JPH04501259A - CRF antagonist - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention relates to peptides and intensive mammalian therapy using the peptides.

More specifically, antagonists of hetetracontapeptide CRF, and pharmaceuticals containing the antagonists. This invention relates to methods of treating mammals using pharmaceutical compositions and analogs.

Background of the invention The hypothalamus plays an important role in regulating the secretory function of corticotropin-producing cells in the adenohypophysis. Experiments and clinical observations suggest that it plays an important role. 25 years or more Kamimae, Guillemin, Rosenberg g), Safran and Schally are German Pituitary glands cultured separately in vitro and pituitary glands maintained in organ culture It has been proposed that a factor exists in the hypothalamus that increases the rate of ACTH secretion by the body.

However, until 1981, physiological corticotropin-releasing factor (CRF) ) The structure of the secretagogue substance that is recognized as being secretagogue was not clarified. In 1981 As disclosed in U.S. Pat. No. 4,415.558, The amino acid sequence of CRF (ocRF) was found to be as follows.

H-Se r-Gl n-G1 u-Pr o-Pr o-I 1 e-8e r-Leu-As p-Leu-Th r-Phe | Hi 5 -Le u-Leu-Ar g-Gl u -Va l -Leu-Gl u -Me　-Th　r-Lys　-Al　t-Asp-G戟@n　- Le　u-Al　a-Gl　n-G1　n-Al　a-Hi　-8e　r- Asn-Arg-Lys-Leu-Leu-As@p- 11e-Ala-NHI.

Sauvagine is similar to this and is produced in South Africa. 4 isolated from the skin of the frog Phyllomedusa sauvaget It is a peptide with 0 residues. Sauvagin was prepared by Erspamer et al. "C Identification FL, Regu-1atory Peptides+2 ( 1981), 1-13. Sauvage has the formula: %formula% It is expressed as Urotensin I is p of Ichikawa et al. True blue as described in ept i de 8 +3.859 (1982) A 41-residue homologous peptide isolated from the urine of urophyses It is. Nvagin, urotensine, and CRF congeners lower blood pressure in mammals. Reported to have biological efficacy in promoting secretion of CTH and β-endorphin It has been done (・ru.

CRF is a 41-residue peptide that is very similar to oCRF. It is expressed as h) Since CRF has the same sequence as this, rCRF and hCR F can be used interchangeably.

Antagonist of CRF homolog consisting of 41 residues represented by the following formula or non-toxic addition thereof Synthesized salt.

Y -R9-RIO-R11-Rho-Jl-Leu-Leu-Arg-J7- R, s-R,, -R,, -R, -RH-J3- R44-RlB -Rt6- RH-JH-RH-Gl n-a l a-R31-R33-As n-Ar g-R36-R3? -Nle-R3g-R<o-R4+-NHtIn the above formula, Y is an acyl group having 7 or less carbon atoms or hydrogen; Ro is Asp or desRl : Rho is Leu or desJo : R11 is Thr, Ser or desR B; R42 is (Q) D - P h e + D - T y r + D −Le u lD −Hr s + D −Na l, D −P a l@ + D - I 1 e + D - Nle, D-Val, D-Met+Phe or Leu; Q is H, 4C1 or 4F ;Rho is His+Tyr or Glu;Rho is Glu, A, in or LyI; Rho is Val, Nle or Met+R+.

and R24 is leu, Ile, ala+Gly+Val, Nle, Phe , Asn and Asn; R2O is Glu or D-Glu; R1, is MeLNva+Ile, ala.

Leu, Nle, Val+Phe or Gin; RH is ala, Thr, As p or Glu; Ru is Arg + Orn, Ha r or LY”: Rzs is Asp or Qlu; R2B is Gin, Asn or LyCGlu; Ru is a I a + Ar g or Lys; R2゜ is Gin or Glu + Rst is His, G 1y+Tyr or a　I　a　; R3s is Ser+　Aan+　Leu, T hr or ala; R36 is L)'s + Orn.

Arg　Har　Har or Leu; R37 is Leu or Tyr; Rso is Glu or is Asp: R40 is Ile+Thr, Glu, ala, Vat, le u+N]e+ Phe, Nva+ Gly or Gin; and R41 is ala, 11e+G1y+Val, leu, Nle+ Phe, Nva or Gin be.

Pharmaceutical compositions of the present invention containing such CRF antagonists or nontoxic addition salts thereof is dispersed in a pharmaceutically or veterinarily acceptable solid or liquid carrier. this The peptide or its pharmaceutically or veterinarily acceptable addition salt is ACT) (1β-endorphin, β-lipotrophin, and other blow opiomelanocols) Chin (Pro-opiomelanocortin) gene product and corti modulate costerone secretion and/or lower stress response and/or or affect emotions, behavior and gastrointestinal function, autonomic nervous system activity. Further C RF analogs are used to evaluate pituitary, metabolic, cardiovascular, gastrointestinal, and central nervous system functions. You can also do that.

Detailed description of preferred embodiments The nomenclature used to define peptides is 5chroner & Lubke. The Peptides”+ Academic Presa (1965) Amino acids less than N are on the left, and amino acids less than C are on the left, according to the conventional designation. Carboxyl groups are shown on the right. α-Amino acid residues are represented using the standard alphabetic character 3. It uses symbols consisting of letters. When amino acid residues have isomers, cleavage is especially important. Rinona - represents the L form as long as possible. For example, if Ser is L-serine and Nle is Norlo Isin, Nva is norvaline, Har is homoarginine, Orn is ornithine. represent. Furthermore, Leu is L-leucine or CaCH, -L-leucine (CM L); ala is L-alanine or CaCH.

-L-alanine (CMA); D-Nal is D-alanine (β carbon is naphthalene) D-Pal (substituted and attached to carbon 1 or 2) is D-alani (the β carbon is attached to the 3-position of pyridine).

The present invention provides a CRF antagonist represented by the following formula (1) and a nontoxic addition salt thereof. provide

Y-R9-RIG-R11Jl-R13-1eu-1eu-Arg-Rho-R ho-Rho-Rm-RH-Ra-R43-RH-R25-Rg-RH-R2g -R2G-Gl n-a l a -Rg-R33-Asn-Arg-Rg-R 31-N　1　e　-R3g　-R46-R4I-N　H2 In the above formula, Y is is an acyl group or hydrogen having 7 or less carbon atoms; Ro is Asp or desRo; Rl g is Lllll or desR4o; Ro is Thr, Ser is desJl: Rlg is (Q) D-4'be, D-Tyr, D-Leu, D-Hi s + D-Na l, D-Pal, D-I l e, D | Nl e + D-Va 1 * D-Me t + P he or Leu; Q is H, 4Cl is 4F: Rlg is His, Tyr or Glu; R+y is Glu , Aan or L)' m: Rlg is Va, l, Nl e or Met; R19 and R, are leu+IIe, ala, Gly+Val+Nle+Phe +Asn and Gln; R9 is Glu or D-Glu; Jl is MeLNva+Ile+a1m+1eu, N1e+Va1. Phe+As n or Gin; RH is ala4'hr, Asp or Glu; R23 is Arg+O rn+Har or LYS: R2S is Asp or Qlu; R26 is Gin.

Asn or Ly 8: R27 is Leu+Ile, ala, Val, Nva, Met, NIe+Phe+Asp, Asn+Gln or Glu; Rzs is a l a * A r g or L)'8; Rto is Gin or G 1 u + R32 is His, G1y+Tyr or ala:Rss is Ser+Asn, Leu, Th r or ala; R, IIl is LyIl, OrrzArg, Har or Leu; R st is Leu or TYr: R2O is Ginnu is Asp; R40 is Ile+ Thr, G1u+ala+Val, Leu, Nle, Phe, Nva, Gxy or is Gln; and R4+ is ala+Ile, Gly, Val+leu+Nle+ Phe+Nvanu is G1. It is n. The antagonist represented by the above formula is natural CRF Binds extremely well to pituitary receptors.

A preferred antagonist is represented by the formula below.

Y -R12-R,, 3-1e u -1e u-Ar g-R, 7-Rlg -J@-RH-Jl -R, 2-R23-R24-R25-R26-R27-R zs-R, 2g-G l na-a 1 a-R32-R33-As n-Ar g-R36-R37-Nle-R3+1-'R4゜-R,,-NHt In the above formula , Y is Ac or hydrogen; R12 is D - P h e + D -Tyr+D-Leu+D-His, D-Nal+ D-Pa l, D-Nl e, D-I 1e + D-Val, D-Me or Phe; R13) iis4'yr or Glu+R+7 is Glu, Asn or Lys; Rlg is Val, Nle or Met; Rag and R24 is l e u + I l e + a l a + G l y + V a l + N 1 ■@r P h e and and Gin; R9 is Glu or D-Glu; R21 is Me t, Nva+I l e + a 1 a + l e u + N 1’ e + V a l + P h e or Gin; R2, is ala, 'Th Chichi AA sp or Glu; R2s is Arg+Orn+Har or L)'s: Rzs is Asp or G l　u　; R26 is Gin.

Asn or Lys: R2? is leu, Ile+ala+Val, Nva, Me t+N1e+Phe+A s p + A a n + G l n or Glu ; Rzs is ala, Arg or Lys; Rte is Gln or G l u r R , 32 is H4s+G1y+Tyr or ala; Rss is Ser, Asn, 1e u+Thr or ala; R311 is Lys+Orn, Arg, Har or le u: R3? is leu or 'ry r: Ra9 is G1 u or Asp ;R,. is Ile, Thr, Glu, s+la, Val, leu, Nle, Ph e+, Nva, Gly or Gin; and R4+ is ala, Ile, Gly, Val, leu, N1e.

Phe, Nva or Gin. Non-toxic salts of the above formula are also used as preferred antagonists. I can do it. Preferred residues include Rlg D-Phe; Phe or D-2Nal: R13 is Hl g: RI? is Glu; R11 is Va l + Rlg and R37 is Leu; R20 is Glu or p-G l u: Rts is Nle; R H is Ala; R23 is Arg; Ru and Rts are AI &; Rts and R5゜ is Glu; R- is Gin; J7 is Leu; Rzs is Gln: Rst is His; R33 is Ser; R36 is Arg, Lys, Har or is Leu; R46 is He; and R21 is Ala or He. especially One of the substances that has been found to be highly effective is [D-Phe”, Nle” ,”)-rCRF(12-41).

The peptides of the present invention can be produced using a complete solid phase method, a partial solid phase method, or a fragment condensate method. synthesized by any suitable method such as fusion or classical liquid phase coupling. Ru. CRF antagonist portions that do not contain D-isomer residues or non-natural amino acid residues are , may be synthesized by recombinant DNA methods developed in recent years.

Synthesis by this recombinant DNA method involves the appropriate structural recombination of the desired form of the CRF homolog. It must be understood that the process of encoding genes is involved. some kind Synthetic CRF peptides include such structural genes as well as promoters and operators. A microorganism carrying an expression vector containing C is transformed from the thus transformed microorganism. Obtained by expressing RF peptide. Published on December 23, 1982. WO32104443 opened and published on May 26, 1983 w.

As described in 83101783, genes are formed using structural genes and Using non-human mammals to synthesize CRF peptides by injecting You can also The CRF peptide synthesized in this way is then extracted from serum etc. It is recovered from mammals by releasing it.

The unstable side chain groups of various amino acid moieties are protected with appropriate protecting groups, and the protecting groups are Preventing chemical reactions from occurring at the site until it is finally removed This is a method often used for synthesis. Also, amino acids and fragments may be part of the carboxyl group. Techniques to protect the α-amino acid of the substance during the reaction at this position are also often used.

Thereafter, the α-amino acid is removed and the next reaction is performed at that position. Therefore, synthesis As a step, the peptide chain contains amino acid residues located in the desired sequence. It is common for intermediates to be formed in which side chain protecting groups are attached to appropriate amino acid residues. That is.

Intermediate (II) represented by the following formula is also considered to be included within the scope of the present invention .

"-Rlg (X) R15CX or X')-Leu-Leu-Arg (X' )-R+7(X', X' or X')-Rlg-Rlg(X')-R2O(X' )-Rst-Rh(X" or X')-R,3(N3 or N6)-R24(X' ) -R25 (N5) -Rzs (X' or x') -R2? (x’ or N5)-R28(N3 or X')-Rn(X' or X')-Gin(X')-a la-R32(X)-R33(N2 or X')-Asn(X')-Arg(X" )-R,6(X' or X')-R37(X)-Nle-R3g(X')-R4 ゜(x　2 or N4 or X')-R41(X')-X'In the above formula, R is As defined.

In the formula, Xl is hydrogen or an α-amino protecting group. α-amino included in Xl Protecting groups include those known to be useful in the field of sequential synthesis of polypeptides. It is. Types of α-amine protecting groups that can be used as Xl include (1) acyl type protecting groups, such as formyl, acrylic (Acr), benzoyl (Bz), Cetyl (Ac), preferably used only at the end of the N bed, (2) aromatic urethane type protecting groups, such as benzyloxycarbonyl (Z), and p-chlorbe p-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-brombe Substitutions such as benzyloxycarbonyl, p-methoxybenzyloxycarbonyl (3) Aliphatic urethane-type protecting group, such as t-butyloxycarbonyl ( BOC), diisopropylmethyloxycarbonyl, isopropyloxycarbo nyl, ethoxycarbonyl, and allyloxycarbonyl; and (4) loalkylurethane type protecting groups, such as fluorenylmethyloxycarbonyl (F MOC) cyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl; (5) thiourethane-type protecting groups, e.g. Contains phenylthiocarbonyl. A preferred amino protecting group is BOC.

N2 is a protecting group for the hydroxyl groups of Thr and Ser, and is acetyl (Ac ), benzoyl (Bz), t-butyl, triphenylmethyl (trityl), t-butyl Trahydropyranyl, benzyl ether (Bzl) Matach, 2,6-dichloro Preferably it is lobenzyl (DCB). The most preferred protecting group is Bzl . N2 may be hydrogen or an atom, in which case there is no protecting group on the hydroxyl group. .

N3 is hydrogen; or a protecting group for the guanidino group of Arg or Har; Nitro, p-1 luenesulfonyl (TO8), Z, adamantyloxycarbonyl Preferably, it is BOC or BOC.

N4 is hydrogen; or a protecting group for the amino group of Asn or Gin; Preferably it is Xan.

N5 is hydrogen; or for the β or γ-carboxyl group of Asp or Glu is an ester-forming protecting group for hensyl (OBzl), 2,6-dichlorobendi Preference is given to using methyl, ethyl and tert-butyl esters. most preferred The correct one is 0Bzl.

N6 is a hydrogen atom; or suitable protection for the side chain amino group of Lys or Orn It is the basis. An example of a suitable side chain amine protecting group is Z, 2-chlorobenzyloxycarbo Nyl (2-CI-Z), Tos, t-amyloxycarbonyl (Aoc), BO C.

It is an aliphatic or aromatic urethane type protecting group as described above.

When His is present, X is hydrogen or Tos or 2,4-dinitropheny It is a protecting group for the imidazole nitrogen, such as (DNP). When Tyr is present, X is hydrogen or a hydroxyl-protecting group such as DCB. When Met is present, the location If desired, the sulfur may be protected with oxygen.

The selection of side chain amino protecting groups can be removed during the removal of alpha amine protecting groups during synthesis. There are no conditions except that it must not be The min protecting group and the side chain alpha protecting group cannot be the same.

N7 is NH, or linkage to a solid resin support used in solid phase synthesis Anchor bonds or protecting groups such as esters. Preferred as a support Ino is -NH-benzhydrylamine (BHA) resin support and -NH-pattern. This is a methylbenzhydrylamine (MEHA) resin support. BI(Ama) Alternatively, the CRF-like amide can be obtained directly by cleavage from the MBHA resin. can. By using N-methyl derivatives of such resins, the same It is possible to make methyl-substituted amides that are considered to be the same.

Intermediate) Structural formula Nioite, X, X'+ X's X"r X'r X', O , lhiX'tf) 5thino1 or more are protecting groups. Selected for each RK determine the presence of the above-mentioned protecting groups, which are well known to those skilled in the art. The selection of specific side chain protecting groups for use in peptide synthesis is performed according to the following rules. ( a) Reaction conditions selected for α-amino, l protecting group removal in each step of the synthesis It must be stable at 1- to the reagent. (b) The protecting group is It must maintain its properties and not decompose under the coupling conditions. (c) Side chain protecting groups are used to modify the peptide chain after completion of the synthesis with the desired amino acid sequence. It must be able to be removed 1.5 times under conditions that do not cause any damage.

The acyl group at the N-terminus represented by Y is acetyl, formyl, acrylyl or or benzoyl.

The present invention also provides methods for making compounds of formula (I). The method is , (a) have few (both have one protecting group; X'l X'l and X6 are each hydrogen or a protecting group, and X7 is a protecting group or a resin support or creates a peptide with formula (II), which is an anchor bond to NH, and ( b) removing the protecting group or anchor bond from the peptide represented by formula (II), (c) Optionally, converting the obtained peptide into a non-toxic addition salt.

Preferably, it is synthesized. However, other known and equivalent synthetic methods are also available as mentioned above. Adoptable. Solid phase synthesis or U.S. patent no. 4.244,946 (R4vier et al.; patented January 21, 1981) Coupling the protected α-amino acid to a suitable resin as generally described in It is common to start synthesis from the C-terminus of the peptide by

The starting material for such an antagonist of CRF is α-amino protected Il. It can be made by bonding e to a BHA resin.

The BOC-protected Ile was prepared using methylene chloride and dimethylformamide (DM Coupling onto BHA resin using F). Then the trichloride in methylene chloride Using fluoroacetic acid (TFA), TFA alone, or HCI in dioxane The α-amino protecting group is removed by removing the α-amino protecting group. At this time, 50% by volume of TFA salt .

Using methylene chloride solution with 0=5% by weight of 1,2-ethanedithiol I prefer it. Removal of protecting groups is carried out at temperatures between approximately 0.degree. C. and room temperature. In addition to this, 5 chroa er & Lubke, “Tbe PepLides” + 1 + pp 7 2-75 (Aeadenvie Press 1965) Electrostatic agents and reagents can be used to remove specific amine protecting groups.

After removing the α-amino protecting group of 11e, the remaining α-amino acid and side chain protected amino acid The amino acids are coupled stepwise in the desired order to obtain the intermediates described below. During synthesis Instead of adding each amino acid separately, add them before adding them to the solid phase reactor. The inner tubes may be coupled to each other. Drugs are selected within the range known to those skilled in the art. Among them, particularly preferable are N, N' -dicyclo-xylcarbodiimide (DCC) and N,N'-diisopropylcarbohydrate Rubodiimide (DICI).

The activation reagents used in solid phase synthesis of peptides are well known in the peptide field. It is. Examples of suitable activating reagents include carbodiimides, such as N,N'-diiso Propylcarbodiimide and N-ethyl-N'-(3'-dimethylamine propyl pill) is a carbodiimide. Other activation reagents and peptide couplings Their use in Each protected amino acid or amino acid sequence is added to the solid phase in approximately 4-fold excess. dimethylformamide (DMF); in a mixed medium of CH2Ch (1:1) or in DMF or CH2Cl, alone It takes place inside. If incomplete coupling occurs, remove the α-amine protecting group Repeat that coupling reaction before coupling the next amino acid let The completion of the coupling reactions at each step of the synthesis, if performed manually, If you are interested, Kaiser et al.'s Anal, Bioche m, 34. Monitored by ninhydrin reaction as described in 595 (1970) It is preferable to do so. If the coupling does not proceed completely, the next amino acid A coupling step is performed prior to removal of the α-amine protecting group prior to coupling. return. Coupling reactions can be performed using automation.

After the desired amino acid sequence is completed, it is treated with a reagent such as liquid hydrogen fluoride to neutralize it. The intermediate peptide is cleaved from the resin support. At this time, liquid hydrogen fluoride binds the peptide. Not only is it separated from the fat, but all remaining side chain protecting groups ’l　X’1 and X6 and α-amino protecting group (if the acyl group is not intended to be present) is also cleaved away to obtain a peptide.

When using hydrogen fluoride for cutting, add hydrogen fluoride as a scavenger to the reaction vessel. Put Nisole or Fresol and methyl ethyl sulfide (in the sequence When Met is present, trifluoroacetic acid is added before cleaving the peptide from the resin. Cleave the BOC protecting group with acid (TFA)/ethanedithiol and proceed with S-alkylation. It can also be avoided.

The following example describes a preferred method for the synthesis of CRF analogs by solid phase methods.

Array below HD-Phe-Hi 5-Leu-Leu -Ar g-Gl u -Va 1-Leu-Gl u -Nl e-Al a-Arg-Ala-Glu-Gl n-Leu-Ala-Gln-Gln-Ala-His-8er-Asn-Ar CD with g-Lys-Leu-Nle-Glu-11e-11e-NHI -P h e “” + N l e “I”] - Human CRF (12-41 ) from Bachern Co., Ltd. Applicable range of approximately 0.1 to Q, 7 m MO Synthesized by resin route method on MBHA hydrochloride resin such as 1/g resin did. This synthesis was performed automatically using a Beckman 990B peptide using an appropriate program. I did it with a synthesizer. Preferred programs are:

2 Wash with methanol (30 ml) (2 times) 33 CI (, CI (80 ml) Wash with (3 times) 34 50% TEA and 5% 1,2-ethanedithiol 12 CH, C12 solution (70 mA) (2 times) 5 Wash with isopropanol (80 ml) Cleaning (2 times) 36 12.5% TEA in CH2Cl, solution (70 ml) (2 times) Wash with 57 1/-# (40ml) (2 times) 28 CHt Clx (8 Wash (3 times) with 39 Boc-amino acids (30 vertical ml of 10 mm) ether, 30-300 DMF or CH, CI, solution [solvent is especially protected Determined by solubility of amino acid] (1 time) and DCCI (10 mmol) CH, CI.

solution By coupling BOC-11e, approximately 0.35 mmol/g of resin He was replaced. Note that all the solvents used were carefully degassed. gas The removal is done by spunning with an inert gas such as helium or nitrogen. It is preferable that

After removing the protecting groups and neutralization, the peptide chains were added step by step onto the resin.

Generally, 1-2 mmol of BOC-protected aminos in methylene chloride per gram of resin. A mixture with tyrene was used. Bzl as the hydroxyl side chain protecting group of Set used. p-nitroph to activate Asn and G1n0 carboxyl terminus. A portion of HOBt was prepared using 1 equivalent of HOBt in a 50% mixture of HOBt and methylene chloride. I let it go. The amino groups of Asn and Gin can be prepared using the DCCI method instead of the activated ether method. When using a coupling, it was protected with Xan. 2-CI-Z is the Lys side chain Used as a protecting group. Tos is the guanide group of Arg and the imidazole group of His. used to protect nitrogen. Also, the carboxyl side chain of Glu is 0Bzl protected by. At the end of the synthesis the following composition was obtained.

BOC-D-Phe-HAs(Tos)-Leu=Leu-Arg(Tos)- Glu(OBzl)-Vai-Lsu-Glu(OBzl)-Nle-AXa- Arg(Tos)-AXa-Glu(OBzl)-Gln(Xan)-L eu-Al　a-Gl　n　(Xan　)-Gl　n(Xan　)”Al　a- His (Tos)’-3er (Bzl)-Asn(Xan)-Arg( Tos)-Lys(2-CI-Z)-Leu-Nle-Glu(OBzl)-1 1e-11e-MBHA resin support Xan to remove α-amino protecting group It could be partially or completely removed by the TFA treatment used.

To cleave the protected peptide-resin and remove the protecting group, peptide-resin 1 1.5 ml of anisole, 0.5 ml of methyl ethyl sulfide, hydrogen fluoride per g CHF) 15+++A at -20°C for 20 minutes and at 0°C for 1.5 hours. Ta. After removing the IF under high vacuum, the resin-peptide was washed with dry diethyl ether and The peptides were washed alternately with chlorine and chloroform, and then the peptides were washed with a degassed 2N acetic acid aqueous solution. Extracted and separated from resin by filtration.

J. Chromatography, 288, 303-328 ( (1984) and purified by IPLC. chromatographic fraction was carefully monitored by HPLC and only fractions showing substantial purity were collected.

The specific rotation of the hCRF peptide synthesized and purified by the above method was determined using Perkinesioma. When measured using a model 241 polarimeter, [G3 v=-3 9,4±1.0 (c=0.5 in 50% acetic acid) (corrected for the presence of water and TFA) ); purity was approximately 95%.

Always boiling HCL to check whether it is synthesized in the correct sequence. , containing 3 μl thioglycol/ml and Nle lnmol as internal standard. The CRF analog was hydrolyzed at 140° C. for 9 hours in a sealed degassing tube. ben 21 fu 1 21. It was confirmed that the peptide consisting of the above 30 amino acids was obtained.

Asx (0.9), Glx (7.1), Ala (3.9), Nle (1 ．． 9), Val (1.1).

5ey (1.1), Ice (2.1), Leu (5.0), Phe (0 ．． 9), Lys(1.0).

His (2.1) and Arg (3.0) Example ■ Array below H-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu- Glu-Nle-Ala-Arg-Al　a-Gl　u-Gl　n　-Le　u -Ala-Gl n-Gln-Al a-Hi 5-8e r-Asn-Arg -Arg-Leu-Nle-Glu-11e-11e-NH2 peptide CD-Phe"+Nle"+"+Argsa]-hCRF (12-41) Synthesized by the method described in Example I.

The specific optical rotation of hCRF peptide synthesized and purified by the above method is Perkin E l-mer Model 24 1 Measured with Polarimeter (d) 22 = -27.7 main 1.0 (c = 0.5: 5 in 0% acetic acid) ( (after correction for the presence of water and TFA). Purity was approximately 99%.

The obtained peptides were found to be uniform due to thin layer chromatography using various solvents. found. Waters HPLC (pore size 3 in 0.46X25Cm column) 001 5 μm G11 silica) I did it. Buffer A contains 1.01 TFA and 42.6% TFA/10100O of solution. A 0.1% TFA solution containing acetonitrile. The operation was performed at room temperature. The flow rate is The current was 2.0 mA/min, and the holding volume was 10.4 mA.

As a result of amino acid analysis of the purified peptide obtained, the above-mentioned peptide consisting of 30 amino acids was found. It was confirmed that it was Petido.

Example■ Regarding the synthetic CRF antagonists obtained in Example I and Example 2, in vitro The effects on ACT) and β-endorphin secretion were tested. Synthetic CRF antagonist based on inhibition of ACTH and β-endorphin secretion by rat pituitary cells. Vale et al.'s Endocrjnology, 9 1 + 5 6 2 (19772).

889-891 (1984), and 1 The 50% inhibitory effect on ACTH secretion by oCRF of M. was determined. US Patent No. 4 , 60 5, 6 4 2, which is a potent CRF antagonist. 41), this peptide was more than 17 times more potent. This is standard Antagonists inhibit GRF-stimulated GH secretion, GnRH-stimulated LH secretion, and FS. No effect on secretion of TSH and prolactone by H or TRF stimulation This can also be explained. Effects of antagonists on oCRF at various concentrations and oCR ACTH secretion stimulated with a constant concentration of F (1 μM) was stimulated by peptides at various concentrations. We also investigated whether Peptide I suppresses temperature and crabs, but none of them showed superiority to Peptide I. Suppression of secretion was observed. Similar tests were conducted on the peptide ■ synthesized in Example ■. It was found to be 41 times more effective than AMC (9-41). .

In vivo testing of CRF antagonists was performed to inhibit spontaneous ACTH secretion in adrenalectomized rats. This was done by examining the system. Intravenously inject BW3/pair (approximately 2.7 mo1.) The amount of ACTH in blood increases considerably by administering the injection, and it lasts effectively for 2 hours. (described in Baer et al., 5science, 213, 1394.1981) method). For non-anesthetized rats, CRF (The dose was 0.09 μmmol, the effect was ). Also, by treating with ether, most (but not all) In this case, the increase in ACTH was suppressed.

These results suggest that corticosteroid phytochemistry can be improved by administering CRF antagonists. Spontaneous secretion of ACTH, which is quantified after debugging, is suppressed and CRF blocks ACTH secretion induced by stress in untreated rats. This indicates that the secretion of ACTH is almost suppressed. These data are -ra's 5clence. 218, 377-9 (1982), endogenous This is consistent with the data obtained with CRF antiserum showing the effect of CRF on regulating ACTH secretion. It matches.

Synthetic hCRF inhibits in vivo ACTH secretion and β-endorph in prepared rats. It has been shown that it has high potency against fin-like (β-END4LI) immune activation. Ta. Menpthal anesthetized rats using an intravenous cannula on quiescent male or female rats. ACTH and β-END-LI blood levels 5 to 20 minutes after hCRF administration. The concentration increased. Additionally, hCRF significantly lowers blood pressure in rats and dogs. It has also been shown that it has some effects. These antagonists prevent such effects .

Example■ Array below H-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Gl u-Nle-Ala-Arg-Al　a-Gl　u-Gl　n-Le　u-Al a-Gl n-G1 n-Al a-Hi 5-8er -As n-Arg -Lys-Leu-Nle-Glu-11e-11e-NHI peptide [N1e”・”)-hCRF(12-41) by the method described in Example I Synthesized.

The specific optical rotation of hCRF peptide synthesized and purified by the above method is Perkin E Measured with l-mer Model 241 Polarimeter [d) D--27.2 1,0 (c=o.5; 5 in 0% acetic acid) (water (after correction for the presence of TFA).

Purity was approximately 99%.

The obtained peptides were confirmed to be uniform by thin layer chromatography analysis using various solvents. found. Waters HP LC (0.4 6 x 2 5cm column Filled with 5 μm G13 silica with a pore size of 3 OO; ROMAT analysis was performed. Buffer A is 1.0 mA of TFA per 10,100 O of solution. and 36.0% acetonitrile in 0.1% TFA solution. Operation at room temperature went. The flow velocity is z. ornlV 1 minute, and the holding volume was 9.5 ml.

As a result of amino acid analysis of the purified peptide obtained, the above-mentioned peptide consisting of 30 amino acids was found. It was confirmed that it was Petido.

As a result of testing according to the general method described in Example ■, the peptide of this example It is clear that tide also suppresses the secretion of ACTH and β-endorphin. became. The peptide of this example showed 12 times the potency of AMC(9-41).

Array below H-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val-Leu-Gl’u-Nl e-Al a-Arg-Al a-G l u-Gl n-Leu-Al a-Gl n-G1 n-Al a-Hi s@− 8er-Asn-Arg-Lys-Leu-Nle-Glu-11e-11e- Peptide I:N1e"")-hCRF(941) with N% was synthesized.

The specific optical rotation of hCRF peptide synthesized and purified by the above method is perkin E The l-0 purity was approximately 99%.

The obtained peptides were confirmed to be uniform by thin layer chromatography analysis using various solvents. found. Waters HPLC (pore size 3 in 0,46X25cr IL column) Especially for reverse-layer high-pressure liquid chromatography analysis using I did it. Buffer A contains 1.01 d of TFA and 39.6% per 10,100 O of solution. 0.1% TFA solution containing acetonitrile. The operation was performed at room temperature. flow rate was 2.0 m11 minutes, and the holding volume was 10.1 m higher.

As a result of amino acid analysis of the purified peptide obtained, the above-mentioned peptide consisting of 33 amino acids was found. It was confirmed that it was Petido.

It is clear that putido similarly suppresses the secretion of ACTH and β-endorphin. Became. The peptide of this example showed 6 times the potency of AMC(9-41).

Example■ Array below H-Phe-His-Leu-Leu-Arg-Asn-Met-I l　e　-Gl　u　-Me　t-Al　a　-Arg-Asn-Glu-As n-Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Ar g-T, ys-Tyr　-Nl　e　-As　p-Gl　u　-Va　1-NH The peptide [N1e”)-Keuurotensin-e (12-41) with 2 was synthesized. Ta.

As a result of testing according to the method described in Example ■, the peptide of this example also showed A CT) (and was found to similarly suppress the secretion of β-endorphin) .

Example■ Array below H-Asp-Leu-Th　r-Phe-Hi　a　-Le　u　-Le　u- Arg-Gl u - Va l - Le u - Glu-Nle-Ala-Ar g-Ala-Glu-Gln-Leu-AXa-Gln-Gln-Alt-Hi s-8er-Asn-Arg-Arg-Leu-Nle-Glu-Tle-Il Peptide with e-NH2 [Nle”I36]-h CRF (9-41) Synthesized.

The specific optical rotation of hCRF peptide synthesized and purified by the above method is Perkin E Measured with l-mar Model 241 Polarimeter [d)”, :=-16,0±1.0 (c=0.5: in 50% acetic acid) (water (after correction for the presence of TFA). Purity was approximately 99%.

The obtained peptides were confirmed to be uniform by thin layer chromatography analysis using various solvents. found. Waters HPLC (3oo pore size in 0,46X25Cm column) In particular, perform reverse phase high pressure liquid chromatography analysis using 5μmc1g silica of A). It was. Buffer A is 1.0 mA per 1000 mA of solution. ON TFA and 39.0% ace A 0.1% TFA solution containing tonitrile. The operation was performed at room temperature. The flow rate is 2. 0 d/min, and the holding volume was 9.1 ml.

As a result of amino acid analysis of the purified peptide obtained, the above-mentioned peptide consisting of 33 amino acids was found. It was confirmed that it was Petido.

As a result of testing according to the general method described in Example ■, the peptide of this example It is clear that tide also suppresses the secretion of ACTH and β-endorphin. became. The peptide of this example showed twice the potency of A)(C(9-41)).

Example■ Array below H-Phe -Hi 5-Leu -Le u-Arg-Asn -Nl e- I l e-Gl u-Nle-Al a-Arg-Asn-Glu-Asn- Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg- Peptide with Lys-Tyr-Nle-Asp-Glu-Val-NH2 ( Nle”・21.5]-Carp urotensin I (12-41) was synthesized. Example As a result of testing according to the method described in ①, the peptide of this example also showed ACTH. It was revealed that the secretion of β-endorphin and β-endorphin was similarly suppressed.

Example■ Array below H-Phe-Hi　s-Leu-Leu-Arg-Glu-Val-Leu-G lu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-G ln-Gln-Ala-Hi s-8er-Asn-Arg-Arg-Leu- Peptide CN1e""+A with Nle-Gln5-8er-Asn-Ar rg'')-hCRF (1241) was synthesized and purified using the method described above. The specific optical rotation of the hCRF peptide is determined by Perkin El-mer Model 2. 41 Measured with Polarimeter: Cd]”2--23.7 ．． 0 (c=o, 5: in 50% acetic acid) (after correction for the presence of water and TFA) there were.

The purity was about 9g%. The obtained peptide was uniformly determined by thin layer chromatography analysis using various solutions. The power of power 548 II was revealed. Watera HP LC (0,46X Z 5a The reverse layer height is particularly high using 5 μm (H silica, filled) with a pore size of 300λ in the rL cuff. Pressure liquid chromatography analysis was performed. Buffer A is 1.0% per 10100O of solution. Qml A 0.1% TFA solution containing TFA and 36.6% acetonitrile. The operation is Performed at room temperature. The flow rate was 2.0 m11 min, and the holding volume was 10.1 mA.

As a result of amino acid analysis of the purified peptide obtained, the above-mentioned peptide consisting of 30 amino acids was found. It was confirmed that it was Petido.

As a result of testing according to the general method described in Example ■, the peptide of this example It is clear that tide also suppresses the secretion of ACTH and β-endorphin. became. The peptide of this example showed 6 times the potency of AHC(9-41).

Example X Array below H-D-2Nal-His-Leu-Leu-Arg-Glu-Val-Leu -Glu-Nle-AIa-Arg-Ala-Glu-Gln-Leu-Ala -Gln-Gln-Ala-His-Leu-Asn-Arg-Lys-Leu -Nle-Glu-11e-11e-NH2 (D-2Na 112+N1. e"+")-hCRF (1241) was synthesized.

The specific optical rotation of hCRF peptide synthesized and purified by the above method is Perkin E When measured with l-mer Model 241 Porartmeter [ : d]", p=-26, 8 1.0 (c=o, 5 2 in 50% acetic acid) (with water (after correction for the presence of TFA).

Purity was approximately 99%.

The obtained peptides were confirmed to be uniform by thin layer chromatography analysis using various solvents. found. Waters HPLC (3oo pore size in 0,46X25Cm column) 5 μm c16 silica) was used for reverse phase high pressure liquid chromatography analysis. It was. Buffer A is 1.0% per 10100O of solution. Ol' TFA and 39% acetonate It is a 0.1% TFA solution containing tolyl. The operation was performed at room temperature. Flow velocity is 2.0m l/min, and the holding volume was 8.9 m.

To steal? =Results of amino acid analysis of refined jLM fruit 6 - the above 30 amino acids? It was confirmed that there are peptides.

As a result of testing according to the general method described in Example ■, the peptide of this Example It is clear that tide also suppresses the secretion of ACTH and β-endorphin. became. The peptide of this example showed 9 times the potency of AHC(9-41).

Performing a handstand Array below HD-Phe-Hi a-Ls u-Leu-Arg-Gl u-Va l -Le Ll-Gl u -Nl e-Alm-Arg-Al a-Gl u-Gl n-Le u-Al a-Gl n-G1 n-AX a-Hi 5-8e r-Asn-Arg-Leu-Leu-Nle-Glu-11e-1 Peptide represented by 1e-NH2 [D-PheIt, Nle"+", Leu3 1′)-hCRF(12-41) was synthesized.

The specific optical rotation of the hCR,F peptide synthesized and purified by the above method was determined by Perkin Measured with El-mer Model 241 Polarmeter [d E 22=-22,7±1.0 (c=0.5: in 50% acetic acid) (water (after correction for the presence of TFA).

Purity was approximately 68.2%.

The obtained peptides were confirmed to be uniform by thin layer chromatography analysis using various solvents. found. Waters HP LC (0,46X 25cm column with pore size 3 Especially for reverse-layer high-pressure liquid chromatography analysis using 5μm 016 silica of I did it. Buffer A contains LO ml of TFA and 45.0% acetic acid per solution ZOOM. A 0.1% TFA solution containing tonitrile. The operation was performed at room temperature. The flow rate is 2. 07 M/min, and the holding volume was 9.0 ml.

As a result of amino acid analysis of the purified peptide obtained, the above-mentioned peptide consisting of 30 amino acids was found. It was confirmed that it was Petido.

As a result of testing according to the general method described in Example ■, the peptide of this example It is clear that tide also suppresses the secretion of ACTH and β-endorphin. became. The peptide of this example showed three times the potency of AHC(9-41).

Array below Ac-Asp-Leu-Thr-Pbe-His-Leu-Leu-Arg-G lu-Val-Gly-Glu-Met-Ala-Arg-Ala-Glu-G ln-Leu-Ala-Gln-Gln-Ala-His-8er-Asn-A Pep with rg-Lys-Leu-Nle-Asp-Nva-Ile-NH2 Tido [Acetyl-Aap'+Gly"+Nle"+Asp", Nva4o) -hCRF(9-41) was synthesized. Test according to the method described in Example ■. As a result of experiments, the peptide of this example also stimulated the secretion of ACTH and β-endorphin. It was revealed that it was suppressed in a similar manner.

Example■ Array below )1-Phe-)tis-Leu-Leu-Arg-Glu-Val-Gln- Glu-Met-ANa-Lys-Val-Glu-Gln-Leu-Ala- Gln-Gln-Ala-His-CMA-Asn-Arg-Lys-Leu- Peptide with Nle-Glu-11e-11e-NHl [:Gln”, Ly s”, Val” + CMA”, Nle”: ]-hCRF (12-41) was synthesized. Ta. Results of testing according to the method described in Example ■.

The peptide of this example also inhibits the secretion of ACTH and β-endorphin. It became clear.

Example XV Array below H-Leu-Thr-Phe-His-Leu-Leu-Arg-C1u-Va l-Leu-Glu-Nl e-Al a-Ar g-Gl y-Gl u- Gl n-Le u-Al a-Gl n-G1'n-Al a -Ty r- 8er-Asn-Arg-Orn-Leu-Nle-Glu-11e-11e- Peptide CN1e”+”+Gly”+Tyr” with NHI, 0rn31′ ': 1-hCRF (1O-41) was synthesized. The method described in Example ■ was used. Therefore, as a result of the test, the peptide of this example also showed the effects of ACTH and β-endorphin. It was revealed that secretion was similarly suppressed.

Example XVI Array below H-Se r -Le u-Gl u -Le u-Leu-Arg-Lys -Me t-I 1 e-Al a-Lys -Gln-Glu-Lys-Gl u-Lys-Gln-Gln-Ala-Ala-Asn-Aan-Arg-Le Peptide with u-Leu-Nle-Asp-Tbr-Gin-NH2 [Al t”, Nle”, Gin”)-sauvazine (10-40) was synthesized. Example ■ As a result of testing according to the method described in The following sequence was shown to similarly suppress the secretion of beta- and beta-endorphin. H-Leu-Glu-Leu-Leu-Arg-Lys-Met-11e-Gl u-Ala-Glu-Har-Gln-Glu-Lya-Glu-Lys-Gl n-Gln-Ala-Als-Asn-Asn-Arg-Leu-Leu-Nl Peptide with e-Asp-Phe-11e-NH2 [Ala”, Har”+ N1e3), phe”]-sauvazine (11-40) was synthesized. As a result of testing according to the method described, the peptide of this example also showed ACTH and It was revealed that it similarly suppresses the secretion of β-endorphin.

Example X■ Array below H-Leu-Glu-Leu-Leu-Arg-Lys-Met-Val-Gl u-Val-Glu-Lys-Gln-Glu-Lys-11e-Lys-Gl n-Gln-Ala-Ala-Aso-Asn-Arg-Leu-Leu-Nl Peptide with e-Asp-Thr-Gly-NH2 [Val”+”, l1e 2fl+Nle'', Gly'')-unvazine (11-40) was synthesized. implementation As a result of testing according to the method described in Example ■, the peptide of this example also showed ACT. It was found that H and β endorphin secretion was similarly inhibited.

Example XtX Array below H-4FD-Phe-Glu-CML-CML-Arg-Glu-Met-CM L-Glu-Met-CMA-Lys-Ala-Glu-Gln-CML-Al a-Giu-Gln-Ala-CMA-CML-Asn-Arg-Leu-CM L-Nle-Glu-Glu-CMA-NH2 “j”f)” C4 FD-Phe"I CML"l"l"I"!"l"I CMA"I “l” INje”)-AHC (12-41) was synthesized as described in Example ■. As a result of testing according to the method of It was revealed that the secretion of dolphin was similarly suppressed.

Array below H-As p -Le u-Thr-4CID-Phe-Hi 5-Leu - Le u-Arg-Gl e-Nl e-Leu-Glu-Nle-Ala -Lys-Ala-Glu-Gln-Gl’u-Ala-Glu-Gln-Al a-Al　a　-Le　u-As　n-Arg-Leu-Leu　-Nl　e Peptide L 4 CID with -Gl u-Gl u-AN a -NHt -Phe'+Nle"""]-AMC (941) was synthesized. Example As a result of testing according to the method described in In., the peptide of this example also showed ACT The following sequence was shown to similarly inhibit H and β endorphin secretion. H-D-Phe-Hjs-Leu-Leu-Arg-Glu-Met-Leu- Glu-Nle-Ala-Ly 5-Al a-Gl u-Gl n -M, e t-Al a-Gl u-G 1 n-Al a-Al a-Leu-Asn -Arg-Leu-Leu-Nle-Glu-Glu'-Ala-NHI Peptide [D-PheI2+Met”+Nle”T”)-AH C(1241) was synthesized. Results tested according to the method described in Example ■ As a result, unexampled peptides also inhibited the secretion of ACTH and β-endorphin. It became clear that it would.

Example XX■ Array below H-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Gl u-Leu-Ala-Lys-Al a-Gl u-G 1 n-Al a-A l　a　-G　　u-Gl　n-Al　a-Al　a-Le　u-Asn-A rg-Le u-Le u-Nl e-Gl u-Gl u-Al a-NH Peptide with 2 [Nle””’+Leu”+Ala”Ding:)-AHC(1 2-41) was synthesized. 1- Results of testing according to the method described in Example ■ , the peptide of this example also suppresses the secretion of ACTH and β-endorphin. It became clear that

Example xxm Array below H-Le　u-Gl　u-Le　u-Le　u-Arg-Gl　u　-Me　 -Le u-Gl u -Me t-Gl u -Ly 5-Al a-Gl u-Ly 5-Gl u-Al a-Gl u-Gl n-A, l a-Al a-Leu -As n-Arg-Le u-Le u-N1. e-Gl Peptide with u-Gl u-Al a-NH2 [Leu12. Glu””’ , Lys"+Nle", )-AMC(12-41) was synthesized. Described in Example ■ As a result of testing according to the method described above, the peptide of this example also showed ACTH and β It has been shown that it also suppresses the secretion of endorphins.

Example XXIV Array below H-D-Il-e-Tyr-Leu-Leu-Ar g-G1.　 e-Me t-Le u-Gl u-Me L-Alt-Ly 5-Al a-Gl u-Gl n-G 1 u-CMA-Gl u-Gl n-Al a-Al　a-Le　u-Asn-Arg-Leu-Leu-N1. e-Glu -Peptide with Glu-Ala-NH2 [D-mouth, xt, Tyr13+CM A'', Nle'')-AHC (12-41) was synthesized. Persons described in Example ■ As a result of testing according to the method, the peptide of this example also showed ACTH and β-enzyme. It was revealed that the secretion of dolphin was similarly suppressed.

Example XXv Array below H-D -Le u-Gl u-Le u-Le u-A, r g-Gl u -Me t -Le u-Gl u, -Me t-Ala-Ly 5-Al a-Gl u-Gl n-G1 u-AX a-G l u-Gl n-AX a-Al a-Al a-Aa n-Arg-Leu-Leu-Nle-G Peptide with lu-Glu-Ala-NH2 (D-Leu”, Glu”+A la33+Nle":]-AHC (12-41) was synthesized. Described in Example ■ As a result of testing according to the method described above, the peptide of this example also showed ACTH and β It has been shown that it also suppresses the secretion of endorphins.

Example XXVI Array below H-Pb　e-Hi　s-CML-Le　u-Arg-Gl　u　-Me　t- CML-Gl　u　-Me　-Al　a　-Lys-Al　a-Gl　u-G l n-CML-A 1 a-Gl u-Gl n-AX a-Al a-Le u-Asn-Arg-CML-Leu-Nl e-Leu-Gl u-Gl Peptide with u-Ala-NHI [CMLL4. +9. ．． sa, Nl e"]-AHC (12-41) was synthesized. 1- Therefore, as a result of testing, the peptide of this example also showed ACTH and β-endorphin. It was also revealed that the secretion of the drug was similarly suppressed, and blood pressure was also significantly lowered.

Array below H-D -Nl e-His -Le u-Le u-Ar g-Gl u- Nl　e　-As　n-G1　u-Nl　e-Asp-Ly　5-Al　a-G l u-Gl n-Ph e-Al a-Gl u-Gl n-Al a-Al a-Le u-As n-Arg-Leu-Leu-Nle-Glu-Glu -Ala-NHI peptide [D-Nle"+N1e'L"+38. Aa n''+Asp'', Pbe27]-AMC (12-41) was synthesized l-. Example■ As a result of testing according to the method described in It has been shown that the secretion of beta-endorphin and beta-endorphin is similarly suppressed.

Example Xη1 Array below H-Th r-D-Va 1-Hi s-Le u-Le u-Ar g-G l　u　-Va　1-Le　u-Glu-Nle-Al　a-Arg-I　l　 e-Gl u-Gl n-I l e-Al a-Gl n-G1 n-Al a-Hi 5-3e r-Asn-Arg-Lys-Leu-Nle-Glu- Peptide with 11e-Nva-NH2 [D-Val”+Nle”l”, l1 e2413? , Nva”)-hCRF (1l-41) was synthesized. As a result of testing according to the method described, the peptide of this example also showed ACTH and It was revealed that it similarly suppresses the secretion of β-endorphin.

Example XxIX Array below Ae r-Le u-Th r-Pb e-H45-Le u-Le u-Ar g-Gl u-Va l-Le u-Gl e-Me t-Al a-Ar g-Al a-Gl u-Gl n-Va 1-Al a-Gl n-G1 n-Al　a-His-3er-Aan-Arg-Lys-Leu-Nle-G Peptide with lu-Ala-Leu-NH4, Va1 27. Nle”+Ala”,Leu”]-hCRF(10-41) was synthesized . It was found that the test according to the method described in Example ■ .

Example XXX Array below H-D-Ty r-His-Le u-Le u-Arg-Gl u-Va 1-AX a-Gl e-Me t-Ala-Lys-Nl e-Gl u- Gl n-Nva-A’l a-Gl n-G1 n-Al a-Hi 5-3 e r-Asn-Arg-Lys-Le u-Nl e-Gl u-Nl e -I　l　e　-Peptide CD with NHI-Tyr”+Alt”+Lys” , Nle"+"+", Nva")-hCRF (12-41) was synthesized. Example As a result of testing according to the method described in ①, the peptide of this example also showed ACTH. It was revealed that the secretion of β-endorphin and β-endorphin was similarly suppressed.

Example XXX I Array below H-Thr-D-H4a-His-Leu-Leu-Arg-Glu -Va l -CMA-Gl u -CMA -Al a-Arg-CMA -Gl　u-Gl　n　-CMA　-Al　a-Gl　n-G1　n-Al　a -Tyr -Tbr -As n-Arg-Ly s -Le u -Nl Peptide CD-Hl with e-Gl u-Gl n-Il e-NH2 s”, CMA”+”+”+27. Tyr”+Thr”, N1e38°Glu40 )-hCRF(11-41) was synthesized. According to the method described in Example ■ As a result of the test, the peptide of this example also stimulated the secretion of ACTH and β-endorphin. was found to be similarly suppressed.

Example xxxn Array below H-4C1-D-Phe-His-Leu-Leu-Arg-Gl u-Va l -Nl e-D-Glu-Nv a-Al a-Ar g-Le u-Gl u-Gl n-N1 e-Al a-Gl n-G1 n-Al a-Hi s-8er-Asn-Arg-Lys-Leu-Nle-Glu5-8er- Peptide C4C1-D-Phe” with Asn-Ar, N1e19I”lss , D-Glu”+Nva”, Leu”, Gly40.CMA”:]-hCRF( 1241) was synthesized. As a result of testing according to the method described in Example ■, The peptide of this example also inhibits the secretion of ACTH and β-endorphin. It became clear.

Example xxxm Array below Bz-D-Met-His-Leu-Leu-Arg Glu-Val-'Le u-Glu-CML-A1 a-A rg-CML-G l u-Gl n-L e u-Al a-Gl n-G1 n-Al a-Hi I! l-8e r -Asn-Arg-Har-Leu-Nle-Glu-Leu-Phe-NH2 The peptide having benzoyl-D-M et ” + CM L ”’ ”　r　Ha　r　36+　Nl　e　　”+　Leu”, Phe”)-hC RF(1241) was synthesized. Tested according to the method described in Example ■ As a result, the peptide of this example also inhibited the secretion of ACTH and β-endorphin. It became clear that it would be controlled.

Example XXXVI Array below H-D -Hi 5-Hi 5-Le u -Le u-Ar g-Gl u -Va　1-Le　u-Gl　u-Ph　e-Ala-Or　n-Ph　e-G l u-Gl n-As p-Al a-Gl n-G1 n-Al a-Hi 5-8e r-As n-Arg-Lys-Leu-Nle-Glu-CMA Peptide CD-H4s12+Phe"+24+Orn with -Val-NH2 "+Asp27.Nle",CMA"+Val"]-hCRF(1241) Synthesized. As a result of testing according to the method described in Example ■, the paper of this example It is clear that putido similarly suppresses the secretion of ACTH and β-endorphin. Became.

Example xxxv Array below Forr　-D　-Pa　-Hi　-Le　u　-Le　u-Arg-G l　u　-Va　1-Phe-Gl　u-Gl　n　-Th　r　-Ar　g- Al a-Gl u-Gl n-Le u-Al a-Gl n-G1 n-A l a -Ty r-8e r-Asn-Arg-Lys-Leu-Nle-G Peptide with lu-CML-CML-NH2 formyl-D-Pal"+ Phe”+Gln”+Thr22. Tyr”+Nle”, CML”!”)-hC RF(1241) was synthesized. Tested according to the method described in Example ■ As a result, the peptide of this example also inhibited the secretion of ACTH and β-endorphin. It became clear that it would be controlled.

Example xxxvi Array below H-D　-Ph　e-Hi　5-Le　u-Le　u-Ar　g-Gl　u　- Va 1-Le u-Gl u-Nl e-Thr-Lys-Al a-As p-G1 n-T, e u-Al a-Gl n-G1 r+-Al a- H45-8e r-As n-Arg-Lys-Leu-Nle-Asp-11 Peptide with e-Ala-NHI [D-Phe′2+N1e21+”]-o CRF (1241,) was synthesized. Tested according to the method described in Example ■ As a result, the peptide of this example also stimulated the secretion of ACTH and β-endorphin. It was revealed that it was suppressed.

Example XXX■ Array below H-D -Phe-His -Le u-Leu-Ar g -G 1 u -Va I -Le u-Gl u-Nl e-Thr-Ly 5-Al a- As　p-G　n-Le　u-Al　a-Gl　n　-G　n-Al　 a-Hi s-8e r-Asn-Arg-Arg-Leu-Nle-Asp- Peptide CD with Xle-Ala-NH2-P h e ” + N 1 Combine e ” + 38 + A r g 31'3- o CRF (1241) accomplished. As a result of testing according to the method described in Example ■, the peptide of this example It is clear that tide also suppresses the secretion of ACTH and β-endorphin. became.

CRF greatly stimulates the adrenocortical axis of the pituitary gland, and the CRF antagonist is called ACTH or High levels of glucocorticoid production suppress the function of the adrenal cortex in certain patients. It is useful in regulating the function of the pituitary and adrenal cortex in patients suffering from.

Many other regulatory peptides influence the endocrine, central nervous and gastrointestinal systems It is known. ACT) (and β-END-LI secretion is a mammalian response to stress. Since the response of mammals is r　Sir+e　qua　nonJ, CRF It is no surprise that it exerts an effective effect on the brain as a stress response mediator. stomach. Therefore, in the brain, CRF antagonists are present in patients with normal or psychiatric disorders. can be used to improve learning functions and behavior. Also, in the brain, C RF antagonists are responsible for the effects of stress, to which endogenous CRF is thought to contribute. (some types of hypersexuality, dysphonia, hyposexuality, impotence, high (including glycemia). The peripherally administered CRF antagonists were A and C. TH1β endorphin, β lipotoshu! and other blow opiomelanocols. Administration of antagonists has a negative impact on the brain, as it lowers the gene product and cortiosterone levels. Reduce the effects of these substances on memory, mood, pain and more specifically alertness , improves depression and/or anxiety17, regulates the immune system, gastrointestinal disorders, and adrenal cortex function. It would be possible to use it for manufacturing purposes.

All peptides similar to CRF have been shown to have mesenteric vascular bed dilating effects. It's turning into a crab. CRF antagonists reduce blood flow in the gastrointestinal system of mammals (especially humans) It is useful for In addition, oCRF suppresses the production of gastric acid, and CRF antagonists suppress the production of gastric acid. Can be effectively used to suppress gastric acid production and/or adjust gastrointestinal function in mammals .

CRF antagonists or non-toxic salts thereof can be used for pharmaceutical or veterinary purposes to create pharmaceutical compositions. Admixed with a medically acceptable carrier, administered intravenously or subcutaneously to animals, including humans. can be administered intramuscularly, transdermally (e.g. intranasally), or even orally. . These synthetic peptides are at least about 90%, preferably at least about 98% purity. However, mammals other than humans have less purity. is also effective and can be used. In this specification, purity refers to the presence of the intended peptide. represents the indicated weight percent of all peptides and peptide fragments present. means. The peptide of the present invention can lower blood pressure or inhibit glucocorticoid production in the body. The body needs to supply cerebrospinal fluid to the ventricles. Alternatively, the antagonist may be present at the blood-brain barrier. Means should be found to enable passage through the

The stiff peptide (appropriately administered and monitored for biological functions) Hypothalamus pituitary adrenal glands in mammals along with endocrine gland or central nervous system pathology It is also possible to use it for functional evaluation. For example, Cushing ) It can be administered as a diagnostic tool for disease evaluation.

These peptides include zinc, iron, calcium, barium, magnesium, and aluminum. complexes with metals such as Ni (also considered as one of the non-toxic addition salts of the present invention) or Administered in the form of pharmaceutically or veterinarily acceptable non-toxic salts, such as acid addition salts Sometimes. Examples of such salts include hydrochloride, hydrobromide, sulfate, Phosphates, tannates, succinates, fumarates, gluconates, alginates salt, maleate, acetate, citrate, benzoate, succinate, malate , ascorbate, tartrate, etc. Oral administration of active ingredient in tablet form If so, the tablets may contain ingredients such as tragacanth, corn starch or gelatin. binders; disintegrants such as alginic acid; and magnesium stearate. It may contain a lubricant. Sweeteners and/or flavorants if liquid administration is desired It can also be administered intravenously by isotonic saline or phosphate buffer. You can also do this.

This peptide should be administered to humans under the guidance of a physician, and the pharmaceutical composition should be administered to humans under the guidance of a physician. containing the peptide with a conventional pharmaceutically acceptable solid or liquid carrier. Dew. Generally, the dosage will be from about 1 to about 200 my of peptide per body weight of the recipient animal. It would be a black gram. In this specification, all temperatures are expressed in degrees Celsius, and all ratios are expressed in °C. All are volume ratios. For liquids, percentages are also volume %.

Although the invention has been described in the preferred embodiment to the best knowledge of the inventors, , various changes and modifications apparent to those of ordinary skill in the art are hereby incorporated by reference. Original translation submission form (Article 184-8 of the Patent Act) March 4th, 1991 Commissioner of the Patent Office Toshi Ue Matsu Nawate 1 1. Display of patent application PCT/US89104118 2. Name of the invention CRF antagonist 3. Patent applicant Address: 92037 for California, USA. La Hora.

North Torrey Vines Road 10010 Name The Salk Ince Tetuto Fornokiological Address: 2-chome Otemachi, Chiyoda-ku, Tokyo 2-1 Shin Otemachi Building 206 Ward 5. Date of submission of written amendment Article 34 amendment 1. H-R, -R, 1o-R4, -R, □-His-Leu-Leu-Arg -Glu-Val-Leu-R2o-Nle-Ala-Ar　g-Al　a-G lu-Gl n-Leu-Al a-Gin-Gin-Al a-Hi 5-8 e　r-Asn-Ar　g-Rss　-R37-N1e-Gl　u-I　l　e -R41-NH2 (In the above formula, Ro is Asp or des　Re　; Rlo is 1. e u or des R4o; Rat is Thr or desR,; R12 is D-Phe, D-2Nal or Phe; R2o is Glu or D-Gl u; R3e is Lys+Arg, Leu or Har; R, 7 is leu; and R4+ is Al3 or He) or a non-toxic addition salt thereof.

2. Ro is des Re: Rlo is des R4o; Rat is desR H; and R12 is D-Phe or D-2Nal.

3. The compound according to claim 2, wherein Leu at position 37 is C(:tCH3-Leu).

4, RI□ is D - P h e: R2O is G l u p Rsa is Lys and R41 is He. 4. The compound of claim 3, wherein R41 is He.

5 Y-Re R+o Ru-Rat-Hts-15-1eu-1eu-Ar+ 7 RIg-R+, -Rzo-Rzt-Rz2-Rzs-Rz4 Rz5-Rz a-Rzt-Rzs-Rza Gln-alaR32R33-Asn-Arg- Rse R57-Nle-Rso R4o-R4+-NHt (in the above formula, Y is hydrogen or acyl having 7 or less carbon atoms: R0 is Asp or desR, :R+ o is Leu or des R7o: Ro is Thr, Ser or de S Ro : Rat is (Q) D-Phe, D-Tyr, D-Leu, D-Hi s, D-Nal, D-Pa l, D-Ile, D-Nle, D- Val, D-Met, Phe or Leu; Q is H, 4C1 or 4F; R, is H is, Tyr or G Iu; Rl 7 is Glu, Asn or LyS; RI g is Val, Nle or Met; R, g and R24 are leu, IIe, ala , Gly, Val, Nle, Phe, Asn and Gln. R; , is Glu or D-G l　u　; R21 is Met, Nva.

11e, ala, Ieu, Nle, Val, Phe or Gln:R2z is a l a, Thr, Asp or Glu; RH is Arg, Orn, Har or Ly s; R,, is Asp or &LG Iu; R26 is GIn, Asn or L ys; R22 is leu, IIe, ala, Vat, Nva, Met+NIe+ Phe.

Asp, Asn, Gin or Glu; R28 is ala, Arg or I-Y S: R29 is Gin or Glu; R32 is His, Gly4'yr or al a; R33 is Ser, Asn+Leu, Thr or ala; Rv is Lys+Or n+Arg+Har or L e u ; R37 is leu or 'ry r ; Rse is Glu or A8p: R2O is 11e4'hr+Glu+ala, Val +leu, N1e+Phe.

Nva, Gly or Gln; and R4+ is a l a, I l e + G 1 y + V a l + l e u + N], @e + P he, Nva or Gin) or a non-toxic addition salt thereof.

6, R, is H4s; R17 is Glu: Ru is Val; R211 is Gin; 6. The compound of claim 5, wherein R2H is Alm.

7. The compound of claim 6, wherein R, is Nle.

8, R, n is A l a: Rxs is Ar g: J5 is Glu; Ru is H 8. The compound of claim 7, wherein ts; Ru is Ser; and R, is IIs.

9, R,, is Leu: Rza is AIIL; R27 is Le-u; and R39 is 9. The compound of claim 8 which is Glu.

10, R, is deaRg; 'J. is deaR4o: and R11 is de 10. The compound of claim 9 which is sRH.

11. The compound of claim 9, wherein R, □ is D-2NAL.

12. The compound according to claim 10, wherein R, is DPhe.

13. The compound according to claim 11 or 12, wherein R is Lys.

14. The compound according to claim 8, wherein R and □ are Phe.

15. The compound of claim 14, wherein R is Arg.

16. The compound of claim 7, wherein R, is D-Glu.

17. The following sequence H-D-Phe-Hi　a　-Le　u-Le　u-Arg-G　l　u　-V a　-Le　u-Gl　u-Nl　e-Ala-Ar　g-Al　a-Gl u-Gl n-Le u-Al a-Gl n-Gln-Al a-Hi s-8er-Asn-Arg-Lys-Leu-Nle-Glu5-8er-A 6. The compound of claim 5 having sn-Ar.

18. The following sequence H-D -Phe-His-Le u-Le u-Ar g-Gl u-V a　-Le　u-Gl　u-Nl　e　-Al　a-Arg-Al　a-G l u-Gl n-Leu-Al a-Gl n-G1 n-Al a-Hi 5-8e r-Asn-Arg-Leu-Nle-Glu-11e-11e-N 6. The compound of claim 5 having H2.

19. The following sequence HD-2NA L-Ht 5-Leu-Leu-Arg-Glu-Va l -Le u-Glu-Nl e-Al a-Ar g-Al a-G 1 u -G 1 n-Le u-Al a-Gl n-G1 n-Al a- H4s-3er-Asn-Arg-Lys-Leu-Nle-Glu-Ile 6. The compound of claim 5 having -Ile-NH2.

20. Effective amount of the synthetic peptide or non-toxic addition salt thereof of claim 5 and pharmaceutical or veterinary A mammalian stress-reducing composition comprising a medically acceptable liquid or solid carrier. A product.

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Claims (20)

[Claims] 1. [There is an array] (In the above formula, R9 is Asp or desR9; R10 is Leu or desR1 0; R11 is Thr or desR11; R12 is D-Phe, D-2Nal or Phe; R20 is Glu or D-Glu; R36 is Lys, Arg, Leu or Har; and R41 is Ala or He) or a non-toxic addition salt thereof. 2. R9 is desR9; R10 is desR10; R11 is desR11; and 2. A compound according to claim 1, wherein R12 is D-Phe or D-2Nal. 3. Y-R9-R10-R11-R12-R13-Ieu-Ieu-Arg-R 17-R18-R19-R20-R21-R22-R23-R24-R25-R 26-R27-R28-R29-Gln-ala-R32-R33-Asn-A rg-R36-R37-Nle-R39-R40-R41-NH2 (the above formula , Y is hydrogen or acyl having 7 or less carbon atoms; R9 is Asp or desR 9; R10 is Leu or desR10; R11 is Thr, Ser or desR1 1; R12 is (Q)D-Phe, D-Tyr, D-Leu, D-H1s, D-N al, D-Pal, D-Ile, D-Nle, D-Val, D-Met, Phe or Leu; Q is H, 4Cl or 4F; R13 is His, Tyr or Glu; R17 is Glu, Asn or Lys; R16 is Val, Nle or Met; R1 9 and R24 are Ieu, He, ala, Gly, Val, Nle, Phe, selected from the group consisting of Asn and Gln; R20 is Glu or D-Glu; R21 is Met, Nva, He, ala, Ieu, Nle, Val, Phe or is Gln; R22 is ala, Thr, Asp or Glu; R23 is Arg, Or n, Har or Lys; R25 is Asp or Glu; R26 is Gln, Asn or is Lys; R27 is leu, He, ala, Val, Nva, Met, Nle , Phe, Asp, Asn, Gln or Glu; R28 is ala, Arg or Lys; R29 is Gln or GIu, R32 is His, Gly, Tyr or al a; R33 is Ser, Asn, Ieu, Thr or ala; R36 is Lys, O rn, Arg, Har or Ieu; R37 is Ieu or Tyr; R30 is Glu or Asp; R40 is He, Thr, Glu, ala, Val, Ieu, Nl e, Phe, Nva, Gly or Gln; and R41 is ala, He, Gl y, Val, Ieu, Nle, Phe, Nva or Gln) or non-toxic Sex addition salt. 4. R13 is His; R17 is Glu; R18 is Val; R20 is Gln; and 4. The compound of claim 3, wherein R28 is Ala. 5. R22 is Ala; R23 is Arg; R25 is Glu; R32 is His; R3 5. The compound of claim 3 or 4, wherein 3 is Ser; and R40 is He. 6. R19 is Leu; R24 is Ala; R27 is Leu; and R39 is Glu The compound according to any one of claims 3 to 5. 7. 7. A compound according to any one of claims 3 to 6, wherein R21 is Nle. 8. 8. The compound according to any one of claims 3 to 7, wherein R12 is D-2NAL. 9. 8. A compound according to any one of claims 3 to 7, wherein R12 is D-Phe. 10. 8. A compound according to any one of claims 3 to 7, wherein R12 is Phe. 11. The compound according to any one of claims 3 to 10, wherein R36 is Arg. 12. The compound according to any one of claims 3 to 10, wherein R36 is Arg. 13. The compound according to any one of claims 3 to 10, wherein R36 is Lys. 14. 14. The compound according to any one of claims 3 to 13, wherein R20 is D-Glu. 15. R9 is desR9; R10 is desR10; and R11 is desR11 The compound according to any one of claims 3 to 14. 16. Array below [There is an array] 4. The compound of claim 3. 17. Array below [There is an array] 4. The compound of claim 3. 18. Array below [There is an array] 4. The compound of claim 3. 19. An effective amount of the synthetic peptide or non-toxic addition salt thereof according to any one of claims 1 to 18. and a pharmaceutically or veterinarily acceptable liquid or solid carrier. A composition that reduces stress. 20. Stress in a mammal comprising administering to the mammal an effective amount of the composition of claim 19. How to reduce