Nonapeptides and methods for their production

New nonapeptides having the formula R-Gln-Trp-Ser-Tyr-Trp-Pro-Pro-Tyr(benzyl)-Ser(benzyl)-Y wherein R is a protective group and Y is amino, lower alkylamino or di(lower alkyl)amino.

SUMMARY AND DETAILED DESCRIPTION 
The present invention relates to new peptide compounds that are useful as 
luteinizing hormone releasing factor antagonists and to methods for their 
production. More particularly, the invention relates to new nonapeptides 
that are represented by the formula 
EQU R-Gin-Trp-Ser-Tyr-Trp-pro-pro-Tyr(benzyl)-Ser(benzyl)-Y I 
wherein R is a protective group and Y is amino, lower alkylamino or 
di(lower alkyl)amino. 
The preferred compounds of formula I are those wherein R is 
benzyloxycarbonyl, and Y is amino. 
In formula I, the conventional symbols for amino acid residues of peptide 
compounds linked thereto are used and each is intended to have the 
following meaning: Pro, D-prolyl or L-prolyl; Trp, D-tryptophyl or 
L-tryptophyl; Ser; D-seryl or L-seryl; Tyr, D-tyrosyl or L-tyrosyl; Gln, 
D-glutaminyl or L-glutaminyl; Tyr(benzyl), D-tyrosyl(benzyl) or 
L-tyrosyl(benzyl); and Ser(benzyl), D-seryl(benzyl) or L-seryl(benzyl). In 
addition, the term "lower alkyl" is intended to mean a straight, branched 
or cyclic hydrocarbon moiety of up to six carbon atoms, such as methyl, 
ethyl, isopropyl and cyclopropyl. A protective group is intended to mean a 
group usually employed in the area of peptides for protecting an amino 
function, such groups are disclosed in the following texts which are 
incorporated by reference: E. Schroder and K. Lubke, "The Peptides, " Vol. 
I, Chapter 1., Academic Press, 1966 and J. Meienhofer in "Hormonal 
Proteins and Peptides", Vol. II, p. 227., Academic Press, 1973. These 
symbols and terms will also be used in the formulae that follow for other 
compounds and each such symbol or term should be understood to have the 
meaning given above. 
In accordance with this invention, compounds of the formula I, wherein R 
and Y are as previously defined, are produced by reacting a compound of 
the formula 
EQU R-Gln-Trp-Ser-Tyr-Trp-Pro-Pro-Tyr(benzyl)-Ser(benzyl)-lower alkoxy II 
wherein R is as previously defined and "lower alkoxy" is intended to mean 
an alkoxy group having a straight, branched or cyclic hydrocarbon moiety 
of up to 6 carbon atoms, such as methoxy, ethoxy and isopropoxy 
(preferably methoxy), with ammonia, lower alkylamine or di(lower 
alkyl)amine. 
The reactions are conducted at temperatures of from about 5.degree. to 
100.degree. C. for from three hours to four days, preferably about room 
temperature. Generally, a large excess of amine is used (over five fold). 
The reaction is usually carried out in a non-reactive solvent, such as a 
lower alkyl alcohol, preferably methanol or ethanol, an ether such as 
tetrahydrofuran or dioxane, dimethylformamide or mixtures thereof. 
The starting materials of formula II are produced by reacting an azide, 
represented by the formula 
EQU R-Gln-Trp-Ser-Tyr-N.sub.3 III 
with a compound of the formula 
EQU Trp-Pro-Pro-Tyr(benzyl)-Ser(benzyl)-lower alkoxy IV 
in a non-reactive solvent medium, preferably dimethylformamide or a 
dimethylformamide-tetrahydrofuran mixture wherein R is as previously 
defined for formula I. 
The azide of the formula III is prepared and used in situ. 
The two components, III and IV, are generally reacted in approximately 
equimolar amounts at temperatures of from about -30.degree. to about 
30.degree. C. for from 16 to 50 hours, although temperatures of from 
30.degree. to 50.degree. C. may be used with a shortened reaction period. 
The peptide azide compounds of the formula III that are used as a reactant 
in the foregoing process are normally prepared in situ by reacting a 
peptide hydrazide compound represented by the formula 
EQU R-Gln-Trp-Ser-Tyr-NHNH.sub.2 V 
wherein R is as previously described in formula I, with a lower alkyl 
nitrite, preferably isoamyl nitrite in the presence of an acid in an inert 
solvent medium such as dimethylformamide, and the resultant azide is 
reacted further as described above without isolation. The preferred acid 
for use in the azide preparation is a solution of hydrogen chloride in 
dimethylformamide or tetrahydrofuran; between 3 and 6 equivalents of acid 
are used for each equivalent of the hydrazide of formula V. The 
preparation of the azide is carried out at a temperature between 
-60.degree. and 10.degree. C. Following the in situ formation of the azide 
of formula III and prior to the further reaction of the peptide azide with 
the compound of formula IV to form the nonapeptide product I, a tertiary 
amine such as triethylamine is added to the reaction mixture to neutralize 
the acid used. 
The peptide hydrazide compounds of formula V above are prepared by various 
methods. The hydrazide of the formula V, wherein R is as previously 
described in formula I, may be prepared by reacting an ester of the 
formula 
EQU R-Gln-Trp-Ser-Tyr-OCH.sub.3 VI 
wherein R is as previously defined, with excess hydrazine (1:1.1 to 100) 
preferably in the form of its hydrate, in an organic solvent, such as 
dimethylformamide, methanol, ethanol, etc. The reaction is generally 
carried out at room temperature, although temperatures of from 5.degree. 
to 100.degree. C. may be employed for periods of from about 30 minutes to 
about 200 hours, preferably about 72 hours. 
The esters of formula VI are prepared by reacting a compound of the formula 
EQU R-Gln-Trp-NHNH.sub.2 VII 
wherein R is as previously defined, with a compound having the formula 
EQU Ser-Tyr-OCH.sub.3 .multidot.HCl VIII 
in the presence of a lower alkyl nitrite and an acid in the same manner 
that compounds of the formula II are prepared. 
The compounds of the formula VIII is prepared by catalytically reducing the 
known compound of the formula 
EQU benzyloxycarbonyl-Ser-Tyr-OCH.sub.3 IX 
using hydrogen and palladium-on-carbon in an inert solvent. 
The hydrazides of the formula VII are prepared from esters of the formula 
EQU R-Gln-Trp-OCH.sub.3 X 
wherein R is as previously defined according to the procedure for preparing 
the hydrazides of the formula V. 
The compounds of the formula X are prepared by reacting protected glutamine 
with tryptophan methyl ester hydrochloride in the presence of 
diphenylphosphorazidate in a solvent, such as dimethylformamide at 
temperatures of from 5.degree. C. to 25.degree. C. 
The esters of the formula IV are prepared by treating compounds of the 
formula 
EQU t-butoxycarbonyl-Trp-Pro-Pro-Tyr(benzyl)-Ser(benzyl)-O-lower alkyl 
XI 
with a large excess of trifluoroacetic acid in an inert solvent at about 
room temperature. 
The compounds of the formula XI, are produced by removing a compound of the 
formula 
EQU t-butoxycarbonyl-Trp-Pro-Pro-Tyr(benzyl)-Ser(benzyl)-resin XII 
wherein said resin is a resin employed in solid phase peptide syntheses, 
such as those disclosed in a text by Stewart and Young, "Solid Phase 
Peptide Synthesis, " W. H. Freeman & Company, San Francisco, 1969, which 
is incorporated by reference; preferably the resin is a crosslinked 
copolymer comprising 98 to 99 percent polystyrene crosslinked with 1 to 2 
percent divinylbenzene, which is attached to the protected pentapeptide 
through a methyleneoxy bridge wherein the methylene group is attached to 
the polymeric portion of the resin and the oxygen atom is attached to the 
protected pentapeptide; by treating said resin of the formula XII with a 
lower alkyl alcohol in the presence of tertiary amine, such as 
triethylamine or tripropylamine. 
The resin complex is suspended in an excess of the lower alkyl alcohol, 
preferably methanol for periods of from about 10 hours to 4 days, 
preferably 16 to 24 hours, at about 15.degree. to about 35.degree. C. 
While a large excess of the lower alkyl alcohol is preferred, only a 
catalytic amount of tertiary amine is required; however, larger amounts 
are preferred, such as about 10 percent volume/volume based on the amount 
of lower alkyl alcohol employed. 
The complex resins of the formula XII are prepared by coupling a protected 
amino acid of the formula 
EQU t-butoxycarbonyl-Trp-OH XIII 
with complex resins of the formula 
EQU Pro-Pro-Tyr(benzyl)-Ser(benzyl)-resin XIV 
in an organic solvent, such as dichloromethane with the aid of 
dicyclohexylcarbodiimide. The three reactants may be used in about 
equimolar quantities, but excess amounts of the protected amino acid and 
dicyclohexylcarbodiimide are sometimes advantageous. The reaction is 
generally conducted at about room temperature for a period of from about 
fifteen minutes to about 20 hours. 
The complex resins of the formula XIV are prepared by treating complex 
resins of the formula 
EQU t-butoxycarbonyl-Pro-Pro-Tyr(benzyl)-Ser(benzyl)-resin XV 
with a large excess of trifluoroacetic acid utilizing dichloromethane as 
the solvent at temperatures of from 20.degree. to 30.degree. C. for about 
10 minutes followed by neutralization of the trifluoroacetic acid salt 
with a base such as triethylamine. 
The complex resins of the formula XIV are prepared by coupling 
EQU t-butoxycarbonyl-Pro-OH 
to complex resins of the formula 
EQU Pro-Tyr(benzyl)-Ser(benzyl)-resin XVI 
using the reaction procedure described for the preparation compounds of the 
formula XII. 
The complex resins of the formula XVI are prepared by treating complex 
resins of the formula 
EQU t-butoxycarbonyl-Pro-Tyr(benzyl)-Ser(benzyl)-resin XVII 
with trifluoroacetic acid using the reaction procedure for the preparation 
of compounds of the formula XIV. 
The complex resins of the formula XVII are prepared by coupling 
EQU t-butoxycarbonyl-Pro-OH 
to comlex resins of the formula 
EQU Tyr(benzyl)-Ser(benzyl)-resin XVIII 
using the reaction procedure described for the preparation of compounds of 
the formula XII. 
The complex resins of the formula XVIII are prepared by treating the 
complex resins of the formula 
EQU t-butoxycarbonyl-Tyr(benzyl)-Ser(benzyl)-resin XIX 
with trifluoroacetic acid using the reaction procedure for the preparation 
of compounds of the formula XIV. 
The complex resins of the formula XIX are prepared by coupling 
EQU t-butoxycarbonyl-Tyr(benzyl)-OH 
to complex resins of the formula 
EQU Ser(benzyl)-resin XX 
according to the procedure used for the preparation of compounds of formula 
XII. 
The complex resins of the formula XX are prepared by treating the complex 
resins of the formula 
EQU t-butoxycarbonyl-Ser(benzyl)-resin XXI 
with trifluoroacetic acid using the reaction procedure for the preparation 
of compounds of the formula XIV. 
The compounds of this invention can exist in anhydrous forms as well as in 
solvated, including hydrated, forms. In general, the hydrated forms and 
the solvated forms with pharmaceutically-acceptable solvents are 
equivalent to the anhydrous or unsolvated form for the purposes of the 
invention. 
Nonapeptides of this invention were screened for LRF antagonist activity in 
vitro using rat anterior pituitary cell cultures as described by Vale et. 
al. [Endocrinology, 91, 562 (1972)]. The inhibition of LRF (luteinizing 
hormone releasing factor) induced luteinizing hormone (LH) release into 
the culture medium is the endpoint in this in vitro bioassay. 
______________________________________ 
ACTIVITY TABLE FOR IN VITRO TEST IN RAT 
ANTERIOR PITUITARY CELL CULTURES 
% LH 
Molar LH Value Release 
Compound Conc. ng./ml. Inhibition 
______________________________________ 
N.sup..alpha. -Benzyloxycarbonyl-L- 
glutaminyl-L-tryptophyl-L- 
seryl-L-tyrosyl-L-tryptophyl- 
L-prolyl-L-prolyl-O-benzyl- 
L-tyrosyl-O-benzyl-L-serin- 
1.times.10.sup.-7 
52.33 29 
amide 1.times.10.sup.-8 
47.20 37 
LRF Control 5.times.10.sup.-10 
69.51 
Saline Control 9.76 
______________________________________ 
The luteinizing hormone releasing factor is known to be formed in the 
hypothalamus of mammals, from which it is released and transported by way 
of the hypothalamic hypophyseal portal system to the anterior pituitary, 
where it stimulates the secretion of luteinizing hormone. The secretion of 
luteinizing hormone from the anterior pituitary in turn is known to effect 
ovulation in experimental animals. Thus, LRF can be used to induce 
ovulation in animals. For a report of the structure of LRF, which has also 
been referred to as luteinizing hormone releasing hormone, or LH-RH, and 
its biological activity, see Science, Vol. 174, No. 4008, October 29, 
1971, pages 511-512. Thus, the nonapeptides of this invention are useful 
in controlling ovulation and in restricting fertility.

The invention is illustrated by the following examples. 
EXAMPLE 1 
N.sup..alpha. 
-Benzyloxycarbonyl-L-glutaminyl-L-tryptophyl-L-seryl-L-tyrosyl hydrazide, 
0.6 g., is dissolved in 10 ml. of dimethylformamide containing 2 ml. of 
2.5N hydrogen chloride in tetrahydrofuran. The solution is cooled to 
0.degree. C. and 0.1 g. of isopentylnitrite is added. The mixture is 
stirred at 0.degree. C. for one hour and cooled to -60.degree. C., 
triethylamine, 0.3 ml., is added followed by 0.6 g. of 
L-tryptophyl-L-prolyl-L-prolyl-O-benzyl-L-tyrosyl-O-benzyl-L-serinamide 
trifluoroacetate in 10 ml. of dimethylformamide. After three days at 
0.degree. C., the product N.sup..alpha. 
-benzyloxycarbonyl-L-glutaminyl-L-tryptophyl-L-seryl-L-tyrosyl-L-tryptophy 
l-L-prolyl-L-prolyl-O-benzyl-L-tyrosyl-O-benzyl-L-serinamide is obtained 
after evaporation and chromatography on silica gel using 
methanol-acetonitrile (1:9); [.alpha.].sub.D.sup.23 -65.5.degree. (c 1.02, 
methanol). 
N.sup..alpha. 
-Benzyloxycarbonyl-L-glutaminyl-L-tryptophyl-L-seryl-L-tyrosyl hydrazide 
is obtained from the corresponding methyl ester, 58.3 g., using 20 ml. of 
hydrazine hydrate with 500 ml. of dimethylformamide and allowing the 
solution to stand at 25.degree. C. for four days. The product, 52.3 g., is 
isolated by filtration and washing with methanol; m.p. 251-253.degree. C. 
N.sup..alpha. 
-Benzyloxycarbonyl-L-glutaminyl-L-tryptophyl-L-seryl-L-tyrosine methyl 
ester is prepared by deprotecting 62.7 g. of N.sup..alpha. 
-benzyloxycarbonyl-L-seryl-L-tyrosine methyl ester [cf. Shioiri and 
Yamada, Chem. Pharm. Bull., 22, 859 (1974)] in a mixture of 550 ml. of 
methanol and 70 ml. of 2.35N hydrogen chloride in methanol in the presence 
of 3 g. of 5% palladium on carbon by stirring under an atmosphere of 
hydrogen for four hours. The catalyst is filtered and the mixture 
evaporated to yield 50.5 g. of L-seryl-L-tyrosine methyl ester 
hydrochloride which is suitable for use without further purification. 
N.sup..alpha. -Benzyloxycarbonyl-L-glutaminyl-L-tryptophyl hydrazide, 52.8 
g., is dissolved in 800 ml. of dimethylformamide containing 150 ml. of 
2.2N hydrogen chloride in tetrahydrofuran. The solution is cooled to 
-10.degree. C. and 17 ml. of isopentylnitrite is added. The mixture is 
stirred at -15.degree. .+-. 5.degree. C. for one hour, cooled further to 
-30.degree. and triethylamine 65 ml., is added followed by 38.5 g. of 
L-seryl-L-tyrosine methyl ester hydrochloride. After refrigeration 
overnight and evaporation, 58.7 g. of N.sup..alpha. 
-benzyloxycarbonyl-L-glutaminyl-L-tryptophyl-L-seryl-L-tyrosine methyl 
ester is obtained after recrystallization from methanol; m.p. 
238.degree.-241.degree. C. 
N.sup..alpha. -Benzyloxycarbonyl-L-glutaminyl-L-tryptophyl hydrazide is 
obtained from the corresponding methyl ester, 74.2 g., using 37.5 ml. of 
hydrazine hydrate with 150 ml. of dimethylformamide and 1.5 l. of methanol 
and allowing the solution to stand at 25.degree. C. for 20 hours. The 
product, 66.6 g., is isolated by filtration and washing with methanol; 
m.p. 246.degree.-248.degree. C. 
To a stirred mixture of N.sup..alpha. -benzyloxycarbonyl-L-glutamine, 56 
g., and L-tryptophan methyl ester hydrochloride, 56 g., in 300 ml. of 
dimethylformamide at 5.degree. C. is added diphenylphosphorazidate, 47.5 
ml., in 100 ml. of dimethylformamide dropwise over ten minutes followed by 
61.6 ml. of triethylamine over one hour. The mixture is stirred in an ice 
bath for three hours and at room temperature for 18 hours. After 
evaporation the residue is dissolved in 1 l. of ethyl acetate and washed 
successively with 500 ml. of 1N hydrogen chloride solution, 500 ml. of a 
saturated solution of sodium bicarbonate and 500 ml. water. The ethyl 
acetate layer is separated, dried, evaporated and 74.2 g. of N.sup..alpha. 
-benzyloxycarbonyl-L-glutaminyl-L-tryptophan methyl ester is obtained by 
recrystallization from methanol; m.p. 197.degree.-198.degree. C. 
N.sup..alpha. 
-t-Butoxycarbonyl-L-tryptophyl-L-prolyl-L-prolyl-O-benzyl-L-tyrosyl-O-benz 
yl-L-serinamide, 0.6 g., is treated with 10 ml. of trifluoroacetic acid. 
The solution is allowed to stand at room temperature for 30 minutes and 
then the excess trifluoroacetic acid is removed under reduced pressure. 
The residue is suspended in diethyl ether, filtered and washed with 
diethyl ether to yield 0.6 g. of 
L-tryptophyl-L-prolyl-L-prolyl-O-benzyl-L-tyrosyl-O-benzyl-L-serinamide 
trifluoroacetate. Finally the product is dried under reduced pressure at 
50.degree. C. and used without further treatment. 
N.sup..alpha. 
-t-Butoxycarbonyl-L-tryptophyl-L-prolyl-L-prolyl-O-benzyl-L-tyrosyl-O-benz 
yl-L-serine methyl ester, 0.5 g., is treated with 25 ml. of methanol 
saturated with ammonia for three days at room temperature. The product, 
N.sup..alpha. 
-t-butoxycarbonyl-L-tryptophyl-L-prolyl-L-prolyl-O-benzyl-L-tyrosyl-O-benz 
yl-L-serinamide, 0.2 g., is obtained as a white glass after evaporation and 
chromatography on silica gel using methanol-ethyl acetate (1:4); 
[.alpha.].sub.D.sup.23 -69.degree. (c 1.05, methanol). 
N.sup..alpha. 
-t-Butoxycarbonyl-L-tryptophyl-L-prolyl-L-prolyl-O-benzyl-L-tyrosyl-O-benz 
yl-L-serine resin, 20 g., is converted to the methyl ester yielding 
N.sup..alpha. 
-t-butoxycarbonyl-L-tryptophyl-L-prolyl-L-prolyl-O-benzyl-L-tyrosyl-O-benz 
yl-L-serine methyl ester in the form of a glass; [.alpha.].sub.D.sup.23 
-68.degree. (c 1.05, methanol). The above resin is prepared according to 
the General Procedure given below using N.sup.60 
-t-butoxycarbonyl-O-benzyl-L-serine resin which is successively reacted 
with 1) 8.9 g., 0.024 mol., of N.sup..alpha. 
-t-butoxycarbonyl-O-benzyl-L-tyrosine and 5 g., 0.0242 mol., of 
dicyclohexylcarbodiimide, 2) 5 g., 0.0232 mol., of N.sup..alpha. 
-t-butoxycarbonyl-L-proline and 5 g., 0.0242 mol., of 
dicyclohexylcarbodiimide, 3) 5 g., 0.0232 mol., of N.sup..alpha. 
-t-butoxycarbonyl-L-proline and 5 g., 0.0242 mol., of 
dicyclohexylcarbodiimide, 4) 7.3 g., 0.024 mol., of N.sup..alpha. 
-t-butoxycarbonyl-L-tryptophan and 5 g., 0.0232 mol., of 
dicyclohexylcarbodiimide. 
N.sup..alpha. -t-Butoxycarbonyl-O-benzyl-L-serine resin is obtained by 
refluxing 220 g., 0.352 mol., of 2% chloromethylated resin, 120 g., 0.41 
mol., of N.sup..alpha. -t-butoxycarbonyl-O-benzyl-L-serine and 35 g., 0.35 
mol., of triethylamine in 2 l. of absolute ethanol for three days. 
Nitrogen analysis shows 0.00084 mol. per gram. 
General Procedure for the Solid Phase Synthesis of Peptide Resins 
The peptide resin is obtained by attaching an .alpha.-amino-protected amino 
acid to a resin (usually a chloromethylated resin which is commercially 
available from Lab Systems, Inc., San Mateo, California). The peptide 
system is then constructed by de-protecting the .alpha.-amino-protected 
amino acid resin and attaching an .alpha.-amino-protected amino acid. 
Repetition of this process produces the peptide resin having the required 
number and sequence of the desired peptide. The terminal .alpha.-amino 
protection is changed by de-protection and attaching the desired 
carboxylic terminal group. The solid phase synthesis procedure is 
described by J. M. Stewart, "Solid Phase Peptide Synthesis, " W. H. 
Freeman and Co., 1969. 
Each cycle of the procedure folows the scheme: 
1. De-protection with excess 50% trifluoroacetic acid in dichloromethane. 
2. Three washes with dichloromethane. 
3. Neutralization of the trifluoroacetic acid salt with an excess of cold 
10% triethylamine in dichloromethane. 
4. Three washes with dichloromethane. 
5. Fifteen to thirty minutes agitation with the .alpha.-amino-protected 
amino acid which is present in up to a four-fold molar excess based on the 
resin nitrogen analysis. However, when a large excess of the 
.alpha.-amino-protected amino acid is used it is agitated with the resin 
for 15 minutes and the excess recovered by draining the solution from the 
reactor. 
6. Addition of dicyclohexylcarbodiimide at least equivalent to the 
.alpha.-amino-protected amino acid in step 5 in dichloromethane followed 
by agitation for four to twenty hours. In the alternate method, a 3.3-fold 
excess of dicyclohexylcarbodiimide is used relative to the 
.alpha.-amino-protected amino acid resin. 
7. Three washes with dichloromethane.