P-glu&#39; his&#39; n&#39; ala&#39; nh&#39; 2 &#39;and intermediates
United States Patent [191 McGregor Apr. 8, 1975 I p-Glu-His-N-Ala-NH AND INTERMEDIATES [75] Inventor: William H. McGregor, Malvern. Pa.  
  [73] Assignee: American Home Products Corporation, New York. NY. 221 Filed: Oct. 29, 1973 [21] Appl. No.2 410,585  
 [52] US. Cl 260/112.5; 424/177 [51] Int. Cl. C07c 103/52; A6lk 27/00 [58] Field of Search 260/1125 [56] References Cited OTHER PUBLICATIONS Bowers et 111.: Biochem. Biophys. Res. C0mm.. 40. 683-69I (1970).  
 Primary E.ranzinerLewis Gotts Assistant E.\&#39;uminerReginald J. Suyat described. The tripeptide is active in releasing thyrotropin.  
 1 Claim, No Drawings l P-GLUl-llSNALA-NH AND INTERMEDIATES The thyrotropin releasing hormone (TRH) is one of the neurohormones of the hypothalamus gland of mammalian species. Within the five years the structure of TRH has been reported in the literature as the tripeptide LpGluLHis--L-ProNH [See Burgus et al. Ann. Rev. Biochem. 39. pp 504-511 (l97()) U.S. Pat. No. 3.753.969. Guillemin et .11.. \&#39;its. and Hormones. 29. pp l39 (Academic Press 1971 )1. TRH has utility in the diagnostic evaluation of the functioning of the anterior pituitary gland as discussed in column 2. lines 1-12 of U.S. Pat. No. 3.753.969 and is in clinical use as described by Guillemin et al.. supra. at pages 34-35.  
 The present invention relates to a structural .modification of TRH by replacing proline with N-methyl alanine.  
  The novel tripeptides of the present invention are represented by the formula and non-toxic acid addition salts thereof.  
  The present invention also includes within its scope tripeptides of the formula p-GluHis(N&#34;&#34;R)-l l(CH;;)AlaNH and non-toxic acid addition salts thereof. In formula ll. N means the nitrogen atoms of the imidazole ring of histidine and R is a protecting group for the imidazole nitrogens. The selection of such protecting group is not critical except such group should fulfill the following requirements (a) be one that is easily cleaved without alteration or destruction of the tripeptide chain. and (b) not interfere with or otherwise give rise to side reactions during the synthesis of the tripeptide. Illustrative of such protecting groups are tosyl. benzyl. trityl. 2.2.2-trifiuoro-1-benzyloxycarbonylaminoethyl and 2,- 2.2-trifluoro-1-tert-butyloxycarbonylaminoethyl.  
  All chiral amino acid residues identified in formulas l and 11 supra, are of the natural or L-configuration unless specified otherwise.  
  lllustrative of&#39;non-toxic, pharmaceutically aceptable. acid addition salts, contemplated by formulas l and 11. are hydrochloride. hydrobromide. sulfate, phosphate, maleate, acetate. citrate. benzoate, succinate. malate. ascorbate and the like.  
 The tripeptide of the present invention may be synthesized by either the classical route or solid phase method, preferably the classical method is used. The classical method involves converting N-methyl alanine (available from Bachem lnc., Marino. Del. Rey. Calif.) to the corresponding amide via the ammonolysis route in methanol to obtain N(CH;,)AlaNH This compound is then reacted with an azide of the formula p-- Glu-HisN,-; or pGluHis(N&#34;&#34;R)N at a temperature between 40C and about +50C in an aqueous or nonaqueous solvent (e.g. dimethylformamide. tetrahydrofuran. dioxane, chloroform, methylene chloride, toluene, etc.). The azide may be formed in situ without isolation from the reaction medium by reacting a hydrazide of the formula pGluHis-NH or p- Glul-lis(N&#34;-R) NH with a reagent which yields nitrous acid in situ at a temperature between about -10C and +20C to form the corresponding azide and thereafter adding N(CH;;)AlaNH. to the reaction medium to form the tripeptide pGlu-His-N(CH- -.)AlaNH- or pGlu-His(N&#34;&#34;R)-N(CH;.) Ala- NH- Suitable reagents which furnish nitrous acid in situ are lower alkyl nitrites (e.g. tert-butyl nitrite. isoamyl nitrite) or an alkali metal nitrite salt (e.g. sodium nitrite. potassium nitrite) in the presence of a strong acid such as hydrochloric. phosphoric. sulfuric, etc. The reaction forming the tripeptide is preferably carried out in the presence of an acid acceptor. preferably a tertiary amine (e.g. triethylamine. pyridine. quinoline. dimethylaniline. etc.). an alkali metal carbonate or other acid binding agent known in the art. If a protecting group is used on histidine. this being optional. it may be removed by employing a reagent such as Hg/Pd or other reagent described by Schroeder and Lubke. The Peptides. 1. pp 72-74 (Academic Press 1965). to yield a compound of formula 1.  
  The following examples are illustrative of the preparation of the compounds of the present invention.  
 EXAMPLE 1 Anal. Calcd. for (.H DN C. 47.03. H 9.87. l\&#39; 27.43 Found C. 46.53. H 10.53. N 25.76  
 EXAMPLE 2 L-(5-Oxoprolyl)-L-histidine methyl ester L-5-Oxoproline (13 gr, 0.1 mole) and L-histidine methyl ester dihydrochloride (22.8 gr, 0.1 mole) are suspended in dimethylformamide (200 ml). The mix ture is treated with triethylamine (27 ml, 0.2 moles and cooled at -5C., then dicyclohexylcarbodiimide (20.6 gr. 0.1 mole) is added and the mixture is stirred for 2 hours at -5C and overnight at room temperature. The dicyclohexylurea which separates is filtered off and the filtrate is evaporated to dryness. The residue is triturated with water and the insoluble solid is filtered off. The filtrate is evaporated to dryness and the residue dried twice with abs. ethanol. The oily residue crystallizes from abs. EtOl-l to afford the above titled dipeptide methyl ester 13 g (45%), mp. 198l99C; [b11 4.3 (cl, Methanol); R,(n-Butanol-water-acetic acid 4:1:1) 0.30; R,(n-Butanol-water-acetic acid-pyridine 30:24:6220) 0.50; single spot with Pauly and 1 reagents.  
  Anal. Cald. for C H N O (280.28): C, 51.42; H, 5.75; N, 19.99; Found: C, 51.34; H, 6.04; N, 20.12.  
 EXAMPLE 3 L-( S-Oxoprolyl )-L-histidyl hydrazide L-( S-Oxoprolyl)-L-histidine methyl ester of Example 2 gr. 0.036 moles) is dissolved in methanol (150 ml) and treated with hydrazine hydrate 99% (8 ml) at 10C for one hour then at room temperature overnight. The white solid which separates is filtered and washed with methanol and then with ether. Recrystal1i zation from water-ethanol affords the above-titleddipeptide hydrazide 8.8 gr (8892 Recrystallization from water-ethanol [78%] mp. 24l.5242C: [11],} l4.50 (c 1. H O): R,(n-Butanol-water-acetic acid 4:121) 0.05; RAn-Butanol-water-acetic acid-pyridine 30:24:6120) 0.10; singles-pot with Pauly and lrea gents.  
 EXAMPLE 4 L-(S-oxoprolyl)-L-histidyl-L-N-methyl-alanine amide acetate 1 4 L-(S-oxoprolyl)-L-histidine hydrazide 1.07 g. 3.8 m mole) is dissolved in 25 ml dimethylformamide and 5 ml 2.41 N-hydrochloric acid in tetrahydrofuran added and cooled to Z5C. lsoamyl nitrite (0.81 ml) is added. stirred min. at -C followed by 3.3 ml of triethylamine and 0.4 g (4 mmole) of a cold solution of L-N-methyl-alanine amide. The reaction is allowed to proceed for two days at 4C. The solvent is evaporated under reduced pressure C) and the resulting oil chromatographed on silica gel using methanol as solvent. Crude product (asjudged by TLC) 350 mgs is obtained which is further purified by partition chromatography on Sephadex G-25 F in the system n-butanolacetic acid-water (4:115). One ml. fractions are collected and the fraction in tubes296-326 is pooled and lyophylized to yield mg of the above titled product.  
 N-Mc-Ala 1.13 CHCl-t/MeOH His NH 1.01 1.13 0.67  
 Amino acid analysis:  
  Compound of Thyrotropin Exp. Example 4 TRH Secreted No. tag/ml) lug/ml) (ng/ml) l x44 Z 50 5(1 1136* 3 50 813fi-W 4 25:18 5 1 l76:75+ (w l 316x91 7 0.1 91:81 h 0.1 287:1 28+ 9 0.05 25813 1 l0 0.01 2l8i86+ As shown in the foregoing table. at 50 ug/ml. 1.0 ug/ml and 0.1 Lg/ml. the compound of Example 4 induced the secretion of more thyrotropin than did TRH. Thus. the tripeptide of the present invention is useful as a diagnostic agent in evaluating the functioning of the anterior pituitary gland in mammals including hurans. The compound may be administered to mammals by intravenous or subcutaneous injection or orally. A practical dose range for intravenous injection in a isotonic saline solution is believed to be between about 200 pg and &#39;1 mg of the compound. The effective dose for oral will be somewhat higher than for intravenous injection. Oral administration can be in the form of a tablet. capsule. etc. The dosage forms may additionally include excipients. binders. fillers and other therapeutically insert ingredients. A typical tablet may contain starch. calloidal silica and magnesium stearate.  
 What is claimed is l. A compound selected from L-(5-oxoprolyl)-L- histidyl-L N-methyl-alanine amide and its non-toxic