CA1341051C - Crf antagonists - Google Patents

Abstract

Several known members of the corticotropin releasing factor (CRF) family have been synthesized and tested, including human and rat CRF which have the formula: H-Ser-G:Lu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. Peptides are herein disclosed that are potent competitive antagonists of CRF in mammals. One which has been found to be particularly potent is:
H-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH2. These antagonists or pharmaceutically or veterinarily acceptable salts thereof, dispersed in a pharmaceutically or veterinarily acceptable liquid or solid carrier, can be administered to mammals, including humans, to achieve a prevent elevation of ACTH, D-endorphin, .beta.-lipotropin, other products of the pro-opiomelanocortin gene and corticosterone levels and/or a lowering of brain mediated responses to stress over an extended period of time.
They may also be used to affect mood, memory and learning, as well as diagnostically.

Description

CRF ANTAGONISTS
This invention is directed to peptides and to methods for pharmaceutical treatment of mammals using such peptides. More specifically, the invention relates to antagonists of the hentetracontapeptide CRF, to pharmaceutical compositions containing CRF antagonists and to methods of treatment of mammals using CRF
antagonists.
BACKGROUND OF THE INVENTION
i0 Experimental and clinical observations have supported the concept that the hypothalamus plays a key role in the regulation of adenohypophysial corticotropic cells secretory :functions. Over 25 years ago, Guillemin, Rosenberg and Sa:ffran and Schally independently demonstrated the presence of factors in hypothalamus which would increase the rate of ACTH secretion by the pituitary gland :incubated in vitro or maintained in an organ culture. None of the secretagogs characterized met the criteria expected of a physiologic corticotropin releasing fa~~tor (CRF) until ovine CRF (oCRF) was characterized in 1981 and, as disclosed in U.S. Patent No. 4,415,55;x, was found to have the formula:
H-Ser-Gln-Gl~.x-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-'~al-Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-llis-Ser-Asn-Arg-Lys-Leu-Leu-Asp-Ile-Ala-NH2.
Sau'~agine is a 40-residue, amidated generally similar peptide which was isolated from the skin of the South American frog Phyllomedusa sauvagei. It was characterized by Erspamer et al. and was described in Regulatory Peptides, Vol. 2 (1981), pp. 1-13. Sauvagine has the formula: ;~pGlu-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-heu-Leu-Arg-Lys-Met-Ile-Glu-Ile-Glu-Lys-Gln-Glu-Lys-Glu-7~ys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Thr-Ile-2JH2. l;lrotensin I is a homologous 41-residue peptide which was isolated from the urophyses of teleost fish as reported in Ichikawa, et al. Peptides, 3, 859 (1982). Sauvagine, Urotensin I, and members of ~ 34 ~ ~5 1 the CRF family have been reported to have biological activity in lowering blood pressure in mammals and in stimulating the secretion of ACTH and !3-endorphin.
Rat: CRf has been characterized as a 41-amino acid peptide having high homology with oCRF and the formula: H-~~er-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-G.'Ln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2. Human CRF has the same structure, and the abbreviations rCRF and hCRF are used interchangeably.
SUMMARY OF THE INVENTION
Competitive antagonists of the 41-residue CRF
family of peptides have been synthesized which have the following formula: Y-R9-R10-R11 R12 R13-leu-leu-Arg-R17-is R18 R19-R20 R21 R22 R23 R24-R25 R26-R27-R28-R29-Gln-ala-R32-R33-Asn-Ang-R36-R37-Nle-R39-R40-R41-NH2 wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R9 is Asp or desR9; R10 is Leu or desRlO~
R11 is Thr, Ser oz- desRll; R12 is (Q)D-Phe, D-Tyr, D-Leu, D-His, D-Nal, D-Pal, D-Ile, D-Nle, D-Val, D-Met, Phe or Leu; Q is H, 4C1 or 4F; R13 is His, Tyr or Glu; R17 is Glu, Asn or Lye>; R18 is Val, Nle or Met; R19 and R24 are selected from the group consisting of leu, Ile, ala, Gly, Val, Nle, Phe, Asn and Gln: R20 is Glu or D-Glu; R21 is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R22 is ala, Thr, Asp or Glu; R23 is Arg, Orn, Har or Lys; R25 is. Asp or Glu; R26 is Gln, Asn or Lys; R27 is leu, I:le, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R28 is ala, Arg or Lys; R29 is Gln or Glu, R32 i~; His, Gly, Tyr or ala: R33 is Ser, Asn, leu, Thr or ala; R36 is Lys, Orn, Arg, Har or leu;
R37 is leu or Tyr; R39 is Glu or Asp; R40 is Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln; and R41 is ala, Ile, Gly, Val, leu, Nle, Phe, Nva or Gln;
or a nontoxic addition salt thereof.
Pharmaceutical compositions in accordance with the invention include such CRF antagonists, or nontoxic ~ 34 ~ ~5 1 addition salts thereof, dispersed in a pharmaceutically or veterinarily ac:ceptable liquid or solid carrier. The administration of such peptides or pharmaceutically or veterinarily acceptable addition salts thereof to mammals, particularly humans, in accordance with the invention may be carried out for the regulation of secretion of ACTH, B-endorphin, 13-lipotropin, other products of the pro-opiomelanocortin gene and corti-costerone and/or for the lowering of stress responses and/or for affecting mood, behavioral metabolic and gastrointestinal functions and autonomic nervous system activities. Furthermore CRF antagonists may be used for the evaluation of the status of pituitary, metabolic, cardiovascular, gastrointestinal or central nervous system functions.
DETAILED DESfRIPTION OF THE PREFERRED EMBODIMENTS
The nomenclature used to define the peptides is that specified by Schroder & Lubke, "The Peptides", Academic Press (1965) wherein, in accordance with conventional representation, the amino group appears to the left and the carboxyl group to the right. The standard 3-letter abbreviations to identify the alpha-amino acid residues, and where the amino acid residue has isomeric forms, it is the L-form of the amino acid that is rep:reaented unless otherwise expressly indicated, e.g. Se:r = L-serine, Nle = L-norleucine, Nva =
L-norvaline, Har =~ L-homoarginine, Orn = L-ornithine, etc. In addition the following abbreviations are used:
leu = either L-leucine or C°~CH3-L-leucine (CML); ala =
either L-alanine c~r C°~CH3-L-alanine(CMA); D-Nal =
D-alanine, the !3~-carbon of which is substituted with napthalene and linked to the 1- or 2-carbon thereon, and D-Pal = D-alanine, the !3-carbon of which is linked to the 3-position carbon of pyridine.
The invention provides antagonists of CRF having the following Formula (I): Y-R9-R10-R11-R12-R13-leu-leu-Arg-R17-R18 R19-R20-R21 R22-R23-R24-R25-R26 R27-~ 34 1 05 ~

R28-R29-Gln-~ala-R32-R33-Asn-Arg-R36 R37-Nle-R39-R40-R41 NH2 wherein Y is an acyl group having 7 or less carbon atoms or hydrogen; R9 is Asp or desR9; R10 is Leu or desRlO: F;11 is Thr, Ser or desRll: R12 is (Q)D-Phe, D-~Tyr, I)-Leu, D-His, D-Nal, D-Pal, D-Ile, D-Nle, D-Val, D-Meet, Phe or Leu; Q is H, 4C1 or 4F: R13 is His, Tyr or Glu; R17 is Glu, Asn or Lys; R18 is Val, Nle or Met; R19 and R24 are selected from the group consisting of leu, Ile, ala, Gly, Val, Nle, Phe, Asn and Gln; R20 is Glu or D-Glu; R21 is Met, Nva, Ile, ala, leu, Nle, Val, Phe, Asn or Gln; R22 is ala, Thr, Asp or Glu; R23 is Arg, Orn, Har or Lys; R25 is Asp or Glu; R26 is> Gln, Asn or Lys; R27 is leu, Ile, ala, Val, Nva, Met:, Nle, Phe, Asp, Asn, Gln or Glu; R28 is ala, Arg or Lys; R29 is Gln or Glu, R32 is His, Gly, Tyr or ala; Ft33 is Ser, Asn, leu, Thr or ala;
R36 is Lys, Orn, P,rg, Har or leu; R37 is leu or Tyr;
R39 is Glu or Asp; R40 is Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln; and R41 is ala, Ile, Gly, Val, leu, Nle, Phe, Nva or Gln; or a nontoxic addition salt thereof. Antagonists in accordance with this formula exhibit excellent binding to pituitary receptors for native CRF.
A preferred group of antagonists are those having the formula.: Y-R12-R13-leu-leu-Arg-R17-R18 R19-R20 R21-R22 R23 R24 R25 R26 R27-R28 R29-Gln-ala-R32-R33-Asn-Arg-R36-R37-Nle-R39-R40-R41 NH2 wherein Y is Ac or hydrogen; R12 is D-Phe, D-Tyr, D-Leu, D-His, D-Nal, D-Pal, D-Nle, D-Ile, D-Val, D-Met or Phe; R13 is His, Tyr or Glu; R.17 is Glu, Asn or Lys; R18 is Val, Nle or Met; R19 anal R24 are selected from the group consisting of leu, Ile, ala, Gly, Val, Nle, Phe and Gln;
R20 is Glu or D-Glu; R21 is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R22 is ala, Thr, Asp or Glu;
R23 is Arg, Orn, Har or Lys; R25 is Asp or Glu: R26 is Gln, Asn or Lys; R27 is leu, Ile, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R28 is ala, Arg or Lys; R29 is Gln ar Glu, R32 is His, Gly, Tyr or ala;
R33 is Ser, Asn, leu, Thr or ala; R36 is Lys, Orn, Arg, Har or leu; R37 is leu or Tyr; R3g is Glu or Asp; R40 is Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln; and R41 is ala, Ile, Gly, Val, leu, Nle, Phe, Nva or Gln; or a nontoxic addition salt thereof. A particularly preferred subgroup of this group of antagonists .includes the following: R12 is D-Phe, Phe or D-2Nal, R13 is His, R17 is Glu, R18 is Val, R19 and R37 are Leu, R20 is Glu or D-Glu, R21 is Nle, R22 is Ala,, R23 is Arg, R24 and R28 are Ala, R25 and R39 are Glu, R26 is Gln, R27 is Leu, R29 is Gln, R32 is His, R33 is Ser, R36 is Arg, Lys, Har or Leu, R40 is Ile and R41 is Ala or Ile. One analog which has been found to be particularly potent is: [D-Phe~l2, N1e21,38~-rCRF(12-41).
Thca peptides are synthesized by a suitable method, such as b;y exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution addition. Certain CRF
antagonist :sections which do not include D-isomer residues or unnatural amino acid residues may also be synthesized by recently developed recombinant DNA
techniques.
Synthesis by the use of recombinant DNA
techniques, for purposes of this application, should be understood t:o inc:Lude the suitable employment of a structural gene coding for the desired form of CRF
analog. Thus ceri~ain synthetic CRF peptides may be obtained by transforming a microorganism using an expression vector including a promoter and operator together with such structural gene and causing such transformed microorganism to express the CRF peptide. A
non-human animal may also be used to produce certain CRF
peptides by gene-farming using such a structural gene and the microinjection of embryos as described in W083/01783 published 26 May 7.983 and W082/04443 published ~ 34 1 05 1 23 December 1982. Such synthetic CRF peptides are then suitably recovered from the animal by extraction from sera or the like.
Common to chemical syntheses of peptides is the protection of the labile side chain groups of the various amino acid moieties with suitable protecting groups which will prevent: a chemical reaction from occurring at that site until t:he group is ultimately removed. Usually also common is tree protection of an alpha-amino group on an amino acid or a fragment while that entity reacts at the 1. 0 carboxyl group, followed by the selective removal of the alpha-amino protecting group to allow subsequent reaction to take place at. i~hat location. Accordingly, it is common that, as a step in the synthesis, an intermediate compound is produced which includes each of the amino acid residues located in its desired sequence in the peptide chain with various of these residues having side-chain proteci:ing groups.
Also considered to be within the scope of the present invention are intermediates of the Formula (II):
X1 R12(X) R1.3(X or X5)-leu-leu-Arg(X3)-R17(X4,X5 or X6)-R18-R19 (X4 ) ~R20 (X~~) -R21 R22 (X2 or X5) -R23 (X3 or X6) -R24(X4)-R25(X5)-R~,6(X4 or X6)-R27(X4 or X5)-R28(X3 or X6)-R29(X4 or X5')-Gln(X4)-ala-R32(X)-R33(X2 or X4)-Asn(X4)-Arg(X3) R36(X3 or X6)-R37(X)-Nle-R39(X5)-R40(X2 or X4 or X5)-R41(X4)-X7 wherein: the R-groups are as hereinbefore: defined.
Xl is either hydrogen or an alpha-amino protecting group. The alpha-amino protecting groups contemplated. by X~~ are those known to be useful in the art in the step-wise synthesis of polypeptides. Among the classes of alpha-amino protecting groups covered by X1 are (1) acyl-type protecting groups, such as formyl, acrylyl(Acr), benzoyl(Bz) and acetyl(Ac) which are preferably used only at the N-terminal; (2) aromatic urethan-type protecting groups, such as benzyloxycarbonyl(Z) and substituted Z, such as ~ 34 ~ ~5 1 _,-p-chloroben;.yloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenz~~loxycarbonyl, p-methoxybenzyloxycarbonyl; (3) aliphatic urethan protecting groups, such as t-butyloxyc<~rbonyl (BOC), diisopropylmethoxycarbonyl, isopropylox~~carbo:nyl, ethoxycarbonyl, allyloxycarbonyl;
(4) cycloallcyl urethan-type protecting groups, such as fluorenyl mcahylo:xycarbonyl (FMOC), cyclopentyloxy-carbonyl, a<iamant:yloxycarbonyl, and cyclohexyloxy-carbonyl; and (5) thiourethan-type protecting groups, such as phenylthiocarbonyl. The preferred alpha-amino protecting croup is BOC.
X2 is a protecting group for the hydroxyl group of Thr and Ser and is preferably selected from the class consisting of acetyl(Ac), benzoyl(Bz), tert-butyl, triphenylmet:hyl(t:rityl), tetrahydropyranyl, benzyl ether(Bzl) and 2:,~5-dichlorobenzyl (DCB). The most preferred protecting group is Bzl. X2 can be hydrogen, which means there is no protecting group on the hydroxyl group.
X3 ~.s a protecting group for the guanidino group of Arch or Har preferably selected from the class consisting of ni.tro, p-toluenesulfonyl(Tos), Z, adamantylox~~carbonyl and BOC, or is hydrogen. Tos is most preferred.
X4 is hyc9rogen or a protecting group, preferably ~;anthyl(Xan), for the amido group of Asn or Gln.
X5 is hydrogen or an ester-forming protecting group for the f3- or ~-carboxyl group of Asp or Glu, preferably ~:electE~d from the class consisting of benzyl, 2,6-dichlorobenzyl, methyl, ethyl and t-butyl ester.
OBzl is most: pre.fe~rred.
X6 is hydrogen or a protecting group for the side chain amino substituent of Lys or Orn. Illustrative of suitable side chain amino protecting groups are Z, 2-chlorobenz;yloxyc:arbonyl(2-C1-Z), Tos, t-amyloxycarbonyl(Aoc), BOC and aromatic or aliphatic _ _ ~ 34 ) 05 ) urethan-typs~ protecting groups as specified hereinbefore.
When His is present, X is hydrogen or a protecting c~roup~ for the imidazole nitrogen such as Tos or 2,4-dinit:rophenyl(DNP), and when Tyr is present, X is hydrogen or a pros=ecting group for the hydroxyl group such as DCB. When Met is present, the sulfur may be protected, i.f desired, with oxygen.
The selecaion of a side chain amino protecting group is not: critical except that it should must be one i0 which is not: remo~red during deprotection of the alpha-amino group:a during the synthesis. Hence, the alpha-amino protecting group and the side chain amino protecting croup cannot be the same.
X~ is NHS,, a protecting group such as an ester or an anchoring bond used in solid phase synthesis for linking to a :solid resin support, preferably one represented by the formulae:
-NH-~benzhydrylamine (BHA) resin support and -NH-para.methy7.benzhydrylamine (MBHA) resin support.
Cleavage from a BHA or MBHA resin directly gives the CRF
analog amide:. By employing a methyl-derivative of such a resin, a methyl-substituted amide can be created, which is considered to be the equivalent thereof.
In the formula for the intermediate, at least one of X, X1, X2, X3, X4, X5 and X6 is a protecting group. The particular amino acid chosen for each the R-group dete:rmine:a whether there will also be a protecting group attached as specified hereinbefore and as generally known in the art. In selecting a particular side chain p~rotect:ing group to be used in the synthesis of the peptides, t:he following rules are followed: (a) the protecting group should be stable to the reagent and under the reaction conditions selected for removing the alpha-amino protecaing group at each step of the synthesis, (b) the: protecting group should retain its protecting p~ropert:ies and not be split off under coupling conditions and (c) the side chain protecting group must be removable, upon the completion of the synthesis containing the desired amino acid sequence, under reaction conditions that will not alter the peptide chain.
For the acyl group at the N-terminal represented by Y, acetyl., forrnyl, acrylyl and benzoyl are preferred.
Thus, t.hca present invention is also considered to provide a process for the manufacture of compounds defined by i:he F'o:rmula (I) comprising (a) forming a Peptide hav:lng at least one protective group and having the Formula (II) wherein: X, X1, X2, X3, X4, X5 and X6 are each either hydrogen or a protective group, and X~ is either .a protective group or an anchoring bond to resin support or NH2 and (b) splitting off the Protective ~~roup or groups or anchoring bond from said peptide of 'the Formula (II) and (c) if desired, converting a resulting peptide into a nontoxic addition salt thereof.
When the peptides are prepared by chemical synthesis, they are preferably prepared using solid phase synthesis, such as that described by Merrifield, J. Am.
Chem. Soc., 85, p 2149 (1964), although other equivalent chemical syntheses known in the art can also be used as previously mentioned. Solid-phase synthesis is commenced from the C-terminus of the peptide by coupling a protected alpha-amino acid to a suitable resin as - generally set forth in U.S. Patent No. 4,244,946 issued Jan. 21, 1981 to Rivier et al. Such a starting material for an antagonist based upon human CRF can be prepared by attaching alpha-<~mino-protected Ile to a BHA resin.
Il.e protected by BOC is coupled to the BHA resin using methylene chloride and dimethylformamide (DMF) .
Following i:he coupling of BOC-Ile to the resin support, the alpha-amino protecting group is removed, as by using trifluoroac~etic acid (TFA) in methylene chloride, TFA

~3'~~~51 alone or with HC1 in dioxane. Preferably 50 volume % TFA
in methylene chloride is used with 0-5 weight ~ 1,2 ethanedithiol. The deprotection is carried out at a temperature between about 0°C and room temperature. Other standard clE~aving reagents and conditions for removal of specific alpha-amino protecting groups may be used as described in Schroder & Lubke, "The Peptides", 1 pp 72-75 (Academic Press 1965) .
After removal of the alpha-amino protecting group of Ile, the remaining alpha-amino- and side chain-protecaed amino acids are coupled step-wise in the desired ordE:r to obtain the intermediate compound defined hereinbefore. As an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to addition to the solid phase reactor. The selection of an appropriate coupling reagent is within the skill of the art. Particularly suitable as coupling reagents are N,N'-dicyclohexyl carbodi.imide(DCC) and N,N'-diisopropyl carbodi.imide(DICI;I.
The act.i~~ating reagents used in the solid phase synthesis of: the peptides are well known in the peptide art. Examples of :suitable activating reagents are carbodiimides, such as N,N'-diisopropyl carbodiimide and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Other activating reagents and their use in peptide coupling are described b~~ Schroder & Lubke, supra, in Chapter III and by Kapoor, J~. Phar. Sci., 59, pp 1-27 (1970).
Each prot:ected amino acid or amino acid sequence is introduced into the solid phase reactor in about a fourfold excess, and the coupling is carried out in a medium of dimethylformamide(DMF):CH2C12 (1:1) or in DMF or CH2C7.2 alone. In instances where the coupling is carried out manually, the success of the coupling reaction at each stage of the synthesis is monitored by the ninhydrin rea<aion, as described by E. Kaiser et al., Anal. Bioche~m. 34,, 595 (1970). In cases where incomplete ~ 3~ ~ ~5 coupling occurs, the coupling procedure is repeated before removal of 'the alpha-amino protecting group prior to the coupling of the next amino acid. The coupling reactions can be performed automatically, as on a Beckman 990 automati~~ synthesizer, using a program such as that reported in :Rivier et al., Biopol~mers, 1978, 17, pp.1927-1938.
After the desired amino acid sequence has been completed, the intermediate peptide is removedwfrom the resin support by treatment with a reagent, such as liquid hydrogen fluoride, which not only cleaves the peptide from the resin but also cleaves all remaining side chain protecting groups X2, X3, X4, X5 and X6 and the alpha-amino protecting group X1 (unless it is an acyl group which is intended to be present in the final peptide), to obtain the peptide. When using hydrogen fluoride for cleaving, anisole or cresole and methylethyl sulfide are included in the reaction vessel as scavengers. When Met is present in the sequence, the BOC
Protecting group may be cleaved with trifluoroacetic acid(TFA)/ethanedi.thiol prior to cleaving the peptide from the resin to eliminate S-alkylation.
The following Example sets forth the preferred method for synthesizing CRF antagonists by the solid-phase technique.
EXAMPLE I
The: synthesis of the [D-Phel2, N1e21,38~_hL~man CRF(12-41) having the formula:
H-D-Phe-His-~Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-~Gln-LEau-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-G:Lu-Ile-Ile-NH2 is conducted in a stepwise mariner on a MBHA hydrochloride resin, such as available from Sachem, Inc., having a substitution range of about 0.7. to 0..7 mmoles/gm. resin. The synthesis is .*
performed on an automatic Beckman 990B peptide synthesizer using a suitable program, preferably as follows:
*Trade-mark ~ 34' ~5 1 STEP - REAGENTS AND OPERATIONS MIX TIMES MIN.
1 CH2C1.2 wash-80 ml. (2 times) 3 2 Methanol (MEaOH) wash-30 ml. (2 times) 3 3 CH2C12 wash-80 ml. (3 times) 3 4 50 percent TFA plus 5 percent 1,2-ethane dithiol in CH2C12-70 ml. (2 times) 12 5 Isopropano7. wash-80 ml. (2 times) 3 6 TEA 12.5 percent in CH2C12-70 ml.
(2 times) 5 7 MeOH wash-X60 ml. (2 times) 2 8 CH2C12 wash-80 ml. (3 times) 3 9 Boc-amino acid (10 mmoles) in 30 ml. of either DMF or CH2C:12, depending upon the solubility of the particular protected amino acid, (1 time) plus DCC (7.0 mmoles) in CH2C12 30-300 Coupling of BOC-Ile results in the substitution of about 0.35 mmol.. Ile per gram of resin. All solvents that are used are carefully degassed, preferably by sparging with an inert gas, e.g. helium or nitrogen.
After deprotection and neutralization, the peptide chain is built step-by-step on the resin.
Generally, one to two mmol. of BOC-protected amino acid in methylene chloride is used per gram of resin, plus one equivalent of 2 molar DCC in methylene chloride, for two hours. When BOC-Arg(Tos) is being coupled, a mixture of 50% DMF and methyl.ene chloride is used. Bzl is used as the hydroxyl side-chain protecting group for Ser.
P-nitrophenyl estE~r(ONp) can be used to activate the carboxyl end of .Assn or Gln, and for example, BOC-Asn(ONp) can be coupled overnight using one equivalent of HOBt in a 50% mixture of DMF and methylene chloride. The amido group of Asn or Gl.n is protected by Xan when DCC coupling is used instead of the active ester method. 2-C1-Z is used as the protecaing group for the Lys side chain. Tos is used to protect: the guanidino group of Arg and the imidazole group of His, and the side chain carboxyl group of Glu is protected by OBzl. At the end of the . -13- . . ~34~~5 synthesis, thE~ following composition is obtained BOC-D-Phe-His(Tos}-Leu-Leu-Arg(Tos)-Glu(OBzl)-Val-Leu-Glu(OBzl)-Nle-Ala-Arg(Tos)-Ala-Glu(OBzl)-Gln(Xan)-Leu-Ala-Gln(Xan)-Gln(Xan)-Ala-His(Tos)-Ser(Bzl)-Asn(Xan)-Arg(Tos)-Lys(~-C1-Z)-Leu-Nle-Glu(OBzl)-Ile-Ile-MBHA resin support. Xan may have been partially or totally removed by TFA treatment used to deblock the alpha-amino protecting gr~~up .
In order to cleave and deprotect the resulting Protected peptide-resin, it is treated with 1.5 ml.
anisole, 0.5 ml. of methylethylsulfide and 15 ml.
hydrogen fluoride (HF) per gram of peptide-resin, first at -20C. for :20 min. and then at 0°C. for one-half hour.
After elimination of the HF under high vacuum, the resin-peptide is washed alternately with dry diethyl ether and chloroform, and the peptides are then extracted with de-gassed 2N aqueous acetic acid and separated from the resin by filtration.
The peptide is purified by gel permeation followed by semi-preparative HPLC as described in Rivier et al., Peptides: Structure and Biological Function (1979) pp. 125-128, and Rivier et al. J. Chromatography (1983). The chromatographic fractions are carefully monitored by HPLC, and only the fractions showing substantial purity are pooled.
Specific optical rotation of the hCRF peptide, which is synthesized and purified in the foregoing manner, is measured on a Perkin Elmer Model 241 Polarimeter as [p~]h2 = -39.4 + 1.0 (c=0.5 in 50% acetic acid) (with correction for the presence of H20 and TFA): it has a purity of about 95%. To check whether the precise sequence i;~ achieved, the CRF peptide is hydrolyzed in sealed evacuated tubes containing constant boiling HC1, 3 ~,1 of thioglycol/ml. and 1 nmol of Nle (as.
an internal standard) for 9 hours at 140°C.* Amino acid analysis of the hydrolysate using a Beckman 121 MB amino acid analyzer shows the following amino acid ratios:

Asx(0.9), Glx(7.1), Ala(3.9), Nle(1.9), Val(1.1), Ser(1.1), Ile(2.1), Leu(5.0), Phe(0.9), Lys(1.0), His(2.1) and Arg(3.0), which confirms that the 30-residue peptide structure has been obtained.
EXAMPLE II
The syntheaic peptide [D-Phel2, N1e21,38 Arg36]-hCRF(12-41) having the formula: H-D-Phe-His-Leu-Leu-Arg-Glu-Val.-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Alai-His-Ser-Asn-Arg-Arg-Leu-Nle-Glu-Ile-Ile-NH2 is synthesized generally in accordance with the procedure set forth in Example I.
Specific optical rotation of the hCRF peptide, which is synthesized and purified in the foregoing manner, is measured on a Perkin Elmer Model 241 Polarimeter as [d]I~2 = -27.7 + 1.0 (c=0.5 in 50% acetic acid) (with correction for the presence of H20 and TFA); it has a purity of about 99%.
The peptide is judged to be homogeneous using thin layer chromatography and several different solvent systems. It is spEacifically subjected to isocratic reversed-phase high pressure liquid chromatography using the Waters*HF~LC sy:~tem described above with a 0.46 x 25 cm column packed with 5~,m C18 silica, 300A pore size.
The buffer used is an aqueous 0.1% trifluoroacetic acid solution cons~istinc~ of 1.0 ml. of TFA per 1000 ml. of solution plu~~ 42.6% acetonitrile. The determination is run at room temperature. The flow rate is 2.0 ml. per minute, and the rei=ention volume is 10.4 ml.
Amino acid analysis of the resultant, purified Peptide is consistsant with the formula for the prepared peptide and confirms that the 30-residue peptide structure is obtained.
EXAMPLE III
The synthetic CRF antagonists from Examples I
and II are a}:amined for their effects on the secretion of ACTH and B-endorphin in vitro. The effectiveness of synthetic CR~~ antagonists to block the secretion of ACTH
C$

and 8-endorphin by cultured rat pituitary cells is measured u~;ing the procedure as generally set forth in Vale et al., Endocrinolocty, 91, 562 (1972).
The antagonist Peptide No. I, prepared in Example I, is tested using the procedure set forth in detail in J'. Rivier et al., Science, 224, 889-891 (1984) to determir.~e its effect in blocking by 50% the secretion of ACTH stimulatEad by a 1 nM dose of oCRF. Compared to AHC(9-41), a potent CRF antagonist disclosed in U.S.
Patent No. 4,605,642, this peptide was more than 17 times as potent. The ~~pecificity of this inhibition is demonstrated by t:he finding of no effect of the standard antagonist on GRF'-stimulated secretion of GH, GnRH-stimulated ssecretion of LH and FSH or TRF-stimulated secretion of TSH and prolactin. The effects of the antagonist on a number of different concentrations of oCRF and the ability of several different concentrations of Peptide No. I to inhibit ACTH secretion stimulated by a constant dose of oCRF (1 nM) are considered to demonstrate competitive inhibition. Similar testing shows that Peptide No. II, prepared in Example II, is more than 15 times as potent as AHC(9-41).
The in yivo effect of CRF antagonists is tested on the spontaneous ACTH release by adrenalectomized rats. The iv injection of 3 mg/kg BW (about 2.7 nmole) causes a marked decrease in plasma ACTH levels (measured as described in Vale et al. Science, 213, 1394, 1981), which is statistically significant for 2 hours. In the intact, non-anesthetized rats, the antagonists induce a dose-related in:hi.bition of CRF-induced ACTH secretion, which is significant at the 0.09 ~cmole dose level, and prevent most, but: not all, of the ACTH rise due to ether-exposure.
These results indicate that administration of CRF antagonists reduces the spontaneous ACTH release observed after removal of the corticosteroid feedback, totally blocks the ACTH secretion caused by CRF, and ~ 34 ~ p ' - 16 -inhibits most of the stressor-induced ACTH release in intact rats. Such. data are comparable to those previously obtained with an antiserum to CRF which demonstrate the role played by endogenous CRF in regulating ACTH secretion, Rivier, C. et al., Science, 218, 377-9(1982).
Synthetic: hCRF has been shown to be a powerful stimulator of secretion of ACTH and f3-endorphin-like (I3-END-LI) immunoactivities in vivo in several~~rat preparations. Plasma levels of ACTH and !3-END-LI are elevated for at lE:ast 5-20 minutes following the intraveneous administration of hCRF to nembutal-anesthesized male rats and to quiescent male or female rats with indwelling intravenous cannulae. In addition, hC'RF is found to have a dramatic effect to lower blood pressure in rats and dogs. These antagonists should counteract such effects.
EXAMPLE IV
The: peptide [N1e21,38]_hCRF(12-41) having the formula: H-F~he-Hi:>-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-LE:u-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-I7.e-NH2 is synthesized.
Specific optical rotation of the hCRF peptide, which is synthesized and purified in the foregoing manner, is measurEad on a Perkin Elmer Model 241 as [d]22 = -27.2 ~ 1.,0 (c=0.5 in 50~ acetic acid) (with D
correction for thE~ presence of H20 and TFA), and it has a purity of about 99~.
Ths: peptide is judged to be homogeneous using thin layer c:hromai~ography and several different solvent systems. It: is specifically subjected to isocratic reversed-phase high pressure liquid chromatography using the Waters*HPLC system described above with a 0.46 x 25 cm column packed with 5~cm C18 silica, 300A pore size.
The buffer used i:~ an aqueous 0.1~ trifluoroacetic acid solution consisting of 1.0 ml. of TFA per 1000 ml. of solution plus 36.0 acetonitrile. The determination is 1 34 ~ 05 ~

run at room temperature. The flow rate is 2.0 ml. per minute, and the retention volume is 9.5 ml.
Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared peptide and confirms that the 30-residue peptide structure had been obtained.
Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of.,~CTH and 13-END-LI. The peptide is more i0 than 12 timE~s as potent as AHC(9-41).
EXAMPLE V
Then peptide [N1e21,38~_hCRF(9-41) having the formula: H-~~sp-Leo-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala--Arg-A:la-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys--Leu-N:le-Glu-Ile-Ile-NH2 is synthesized.
SpE;cifi_c optical rotation of the hCRF peptide, which is synthesi;aed and purified in the foregoing manner, is measured on a Perkin Elmer Model 241 as [~22 = _17..6 + 1.0 (c=0.5 in 50~ acetic acid) (with D
correction i:or the presence of H20 and TFA), and it has a purity of about 99°s.
Ths~ peptide is judged to be homogeneous using thin layer c:hromaitography and several different solvent systems. It: is specifically subjected to isocratic reversed-phase high pressure liquid chromatography using the Waters FiPLC s!~stem described above with a 0.46 x 25 cm column packed with 5~.m C18 silica, 300A pore size.
The buffer used is an aqueous 0.1~ trifluoroacetic acid solution consisting of 1.0 ml. of TFA per 1000 ml. of solution plus 39.60 acetonitrile. The determination is run at room temperature. The flow rate is 2.0 ml. per minute, and the rcatention volume is 10.1 ml.
Amino acid analysis of the resultant, purified peptide is c:onsisi~ent with the formula for the prepared Peptide and confirms that the 33-residue peptide structure has been obtained.

13,~ ~ ~5 _ Testing .in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and l3-END-LI. The peptide is more than 6 timer as potent as AHC(9-41).
EXAMPLE VI
The' peptide ( N1e38 ] -Carp Urotensin I ( 12-41 ) having the i:ormula: H-Phe-His-Leu-Leu-Arg-Asn-Met-Ile-Glu-Met-Ala--Arg-A:~n-Glu-Asn-Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys--Tyr-N:Le-Asp-Glu-Val-NH2 is synthesized.
Testing in accord<~nce with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and f3-END-LI.
EXAMPLE VII
The: peptide [N1e21,38~ Arg36]_hCRF(9-41) having the formula: H-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Nle-Glu-Ile-Ile-NH2 is synthesized.
Specific optical rotation of the hCRF peptide, which is synthesized and purified in the foregoing manner, is measurE~d on a Perkin Elmer Model 241 as [p~]p2 = -16.0 + 1,.0 (c=0.5 in 50~ acetic acid) (with correction for thE~ presence of H20 and TFA); it has a purity of ax>out 99%.
The: peptide is judged to be homogeneous using thin layer chromatography and several different solvent systems. It: is specifically subjected to isocratic reversed-phase high pressure liquid chromatography using the Waters HPLC system described above with a 0.46 x 25 cm column packed with 5~m C18 silica, 3001 pore size.
The buffer used i:~ an aqueous 0.1~ trifluoroacetic acid solution consisting of 1.0 ml. of TFA per 1000 ml. of solution plus 39.0 acetonitrile. The determination is run at room temperature. The flow rate is 2.0 ml. per minute, and the rEaention volume is 9.1 ml.
Amino acid analysis of the resultant, purified peptide is c:onsist:ent with the formula for the prepared 5~

peptide and confirms that the 33-residue peptide structure has been obtained.
Te:~ting .in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and J3-END-LI. The peptide is about twice as potent a:a AHC(9-41) .
EXAMPLE VIII
The peptide [N1e18,21,38]-Carp Urotensin I(12-41) having the formula: H-Phe-His-Leu-Leu-Arg-Asn-Nle-Ile-~Glu-Nle-Ala-Arg-Asn-Glu-Asn-Gln-Arg-Glu-Gln-Ala-Gly-Leu-Asn-Arg-Lys-Tyr-Nle-Asp-Glu-Val-NH2 is synthesized. Tesi~ing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secrE~tion of ACTH and !3-END-LI.
1.5 EXAMPLE IX
The peptide [N1e21,38~ Arg36]-hCRF(12-41) having the formula: H-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Al.a-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Nl.e-Glu-Ile-Ile-NH2 is synthesized.
Specific optical rotation of the hCRF peptide, which is synthesized and purified in the foregoing manner, is m,easurs~d on a Perkin Elmer Model 241 as [o(]D2 = -23.7 ~ 1.0 (c=0.5 in 50% acetic acid) (with correction for the presence of H20 and TFA), and it has a purity of about 98%.
The peptide is judged to be homogeneous using thin layer chromatography and several different solvent systems. It is specifically subjected to isocratic reversed-phase high pressure liquid chromatography using the Waters HPLC system described above with a 0.46 x 25 cm column packed with Sum C18 silica, 300A pore size.
The buffer used is, an aqueous 0.1% trifluoroacetic acid solution consisting of 1.0 ml. of TFA per 1000 ml. of solution plus 36.6% acetonitrile. The determination is ~n at room temperature. The flow rate is 2.0 ml. per minute, and the reaention volume is 10.1 ml.

?34~ ~5 1 Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared peptide and conf.ir~ms that the 30-residue peptide structure has been. obtained.
Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and B-END-LI. The peptide is more than 6 times as potent as AHC(9-41).
EXAMPLE X
The peptide [D-2Na112, N1e21,38~-hCRF(12-41) having the formula: H-D-2Na1-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-.Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Leu-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH2 is synthesized.
Specific optical rotation of the hCRF peptide, which is synthesized and purified in the foregoing manner, is measured on a Perkin Elmer Model 241 as [~22 = _26.8 + :1.0 (c=0.5 in 50% acetic acid) (with D
correction for the presence of H20 and TFA), and has a purity of about 600.
The peptide is judged to be homogeneous using thin layer chromatography and several different solvent systems. It is specifically subjected to isocratic reversed-phase high pressure liquid chromatography using the Waters HPLC system described above with a 0.46 x 25 cm column packed with 5~m C18 silica, 300A pore size.
The buffer used is an aqueous 0.1~ trifluoroacetic acid solution consisting of 1.0 ml. of TFA per 1000 ml. of solution plus 39% acetonitrile. The determination is run at room temperature. The flow rate is 2.0 ml. per minute, and the retention volume is 8.9 ml.
Amino acid analysis of the resultant, purified peptide is consistent with the formula for the prepared peptide and confirmed that the 30-residue peptide structure had been obtained.
Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and B-END-LI. The peptide is about 9 times as potent as AHC(9-41).

21 - ~ ~ ~ i EXAMPLE XII

N1e21,38 Leu36]-The peptide [D-Phel2 ~
, hCRF(12-41) having the formula: H-D-Phe-His-Leu-Leu-Arg-Glu-Val-~Leu-G:~Lu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-~Ser-A:an-Arg-Leu-Leu-Nle-Glu-Ile-Ile-NH2 is synthesized.

Specific optical rotation of the hCRF peptide, which is synthesi::ed and purified in the foregoing manner, is nieasurE~d on a Perkin Elmer Model 241 as '' 10 [~]p2 = -22.7 1..0 (c=0.5 in 50~ acetic acid) (with ! correction for the presence of H20 and TFA), and it has a purity of about 68.20.

They peptide is judged to be homogeneous using thin layer c:hromat:ography and several different solvent systems. It: is specifically subjected to isocratic reversed-phase high pressure liquid chromatography using the Waters HPLC system described above with a 0.46 x 25 cm column packed with 5~.m C18 silica, 3001 pore size.

The buffer Used is an aqueous 0.1~ trifluoroacetic acid solution consisting of 1.0 ml. of TFA per 1000 ml. of solution plus 45.0% acetonitrile. The determination is run at room temperature. The flow rate is 2.0 ml. per minute, and the rE~tention volume is 9.0 ml.

Amino acid analysis of the resultant, purified Peptide is c:onsist:ent with the formula for the prepared peptide and confirms that the 30-residue peptide structure ha,d been obtained.

Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and f3-END-LI. The peptide is about 3 times as potent as AHC(9-41).

EXAMPLE XIII

The peptide [Acetyl-Asp9, G1y19, N1e38, Asp39, Nva40]-hCRF(9-41) having the formula: Ac-Asp-Leu-Thr-Phe-His-Leu-~Leu-Arg-Glu-Val-Gly-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Asp-Nva-Ile-NH2 is synthesized. Testing in accordance with 1 34 1 ~5 T

the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and l3-END-LI.

EXAMPLE XIV

Ths~ peptide [Glnl9, Lys23, Va124, CMA33~

N1e38]-hCRFl;l2-91) having the formula: H-Phe-His-Leu-- Leu-Arg-Glu--Val-G:Ln-Glu-Met-Ala-Lys-Val-Glu-Gln-Leu-Ala-Gln-Gln-Ala--His-CMA-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH2 is synthesi~:ed. Testing in accordance with the general procedure sEa forth in Example III shows that it likewise inhibits the: secretion of ACTH and B-END-LI.

EXAMPLE XV

The' peptide [N1e21,38~ G1y24~ Tyr32~ Orn36]-hCRF(10-41) havi.nc~ the formula: H-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu--Val-LE~u-Glu-Nle-Ala-Arg-Gly-Glu-Gln-Leu-Ala-Gln-Gln-Ala--Tyr-Ser-Asn-Arg-Orn-Leu-Nle-Glu-Ile-Ile-NH2 is synthesis:ed. '.t'esting in accordance with the general procedure set fari;,h in Example III shows that it likewise inhibits the secrE_tion of ACTH and f3-END-LI.

EXAMPLE XVI

G1n40]-ThE~ peptide [A1a21 Nle3~

, , sauvagine(10-40) having the formula: H-Ser-Leu-Glu-Leu -Leu-Arg-Ly:~-Met-:Cle-Glu-Ile-Ala-Lys-Gln-Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala-A:an-Asn-Arg-Leu-Leu-Nle-Asp-Thr-Gln-NH2 is synthesis:ed. '.t'esting in accordance with the general Procedure sea forth in Example III shows that it likewise inhibits the secretion of ACTH and f3-END-LI.

EXAMPLE XVII

Nle3~
Phe39]-The' peptide [A1a20 Har22 , , , sauvagine(17.-40) having the formula: H-Leu-Glu-Leu-Leu-Arg-Lys-Met--Ile-G:lu-Ala-Glu-Har-Gln-Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala--Asn-A:an-Arg-Leu-Leu-Nle-Asp-Phe-Ile-NH2 is synthesized. Tesi~ing in accordance with the general procedure sea forth in Example III shows that it likewise inhibits the secretion of ACTH and B-END-LI.

EXAMPLE XVIII

The' peptide [Va118.20~ I1e26, Nle3~, G1y40]-sauvagine(:L1-40) having the formula: H-Leu-Glu-1 34 ~ 0~ ~

Leu-Leu-Arg-Lys-Met-Val-Glu-Val-Glu-Lys-Gln-Glu-Lys-Ile-Lys-Gln-Gln-Ala-Al.a-Asn-Asn-Arg-Leu-Leu-Nle-Asp-Thr-Gly-NH2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and f3-END-LI.
EXAMPLE XIX
The peptide [4FD-Phel2, CML14,15,19,27,33,37 C~22,32,41~ N1e38']-AHC(12-41) having the formula:
H-4FD-Phe-Glu-CML-~CML-Arg-Glu-Met-CML-Glu-Met-CMA-Lys-Ala-Glu-Gln-CML-Ala-Gl.u-Gln-Ala-CMA-CML-Asn-Arg-Leu-CML-Nle-Glu-Glu-CMA-NH2. Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and !3-END-LI.
EXAMPLE XX
The peptide [4C1D-Phel2, N1e18,21,38~_AHC(9-41) having the formula:
H-Asp-Leu-Thr-4C1D-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Nle-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Nle-Glu-Glu-Ala-NH2 is synthesized.
Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and 13-END-LI.
EXAMPLE XXI
The peptide [D-Phel2, Met27, N1e21,38~-AHC(12-41) having the formula: H-D-Phe-His-Leu-Leu-Arg-Glu-Met-Leu-~~lu-Nle-Ala-Lys-Ala-Glu-Gln-Met-Ala-Glu-Gln-Ala-Ala-Leu-.Asn-Arg-Leu-Leu-Nle-Glu-Glu-Ala-NH2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and l3-END-LI.
EXAMPLE XXII
The peptide [N1e18,38, Leu2l, A1a27)-AHC(12-41) having the formula: H-Phe-His-Leu-Leu-Arg-Glu-Nle-Leu-Glu-Leu-Ala-Lys-Ala-Glu-Gln-Ala-Ala-Glu-Gln-Ala-Ala-Leu-;Asn-Arg-Leu-Leu-Nle-Glu-Glu-Ala-NH2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and 13-END-LI.

EXAMPLE XXIII
The peptide [Leul2, G1u13,22~ Lys26~ N1e38 ]-AHC(12-41) having the formula: H-Leu-Glu-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Glu-Lys-Ala-Glu-Lys-Glu-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Nle-Glu-Glu-Ala-NH2.
Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and f3-END-LI.
EXAMPLE XXIV
The synthetic peptide [D-I1e12, Tyrl3, CMA28~
N1e38]-AHC(12-41) having the formula: H-D-Ile-Tyr-Leu-Leu-Arg-Glu-:Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-CMA-Glu-Gln-Ala-.Ala-Leu-Asn-Arg-Leu-Leu-Nle-Glu-Glu-Ala-NH2 is synthesized. Testing in accordance with the general Procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and B-END-LI.
EXAMPLE XXV
The peptide [D-Leul2, G1u13, A1a33, N1e38]-AHC(12-41) having the formula: H-D-Leu-Glu-Leu-Leu-Arg-Glu-Met-Leu-Glu-Met-Ala-Lys-Ala-Glu-Gln-Glu-Ala-Glu-Gln-Ala-Ala-,Ala-Asn-Arg-Leu-Leu-Nle-Glu-Glu-Ala-NH2.
Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and f3-END-LI.
EXAMPLE XXVI
The peptide [CML14,19,27,36~ N1e38]-AHC(12-41) having the formula: H-Phe-His-CML-Leu-Arg-Glu-Met-CML-Glu-Met-Ala-Lys-Ala-Glu-Gln-CML-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-CML-Leu-Nle-Leu-Glu-Glu-Ala-NH2. Testing in accordance with the general procedure set forth in Example III shows that it likewise stimulates the secretion of ACTH and l3-END-LI and causes a very significant lowering of blood pressure.
EXAMPLE XXVII
The peptide [D-N1e12, N1e18,21,38~ Asnl9 Asp22, Phe27]-AHC(12-41) having the formula:
H-D-Nle-His-Leu-Leu-Arg-Glu-Nle-Asn-Glu-Nle-Asp-Lys-Ala--25- 134~~51 Glu-Gln-Phe-Ala-Glu-Gln-Ala-Ala-Leu-Asn-Arg-Leu-Leu-Nle-Glu-Glu-Ala-NH2 :is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and B-END-LI.
EXAMPLE XXVIII
Th.e peptide [D-Va112, N1e21,38~ I1e24,27~
Nva41]-hCRF'(11-47.}having the formula: H-Thr-D-Val-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ile-Glu-Gln-Ile-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Nva-NH2 is synthesized. Testing in accordance with the general procedure: set forth in Example III shows that it likewise inhibits the secretion of ACTH and B-END-LI.
EXAMPLE XXIX
The peptide [Acrylyl-LeulO, Va127, N1e38, A1a40, Leu41]-hCRF(10-41) having the formula: Acr-Leu-Thr-Phe-His-Leu-Leu~-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Val-Ala-Gln~-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ala-Leu-NH2 is ;synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and !3-END-LI.
EXAMPLE XXX
The peptide [D-Tyrl2, A1a19, Lys23, N1e24,38,40 Nva27,]-hCRF(12-41) having the formula:
H-D-Tyr-His--Leu-Lc~u-Arg-Glu-Val-Ala-Glu-Met-Ala-Lys-Nle-Glu-Gln-Nva--Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Nle-Ile-NH2 is synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and -END-LI.
EXAMPLE XXXI
The peptide [D-Hisl2, C~19,21,24,27~ Tyr32~
Thr33, N1e38, G1n40]-hCRF(11-41) having the formula:
H-Thr-D-His-His-Leu-Leu-Arg-Glu-Val-CMA-Glu-CMA-Ala-Arg-CMA-Glu-Gln-CMA-Al.a-Gln-Gln-Ala-Tyr-Thr-Asn-Arg-Lys-Leu-Nle-Glu-Gln-Ile-NHf2 is synthesized. Testing in accordance with th.e general procedure set forth in ~ 34 ~ 05 1 Example III shows that it likewise inhibits the secretion of ACTH and f3-END--LI.
EXAMPLE XXXII
The: peptide [4C1-D-Phel2, N1e19~27,38 D-Glu2~, Nva.2l, Leu24, Gly4~, CMA41]-hCRF(12-41) having the formula: H-4C1-D-Phe-His-Leu-Leu-Arg-Glu-Val-Nle-D-Glu-Nwa-Ala--Arg-Leu-Glu-Gln-Nle-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Lsau-Nle-Glu-Gly-CMA-NH2 is synthesized. Testing in accordance with the general Procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and f3-END-LI.
EXAMPLE XXXIII
The peptide [Benzoyl-D-Metl2, CML21,24~ Har36 N1e38, Leu40, Pheq'1]-hCRF(12-41) having the formula: ' Bz-D-Met-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-CML-Ala-Arg-CML-Glu-Gln-Leu-Ala-Gl.n-Gln-Ala-His-Ser-Asn-Arg-Har-Leu-Nle-Glu-Leu-Phe-NH2 i~; synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and B-END-LI.
EXAMPLE XXXIV
The peptide [D-Hisl2, phe21,24~ Orn23, Asp27, N1e38, CMA4~, Va141]-hCRF(12-41) having the formula:
H-D-His-His-Leu-Le:u-Arg-Glu-Val-Leu-Glu-Phe-Ala-Orn-Phe-Glu-Gln-Asp-Ala-G1n-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-CMA-Val-NH2 is, synthesized. Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and B-END-LI.
EXAMPLE XXXV
The peptide [formyl-D-Pa112, Phel9, G1n21, Thr22, Tyr32, N1e38, CML40,41]-hCRF(12-41) having the formula: For-D-Pal-His-Leu-Leu-Arg-Glu-Val-Phe-Glu-Gln-Thr-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-Tyr-Ser-Asn-Arg-Lys-Leu-Nle-Glu-CML-CML-NH2 is synthesized.
Testing in accordance with the general procedure set forth in Example III shows that it likewise inhibits the secretion of ACTH and !3-END-LI.

1 34 1 05 1 _ EXAMPLE XXXVI

N1e21,38~-oCRF(12-41) ThE~ peptide [D-Phel2 , having the i_ormul;a: H-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Thr--Lys-A:la-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg--Lys-Leu-Nle-Asp-Ile-Ala-NH2 is synthesized.. Testing in accordance with the general procedure ssa forth in Example III shows that it likewise inhibits the secretion of ACTH and f3-END-LI.

EXAMPLE XXXVII

I Arg36~-0 N1e21,38 The peptide [D-Phel2 . ~
, oCRF(12-41) havin<~ the formula: H-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu--Glu-N:Le-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Nle-Asp-Ile-Ala-NH2 is synthesized. Tesi~ing in accordance with the general procedure set fori;.h in Example III shows that it likewise inhibits the secretion of ACTH and f3-END-LI.

CRf profoundly stimulates the pituitary-adrenalcorti.cal a:~cis, and CRF antagonists should be useful to inhibit the functions of this axis in some tYPes of patients with high ACTH and endogenous glucocorticoid production. For example, CRF antagonists may be useful in ~_regulating pituitary-adrenal function in patients having pituitary Cushings disease or any CRF-sensitive tumor.

Mo~;t other regulatory peptides have been found to have effects upon the endocrine system, the central nervous system and upon the gastrointestinal tract.

Because ACT~~ and 13-END-LI secretion is the "sine qua non"

of mammal's response to stress, it was not surprising that CRF has. significant effects on the brain as a mediator of many of the body's stress responses.

Accordingly, CRF antagonists delivered to the brain should also find application in modifying the mood, learning ands behavior of normal and mentally disordered individuals. Furthermore, CRF antagonists in the brain could ameliorate :stress-induced conditions to which endogenous C'RF might contribute, including some types of ~3'~~ 05 ~

hypertension, infertility, decreased libido, impotency and hyperglycemia. Because peripherally administered CRF
antagonists reduce' the levels of ACTH, B-END, B-lipotropin, other pro-opiomelanocortin gene products and corticosterone, administration of the antagonists may be used to reduce the effects of all of these substances on the brain to thereby influence memory, mood, pain appreciation, etc., and more specifically, alertness, depression and/or anxiety, as well as to modulate the immune system, ga~;trointestinal tract and adrenalcortical growth and function.
All CRF related peptides have been shown to dialate the mesenteric vascular bed. CRF antagonists may also be of use for decreasing blood flow to the gastrointestinal tract of mammals, particularly humans.
Also, oCRF influences gastric acid production, and CRF
antagonists are expected to also be effective to modulate gastrointestinal functions.
CRF antagonists or the nontoxic addition salts thereof, combined with a pharmaceutically or veterinarily acceptable carrier' to form a pharmaceutical composition, may be administered to mammals, including humans, either intravenously, sub~cutaneously, intramuscularly, percutaneously, e.g. intranasally, intracerebroventricularly or orally. The peptides should be at least about 90% pure and preferably should have a purity of at least about 98%; however, lower purities are effective and may well be used with mammals other than humans. This purity means that the intended peptide constitutes the stated weight % of all like peptides and peptide fragments present. Administration to humans may be employed by a physician to inhibit endogenous gluco-corticoid production or for possible uses outlined above. The required dosage will vary with the particular condition being treated, with the severity of the condition arnd with the duration of desired treatment. In order to block the stress-related effects of endogenous 1341051.

CRF within the central nervous system, it may be necessary to deliver the CRF antagonists into the cerebral ventricle or spinal fluid. Alternatively, a means of modifying the antagonists so that they could penetrate the blood-brain barrier should be found.
These peX>tides may also be used to evaluate hypothalamic pituitary adrenal function in mammals with suspected endocrine or central nervous system pathology by suitable administration followed by monitoring body functions. For e~s:ample, administration may be used as a diagnostic tool tc> evaluate the basis of Cushings disease.
Such peptides are often administered in the form of pharmaceutically or veterinarily acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g., with zinc, iron, calcium, barium, magnesium, aluminum or the like (which are considered as addition salts for purposes, of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, tannate, oxalate, fumarate, gluconate, alginate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder, such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid: and a lubricant, such as magnesium stearate. If administration in liquid form is desired, sweetening and/or flavoring may be used, and intravenous administration in isotonic saline, phosphate buffer solutions o~r the like may be effected.
The peptides should be administered under the guidance of a physician, and pharmaceutical compositions will usually contain the peptide in conjunction with a conventional, pharmaceutically or veterinarily-acceptable carrier. Usually, the dosage will be from about 0.01 to about 10 milligrams of the peptide per kilogram of the body weight of the host animal. As used ~ 3 '~ ~ ~ 5 1 herein all temperatures are °C. and all ratios are by volume. Pencentac~es of liquid materials are also by volume.
Although the invention has been described with regard to it.s preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious t:o one having the ordinary skill in this art may be made without departing from the scope of the invention which is> set forth in the claims appended hereto. For example, substitutions and modifications at other positions in the CRF peptide chain can be made in accordance with present or future developments without detracting from the potency of the antagonists. For instance, instead of the simple amide at the C-terminal, a lower alkyl-sub~;tituted amide, e.g. 1-4 carbon atoms, i.e. methylamide, ethylamide, etc, may be incorporated.
Such peptides are considered as being within the scope of the invention.
Various features of the invention are emphasized in the claims which follow.

Claims (25)

1. A corticotropin releasing factor (CRF) antagonist which includes at least one D-isomer and has the formula:

Y-R9-R10-R11-R12-R13-leu-leu-Arg-R17-R18-R19-R20-R21-R22-R23-R24-R25-R26-R27-R28-R29-Gln-ala-R32-R33-Asn-Arg-R36-R37-Nle-R39-R40-R41-NH2 wherein Y is hydrogen or an acyl group having 7 or fewer carbon atoms; R9 is Asp or desR9; R10 is Leu or desR10; R11 is Thr, Ser or desR11; R12 is (Q) D-Phe, D-Tyr, D-Leu, D-His, D-2Nal, D-1Nal, 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; R18 is Val, Nle or Met; R19 and R24 are selected from the group consisting of leu, Ile, ala, Gly, Val, Nle, Phe, Asn and Gln; R20 is Glu or D-Glu;
R21 is Met, Nva, Ile, ala, leu, Nle, Val, Phe or Gln; R22 is ala, Thr, Asp or Glu; R23 is Arg, Orn, Har or Lys; R25 is Asp or Glu; R26 is Gln, Asn or Lys; R27 is leu, Ile, ala, Val, Nva, Met, Nle, Phe, Asp, Asn, Gln or Glu; R28 is ala, Arg or Lys; R29 is Gln or Glu, R32 is His, Gly, Tyr or ala; R33 is Ser, Asn, leu, Thr or ala; R39 is Lys, Orn, Arg, Har or leu; R37 is leu or Tyr; R39 is Glu or Asp; R40 is Ile, Thr, Glu, ala, Val, leu, Nle, Phe, Nva, Gly or Gln; and R41 is ala, Ile, Gly, Val, leu, Nle, Phe, Nva or Gln: wherein either R12 or R20 or both is a D-isomer or a nontoxic addition salt thereof.

2. The CRF antagonist of claim 1 wherein R13 is His, R17, is Glu, R18 is Val, R20 is Gln and R28 is Ala.

3. The CRF antagonist of claim 2 wherein R22 is Ala, R23 is Arg, R25 is Glu, R32 is His, R33 is Ser, and R40 is Ile.

4. The CRF antagonist of any one of claims 1, 2, 3 or 4 wherein R21 is Nle.

5. The CRF antagonist of any one of claims 1 to 4 wherein R19 is Leu, R24 is Ala, R27 is Leu and R39 is Glu.

6. The CRF antagonist of any one of claims 1 to 5 wherein R12 is D-2NAL.

7. The CRF antagonist of any one of claims 1 to 5 wherein R12 id D-Phe.

8. The CRF antagonist of any one of claims 1 to 7 wherein R36 is Arg.

9. The CRF antagonist of any one of claims 1 to 8 wherein R36 is Lys.

10. They CRF antagonist of any one of claims 1 to 5 wherein R12 is Phe and R20 is D-Glu.

11. Then CRF antagonist of any one of claims 1 to 5 wherein R12 is Phe, R20 is D-Glu and R36 is Arg.

12. The CRF antagonist of any one of claims 1 to 11 wherein R9 is desR9, R10 is desR10, and R11 is desR11.

13. The CRF antagonist of any one of claims 1 to 12 wherein R20 is D-Glu.

14. The CRF antagonist of claim 1 having the formula:
H-D-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-heu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-I1e-NH2.

15. The CRF antagonist of claim 1 having the formula:
H-D-Phe-His-heu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Arg-Leu-Nle-Glu-Ile-Ile-NH2.

16. The CRF antagonist of claim 1 having the formula:
H-D-2NAL-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Nle-Glu-Ile-Ile-NH2.

17. A composition for lowering stress in mammals comprising an effective amount of a synthetic CRF
antagonist on a nontoxic addition salt thereof in accordance with claim 5 and a pharmaceutically or veterinarily acceptable liquid or solid carrier therefor.

18. A corticotropin releasing factor (CRF) antagonist which includes at least one D-isomer and has the formula:
H-R9-R10-R11-R12-His-Leu-Leu-Arg-Glu-Val-Leu-R20-Nle-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-R36-R37-Nle-Glu-ILe-R42-NH2 wherein R9 is Asp or desR9, R10 is Leu or desR10, 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; R37 is leu; and R41 is Ala or Ile; wherein at least one of R12 and R20 is a 17-isomer or a nontoxic addition salt thereof.

19. The CRF antagonist of claim 18 wherein R9 is desR9, R10 is desR10, R11 is desR11 and R12 is D-Phe or D-2NAL.

20. The CRF antagonist of claim 18 wherein R20 is D-Glu.

21. The CRF antagonist of any one of claims 18, 19 or 20 wherein R12 is D-2NAL.

22. The CRF antagonist of any one of claims 18, 19 or 20 wherein R12 is D-Phe.

23. The CRF antagonist of any one of claims 18 to 22 wherein R36 is Arg.

24. The CRF antagonist of any one of claims 18 to 23 wherein R41 is Ile.

25. The CRF antagonist of any one of claims 18 to 23 wherein R41 is Ala.