The Salk Institute for Biological Studies

John S Porter

Jean E Rivier

Carl A Hoeger

Paula G Theobald

Catherine L Rivier

Wylie W Vale, Jr

Wayne D Kornreich

Harry A Anderson

Jean E F Rivier

Wylie Walker Vale, Jr

Guangcheng Jiang

Steven C Koerber

Peptides which include unnatural amino acids and which either promote or inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount of such peptides that are GnRH antagonists prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads and may be used to treat steroid-dependent tumors. The agonists can be used for control of reproduction processes, to treat precocious puberty, endometriosis, and the like. The peptides are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-, 5-, 6- and/or 8-positions. Unnatural amino acids having the following formula are incorporated in a preferred group of synthesized peptides:

Peptides intermediates for making GnRH antagonists

Compounds termed 'betides' mimic peptides and contain one or more residues of aminoglycine, C.sup..alpha. -aminoalanine, aminosarcosine or the like wherein the side chain amino group has been acylated and optionally also alkylated. Generally, betides have the formula: X.sub.N -X.sub.1 -X.sub.2 -X.sub.3 -X.sub.m -X.sub.4 -X.sub.5 -X.sub.6 -X.sub.C, where X.sub.N is an acyl or other N-terminal group or a peptide up to about 50 amino acids in length having such a group; X.sub.C is OH, NH.sub.2 or other C-terminal group or a peptide up to about 50 amino acids in length having such a group; and X.sub.1 -X.sub.6 are each independently a betidamino acid or .alpha.-amino acid or des-X; and X.sub.m is a peptide up to about 50 amino acids or des-X; provided however that at least one of X.sub.1 -X.sub.6 is a betidamino acid residue having the formula: ##STR1## wherein R.sub.0 is H or CH.sub.3, R and R.sub.2 are H or lower alkyl, and R.sub.3 is an acyl group, an isocyanate or isothiocyanate group, a sulfonyl group or the like. To make a betide, an aminoglycine residue is subjected to side chain acylation, and optionally also alkylation, after it is coupled into a peptide intermediate. By synthesizing betides with multiple substituents at one or more positions in an otherwise peptidic chain, efficient screening of betides which mimic peptides having a large number of different natural or unnatural amino acid substituents at a particular position, and optionally both D- and L-isomers thereof, is possible.

Methods of making and screening betide libraries

Peptides which include unnatural amino acids and which either promote or inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount of such peptides that are GnRH antagonists prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads and may be used to treat steroid-dependent tumors. The agonists can be used for control of reproduction processes, to treat precocious puberty, endometriosis, and the like. The peptides are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-, 5-, 6- and/or 8-positions. Unnatural amino acids having the following formula are incorporated in a preferred group of synthesized peptides: ##STR1## Methods for synthesizing such peptides having the triazole side chains are disclosed wherein one side chain modification (or two simultaneously) is carried out on an amino-substituted phenylalanine residue in a peptide chain which is a part of a peptidoresin.

GnRH analogs

Methods of making unnatural amino acids are provided which unnatural amino acids can be incorporated into peptides which either inhibit or promote the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. These unnatural amino acids are useful in the synthesis of peptides and have the formula (a): ##STR1## where W is (CH.sub.2) or ##STR2## n is an integer from 1 to 6; j=1, 2 or 3, and preferably, Y is N--CN, X is NH and R.sub.2 is alkyl, modified alkyl, alkenyl, alkynyl, aryl or methyl pyridyl. Disclosed are peptides that are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-, 5-, 6- and/or 8-positions.

Amino acids and processes for making peptides using same

Compounds are provided termed 'betides' which mimic peptides and which contain one or more residues of aminoglycine, C.sup..alpha. -aminoalanine, aminosarcosine or the like wherein the side chain amino group has been acylated and optionally also alkylated. Generally, betides have the formula: X.sub.N --X.sub.1 --X.sub.2 --X.sub.3 --X.sub.m --X.sub.4 --X.sub.5 --X.sub.6 --X.sub.C, where X.sub.N is an acyl or other N-terminal group or a peptide up to about 50 amino acids in length having such a group; X.sub.C is OH, NH.sub.2 or other C-terminal group or a peptide up to about 50 amino acids in length having such a group; and X.sub.1 -X.sub.6 are each independently a betide amino acid or .alpha.-amino acid or des-X; and X.sub.m is a peptide up to about 50 amino acids or des-X; provided however that at least one of X.sub.1 -X.sub.6 is a betide amino acid residue having the formula: ##STR1## wherein R.sub.o is H or CH.sub.3, R and R.sub.2 are H or lower alkyl, and R.sub.3 is an acyl group, an isocyanate group, an isothiocyanate group or a sulfonyl group. In methods for making betides, an aminoglycine residue can be subjected to side chain acylation, and optionally also alkylation, after it has been incorporated into a peptide intermediate. This method can be used as a valuable tool to synthesize and screen multiple substituents at one or more positions in a peptide, permitting simultaneous screening of betides which mimic peptides having a large number of natural or unnatural amino acid substituents at a particular position, and optionally both D- and L-isomers of those substituents.

Betides and methods for screening peptides using same

Methods of making unnatural amino acids are provided which unnatural amino acids can be incorporated into peptides which either inhibit or promote the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. These unnatural amino acids are useful in the synthesis of peptides and have the formula (a): ##STR1## where W is (CH.sub.2).sub.n or ##STR2## n is an integer from 1 to 6; j=1, 2 or 3, and preferably, Y is N--CN, X is NH and R.sub.2 is alkyl, modified alkyl, alkenyl, alkynyl, aryl or methyl pyridyl. Disclosed are peptides that are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-, 5-, 6- and/or 8-positions.

Preparation of phenylalanine derivatives bearing a 1,2,4-triazole moiety

Analogs of the decapeptide GnRH which include two significantly modified amino acids at positions 5 and 6 inhibit the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount of such GnRH antagonists prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads and may be used to treat steroid-dependent tumors. Particularly effective peptides which are soluble in water at physiologic pH and which have a low tendency to gel when administered in vivo have the following formula:

GNRH antagonists XIII

Peptides which have substantial bioactivity to inhibit the secretion of gonadotropins by the pituitary gland and to inhibit the release of steroids by the gonads. Administration of an effective amount of such GnRH antagonists prevents ovulation and/or the release of steroids by the gonads. They may also be used to treat steroid-dependent tumors, such as prostatic and mammary tumors. The peptides are bicyclic analogs of the decapeptide GnRH having two covalent bonds, between the residues in the 4- and 10-positions and the residues in the 5- and 8-positions. The latter linkage includes peptide bonds between a residue of an .alpha.-amino acid outside of the main chain and a side-chain carboxyl group of the 5-position residue and a side-chain amino group of the 8-position residue.

Bicyclic GnRH antagonists and a method for regulating the secretion of

Unnatural amino acids are provided which can be incorporated into peptides which either inhibit or promote the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. These unnatural amino acids are useful in the synthesis of peptides and have the formula (a): ##STR1## where W is (CH.sub.2).sub.n or ##STR2## n is an integer from 1 to 6; j=1,2 or 3, and preferably, Y is N--CN, X is NH and R.sub.2 is alkyl, modified alkyl, alkenyl, alkynyl, aryl or methyl pyridyl; or the formula (b): ##STR3## where R.sub.11 is H or acyl and W is as defined in (a), and preferably R.sub.11 is H and W is ##STR4## Disclosed are peptides that are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-, 5-, 6- and/or 8-positions.

Amino acids useful in making GnRH analogs

Peptides which include unnatural amino acids and which either inhibit or promote the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount of such peptides that are GnRH antagonists prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads. The antagonists may be used to treat steroid-dependent tumors, such as prostatic and mammary tumors. The peptides are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-, 5-, 6- and/or 8-positions. Such unnatural amino acids are useful in the synthesis of peptides and have the formula U.sup.* : ##STR1## where W is (CH.sub.2).sub.n or ##STR2## n is an integer from 1 to 6; and j=1, 2 or 3. Preferably, either Y is N--CN, X is NH and R.sub.2 is alkyl, modified alkyl, alkenyl, alkynyl, aryl or methyl pridyl or ##STR3## where R.sub.11 is H or acyl.

Peptides which include unnatural amino acides and which either inhibit or promote the secretion of gonadotropins by the pituitary gland and inhibit the release of steroids by the gonads. Administration of an effective amount of such peptides that are GnRH antagonists prevents ovulation of female mammalian eggs and/or the release of steroids by the gonads. The antagonists may be used to treat steroid-dependent tumors, such as prostatic and mammary tumors. The peptides are analogs of the decapeptide GnRH wherein there is at least one residue of an unnatural amino acid in the 3-position, the 5-position, the 6-position and/or the 8-position. Such unnatural amino acids are useful in the synthesis of peptides and have the formula U*: ##STR1## where n is an integer from 1 to 6; Y is N--CN, N--CONHR.sub.9, S, O or CH--NO.sub.2 ; R.sub.9 is H, Ac, lower alkyl, aromatic or heterocyclic; X is NH, O, S, M.sub.1 (CH.sub.q).sub.p M.sub.2 or M.sub.1 --(CH.sub.2).sub.p' --M.sub.2 (CH.sub.2).sub.p' --M.sub.3, where M.sub.1 is NR.sub.10, O, S or CHR.sub.3 wherein R.sub.3 is methyl, ethyl, propyl, phenyl, pyridinyl, pyrimidinyl or purinyl, q is 1 or 2; p, p' and p' are integers between O and 6; R.sub.10 is H, lower alkyl or the like, and M.sub.2 and M.sub.3 are M.sub.1, COOH, CONH.sub.2, COOR.sub.3 or CN; R.sub.1 is H, alkyl, modified alkyl, alkenyl, alkynyl, aryl or a direct bond to X; R.sub.2 is R.sub.1, OH, NH.sub.2, NHR.sub.1, or heterocycle.

Methods for making peptides of a suitable length for solid phase synthesis, which peptides include in their sequence a pair of residues of a different character which have acylated side chains and a residue which has an N-alkylated side chain. A peptide intermediate is constructed on the resin using commercially available starting materials. The N-terminus can be acylated by removing the .alpha.-amino protecting group and acylating under standard conditions. First primary amino protecting groups included in those residues to be acylated are removed, and acylation is effected, preferably by using a carboxypyridine or a similar heterocyclic acylating agent. Following such side chain acylation, a second protecting group included in the residue to be N-alkylated is removed, and the N-alkylation reaction is carried out while the peptide remains on the resin using a borohydride and an appropriate aldehyde or ketone. Following cleavage from the resin and removal of any protecting groups still remaining, the peptide is appropriately purified, thus requiring only a single purification to be carried out while forming a synthetic peptide including residues for which the modified amino acids are not readily commercially available.

Method of making peptides

Peptide N-alkylamides, and other C-terminal N-substituted amides, can be synthesized using solid-phase synthesis on a benzene-containing resin which is suitably methylated. Reactive amino groups are attached directly or indirectly to the methyl groups, for example, such as by reacting commercially available chloromethylated polystyrene resins with an alkylamine to create a resin-amine. The C-terminal amino acid of the desired peptide is linked to the resin-amine via an amide linkage, and the peptide is thereafter built in normal fashion. Treatment of the completed peptide intermediate with HF is effective to both effect deprotection and cleave the peptide from the resin in the form of the N-substituted amide. Examples include peptide N-ethylamides, N-fluoroethylamide, N-anilides and other substituted N-benzylamides.

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Leucadia, CA, United States of America

John S Porter