu-Arg-Pro-D-Ala-NH ₂ (VIb),

Ac-D-Nal (2) -D-Phe (4Cl) -D-Trp-Ser-Arg-D-LysArg-Pro-D-Ala-NH ₂ (VIc) g (about 1 mmol) containing 1 mEq of amino group to neutralized benzhydrylamine resin (Advanced Chemtech, Louisville, KY) with N, N -diisopropylcarbodiimide and 1-hydroxybenzotriazole, ca. Boc-D-Ala was coupled in methylene chloride or dimethylformamide at room temperature for 2 hours. The sequential coupling of the protected amino acids is carried out in a solid-phase synthesis flask using a 2.5 to 3.0 molar excess of protected amino acids according to the following program:

Step Reagent and operation Mixing time (minutes) First Coupling: Boc-amino acid, depending on solubility of protected amino acid in DCM or DMF, + D1C 60-90 Second iPrOH (or DMF then iPOH), wash 2 Third DCM, wash 2 4th iPrOH, wash 2 5th DCM, wash (3 times) 2 6th Deprotection: 50% TFA In DCM (twice) 5 and 25 7th DCM, wash 2 8th iPrOH, wash 1 9th Neutralization: 10% TEA in DCM 2 10th iPrOH, wash 1 11th Neutralization: 10% TEA in DCM 2 12th iPrOH, wash 1 13th DCM, wash (3 times) 2

After coupling Boc-ALA to the resin, the following sequence of events is coupled with the following amino acids: Boc-Pro, Boc-Arg (Tos), Boc-Leu,

Boc-D-Lys [Z (2-Cl)], Boc-Tyr (Bzl), Boc-Ser (Bzl),

Boc-D-Pal (3), Boc-D-Phe (4Cl) and Boc-D-Nal (2).

Using Boc-Arg (Tos) instead of Boc-Tyr (Bzl), the structure of the peptide resin will be: Boc-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser ( Bzl) -Arg (To s) -D-Lys [Z (2-Cl)] - Leu-Arg (Tos) -Pro-D-Ala-NH resin. Similarly, when Boc-D- (Pal3) is substituted at position 3 with D-Trp, Boc-D-Nal (2) -D-Phe (4Cl) -D-TrpSer (Bzl) -Arg (Tos) The product obtained is -D-Lys [Z (2-Cl)] -Leu-Arg (Tos) -Pro -D-Ala-NH-resin.

3-3.5 g of the decapeptide resin containing the free amino group at the N-terminus is reacted with 50 times excess acetic anhydride and triethylamine in 30 ml of dimethylformamide for 30 minutes. The acylated peptide resin was then washed 3 times with dimethylformamide, 3 times with isopropyl alcohol and 3 times with dichloromethane and then dried under vacuum. Deprotection and cleavage of the decapeptide from the resin is accomplished by treating 1.52.0 g with 3 ml of anhydrous hydrogen fluoride in 3 ml of anisole at 0 ° C for 45 minutes. Hydrogen fluoride was removed by a stream of nitrogen and the peptide was precipitated by addition of diethyl ether. The peptide was extracted 3 times with 50% aqueous acetic acid, separated from the resin by filtration, diluted with water and lyophilized.

The crude peptides in column A were made from the components of the solvent system i) eluting with a linear gradient of 40-70% B) for Compound (Via) over 60 minutes, and a linear gradient of 20-60% B for VIb and VIc. gradient elution with gradient mixture for 80 minutes. The retention time of (Via) (837 mg) is 25.5 minutes, the retention time of (VIb) (540 mg) is 11.4 minutes and the retention time of (VIc) (521 mg) is 18.8 minutes for the components of solvent system i). Eluting with a linear gradient mixture of -60% B) for 30 minutes. Amino acid analysis gives the expected result.

VII. production

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser (Bzl) -Tyr (Bzl) D-Lys (A ₂ pr) -Leu-Arg (Tos) -Pro- D-Ala-NH resin (VIIa),

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser (Bzl) -Arg (Tos) D-Lys (A ₂ pr) -Leu-Arg (Tos) -Pro- D-Ala-NH resin (VIIb),

Ac-D-Nal (2) -D-Phe (4Cl) -D-Trp-Ser (Bzl) -Arg (Tos) -DLys (A ₂ pr) -Leu-Arg (Tos) -Pro-D-Ala- NH Resin (VIIc)

The preparation of (VIIa) is described in Example VI. is carried out by solid phase peptide synthesis. The adecapeptide is assembled in 10 consecutive steps, first with 1 g of Boc-D-Ala, followed by Boc-Pro, Boc-Arg (Tos), Boc-Leu, Boc-DLys [A ₂ pr (FMOC) ], Boc-Tyr (Bzl), Boc-Ser (Bzl), BocD-Pal (3), Boc-D-Phe (4-Cl) and Boc-D-Nal (2). The N-terminal acylation was carried out with a 50-fold excess of acetic anhydride in dimethylformamide for 30 minutes. The FMOC protecting groups on the A ₂ pr moiety are removed so that the peptide

The reaction mixture was treated with 20 ml of 50% piperidine in dimethylformamide for 18 minutes, washed 3 times with dimethylformamide, 3 times with isopropyl alcohol and 3 times with dichloromethane and kept in a desiccator until further reaction. .

Similarly, but incorporating Boc-Arg (Tos) at position 5 with BocTyr (Bzl) instead of Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser (Bzl) Arg (Tos) -D-Lys (A ₂ pr) -Leu-Arg (Tos) -Pro-D-Ala-NH resin (VIIb) is obtained. By inserting the Boc-DPal (3) at the 3-position with Boc-D-Trp and the 5-position with Boc-Arg (Tos) instead of the Ac-D-Nal (2) -D-Phe ( 4Cl) -D-Trp-Ser (Bzl) Arg (Tos) -D-Lys (A ₂ pr) -Leu-Arg (Tos) -Pro-D-Ala-NH resin (VHc) is obtained.

Vili. production

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys (A ₂ prj-Leu-Arg-Pro-D-Ala-NH ₂ (Villa), Ac -D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-DLys (A ₂ pr) -Leu-Arg-Pro-D-Ala-NH ₂ (VIIIb), Ac -D-Nal (2) -D-Phe (4Cl) -D-Trp-Ser-Arg-DLys (A ₂ pr) -Leu-Arg-Pro-D-Ala-NH ₂ (VIIIc)

The peptides (VIIIa), (VIIIb) and (VIIIc) on the benzhydrylamine hydrochloride resin were prepared by the solid phase method as described in Example VI. is prepared as described in Preparation.

0.5 g. 0.5 mmol amino group resin for 10 cycles of Boc-D-Ala, Boc-Pro, BocArg (Tos), Boc-Leu, Boc-Lys [A ₂ pr (Z) ₂ ], Boc-Tyr (Bzl), Boc- Ser (Bzl), Boc-D-Pal (3), Boc-D-Phe (4Cl), Boc-DNal (2) are reacted with acetic anhydride / imidazole to give a peptide resin product which is treated with hydrogen fluoride and anisole free peptide (W1) containing D-Lys (A ₂ pr) moiety.

In a similar manner, however, 500 mg of free Boc-D-Trp is incorporated into the 3-position instead of Boc-D-Pal (3). The peptide (VIIIc) containing the D-Lys (A ₂ pr) moiety is obtained.

Alternatively, peptides (VIIIa), (VIIIb) and (VIIIc) can be acylated with the Boc ₂ A ₂ pr amino acids of the products (VIa), (VIb) and (VIc) in a carbodiimide reaction in the presence of HOBt, followed by 50% TFA solution in methylene chloride, the peptide was precipitated with diethyl ether, filtered and dried in vacuo.

The crude peptides were purified on column A using a gradient mixture of solvent system components i) (20-60% B, 80 min). Compounds (VIIIa), (VIIIb) and VIIIc have HPLC retention times of 15.1, 10.1 and 17.5 minutes using a gradient mixture of solvent system component (i) (30-50% B).

IX. production

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-D-Lys (DL-A ₂ bu) -Leu-Arg-Pro-D-Ala-NH ₂

Compound (IX) was prepared by solid phase peptide synthesis as described for (Villa) except that at position 6, Boc-D-Lys [A ₂ pr (Z) ₂ ] was replaced by Boc-D-Lys [A ₂ pr (Z) ₂ ]. DL-A ₂ bu (Z) ₂ ] was introduced. The HPLC retention time of compound (IX) was 10.4 minutes using a linear gradient mixture (35-50% B, 30 minutes) of the solvent system components (i) as eluent.

Example 1

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-D-Lys [A ₂ pr (Ac) ₂ ] -Leu-Arg-Pro-D-Ala- The NH ₂ 8 peptide is prepared on a solid phase by acylation of 0.3 g of free amino groups on the A pr ₂ -substituted Lys side chain of compound VII with 470 microliters of acetylimidazole in the presence of 700 microliters of triethylamine. Deprotection of the peptide and cleavage of the peptide from the resin is carried out in one step using liquid hydrogen fluoride as described for the preparation of compound (VI). The crude peptide was purified by column B using HPLC eluting with a linear gradient mixture of solvent system components i) (25-50% B9, 45 min).

Example 2

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A ₂ pr- (Bz) ₂ ] -Leu-Arg-Pro-D-Ala- Peptide NH ₂ and Ac-D-Nal (2) -D-Phe (4Cl) -Ser-Arg-DLys [A ₂ pr (Bz) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ Synthesis of peptide 6 of formula VIa is carried out by coupling compounds (VIa) and (VIb) with 2,3-bis-benzoyl-diaminopropionic acid (Ia) by the carbodiimide process. A solution of 7 mg (Ia) and 3 mg HOBt in 200 microliters of dimethylformamide was cooled to 0 ° C and reacted with 3.5 microliters of diisopropylcarbodiimide for 15 minutes. thereafter

Compound (Via) (36.3 mg) was dissolved in 200 ml of dimethylformamide, neutralized with triethylamine and mixed with the above active ester solution and kept at 0 ° C for 18 hours. A similar reaction was carried out with compound VIb. The reaction mixture was directly injected onto a C column and eluted with the solvent system i) to give 17.6 mg of 13 and 18 mg of peptide 6.

Following the same procedure, but acylating compounds (VIb) and (VIc) with the A ₂ pr (Ac) ₂ derivative, 19.1 mg of Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-D-Lys [A ₂ pr (Ac) _2] -Leu-Arg-Pro-D-Ala-NH ₂ formula 2 peptide and

17.2 mg of Ac-D-Nal (2) -D-Phe (4Cl) -D-Trp-Ser-Arg-DLys [A ₂ pr (Ac) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ 23 peptides were prepared.

When acylating compounds (Via) and (VIb) with 2,3-bis-cyclohexanoyl-diaminopropionic acid (IIa), 14.8 mg of Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-D-Lys [A ₂ pr (CHC) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ and

15.3 mg Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-TyrD-Lys [A ₂ pr (CHC) ₂ ] -Leu-Arg-Pro-D-Ala- This yields 72 peptides of the formula NH ₂ .

When compound (Via) is reacted with 2,4-bis-benzoyl-diaminobutyric acid (1b), 2,4-bis-cyclohexanoyl-diaminobutyric acid (Hb) or 2,4-bis-lauroyl-diaminobutyric acid, 16.6 mg of Ac-D -Nal (2) -D-Phe (4Cl) -DPal (3) -Ser-Tyr-D-Lys [DL-A ₂ bu (Bz) ₂ ] -Leu-Arg-Pro-DAla-NH ₂ , 14.7 mg Ac-D-Nal (2) -DPhe (4Cl) -D-Pal (3) -Ser-Arg-D-Lys [A ₂ bu (CHC) ₂ ] -Leu -Arg-Pro-D 27-peptide -Ala-NH ₂ and 8 mg of Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-D-Lys [D

HU 211 932 A9

There are obtained 19 peptides of formula LA ₂ bu (LAU) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ .

Example 3

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu (Car) ₂ ] -Leu-Arg-Pro-D-Ala The 16 peptides of the formula -NH ₂ are prepared by carbamoylating the intermediate peptide (IX) containing the two free amino groups. 37 mg of compound (IX) are dissolved in 100 microliters of dimethylformamide and the solution is buffered with 15 microliters of dimethylamine and 30 microliters of acetic acid. To this mixture was added a solution of potassium cyanate (48 mg) in 100 microliters of water and the reaction mixture was kept at ambient temperature for 48 hours. 15.8 mg of the title peptide was isolated by HPLC purification on a C column using (i) a solvent system.

In a similar manner but starting from compounds (VIIa), (VIIIb) and VIIIc, 12.2 mg of Ac-D-Nal (2) D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-D -Lys [A ₂ pr (Car) ₂ ] -Leu -Arg-Pro-D-Ala-NH ₂ peptide, 14.7 mg AcD-Nal (2) -D-Phe (4Cl) -D-Pal ( 3) Preparation of 22 peptides of the formula -Ser-Arg-D-Lys [A ₂ pr (Car) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ .

Example 4

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [DL-A ₂ bu (PRL) ₂ ] -Leu-Arg-Pro-D-Ala The peptide 18 -NH ₂ is obtained by propionylation of the two free amino groups on the substituted Lys side chain of the compound of formula IX. 37 mg of intermediate peptide were dissolved in 100 microliters of dimethylformamide, neutralized with 7 microliters of triethylamine and treated with 50 microliters of prepared propionyl imidazole reagent for 24 hours at room temperature. The reaction mixture was purified by column C HPLC using a solvent system (i) as eluent. Fractions containing pure peptide were lyophilized to give 13 mg of the title compound.

14.1 mg of Ac-D-Nal (2) -DPhe (4 Cl) -D-Pal (3) -Ser- were used as described above, but starting from compounds (VIIIa) and (IX), and using acetylimidazole as the acylating agent. Tyr-D-Lys [A ₂ pr (Ac) ₂ ] -Leu-A rg-Pro-D-Ala-NH ₂ peptide and 12.8 mg AcD-Nal (2) -D-Phe (4Cl) - Peptide 14 of formula D-Pal (3) -Ser-Tyr-D-Lys [DL-A ₂ bu (Ac) ₂ ] -Leu-Arg-Pro-D-Ala-NH _{2 was} prepared.

Example 5

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DL y [A ₂ pr (PRL) ₂ ] -Leu-Arg-Pro-D-Ala-NH Peptide _{2 of} Formula ₂ is prepared by solid phase peptide synthesis on benzhydrylamine resin (1 g = 1 mmol). in the manner described in the preparation. The decapeptide is constructed by sequential linkage of the following amino acids or derivatives: Boc-Ala, Boc-Pro-, Boc-Arg (Tos), Boc-Leu, Boc-D-Lys [A ₂ pr (PRL) ₂ ], Boc- Tyr (Bzl), Boc-Ser (Bzl), Boc-D-Pal (3), Boc-D-Phe (4Cl) and Boc-D-Nal (2). The acylation of the N-terminal amino group, deprotection and cleavage of the decapeptide from the resin are described in Table VI. . The crude lyophilized peptide was purified by column C.

Example 6

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-ArgD-Lys [A ₂ pr (For) ₂ ] -Leu-Arg-Pro-Ala-NH ₂ Synthesis of peptide 3 is accomplished by formylation of the free amino group of the peptide of intermediate (Villa) with mixed anhydride formic acid and acetic acid. To prepare the anhydride, 960 microliters (10 mmol) of acetic anhydride was reacted with 390 microliters (10 mmol) of formic acid at 0 ° C for 30 minutes. A solution of 37 mg (Villa) in 100 microliters of dimethylformamide was treated with 7 microliters of triethylamine and 6.7 microliters (50 micromoles) of the reagent prepared above and kept at 0 ° C for 1 hour. Peptide 3 was purified by HPLC on C column eluting with solvent system i) to give 22.8 mg of product.

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A2pr (For) _2] -Leu-Arg-Pro-D-Ala-NH ₂ Formula 10 peptides, Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -SerTyr-D-Lys [DL-A ₂ bu (For) ₂ ] -Leu-Arg-Pro- 75-peptide D-NH ₂ and Ac-D-Nal (2) -D-Phe (4Cl) -D-TrpSer-Arg-D-Lys [A ₂ pr (For) ₂ ] -Leu-Arg-Pro- peptide D-Ala 24 -NH ₂ was synthesized in the same manner, except that the starting material, Ac-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-D-Lys ( A2pr) -Le u -Arg-Pro-D-Ala-NH ₂ , Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr- D-Lys (D LA ₂ bu) -Leu-Arg-Pro-D-Ala-NH ₂ and Ac-D-Nal (2) -D-Phe (4Cl) -D-Trp-Ser Arg-DLys (A ₂ pr) -Leu-Arg-Pro-D-Ala-NH ₂ (VIIIc) is used.

Example 7

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-DLys [A ₂ pr (EtCar) ₂ ] -Leu-Arg-Pro-D-Ala-NH _The synthesis of peptide ₂ is accomplished by synthesizing the intermediate Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-DLys (A ₂ pr) -Leu-Arg-. The pro-D-Ala-NH ₂ peptide (VIIIb) is reacted with N-ethyl isocyanate. The intermediate peptide (36 mg, 20 micromoles) was dissolved in 100 microliters of dimethylformamide, the pH of the solution was adjusted to 14 microliters (100 micromoles) of triethylamine, and the peptide was reacted with 3.5 microliters of N-ethylisocyanate at 0 ° C. For 10 hours. The reaction mixture was injected onto column C and eluted with i) the solvent system. so

21.8 mg of the desired peptide are obtained.

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Tyr-DLys [A2pr (EtCar) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ Peptide 9 and Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -SerTyr-D-Lys [DL-A ₂ bu (EtCar) ₂ ] -Leu-Arg-Pro- The peptide 17 of formula D-Ala-NH ₂ is prepared in a similar manner starting from Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -SerTyr-D-Lys (A ₂ pr) - Leu-Arg-Pro-D-Ala-NH ₂ peptide (VIHa) and Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) Ser-Tyr-D-Lys (DL-). Peptide IX of formula ₂ bu) -Leu-Arg-Pro-D-Ala-NH _{2 was} used.

HU 211 932 A9

Table 1

Methods of making and purifying LHRH antagonists

Number of peptide Synthesis Method Gradient (% B / min) Retention time (minutes) cleaning analysis 1 3 25-50 / 40 30-45 / 15 12.5 2 2 30-45 / 45 35-50 / 15 10.0 3 6 20-50 / 50 35-50 / 15 8.7 4 7 25-45 / 50 35-50 / 15 11.0 5 2 30-50 / 50 45-60 / 15 12.0 6 2 35-55 / 60 45-60 / 15 9.5 7 3 30-60 / 60 35-50 / 15 8.2 8 1 and 4 25-50 / 50 35-50 / 15 12.5 9 7 25-45 / 50 40-55 / 15 10.6 10 6 30-45 / 45 35-50 / 15 10.3 11 4 30-55 / 50 35 to 50/15 14.8 12 2 25-45 / 50 55-70 / 15 11.95 13 2 25-45 / 50 35 to 50/15 12.3 14 4 26-45 / 50 35 to 50/15 12.8 15 6 25-40 / 45 35 to 50/15 12.3 16 3 35-50 / 45 35 to 50/15 11.8 17 7 25-50 / 50 35 to 50/15 13.9 18 4 30-50 / 40 35-65 / 15 14.7 19 2 50-90 / 60 80-95 / 15 12.0 20 2 35-50 / 45 45-60 / 15 8.0 21 2 50-80 / 60 50-65 / 15 10.9 22 3 30-50 / 40 45-60 / 15 8.0 23 2 35-55 / 40 45-60 / 15 9.3 24 6 35-55 / 40 40-55 / 15 12.0 25 Fork production 20-50 / 60 35 to 50/15 9.3 26 IX. production 20-50 / 60 35 to 50/15 9.1

Table 2

Ac-D-Nal (2) -D-Phe (4Cl) -R ³ -Ser-Arg-D-Lys [A ₂ pr (Y) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ General Ovarian Inhibitory Effect of Peptides of Formula I and Affinity on Membrane Receptors of Human Breast Cancer Cells

Number of peptide The peptide % Inhibition of ovulation Affinity is constant R ³ Y 0.75 pg 1.5 pg K _a l ntVT ¹ K _a 2 pM- ¹ 1 D-Pal (3) tsar 7.07 3.15 2 D-Pal (3) Ac 16.35 0.32 3 D-Pal (3) for NB 4 D-Pal (3) EtCar 9.71 0.05 5 D-Pal (3) CHC 40 NB 6 D-Pal (3) BZ 10 20 NB 22 D-Trp tsar 4.09 2.67 23 D-Trp Ac 6.87 0.14 24 D-Trp for 50 NB

'' 1D-TrpfLHRH was used as labeled ligand. NB does not bind

HU 211 932 A9

Table 3

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-D-Lys [A (Y) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ Ovarian Inhibitory Effect of Peptides of Formula I and Affinity on Membrane Receptors of Human Breast Cancer Cells

Number of peptide The peptide % Inhibition of ovulation Affinity] constant THE Y 0.75 pg 1.5 Pg K _a l nM ^-1 K _a 2pM> 7 A ₂ pr tsar 6.27 5.72 8 A ₂ pr Ac 1.57 6.16 9 A ₂ pr EtCar 20 50 30,92 8.57 10 A ₂ pr for 67 100 48.29 2.11 12 A ₂ pr CHC 1.68 3.57 14 DL-A ₂ bu Ac 20 4.83 0.26 15 DL-A ₂ bu for (25 **) 100 NB 16 DL-A ₂ bu tsar 33 NB 18 DL-A ₂ bu PRL 75 21.18 6.17 19 DL-A ₂ bu LAU 0 0 21 DL-A ₂ bu CHC NB 25 A ₂ pr 3.49 1.29 26 DL-A ₂ bu NB

* '' '1-ID-TrpjYHRH was used as labeled ligand · * dose 0.375 microgram NB not bound

Table 4

Ac-D-Nal (2) -D-Phe (4Cl) -R ³ -Ser-Arg-D-Lys [A ₂ pr (Y) ₂ ] -Leu-Arg-Pro-D-Ala-NH ₂ General LHRH Inhibitory Activity of Antagonists of Formula I in Perfused Rat Pituitary Cell System at Different Antagonist / LHRH Molar Ratios

Number of peptide % Inhibition of LH response antagonist: LHRH = 1: fly (min) antagonist: LHRH = 3: fly (min) 0 30 60 90 0 30 60 90 1 80 46 31 95 59 55 52 2 26 27 29 25 88 41 13 3 93 43 26 24 4 91 50 42 41 5 95 60 60 60 6 50 40 35 30 82 63 60 60 22 64 22 24 23 52 22 21 22 24 36 11 0 9 58 5 18 27

Table 5

Ac-D-Nal (2) -D-Phe (4Cl) -D-Pal (3) -Ser-Arg-D-Lys [A ₂ pr (Y) ₂ ] -Leu-Arg-Pro-D-Ala LHRH Inhibitory Effect of Antagonists of Formula -NH ₂ on Peripheral Rat Pituitary Cell System Using Different Antagonist / LHRH Molar Ratios

Number of peptide % Inhibition of LH response antagonist: LHRH = 1: lid <5 (min) antagonist: LHRH = 3: fly (min) 0 30 60 90 0 30 60 90 7 57 51 45 41 96 78 70 58 8 64 39 27 22 99 66 47 37 9 90 72 14 80 63 51 53

HU 211 932 A9

Number of peptide % Inhibition of LH response antagonist: LHRH = 1: fly (min) antagonist: LHRH = 3: fly (min) 0 30 60 90 0 30 60 90 15 44 41 22 23 99 60 34 21 16 52 33 24 28 18 90 71 58 54 19 20 20 0 0 25 57 49 43 85 69 62 57 26 70 52 37 90 75 62 57

Table 6

Effect of peptide 8 at 25 micrograms / day on Dunning R3327 prostate cancer in rats

Time (weeks) Size of tumor (mm ³ ) Control group Managed group 0 4326 + 1891 * 4001 ± 1617 1 6901 ± 2968 5459 ± 1863 2 9174 ± 4507 5892 ± 1938 3 9465 ± 4349 6357 ± 2327 4 12582 ± 6659 6237 ± 2974 ** 5 12230 ± 4848 7447 ± 3481 ** 6 14732 ± 6597 8038 ± 4374 ** 7 17796 ± 8602 8129 ± 3525 ***

* SD * 'p 0.05 * · * p 0.01

Claims (21)

PATENT CLAIMS CLAIMS 1. Compounds of the formula X-R'-RLR'-Ser-RNR ^ AYnl-Leu-Arg-Pro-DAla-NH ₂ and pharmaceutically acceptable salts thereof. in the formula R ^{1 is} D-Phe, D-Phe (4Cl), D-Nal (1) or D-Nal (2), R ^{2 is} D-Phe or D-Phe (4Hl), R ^? D-Trp or D-Pal (3), R ^{5 is} Tyr or Arg, R ^{6 is} D-Lys or D-Orn, Hl is fluorine, chlorine or bromine, X is a C 2 -C 5 alkanoyl group, H ₂ N-CH ₂ - (CH ₂ ) _m -CH (NH ₂ ) - (CH ₂ ) _n -CO- (II) diaminoacyl wherein m is 0 or 1 and n is 0 or 1 Y is Y ¹ or Y ² where Y ^{1 is an} acyl group derived from straight or branched aliphatic or alicyclic carboxylic acids having from 3 to 12 carbon atoms or from aromatic carboxylic acids containing 6 or 10 ring carbon atoms, ^Y2 or substituted carbamoyl C1-3 alkyl, N- _(CH2) "- NH-CO (III): wherein n is 0-3 in the image. The peptide of claim 1, wherein Y is Y ¹ and Y ¹ is formyl, acetyl, propionyl, butyryl, isobutyryl, cyclohexanoyl or benzoyl. The peptide of claim 2, wherein R ¹ is D-Nal (2), R ² is D-Phe (4 Cl), R ³ is D-Pal (3), R ⁵ is Tyr, R ⁶ is D- Lys, X is acetyl and A is 2,3-diaminopropionyl. The peptide of claim 3, wherein Y ¹ is formyl. The peptide of claim 3, wherein Y ¹ is acetyl. The peptide of claim 3, wherein Y ¹ is propionyl. The peptide of claim 2, wherein R ¹ is D-Nal (2), R ² is D-Phe (4 Cl), R ³ is D-Pal (3), R ⁵ is Tyr, R ⁶ is D- Lys, X is acetyl and A is 2,4-diaminobutyryl. The peptide of claim 7, wherein Y ¹ is formyl. The peptide of claim 7, wherein Y ¹ is acetyl. The peptide of claim 7, wherein Y ¹ is propionyl. The peptide of claim 2, wherein R ¹ is D-Nal (2), R ² is D-Phe ( ⁴ Cl), R ³ is DPal (3), R ⁵ is Arg, R ⁶ is D-Lys, X is acetyl and A is 2,3-diaminopropionyl. The peptide of claim 11, wherein Y ¹ is formyl. HU 211 932 A9 13. A peptide according to claim 11, wherein ^Y1 is acetyl. The peptide of claim 11, wherein Y ¹ is propionyl. The peptide of claim 2, wherein R ¹ is D-Nal (2), R ² is D-Phe (4 Cl), R ³ is DTrp, R ⁵ is Arg, R ⁶ is D-Lys, X is acetyl. and A is 2,3-diaminopropionyl. 16. A peptide according to claim 15, wherein ^Y1 is formyl. 17. The peptide of claim 15, wherein Y ¹ is acetyl. 18. The peptide of claim 1 which is ² -t YY, and ^Y2 is carbamoyl, N-methylcarbamoyl or N-ethylcarbamoyl group. 19. The peptide of claim 18, wherein R ¹ 5 is D-Nal (2), ^R2 is D-Phe (4Cl), ^R3 is D-Pal (3), ^R5 is Tyr or Arg, ^R6 is D-Lys, X is acetyl and A is 2,3-diaminopropionyl or 2,4-diaminobutyryl. The peptide of claim 19, wherein Y ² 10 is carbamoyl. 21. The peptide of claim 19, wherein Y ² is ethylcarbamoyl.