# US 5,710,249 A · Amino acids and processes for making peptides using same

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- Number: US 5,710,249 A
- Document ID: US5710249A
- Status: Granted
- Kind: A
- Application number: 08/465,854
- Filed: 1995-06-06
- Granted: 1998-01-20
- Earliest claimed: 1989-10-30
- Assignees: The Salk Institute for Biological Studies
- Inventors: Carl A. Hoeger; Jean E. F. Rivier; John S. Porter
- Web page: https://patentgrep.com/patent/US5710249A

## CPC

- C07C 279/24: Y being a hetero atom
- C07C 277/08: of substituted guanidines
- C07C 279/28: having nitrogen atoms of guanidine groups bound to cyano groups, e.g. cyanoguanidines, dicyandiamides
- C07C 313/30: Y being a hetero atom
- C07D 249/14: Nitrogen atoms
- C07K 1/006: {of peptides containing derivatised side chain amino acids}
- C07K 7/02: Linear peptides containing at least one abnormal peptide link
- C07K 7/23: Luteinising hormone-releasing hormone [LHRH]; Related peptides
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07C 2601/14: The ring being saturated
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

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.

## Claims

### Claim 1

An amino acid having the formula: ##STR51## wherein J is 1, 2 or 3, Y is N--CN, CH--NO.sub.2 or N--CONH.sub.2, and R.sub.2 is alkyl (C.sub.1 to C.sub.6), modified alkyl (C.sub.1 to C.sub.5 with terminal substitution by NH.sub.2, OH, Cl, Br, F or F.sub.3), alkenyl (C.sub.2 to C.sub.4), alkynyl (C.sub.2 to C.sub.4), benzyl, tolyl, p-amino-benzyl, p-chloro-benzyl, or methylpyridyl.

### Claim 2

An amino acid having the formula: ##STR52## wherein j is 1, 2 or 3, Y is N--CN, CH--NO.sub.2 or N--CONH.sub.2, and R.sub.2 is alkyl (C.sub.1 to C.sub.6), modified alkyl (C.sub.1 to C.sub.5 with terminal substitution by NH.sub.2, OH, Cl, Br, F or F.sub.3), alkenyl (C.sub.2 to C.sub.4), alkynyl (C.sub.2 to C.sub.4), benzyl, tolyl, p-amino-benzyl, p-chloro-benzyl, or methylpyridyl.

### Claim 3 (depends on 1, 2)

An amino acid according to claim 1 or 2 wherein j is 1.

### Claim 4 (depends on 1, 2)

An amino acid according to claim 1 or 2 wherein Y is N--CN.

### Claim 5 (depends on 5)

An amino acid according to claim 5 wherein X is NH and R.sub.2 is methylpyridyl.

### Claim 6 (depends on 5)

An amino acid according to claim 5 wherein X is NH and R.sub.2 is butyl.

### Claim 7

A method of synthesizing a peptide by chain elongation from the C-terminus thereof, which method comprises
subjecting an .alpha.-amino acid having the formula: ##STR53## to nitration conditions by forming a reaction mixture which includes said amino acid, concentrated sulfuric acid and concentrated nitric acid having at least about 2 moles of HNO.sub.3 for each mole of .alpha.-amino acid and maintaining said reaction mixture at a temperature of about 5.degree. C. or below to cause said nitration to occur predominantly at the 4-position,
reacting said 4NO.sub.2 -substituted product with an appropriate reagent to add an amino-protecting group to said .alpha.-amino acid,
treating said protected amino acid to hydrogenate said substituted nitro moiety and transform it to an amino moiety,
dissolving said hydrogenated .alpha.-amino acid in a suitable solvent and reacting it with diphenylcyanocarbonimidate to form a cyanoguanidino intermediate,
thereafter reacting said cyanoguanidino intermediate with hydrazine to create a 3-amino, 1,2,4 triazole moiety attached to the 4-position of the phenyl ring of the side chain of said .alpha.-amino acid,
forming a peptide intermediate with a protected .alpha.-amino group at its N-terminus, and
deprotecting the .alpha.-amino group of said peptide intermediate and reacting same to form an amide bond with said .alpha.-amino acid having said triazole side chain.

### Claim 8 (depends on 1)

A method of synthesizing a peptide by chain elongation from the C-terminus thereof, which method comprises
forming a peptide intermediate having a protected .alpha.-amino group at its N-terminus,
deprotecting the .alpha.-amino group of said peptide intermediate and
reacting said deprotected peptide intermediate to form an amide bond with an .alpha.-amino acid according to claim 1, having the formula: ##STR54## wherein the .alpha.-amino group is protected.

### Claim 9

A method of synthesizing a peptide by chain elongation from the C-terminus thereof, which method comprises
forming a peptide intermediate which includes the amino acid sequence Leu-Lys(Ipr)-Pro-D-Ala,
causing a first reaction between the .alpha.-amino group of Leu at the N-terminus of said peptide intermediate with the .alpha.-carboxyl group of Boc-D-Aph(3-amino, 1,2,4 triazole) to elongate said peptide intermediate,
removing said Boc protecting group from said elongated peptide intermediate,
carrying out a second reaction with the e-carboxyl group of Boc-L-Aph(3-amino, 1,2,4 triazole) to further elongate said elongated peptide intermediate,
removing said Boc protecting group and sequentially adding Boc-Ser, Boc-D-3PAL, Boc(4Cl)D-Phe and Boc-.beta.-D-2Nal to create a decapeptide intermediate,
removing the Boc protecting group from the N-terminus of said decapeptide intermediate and acetylating said deprotected N-terminus, and
removing any remaining protecting groups to provide the decapeptide Ac-.beta.-D-2NAL-(4Cl)D-Phe-D-3PAL-Ser-Aph(3-amino, 1,2,4 triazole)-D-Aph(3-amino, 1,2,4 triazole)-Leu-Lys(isopropyl)-Pro-D-Ala-NH.sub.2.

### Claim 10 (depends on 9)

A method according to claim 9 wherein Boc-D-4Aph(3-amino, 1,2,4 triazole) is used in said first reaction and Boc-L-4Aph(3-amino, 1,2,4 triazole) is used in said second reaction.