CYCLIC HEXAPEPTIDES AS TACHYQUININ ANTAGONISTS, THEIR PREPARATION AND PHARMACEUTICAL COMPOSITIONS THEREOF

Abstract: A description is given of hexapeptide analogues of tachyquinines and their pharmaceutically acceptable salts as per general formula (I) effective in the treatment of diseases, where tachyquinines play a pathogenetic role, in particular in the treatment of arthritis, asthma, inflammations, tumoral growth, gastrointestinal hypermotility, Huntington's disease, neuritis, neuralgia, migraine, hypertension, incontinence of urine, urticaria, carcinoid syndrome symptoms, influenza, and cold.

Description: CYCLIC HEXAPEPTIDES AS TACHYKININ ANTAGONISTS, THEIR PREPARATION AND PHAR CEUTICAL COMPOSITIONS THEREOF Field of the invention 5 The invention refers to cyclic hexapeptide analogues of tachykinines of general foraula (I) (I) r p--WWHH--CHR j^ - -CHRj-CO-Ai^-Aj-pAla- where R^ ■ H, linear or branched C^.^ alkyl 10 R3 * H, natural or not natural amino acid free or protected side chain or wherein 15 n « 1. 2, 3, 4. 5 R" = cyclooctyl, adamantyl, cyclohexyl, naphthyl R" = phenyl when n is other than 1 R" = a substituted carboxyaaide group when n » 1, 2 A - Gin, DGln 20 A 2 » Trp, DTrp A •> Phe, DPhe W * CO-NR' , CH 2 -NR' where R' « H, CHo and their pharmaceutically acceptable salts with acids or organic or inorganic bases. Tachykinins antagonist coapounds of foraula (I) prove to be effective in the treatment of diseases where tachykinins play a pathogenic role, in particular in the treataent of arthritis , asthaa, inflammations, tumor growth, gastrointestinal hyperaotility, Huntington's disease, neuritis, neuralgia, migraine, hypertension, incontinence of urine, urticaria, carcinoid syndrome symptoms , influenza, and cold. State of the Art Tachykinins are a family of peptides characterized by the following common C- terminal sequence: Phe-X-dy-Leu-Met-NH 2 where X stands for an aaino acid characterizing each of the tachykinins. As far as mammals are concerned, the three tachykinins were called substance P (SP) (where X = Phe) , neurokinin A (NKA) (where X * Val) and neurokinin B (NKB) (where X = Val) and their neuro transmitter role, both at peripheral and central levels, was acknowledged (J.E. Haggio, Peptides. 1985. 6, 237-245 and P.C. Eason et al. , Neuropep tides and their peptidases, A.J. Turner and Ellis Horwood, England. 1987. PP. 87-106) . The pharmacological and biochemical results conveyed by the literature show that the biological activity of tachykinins is mediated, in mammals' tissues, by three distinct receptors at least, called NK-1, NK-2, NK-3. Natural tachykinines exhibit a different affinity with such three receptors. Highly potent tachykinins antagonists seem to be effective to reduce or antagonize pathological effects due to an excess of tachykinins in animals or man. The first generation tachykinins antagonists described, for instance, in US-A-4, 81,139 - scarcely selective - were followed by the second generation ones (EP-A-401,177; EP-A-347,802; GB-Λ- 2,216,529), more selective. Research in the field is anyway aimed at singling out higher affinity and activity antagonists, free from agonist activity on other receptors, hence suitable for therapeutical use. Detailed Description of the Invention This invention refers to cyclic hexapeptide analogues of tachykinins of general formula (I) where R j * H, linear or branched C^ alkyl R * natural or not natural amino acid free or protected side chain or where n « 1.2, 3. .5 R" ■ cyclooctyl, adamantyl, cyclohexyl, naphthyl R" = phenyl when n is other than 1 R" ■ a substituted carboxyamide group when n * 1, 2 A 2 ■ Trp, DTrp Aa « Phe, DPhe « CO-NR* , CH 2 -NR* where R' « H, CH and their pharmaceutically acceptable salts with acids or organic or inorganic bases. According to this invention, linear or branched C j ^ alkyl are selected in the group consisting of : methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl. Natural aaino acid is selected in the group consisting of : glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine , tryptophan, aethionine, serine. threonine, cysteine. tyrosine, asparagine, glutaaine, aspartic acid, gluta ic acid, lyslne , arginine, histidine, in their L or D forms. Not natural aaino acid is selected in group consisting of β-alanine, D or L 2-aainoisobutyric acid, D or L 2,3-diaainopropionic acid, D or L norleucine, D or L alloisoleucine, D or L pyroglutamic acid, L or D 3-hydroxyproline, L or D 4-hydroxyproline, L or D phenylalanine substituted in the ortho, meta, or para position, L or D thienylalanine , L or D pyridylalanine, β(2- or 3~ benzothienylalanine) , 1,2,3.4 tetrahydroisoquinoline-3'carboxyl acid. Among the amino acid chain protectors , the following are given special consideration: Mbs, Mtr, N02, Z. Tos. Pac, For, Ne, Ac. 2- Br-Z, 2-C1-Z, Bzl. 2.6-dichloro-Bzl, S03H, Fmoc. OMe, OBzl, OFm, ONp, OSu. Protected side chain of a natural or not natural aaino acid means, in particular, L or D Arg (Mbs), L or D Arg(Ntr), L or D Arg(N0 2 ) , L or D Arg (Z), L or D Arg(Tos), L or D Arg(Pac), L or D Trp(For), L or D Trp(Mts), L or D Tyr(Me), L or D T r(Ac), L or D Tyr(2-Br-Z) , L or D Tyr(Bzl), L or D Tyr(2,6-dichloro-Bzl) . L or D TyrfSOjH), L or D Ser(Ne), L or D Ser(Ac) , L or D Ser(Bzl). L or D Ser(2,2-dichloro- Bzl), L or D Ser(S03H) . L or D Lys(Ac), L or D Lys(2-Br-Z), L or D Lys(2-Cl-Z), L or D Lys(Fmoc), L or D Lys(Z), L or D Lys(Tos), L or D Lys(Ne), L or D Lys (Bzl), L or D Asp(OMe), L or D Asρ(OBzl), L or D Asp(OFa), L or D Asp(ONρ), L or D Asp(OSu), L or D Glu(OMe). L or D Glu(OBzl). L or D Glu(OFm) , L or D Glu(ONp). L or D Glu(OSu). Substituted carboxaaide group means a CONReRg group, where Re and Rg are equal or different and represent H or a linear or branched or cyclic alkyl, arylalkyl, aryl residue. R and B together with the nitrogen atom can form a 5- or 6- terminal cycle including 4 or 5 carbon atoms or groups - In particular, NReRg can mean the residue of benzylamine, phenylethylamine even substituted with a halogen, 1- or 2- naphthylamine, cyclohexylamine, cyclooctylamine, adamantanamine, adamantyl-methylamine. Among the compounds as per formula (I) of this invention, preference is given to Rl * isobutyl A 2 • Tip R3 * (CH 2 ) n CgH ll f where n ■ 2, 3. 4, 5; (CH 2 ) n -(l-naphthyl) , where n • 2, 3. 4, 5; (CH 2 ) n -(l-adamantyl) , where n - 1, 2, 3. 4, 5; (CH 2 ) n - cyclooctyl, where n « 1, 2, 3, 4, 5; (CH 2 ) n -CPgHe, where n » 2, 3, 4, 5; (CH 2 ) n -C0NHBzl, where n » 1, 2; (CH 2 ) n -C0NMeBzl, where n « 1. 2; (CH 2 ) n -C0 HCH 2 C 6 H u . where n * 1, 2; (CH 2 ) n -C0NMeCH 2 CgH ll t where n * 1, 2; (CH 2 ) n -C0NH-CH 2 (l-adamantyl) , where n * 1, 2; (CH 2 ) n - C0NMe-CH 2 (l-adaaantyl) , where n « 1,2. In particular the following compounds are preferred: cyclo (Leu-Cha-Gln-Trp-Phe-βAla) cydo(Leu-Asp(NHBzl)-Gln-Trρ-Phe-&Ala) cyclo(Leu-Asρ(NMeBzl)-Gln-Trp-Phe-PAla) cyclo( eu-Asp(NHCH 2 C6Hll)-Gln-Trp-Phe-p^la) cyclo(Leu-Asp(NHeCH 2 C6Hll) -Gln-Trp-Phe-PAla) cyclo(Leu-Glu(NHBzl)-Gln-Trρ-Phe-βAla) cyclo(Leu-Glu(NHeBzl)-Gln-Trp-Phe-βAla) cyclo(Leu-Glu(NHCH 2 C6Hll)-Gln-Trp-Phe-βAla) cyclo(Leu-Clu(NHeCH 2 C6Hll)-Gln-Trp-Phe-UAla) cyclo(Leu-Glu(NHCH 2 (1-adamantyl))-Gln-Trp-Phe-PAla) cyclo(Leu-Glu(NMeCH 2 (1-adamantyl))-Gln-Trp-Phe-PAla) cyclo(Leu-Asp(NHCH 2 (1-adaaantyl))-Gln-Trp-Phe-&Ala) cyclo(Leu-Asp(NMeCH 2 (1-adamantyl))-Gln-Trp-Phe- la) cyclo(Leuψ [CH 2 NH3Asρ(NHBzl)-Gln-Trp-Phe-&Ala) cyclo(LeuH* [CH^jAsp(NMeBzl)-Gln-Trp-Phe-&Ala) cyclo(Leuγ [CH2NH]Asp(NHCH 2 C6Hll)-Gln-Trp-Phe-&Ala) cyclo(Leu Y [CH2NH]Asp(NMeCH 2 C6Hll)-Gln-Trp-Phe-pAla) cyclo(Leu « [oyffllGlufNHBzlJ-Gln-Trp-Phe-frAla) cyclo(Leu [OyffljGlu (NMeBzl ) -Gln-Trp-Phe-PAla) cyclo(Uu Y [CH^lGlutNHCH^όHlD-Gln-Trp-Phe-βAla) cyclo(Leu f [CH2NH]Glu(NMeCH 2 C6Hll ) -Gln-Trp-Phe-βAla) cyclo(Uu Y [CH2NH]Glu(NHCH 2 (l-adamantyl) )-Gln-Trp -Phe-βAla) cyclo(Leu Y [CH2NH]Glu(NMeCH 2 (l-adamantyl) )-Gln-Trp-Phe-pAla) cyclo ( Leu V [CH j NHjAsp (NHCH 2 ( 1-adamantyl ) ) -Gln-Trp -Phe-pAla) cyclo(LeuY [CH2NH]Asρ(NMeCH 2 (l-adamantyl) )-Gln-Trρ-Hie-pAla) cyclo(Leu [CH^elAspfNHBzl) -Gln-Trp-Phe-frAla) cyclo (Leu Y [CH^elAsp (NMeBzl ) -Gln-Trp-Phe-&Ala) cyclo(LeuY [CH2NMe]Asp(NHCH C6Hll)-Gln-Trp-Phe-pAla) cyclo(LeuY [CH 2 NMe]Asp(NMeCH 2 C6Hll)-Gln-Trp-Phe-βAla) cyclo(LeuY [CH^eDGlutNHBzD-Gln-Trp-Phe-frAla) cyclo(Leu Y [CH j NMelGlufNMeBzl ) -Gln-Trρ-Phe-&Ala) cyclo(LeuY [CH 2 NMe]Glu(NHOT 2 C6Hll)-Gln-Tι * p-Phe-&Ala) cyclo (Uu Y [CH 2 NMe]Glu (NMeCH 2 C6Hll ) -Gln-Trp-Phe-βAla) cyclo(LeuY [CH2NMe]Glu(NHCH 2 (l-adamantil) )-Gln-Trρ-Phe-pAla) cyclo (Uu Υ [CH2NMe]Glu (NMeCH 2 ( 1-adamantil ) ) -Gln-Trp-Phe-βAla) cydo(UuY [CH 2 N e]Asp(NHCH 2 (i-adaaantil) )-Gln-Trp-Phe-βAla) cyclo(UuY [CH 2 NMe]Asp(NMeCH 2 (l-adamantil) ) -Gln-Trp-Phe-βAla) cyclo(LeuV [CH 2 NH]Asp(0Bzl)-Gln-Trρ-Phe-&Ala) cyclo(LeuY [CH2NMe]Asp(0Bzl)-Gln-Trp-Phe- Λla) cyclo (UuY [CH 2 NMe]Nal-Gln-Trp-Phe-&Ala) cyclo(Leu Ϋ [CH 2 NMe]Cha-Gln-Trp-Phe-βAla) cyclo(LeuY [CH^lCha-Gln-Trp-Phe-flAla) cyclo(LeuY [CH2NH]Nal-Gln-Trp-Phe-pAla) cydo(LeuY [CH 2 NMe]CH( (CH 2 ) 3 Bzl)C0-Gln-Trp-Phe -βAla) cyclo(LeuV [CH 2 NHlCH((CH 2 ) 3 Bzl)CO-Gln-Trp-Phe-pAla) cyclo(Uu-NH-CH( (CH 2 ) 3 Bzl)CO-Gln-Trp-Phe-pAla) The cyclic peptide analogues covered by the present invention can be prepared by known synthetic techniques in the solid phase or in solution. For the obtainaent of linear peptides with the C-terainal carboxyl group in the form of free acid, solid supports such as resin phenylacetamidoaethyl (PAM) or the resin p- hydroxymethylphenoxy ethyl (Hang), can be used. In the case of PAN resin, the amine function of aaino acids is protected by the t- butyloxycabonyl group which can be selectively deprotected by trifluoracetic acid, whilst final deprotection - with simultaneous peptide detachment from the polymer support - is secured by anhydrous hydrofluoric acid. In the case of the Hang resin, the aaino acid aaine function is protected by the 9-fluorenyl- methoxycarbonyl group (F oc). selectively deprotected by piperidine, whilst final deprotonation - with simultaneous peptide detachment from the polymer support - is secured by trifluoracetic acid. In both cases, the trifunctional amino acid side chains can be protected by the known methods described by literature. For the construction of the peptide chain on the insoluble polymer support, each amino acid is made to react in the form of free acid, in the presence of a suitable coupling agent , e . g. dicyclohexyl carbodiiaide (DCC) , used with additives , if - any, such as hydroxybenzothiazole (HOBT) or benzothiazolyl-N- oxytridlmethylaainophosphoniua hexafluorophosphate (BOP) ; as an alternative, the aaino acid can be aade to react in the form of symmetric anhydride, activated ester, or according to any of the other aethods described in literature. Aaino acid coupling reaction completion can be ninhydrin tested, as described by E.T. Kaiser et al. , Anal.Biochea. , 1970, 24. 595- Aaino acids with the R 3 *(CH 2 ) n -C0NReRg group as side chain can be synthesized, e.g. starting from the corresponding acid (where the α- aaino and α-carboxyl groups have been pre-protected) , by condensation with the suitable HNRcRg aaine and the use of activators such as those currently employed in peptide chemistry (BOP. PyBOP. HOBT) . Amino acids whose side chain is represented by the (CH ) π -R" group can be synthesized by known organic chemistry techniques, such as, e.g., those described by Evans et al., J. Am. Chem. Soc., 112 (1990) 4011-4030; G.C. Barret, Chemistry and Biochemistry of the Amino Acids, Ed. G.C. Barret, Chapman & Hall, London, 1985, 246-296. As for the -CH 2 -NR'- bond, it is synthesized according to the procedure described by Sasaki and Coy, Peptides, 1987, 8, 119. Such a procedure was extended to the synthesis in the solid phase according to the Fmoc strategy, as described by Przewosny et al., Peptides, 1990. 370. Specifically (see scheme shown hereunder): N- aethoxymethylamide as per foraula 2 is prepared froa the corresponding N-protected aaino acid. The said aaino acid is dissolved in aethylene chloride; the solution is added with an equiaolar amount of hydroxybenzotriazole and stirred for 20 ainutes. Then, N-0-diaethylhydroxylaaine.HCl dissolved in dichloroaethane and added with an equiaolar amount of a sterically hindered tertiary amine, e.g. diisopropylethylamine, is added to the said solution. The resulting mixture is kept under stirring for about 16 hours, after which it is washed with dilute aqueous HC1, with an NaHC03 saturated solution, as well as with an NaCl saturated solution. The desired product can be purified, e.g. by chroaatography on ailica gel. N-aethoxyaethylamide as per foraula 3 is reduced to produce the corresponding aldehyde as per formula 4. e.g. with equimolar lithium aluminium hydride at 0 * C in an ether solution. On reaction completion, the mixture is treated with a solution of acid potassium sdphate in water. The product is then isolated by extraction, with ether, of the aqueous phase: for this purpose the ether phase is washed with dilute aqueous HC1, with NaCO? saturated solution, and B * Boc, Fmoc R 2 * H; Me The aldehyde as per formula 4 is allowed to react with the coapound as per formula 6, or with the N-terminal end of a pentape tide chain bound to the resin by a β-alanine residue. The initial Schiff base is reduced in situ, e.g. by sodium cyanoborohydride, to give a modified hexapeptide bound to the resin as per formula 7 * After deprotection and detachment, performed as described above, the suitably freeze-dried raw peptide is purified to homogeneity, e.g. by high pressure reversed-phase preparative chromatography. Cyclic peptide synthesis can be obtained via cyclization in solution after preparation - according to one of the aforementioned methods, in the solution * or solid phase - of the linear precursor of the desired cyclic peptide. Cyclization is performed with condensing agents and, if necessary, by activating the C-terainal carboxyl group of the cyclic precursor. EXAMPLE Preparation of the cyclic peptide : cyclo(Uu*f rCH 2 NHlAsp(NHBzl)-Cln-Trp-Phe-pAla) <**> a) Synthesis of the linear peptide having the following sequence: H-UuΥ [CH2NH]Asp(NHBzl)-Gln-Trp-Phe-pAla-0H (i) Synthesis of Boc-Asp(NHBzl)-OH: 323 g Boc-Asp-OBzl (Novabiochea, Switzerland) is solubilized in 70 aL dioxane; then the solution is added with 530 ag BOP, 37μL DIEA and, finally. 107 "g benzylaaine. After 3 hours, the reaction mixture is dried and the residue is purified by chromatography on Iterck silica gel 60 (mesh 70-230) with ethyl acetate- /n-hexane-1 (v/v), as eluent, Rf * 0.3.310 ag yield. Carboxyl group deprotection is obtained by dissolving 300 ag benzyl ester in 40 aL aqueous 952 ethyl alcohol and adding the solution to a suspension of 100 ag Pd/C (10% Pd) in 6 mL 95X aqueous ethyl alcohol. The environment is saturated with hydrogen and the reacting mixture is kept under hydrogen environment for 2 hours. Then, the solution is filtered and dried. 3.0 g Boc-βAla-PAM resin (Bachem, Switzerland), equal to 0.45 aaoles of aaine groups, is fed to a Labortec SP 640 seai-autoaatic peptide synthesis reactor. The resin is washed as described in Table 1, cydes 6-7. For resin coupling to the subsequent aaino acid, symmetric anhydride is prepared by dissolution of 0.48 g Boc-Phe-OH in 5 mL dichloroaethane. The solution temperature is brought to 0*C and added with 0.9 aL of a 1M solution of dicyclohexylcarbodiiaide In dichloroaethane. After 15 ainutes, dlcyclohexylurea is filtered and the resulting solution is added to the deprotected resin. The resin is kept under stirring at ambient temperature for 60 ainutes (cycle 8) . The procedure is completed by washing (cycles 9-12) and the reaction is ninhydrin-tested by the Kaiser method. In case of a negative response, the Boc group is hydrolyzed with 0% TFA (cycles 1-4), before the subsequent aaino acid coupling, which takes place according to the described procedure. The following residues are aade to react in the same order, in the quantities indicated in brackets: Boc-Trp-OH (0.548 g) , Boc-Gln-OH (0.443 g). Boc- Asp(NHBzl)-0H (0.581 g). After deprotection, Boc-Leu-H (0.242 g) dissolved in a dimethylformamide solution containing "L 1% acetic acid is added to the resin; 5 mL of an NaBH CN solution (70 mg) in a dimethylformamide solution containing 5 mL 1% acetic acid is allowed to drip under stirring for 40 ainutes. The resin is kept under stirring at ambient temperature for about 6 hours. The procedure ends with washing (cycles 9-12) after which the ninhydrin test as by the Kaiser aethod is performed. In case of a negative response, the Boc group is hydrolyzed with 50 TFA. Then, the resin is washed (cycles 9-1 ) and dried under vacuum, with the obtalnment of 1.25 S dry product. For peptide detachment from the resin, the product is placed in a Teflon reactor with 1.5 mL anisole and 0.75 mL dimethyl sdphide. The mixture temperature is brought to ~50 * C and 15 aL hydrofluoric acid is distilled therein; then the mixture is kept under stirring for 60 ain. in an ice bath. Hydrofluoric acid is removed by nitrogen blowing. The raw product is dried under suction for about 2 hours, is washed with ethyl ether (15 aL twice), extracted in 50% acetic acid (15 aL three tiaes) and filtered in a fritted disc filter funnel to reaove the exhaust resin. The resdting solution is diluted with water and freeze-dried to yield 0.210 g raw product. Findly, the peptide is purified by reverβed- phase liquid chromatography and characterized by analytical HPLC, Haters Cl8 Deltapack 3-9 x 150 aa coluan with an acetonitrlle gradient containing 0.1% (v/v) trifluoracetic acid (phase B) vs. 0.1% (v/v) aqueous trifluoracetic acid (phase A), as well as 20 to 80% phase B, in 20 ainutes, at a rate of 1 aL/aln.. with 210 na UV monitoring. Retention time (Rt) * 9-2' ; chromatographic purity: > 992. b) Cyclization of the above said peptide (1) into the cyclic peptide cydo(Uu [CHgNHlAsptNHBzD-Gln-Trp-Phe-βAla (ii) 65 mg product (i) is dissolved in 35 aL DMF. The solution is added with 47 mg PyBOP, then 32 μL DIEA. The resulting solution is kept under stirring at aabient temperature for 2 hours , then DMF is removed under vacuum and the resdting mixture freeze-dried. The cyclic peptide (ii) is purified by reversed-phase liquid chromatography and characterized by andyticd HPLC, Haters C18 Deltapack 3 * 9 * 150 aa coluan with an acetonitrlle gradient containing 0.1% (v/v) trifluoracetic acid (phase B) vs. 0.1% (v/v) aqueous trifluoracetic acid (phase A) , as well as 20 to 80% phase B, in 20 ain., at a rate of 1 aL/ain., with 210 na UV aonitoring. Retention tiae (Rt) * 10.6' ; chromatographic purity: >99X- By the procedure described above and using sdtable reagents, the following peptides are obtained: H-UuY [αym]Asp(NH-CH 2 -(l-adamanty1))-Gln-Trp-Phe-&Ala-0H retention time (Rt) * 9-5' ; chromatographic purity: > 99% ' H-Uu [CH 2 NH]Asρ(NH-CH 2 -C 6 H 11 )-Gln-Trp-Phe-PAla-OH retention time (Rt) * 9-0' ; chromatographic purity: > 99%. H-UuV [CH2NH]Glu(NHBzl)-Gln-Trp-Phe-pAla-0H retention time (Rt) * 9-3'; chromatographic purity: > 99%• H-UuV [CH2NH]Asp(NMeBzl)-Gln-Trp-Phe-frAla-OB retention time (Rt) » 9.8' ; chromatographic purity: > 992. H-UuY [CH2NH]CH((CH 2 ) 3 Bzl)-CO-Gln-Trp-Phe-&Ala-0H retention time (Rt) ■ 11'; chromatographic purity: > 99% * H-Uu-Asp(NHBzl)-Gln-Trp-Phe-βAla-OH retention time (Rt) * 9.6' ; chromatographic purity: > 99% * H-Uu-Cha-Gln-Trp-Phe-βAla-OH retention tiae (Rt) * 9.8' ; chromatographic purity: > 99%. H-Uu Y [CH 2 NH]Asp(0Bzl)-Gln-Trp-Phe-pAla-0H retention tiae (Rt) « 10.7' ; chromatographic purity: > 99% H-Uu V [OT 2 NH]Uu-Gln-Trp-DPhe-&Alβ-OH retention time (Rt) ■ 7-7' ; chromatographic purity: > 99% H-Uu Y [OT 2 NH]Lys(Z)-Gln-Trp-Phe-&Ala-0H retention time (Rt) » 8.9* ; chromatographic purity: . > 99% H-LeuV [CH^lCha-Gln-Trp-DPhe-βAla-OH retention time (Rt) « 9-0 ; chromatographic purity: > 99% H-Leu Y [CH 2 NH]Nd-Gln-Trp-Phe-&Ala-0H retention time (Rt) * 9.9' ; chromatographic purity: > 99% H-Leu V tCH 2 NMe]Cha-Gln-Trp-Phe-PAla-0H retention time (Rt) * 11.1' ; chromatographic purity: > 99% Which are cyclized into the following cyclic peptides : cyclo (Leu retention time (Rt) * 11.0' ; chromatographic purity: > 99%. cyclo(UuN' [CH^lAsptNH-CH^βHnJ-Gln-Trp-Phe-PAla) retention time (Rt) * 11.2'; chromatographic purity: > 99%. cydo(UuY [CH2NH]Glu(NHBzl)-Gln-Trp-Phe-ftAla) retention time (Rt) * 10.0'; chromatographic purity: > 99%. cyclo(UuY [CH2ΪJH]Asp(NMeBzl)-Gln-Trp-Phe-βAla) retention tiae (Rt) » 12.5'; chromatographic purity: > 99%. cyclo(Uu [CH2NH]CH((CH 2 ) 3 Bzl)-CO-Gln-Trp-Phe-βAla) retention time (Rt) * 13 * 5'; chromatographic purity: > 99%. cyclo(Uu-Asp(NHBzl)-Gln-Trp-Phe-βAla) retention time (Rt) * 10.6'; chromatographic purity: > 99%. cyclo(Uu-Cha-Gln-Trp-Phe-pAla) retention tiae (Rt) •** 11.2' ; chromatographic purity: > 99%. cydo(UuY[CH 2 NH]Asp(0Bzl)-Gln-Trp-Phe-PAla retention time (Rt) ■ 9.8' ; chromatographic purity: > 99% cyclo ( Leu Y retention time (Rt) » 9-4' ; chromatographic purity: > 99% cydo(UuY [CH2NH]Lys(Z) -Gln-Trp-Pbe-frAla) retention tiae (Rt) * 10.7' ; chromatographic purity: > 99% cy ofUujf' [CH^lCha-Gln-Trp-Phe-flAla) retention tiae (Rt) * 11.1'; chroaatograpdc purity: > 99 % cydo(UuY[CH2NH]Nd-Gln-Trp-Phe-pAla) retention tiae (Rt) * 13.0'; chromatographic purity: > 99% cyclo (UuY [CHgNMelChβ-Gln-Trp-Phe-βAla) retention tiae (Rt) * 12.2'; chromatographic purity: > 99% BIOLOGICAL ACTTVOT The ability of the peptides described in the present invention to interact with the neurokinine A receptor as agodsts or antagodsts was assessed through an in vitro test. The preparation used for the test was characterized by the fact that the biologicd response produced by tachykinins and related peptides was exclusively determined by the neurokinine A receptor (receptor NK-2). The sdd preparation consisted of isolated rabbit pulmonary artery affected by a dose dependent contraction brought about by tachykinins (Rovero et d., Neuropeptides, 1989. £$. 263-270). The determination of peptide activity in the test preparation was based on the use of an NKA concentration (3 nm) causing a response equd to 45% of max. response. The peptides considered herein were added to the preparation in growing concentrations. Their activity was assessed as inhibition of response to NKA. The capacity of the peptides described herein to interact with the P substance receptor (receptor NK-1) as agodsts or antagodsts was assessed through an in vitro test, where the biologicd response produced by tachykinins and related peptides was exclusively deterained at the SP receptor. The test preparation consisted of isolated guinea pig ileua affected by a dose-dependent contraction (Lee et al. , Schnied. Arch. Pharmacol. , 1982, 218, 281-287) . The determination of peptide activity in the test preparation was based on the use of an SP aethyl ester concentration (10 na) causing a response equal to 45% of max. response (S. Dion et al. . Life Sc . 1987. 4l . 2269-2278) . The peptides considered herein were added to the preparation in growing concentrations. Their activity was assessed as inhibition of response to SP with satisfactory resdts. The compounds covered by the invention are suitable for therapeutical adainistration to higher animals and nan by the parenteral. oral, dermic, nasd, inhdatory and sublingud ways, with phaxmaceuticd effects matching the described properties. In case of parenteral adainistration (intravenous, intramuscular, intraderad) , sterile solutions or freeze-dried preparations of the coapounds are to be used. In case of oral adainistration, preparations such as tablets, capsdes and syrups are convedently used. Sdtably dosed ointaents and creams are utllizable by the deraic way. In case of nasal instillation, inhalation, and sublingual adaldstration, the compounds to be used are respectively aqueous solutions, aerosol preparations, or capsdes. Doses for therapeuticd treatment range from 0.1 to 10 mg/kg body wdght. TABLE 1 AUTOMATIC SYNTHESIS PROCEDURE. Boc Cycle Reagent Time Solvent volume: 10-20 mL/g resin

Claims: CLAIMS 1. Cyclic hexapeptides of general forada (I) NNHH--CCHHRR 11 --HW--CCHHRR 33 --CCO0--AA 11 --AA 22 --AA 33 --βpAAll«a-ι where R j » H, linear or branched C^ alkyl R 3 * naturd or not naturd aaino acid free or protected side chain R 3 « (CH 2 ) n -R" where n « 1. 2, 3. 4. 5 R" * cyclooctyl, adaaantyl, cyclohexyl, naphthyl R" • phenyl when n is other than 1 R" * a substituted carboxyamide group when n * 1, 2 A 2 * Trp. DTrp A « Phe. DPhe H • CO-NR' , CH 2 -NR' wherein R* « H, CH and their pharmaceuticdly acceptable sdts dth acids or orgadc or inorgadc bases. 2. Cyclic hexapeptides as per claim 1, wherein: linear or branched C^_^ alkyl are selected in the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl. The natural amino acid is selected in the group consisting of : glycine, alanine , valine, leucine , isoleucine , proline , phenyldanine, tryptophan, aethionine, serine, threonine, cysteine, tyrosine, asparagine, glutaaine , aspartic acid, glutaaic acid , lysine, arginine, dstidine, in their L or D foras. Not naturd aaino acid is selected in the group consisting of:β- danine, D or L 2-aainoisobutyric acid, D or L 2,3-daminoproρiodc acid, D or L nor leucine, D or L dloisoleucine, D or L pyroglutamic acid, L or D 3-hydroxyproline, L or D 4-hydroxyproline, L or D phenyldanine substituted in the ortho, meta. or para position, L or D thienylalanine , L or D pyridyl alanine, β(2- or 3- benzotdenyldanine) , 1.2,3.4 tetrahydroisoquinoline-3-carboxyl add. Aaino acid chain protector is selected in the group consisting of : Mbs. Mtr. N02. Z. Tos, Pac. For. Me, Ac, 2-Br-Z, 2-C1-Z. Bzl, 2.6- dichloro-Bzl , S03H. Faoc. OMe, OBzl, OFm, ONp, OSu. 3. Hexapeptides as per clda 2, wherein the protected side chain of a natural or not natural amino acid is selected in the group consisting of : L or D Arg (Mbs) , L or D Arg(Mtr) , L or D Arg(N0 2 ) , L or D Arg (Z) , L or D Arg(Tos) , L or D Arg(Pmc) , L or D Trp(For) . L or D Trρ(Mts) . L or D Tyr(Me) , L or D Tyr(Ac) , L or D Tyr(2-Br-Z) , L or D Tyr(Bzl) , L or D Tyr(2.6-dichloro-Bzl) . L or D Tyr(S0 3 H) . L or D Ser(Me) . L or D Ser(Ac) . L or D Ser(Bzl) . L or D Ser(2.2-dichloro- Bzl) , L or D Ser(S0 3 H) , L or D Lys(Ac) , L or D Lys(2-Br-Z) , L or< D Lys(2-Cl-Z) , L or D Lys(Fmoc) , L or D Lys(Z) , L or D Lys(Tos) , L or D Lys(Me) , L or D Lys (Bzl) , L or D Asp (OMe) , L or D Asp(OBzl) , L or D Asp(OFa) , L or D Asp(ONp) , L or D Asp(OSu) . L or D Glu(OMe) . L or D Glu(OBzl). L or D Glu(OFm), L or D Glu(ONp). L or D Glu(OSu). 4. Cyclic hexapeptide as per ddm 3. wherein R j ■ isobutyl. 5. Cyclic hexapeptide as per ddm 4, wherein A 2 « Trp. 6. Cyclic hexapeptide as per dm 5. wherein R 3 * (CH 2 ) n -CgH llt where n « 2, 3.4, 5« 7. Cyclic hexapeptide as per claim 5, wherein R 3 ■ (CH 2 ) n -(l- naphthyl), where n * 2, 3. 4. 5- 8. Cyclic hexapeptide as per claim 5, wherein R 3 « (CH 2 ) n -(l- adamantyl). where n * 1, 2, 3.4, 5. 9. Cyclic hexapeptide as per claim 5. wherein R * ^ CH 2^n~ cyclooctyl, where n * 1, 2, 3.4, 5 * lO.Cydic hexapeptide as per claim 5. wherein R 3 * (^^n"^^' where n ■ 2, 3.4, 5. 11. yclic hexapeptide as per cldm 5, wherein R 3 > (CH 2 ) n -C0NHBzl, where n » 1, 2. 12.Cyclic hexapeptide as per ddm 5, wherein R^ « (CH 2 ) n -C0NMeBzl, where n * 1, 2. 13. Cyclic hexapeptide as per claim 5 , wherein R 3 * (CH 2 ) n - C0NHCH 2 C 6 H 11« where n * 1, 2. 14. Cyclic hexapeptide as per claim 5. wherein R 3 * (CH 2 ) n - C0NMeCH 2 CgHn, where n * 1, 2. 15. Cyclic hexapeptide as per claim 5, wherein R 3 ■ (CH 2 ) n -CONH- CH 2 (l-adamantyl) , where n * 1, 2. 16.Cyclic hexapeptide as per claim 5. wherein R 3 * (CH 2 ) n -C0NMe- CH (l-adamantyl) , where n » 1 , 2. 17 * Cyclic hexapeptide of general, formula (I) , as per claia 1 , selected in the group consisting of : cyclo(Uu-Cha-Gln-Trp-Phe-βAla) cydo( u-Asp(NHBzl)-Gln-Trρ-Phe-βAla) cyclo (Uu-Asp (NMeBzl ) -Gln-Trp-Phe-PAla) cyclo( u-Asp(NHCH 2 C6Hll)-Gln-Trp-Phe-βAla) cyclo( u-Asp(NMeCH 2 C6Hll) -Gln-Trp-Phe-p^ϋ.a) cyclo(Uu-Glu(NHBzl)-Gln-Trρ-Phe-&Ala) cyclo ( u-Glu (NMeBzl ) -Gln-Trp-Phe-&Ala) cyclo( u-Glu(NHCH 2 C6Hll)-Gln-Trp-Phe-pAla) cyclo( u-Glu(NMeCH 2 C6Hll)-Gln-Trp-Phe-(JAla) cyclo(Uu-Glu(NHCH 2 (l-adamantyl) )-Gln-Trp-Phe-βAla) cyclo(Uu-Glu(NMeCH 2 (l-adamantyl) )-Gln-Trp-Phe-flAla) cyclo(Uu-Asp(NHCH 2 (l-adamantyl ) ) -Gln-Trp-Phe-^Ala) cyclo (Uu- Asp (NMeCH 2 (l-adamantyl) )-Gln-Trp-Phe-flAla) 18. Cyclic hexapeptide , of general formula (I ) , as per claim 1 , selected in the group consisting of : cyclo(UuY[CT2NH]Asp(NHBzl)-Gln-Trp-Phe-&Ala) cyclo(Uu Y [CH2 H3Asp(NMeBzl) -Gln-Trp-Phe-^Ala) cyclo (UuY [CHgNHlAsp (NHCH 2 C6H11 ) -Gln-Trp-Phe-pAla) cydo(UuY [CH 2 NH]Asp(NMeCH 2 C6Hll)-Gln-Trp-Phe-ftAla) cyclo(UuY [CH 2 NH]Glu(NHBzl)-Gln-Trp-Phe-p\Ala) cyclo( u [CH2NH]Glu(NMeBzl ) -Gln-Trp-Phe-&Ala) cyclo(Uuψ[OT2NH]Glu(NHCH 2 C6Hll)-Gln-Trp-Phe-βAla) cyclo(Uu Y [CH^lGldNMeO^CδHll ) -Gln-Trp-Phe-pAla) cyclo(UuY[CH 2 NH]Glu(NHCH 2 (l-adamantyl) )-Gln-Trp -Phe-pAla) cyclojUu 1 [CH 2 NH]Glu(NMeCH 2 (l-adaaantyl) ) -Gln-Trp-Phe-pAla) cyclofUuYCCH^lAspfNHCH^l-adamantylH-Gln-Trp -Phe-pAla) cyclo(Uuy [CH2NH]Asp(0Bzl) -Gln-Trp-Phe-pAla 19. Cyclic hexapeptide of general formula (I) , as per cldm 1 , selected in the group consisting of: cyclo(UuY[CH 2 NMe]Asp(NHBzl)-Gln-Trp-Phe-pAla) cyclo(UuV cyclo (UuY [OyWejAsp (NMeCH 2 C6Hll) -Gln-Trp-Phe-PAla) cyclo(Uu Y[CH2NMe]Glu(NHBzl) -Gln-Trp-Phe-PAla) cyclo(UuY [CH2NMe]Glu(NMeBzl) -Gln-Trp-Phe-βAla) cyclo(UuY [CH 2 NMe]Glu(NHCH 2 C6Hll ) -Gln-Trp-Phe-pAla) cyclo(UuY[CH2NMe]Glu(NMeCH 2 C6Hll)-Gln-Trp-Phe-PAla) cyclo (UuV [CH2NMe]Glu(NHCH 2 ( 1-adamantyl) ) -Gln-Trp-Phe-βAla) cyclo(UuY[CH 2 NMe]Glu(NMeCH 2 (l-adaaantyl) )-Gln-Trρ-Phe-βAla) cyclo (UuY [CH 2 NMe]Asp (NHCH 2 ( 1-adamantyl) ) -Gln-Trp-Phe-βAla) cyclo (UuY [CH 2 NMe]Asp(NMeCH 2 (l-adaaantyl) )-Gln-Trp-Phe-βAla) cyclo(LeuY[CH 2 NMe]CH((CH 2 ) 3 Bzl)C0-Gln-Trp-Phe -pAla) cyclo(UuY[CH2NMe3Asρ(OBzl)-Gln-Trp-Phe-PAla) cyclo (UuY [CH2NMe3Nd-Gln-Trp-Phe-ftAla) cyclo(Uu I [CH 2 NMe]Cha-Gln-Trp-Phe-βAla) 20. Cyclic hexapeptide, generd formula (I) , as per cldm 1, selected among those of the group formed by cydo(Uu Y[CH 2 NH]CH( (CH 2 ) 3 Bzl)CO-Gln-Trp-Phe-&Ala) cyclo(Uu-NH-CH( (CH 2 ) 3 Bzl)CO-Gln-Trp-Phe-βAla) 21.Peptide preparation process as per cldm 1 including peptide chain solid phase synthesis from C-termind end to N-termind end on an insoluble polymer support, the introduction of the iminomethylene bond, the subsequent detachment from polymer support by hydrolysis in anhydrous hydrofluoric acid and the linear peptide cyclization in polar organic solvents. 22.Peptide preparation process as per ddm 1 including peptide chdn solid phase synthesis from C-termind end to N-termind on an insoluble polymer support, the introduction of the iminomethylene bond, the subsequent detachment from polymer support by hydrolysis in trifluoracetic acid and the linear peptide cyclization in polar organic solvents. 23-Phaπnaceuticd compositions for the treatment of diseases where tachyqddnes play a pathogedc role.

Inventors: ARCAMONE FEDERICO MARIA, GIACHETTI ANTONIO, MAGGI CARLO ALBERTO, PATACCHINI RICCARDO, PESTELLINI VITTORIO, QUARTARA LAURA, ARCAMONE, FEDERICO, MARIA, GIACHETTI, ANTONIO, MAGGI, CARLO, ALBERTO, PATACCHINI, RICCARDO, PESTELLINI, VITTORIO, QUARTARA, LAURA
Applicants: MENARINI FARMA IND, A. MENARINI INDUSTRIE FARMACEUTICHE RIUNITE S.R.L.
Application year: 1992
Main classification: C07K7
IPC codes: A61K38, A61P1, A61P9, A61P11, A61P25, A61P29, A61P35, A61P43, C07K1, C07K7