EP0564587A1 - Solution phase process for synthesis of peptide - Google Patents

Solution phase process for synthesis of peptide

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Publication number
EP0564587A1
EP0564587A1 EP92903706A EP92903706A EP0564587A1 EP 0564587 A1 EP0564587 A1 EP 0564587A1 EP 92903706 A EP92903706 A EP 92903706A EP 92903706 A EP92903706 A EP 92903706A EP 0564587 A1 EP0564587 A1 EP 0564587A1
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EP
European Patent Office
Prior art keywords
boc
trp
lys
phe
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92903706A
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German (de)
French (fr)
Other versions
EP0564587A4 (en
Inventor
David Stevenson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Corp
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Filing date
Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Publication of EP0564587A1 publication Critical patent/EP0564587A1/en
Publication of EP0564587A4 publication Critical patent/EP0564587A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C07K1/064—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for omega-amino or -guanidino functions

Definitions

  • This invention relates to a process for preparing the peptide:
  • This peptide has pituitary growth hormone releasing activity.
  • This invention also relates to intermediates used in the process of the invention.
  • the method of preparing this peptide which is exemplified in U.S. 4,411,890 is a solid phase process in which the starting material amino acid is attached to a resin and is then coupled stepwise with the appropriate amino acids.
  • the intermediates in the solid phase process are peptide-resin compounds.
  • the desired peptide product is cleaved from the resin by treatment with hydrogen fluoride.
  • This invention provides an advantageous process for preparing L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2.
  • the process is a solution phase method which provides solid, recrystallizable intermediates which are readily isolated and purified. These solid intermediates are generally crystalline and may be purified by recrystallization. Solid, recrystallizable intermediates, particularly in all steps of a process, are rare in peptide chemistry and offer an advantage in purification.
  • this invention is a process for preparing a compound of the formula:
  • hich comprises : a) coupling L-Lys (BOO -NH2 with Z-D-Phe; b) removing the Z group and coupling the resulting D- Phe-L-Lys(BOC)-NH 2 with Z-L-Trp-NH 2 ; c) removing the Z group and coupling the resulting L- Trp-D-Phe-L-Lys (BOC)-NH 2 with Z-L-Ala; d) removing the Z group and coupling the resulting L- Ala-L-Trp-D-Phe-L-Lys(BOC)-NH 2 with Z-D-Trp; e) removing the Z group and coupling the resulting D- Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH 2 with (BOC) 2 -L-His; and f) removing the BOC groups to give L-His-D-Trp-L
  • the intermediates are preferably recrystallized at each step in the process to prevent the build up of impurities.
  • the purifi ⁇ * " -.ion of the final product is facilitated.
  • the solution phase method of this invention avoids the use of corrosive hydrogen fluoride which is used in the solid phase procedure to cleave the product from the resin.
  • the solution phase process of the invention requires only one acid treatment (at the end of the reaction process to remove the amino protecting groups) , thus minimizing decomposition of the tryptophan residues; whereas, the solid phase method involves repetitive acid treatments.
  • Protected peptide intermediates in the process of this invention are also a feature of this invention.
  • the intermediates are particularly useful in the process of this invention because they are solids, generally crystalline and are readily purified by recrystallization.
  • the 'process of this invention may be represented as follows:
  • L-Trp L-tryptophan
  • L-Ala L-alanine
  • WSC water soluble carbodiimide [1- (3-dimethylamino- propyl) -3-ethylcarbodiimide hydrochloride]
  • intermediates (I) -(VI) are recrystallized before being used in the subsequent steps.
  • Intermediates (II) -(VI) are prepared by removing the benzyloxycarbonyl group (Z) by catalytic hydrogenolysis and then coupling with the appropriate amino acid.
  • N( ⁇ ) -benzyloxycarbonyl-N( ⁇ ) -t- butyloxycarbonyl-L-lysine amide (I) is prepared from N( ⁇ )- benzyloxycarbonyl-N( ⁇ )-t-butyloxycarbonyl-L-lysine by reacting with ammonia, water soluble carbodiimide [i.e. I- 3 (dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride], and 1-hydroxybenzotriazole.
  • the t-butyloxycarbonyl groups are removed using acid, optionally and a carbonium ion scavenger.
  • Suitable acids for removing the t-butyloxycarbonyl group are well known in the art, and include mineral acids or strong organic acids, such as trifluoroacetic acid.
  • Carbonium ion scavengers are also well known in the art, and include electrophilic aryl compounds and mercaptans, such as n-propylmercaptan.
  • the hydrogenolysis to remove Z protecting groups is performed using an appropriate catalyst, for example palladium on carbon, such as 5-10% palladium on carbon. Preferably, 10% palladium on carbon is. used.
  • the pressure at which the hydrogenolysis is performed is not critical, and may be performed at from atmospheric pressure up to several hundred psi. Typically, it is performed at atmospheric pressure up to 100 psi. Increasing the hydrogen pressure enhances the rate of reaction. Performing the reaction at about 100 psi is preferred. Vigorous stirring, such as about 600 rpm, in the autoclave is very advantageous to increase the reaction rate.
  • Coupling reactions are well known in the art and may be accomplished by activating the carboxyl group of the intermediate, such as by formation of an acyl halide, activated ester or activated anhydride, or coupling with a coupling reagent such as a carbodiimide, for example dicyclohexylcarbodiimide or water soluble carbodiimide, PPA (1-propanephosphonic acid cyclic anhydride) , DPPA (diphenylphosphoryl azide) or BOP reagent (benzotriazol-1- yloxy-tris (dimethylamino)-phosphonium hexafluorophosphate) . Water soluble carbodiimide is preferred.
  • the coupling reaction solutions are poured into aqueous base, for example, in the preparation of intermediates (II)-(V), the coupling reaction solutions are poured into aqueous potassium carbonate or potassium bicarbonate solution with vigorous stirring.
  • the coupling reaction solution is, preferably, poured into aqueous potassium bicarbonate solution.
  • the aqueous potassium bicarbonate is stirred vigorously.
  • the rate of addition should be about 500 mL- 1 L/minute.
  • the BOC protecting groups are removed with acid, optionally in the presence of a carbonium ion scavenger.
  • Trifluoroacetic acid is a suitable acid for removing the BOC group.
  • Use of a carbonium ion scavenger is preferred.
  • Typical carbonium ion scavengers are anisole, dimethoxy benzene and mercaptans.
  • n-Propyl mercaptan is suitable.
  • reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (35 mL) and the filtrate and washings were combined.
  • reaction mixture was filtered through a DMF-washed bed of Celite® and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) .
  • the solid which precipitated was collected on a Buchner funnel and the filter cake washed with deionized water (about 1.2 L) until the eluant was neutral to pH paper.
  • the solid was dissolved in boiling isopropanol (240 mL) . To the boiling solution, deionized water (approximately 240 mL) was added to cloud point; the resulting solution was cooled at 0- 5°C overnight.
  • reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (50 mL) and the filtrate and washings were combined.
  • reaction mixture When the WSC had dissolved, the reaction mixture was allowed to stir at ambient temperature for 2.5 hours. At this time, TLC indicated the absence of D-Phe-L-Lys (BOC)-NH 2 .
  • the reaction mixture was filtered through a DMF-washed bed of Celite® and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) .
  • the product separated as a solid which was collected at the pump and washed with deionized water (1.2 L) until the washings were no longer basic.
  • the damp cake was dissolved in boiling methanol (500 mL) ; deionized water (150 mL) was added to cloud point. The solution was allowed to cool at 0- 5°C overnight .
  • TLC indicated the absence of starting material; HPLC showed a trace to be remaining.
  • the reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were -rinsed with DMF (50-mL) and the filtrate and washings were combined.
  • the reaction mixture was filtered through a DMF-washed bed of Celite® and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) .
  • the product separated as a solid, which was collected at the pump and washed with deionized water (1.6 L) until the washings were neutral.
  • the damp filter cake was dissolved in boiling methanol (700 mL) ; deionized water (300 mL) was added to cloud point. The resulting solution was cooled overnight at 0-5°C.
  • TLC indicated the absence of starting material; HPLC showed a trace to be remaining.
  • the reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (50 mL) and the filtrate and washings were combined.
  • reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (50 mL) and the filtrate and washings were combined.
  • reaction mixture was evaporated to a gum which was dissolved in distilled water (300 mL) and re-evaporated twice. Then, the mixture was dissolved in distilled water (1 L) and the pH was adjusted to approximately 3 by the dropwise addition of dilute aqueous ammonium hydroxide solution.
  • This material was purified by reverse phase liquid chromatography using YMC-type AS-5055, end-capped octadecyl silica, spherical in shape, with a particle size of 50 ⁇ and a pore size of 120 A 0 .
  • the bed size was 5 cm i.d. x 50 cm length, corresponding to a bed volume of 981 mL, packed in a stainless steel HPLC column. There was also a guard column of 5 cm i.d. x 10 cm length containing the same packing material (196 mL) .
  • This was equilibrated with 0.1 M aqueous ammonium acetate solution pH 4.5 (4 L) , utilizing a Beckman Prep-350 HPLC system, monitoring the eluant at 254 nm.
  • the above pH adjusted reaction mixture was further diluted with distilled water (300 mL) and then applied to the column at a flow rate of 100 mL/min.
  • the column was washed with 1.0 M ammonium acetate pH 8 (6.0 L), followed by 0.1 M ammonium acetate pH 4.5 (2.5 L) .
  • the product was removed by step-wise elution with solutions of acetonitrile in 0.1 M aqueous ammonium acetate pH 4.5 as follows: 10% v/v (1 L) , 15% v/v (900 mL) , 17% v/v (1 L) , 20% v/v (2.5 L) , 25% v/v (2.5 L) .
  • Fractions were collected, varying in size from 125 mL to 1 L.
  • the column was washed with 50% v/v acetonitrile in 0.1 M aqueous ammonium acetate pH 4.5 (3.5 L) .
  • A 1/4 v/v acetonitrile/water-0.1M ammonium dihydrogen phosphate- 0.8M phosphoric acid (adjusted to pH 3.0 with triethylamine)
  • B 3/7 v/v acetonitrile/water-0.1M ammonium dihydrogen phosphate-0.8M phosphoric acid (adjusted to pH 3.0 with triethylamine), 0% B for 23 min, 0%-100% B over 37 min, UV detection at 210 nm) .

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Abstract

Procédé pour préparer, en phase de solution, le peptide L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2, qui fait preuve d'une activité libératrice de l'hormone de croissance hypophysaire.Process for preparing, in solution phase, the peptide L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2, which exhibits a liberating hormone activity pituitary growth.

Description

SOLUTION PHASE PROCESS FOR PEPTIDE
This invention relates to a process for preparing the peptide:
L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2. This peptide has pituitary growth hormone releasing activity.
This invention also relates to intermediates used in the process of the invention.
BACKGROUND OF THE INVENTION
In U.S. Patent 4,411,890, the peptide L-His-D-Trp-L-Ala- L-*Trp-D-Phe-L-Lys-NH2 is described as having pituitary growth hormone releasing activity. The peptide is useful to treat symptoms related to growth hormone deficiencies.
The method of preparing this peptide which is exemplified in U.S. 4,411,890 is a solid phase process in which the starting material amino acid is attached to a resin and is then coupled stepwise with the appropriate amino acids. Thus, the intermediates in the solid phase process are peptide-resin compounds. The desired peptide product is cleaved from the resin by treatment with hydrogen fluoride. Although it is stated in U.S. 4,411,890 that solution methods known to the art can be used to prepare the peptides, no solution methods are exemplified. The only intermediates disclosed for solution phase processes are protected hexapeptides . DESCRIPTION OF THE INVENTION
This invention provides an advantageous process for preparing L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2. The process is a solution phase method which provides solid, recrystallizable intermediates which are readily isolated and purified. These solid intermediates are generally crystalline and may be purified by recrystallization. Solid, recrystallizable intermediates, particularly in all steps of a process, are rare in peptide chemistry and offer an advantage in purification.
Accordingly, this invention is a process for preparing a compound of the formula:
L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2
hich comprises : a) coupling L-Lys (BOO -NH2 with Z-D-Phe; b) removing the Z group and coupling the resulting D- Phe-L-Lys(BOC)-NH2 with Z-L-Trp-NH2; c) removing the Z group and coupling the resulting L- Trp-D-Phe-L-Lys (BOC)-NH2 with Z-L-Ala; d) removing the Z group and coupling the resulting L- Ala-L-Trp-D-Phe-L-Lys(BOC)-NH2 with Z-D-Trp; e) removing the Z group and coupling the resulting D- Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 with (BOC)2-L-His; and f) removing the BOC groups to give L-His-D-Trp-L-Ala-L- Trp-D-Phe-L-Lys-NH2.
The intermediates are preferably recrystallized at each step in the process to prevent the build up of impurities. When each intermediate is purified before being used in the next step of the p'rocess, the purifiύ*" -.ion of the final product is facilitated.
Furthermore, it is an advantage that the solution phase method of this invention avoids the use of corrosive hydrogen fluoride which is used in the solid phase procedure to cleave the product from the resin. The solution phase process of the invention requires only one acid treatment (at the end of the reaction process to remove the amino protecting groups) , thus minimizing decomposition of the tryptophan residues; whereas, the solid phase method involves repetitive acid treatments.
Protected peptide intermediates in the process of this invention are also a feature of this invention. The intermediates are particularly useful in the process of this invention because they are solids, generally crystalline and are readily purified by recrystallization.
The 'process of this invention may be represented as follows:
Z-L-Lys(BOC)-NH2 ( i;
BOC-L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 and BOC- -His (BOC)-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 (VI)
1) Recryst.
2) Acid
L-His-D-Trp- -Ala-L-Trp-D-Phe-L-Lys-NH2 (VII)
Intermediate compounds of this invention are represented by structures (I) -(VI) hereabove.
Abbreviations for amino acids/residues used herein follow standard peptide nomenclature. Such abbreviations and other terms used herein are as follows :
L-His = L-histidine
D-Trp = D-tryptophan
L-Trp = L-tryptophan L-Ala = L-alanine
D-Phe = D-phenylalanine -Lys = L-lysine
Z = benzyloxycarbonyl
BOC = t-butyloxycarbonyl TFA = trifluoroacetic acid
WSC = water soluble carbodiimide [1- (3-dimethylamino- propyl) -3-ethylcarbodiimide hydrochloride]
DMF = dimethylformamide
In the steps of the process outlined hereabove, intermediates (I) -(VI) are recrystallized before being used in the subsequent steps. Intermediates (II) -(VI) are prepared by removing the benzyloxycarbonyl group (Z) by catalytic hydrogenolysis and then coupling with the appropriate amino acid.
The starting material, N(α) -benzyloxycarbonyl-N(ε) -t- butyloxycarbonyl-L-lysine amide (I), is prepared from N(α)- benzyloxycarbonyl-N(ε)-t-butyloxycarbonyl-L-lysine by reacting with ammonia, water soluble carbodiimide [i.e. I- 3 (dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride], and 1-hydroxybenzotriazole. After removal of the benzyloxycarbonyl group from intermediate (I) by catalytic hydrogenolysis, the resulting amine is coupled to benzyloxycarbonyl-D-phenylalanine using water soluble carbodiimide and 1-hydroxybenzotriazole. This sequence of deprotection (removal of the Z group) and coupling is repeated with benzyloxycarbonyl-L-tryptophan, benzyloxycarbonyl-L-alanine, benzyloxycarbonyl-D-tryptophan and N(α), N(im)-di-t-butyloxycarbonyl-L-histidine to afford a mixture of the di-t-butyloxycarbonyl hexapeptide and the tri- t-butyloxycarbonyl hexapeptide (VI) . Removing the t- butyloxycarbonyl groups with acid gives the desired peptide product (VII) . The peptide product (VII) is purified, for example, by using reverse phase liquid chromatography.
The t-butyloxycarbonyl groups are removed using acid, optionally and a carbonium ion scavenger. Suitable acids for removing the t-butyloxycarbonyl group are well known in the art, and include mineral acids or strong organic acids, such as trifluoroacetic acid. Carbonium ion scavengers are also well known in the art, and include electrophilic aryl compounds and mercaptans, such as n-propylmercaptan.
The hydrogenolysis to remove Z protecting groups is performed using an appropriate catalyst, for example palladium on carbon, such as 5-10% palladium on carbon. Preferably, 10% palladium on carbon is. used. The pressure at which the hydrogenolysis is performed is not critical, and may be performed at from atmospheric pressure up to several hundred psi. Typically, it is performed at atmospheric pressure up to 100 psi. Increasing the hydrogen pressure enhances the rate of reaction. Performing the reaction at about 100 psi is preferred. Vigorous stirring, such as about 600 rpm, in the autoclave is very advantageous to increase the reaction rate.
Coupling reactions are well known in the art and may be accomplished by activating the carboxyl group of the intermediate, such as by formation of an acyl halide, activated ester or activated anhydride, or coupling with a coupling reagent such as a carbodiimide, for example dicyclohexylcarbodiimide or water soluble carbodiimide, PPA (1-propanephosphonic acid cyclic anhydride) , DPPA (diphenylphosphoryl azide) or BOP reagent (benzotriazol-1- yloxy-tris (dimethylamino)-phosphonium hexafluorophosphate) . Water soluble carbodiimide is preferred. Additional reagents may be added to facilitate the coupling reaction with carbodiimides, such as N-hydroxy-benzotriazole. Upon completion, the coupling reaction solutions are poured into aqueous base, for example, in the preparation of intermediates (II)-(V), the coupling reaction solutions are poured into aqueous potassium carbonate or potassium bicarbonate solution with vigorous stirring. In the preparation of the di-BOC and tri-BOC intermediates (VI) , the coupling reaction solution is, preferably, poured into aqueous potassium bicarbonate solution. During the addition of the coupling solution, the aqueous potassium bicarbonate is stirred vigorously. Preferably, the rate of addition should be about 500 mL- 1 L/minute.
When the intermediates (VI) are collected by filtration, they should be washed well until the filtrate is neutral. The damp product should be dried to constant weight before proceeding with recrystallization and/or removal of the BOC protecting groups.
The BOC protecting groups are removed with acid, optionally in the presence of a carbonium ion scavenger. Trifluoroacetic acid is a suitable acid for removing the BOC group. Use of a carbonium ion scavenger is preferred. Typical carbonium ion scavengers are anisole, dimethoxy benzene and mercaptans. n-Propyl mercaptan is suitable.
The following example illustrates the specific practice of this invention but is not intended to limit its scope.
EXAMPLE 1
Preparation of Z-L-Lys (BOC) -NH2
Z-L-Lys(BOC)-OH (682.0 g, 1.79 mol) and 1-hydroxy- benzotriazole hydrate (330.4 g, 2.15 mol) were dissolved sequentially in DMF (1.06 L) in a round-bottomed flask equipped with an overhead stirrer. The resulting solution was cooled in an ice bath to 0-5°C and WSC (377.0 g, 1,98 mol) was added through a powder funnel at such a rate that the temperature did not exceed 12°C. After the addition, the ice bath was removed and the mixture was stirred for thirty minutes at room temperature. Next, this was cooled again in an ice bath to 0-5°C and a solution of concentrated ammonium hydroxide (225 mL, 3.59 moles NH3) in DMF (500 mL) was added at such a rate that the temperature was kept below 25°C. The resulting solution was stirred at room temperature for 1.5 hours and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5.6% w/v, 36 L) . The white solid, which separated, was collected at the pump, washed with deionized water (approximately 25 L) until the washings were neutral to pH paper, and air-dried overnight. This solid was recrystallized from a mixture of isopropanol (3.95 L) and deionized water (5.55 L) to afford white crystals which were collected at the pump, washed with isopropanol-water (2:3 v/v, 2 L) and dried to a constant weight in vacuo at 45°C (436 g, 64.1%) : 1H NMR(DMSO-d6, 360 MHz) 5 7.4-7.15 (m, 6H) , 6.93 (s, 2H) , 6.72 (br t, 1H) , 5.02 (s,2H), 3.93-3.83 (m, 1H) , 2.91-2.80 (m,2H), 1.65-1.20 ( , 6H) , 1.36 (s,9H) ; MS (FAB) m/e 380.3 [M+H]+; TLC Rf 0.57 (silica,
90:8:2 chloroform:met anol:acetic acid); HPLC RT 7.0 min (5μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml/min, gradient, A: 1/9 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate B: 1/1 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate, 0%-100% B over 10 min, 100% B 35 min, UV detection at 220 nm) .
EXAMPLE 2
Preparation of Z-D-Phe-L-Lys (BOC)-NH2
Z-L-Lys (BOC)-NH2 (20.0 g, 53 mmol) was dissolved in DMF (100 mL) . This solution was transferred to an autoclave suitable for hydrogenation reactions (capacity 300 mL) . To this was added a suspension of 10% palladium on carbon catalyst (0.67 g) in DMF (20 mL) . The autoclave was filled with hydrogen at 100 psi and the turbine agitator turned on at approximately 600 rpm. The temperature was maintained at 22-24°C. After 2 hours, stirring was stopped and the autoclave was vented. HPLC indicated the absence of starting material.
The reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (35 mL) and the filtrate and washings were combined.
This entire product, consisting of a solution of L- Lys(BOC)-NH2 (53 mmol) and toluene in DMF (155 mL) , was placed in a round-bottomed flask equipped with an overhead stirrer and cooled to 0-5°C. Z-D-Phe (17.1 g, 58 mmol) and 1-hydroxybenzotriazole hydrate (9.6 g, 63 mmol) were dissolved sequentially in this solution. WSC (12.8 g, 67 mmol) was added with vigorous stirring through a powder addition funnel at such a rate as to keep the temperature below 10°C. When the WSC had dissolved, the reaction mixture was allowed to stir at ambient temperature for one hour. The reaction mixture was filtered through a DMF-washed bed of Celite® and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) . The solid which precipitated was collected on a Buchner funnel and the filter cake washed with deionized water (about 1.2 L) until the eluant was neutral to pH paper. The solid was dissolved in boiling isopropanol (240 mL) . To the boiling solution, deionized water (approximately 240 mL) was added to cloud point; the resulting solution was cooled at 0- 5°C overnight. The solid was collected on a Buchner funnel and washed with aqueous isopropanol (1:1 v/v, 100 mL) . The solid, was dried in vacuo at 40°C (26.3 g, 95%) : -4. NMR(DMSO- d6), 360 MHz) 5 8.09 (d, 1H,J=7Hz) , 7.54 (d, 1H,J=7Hz) , 7.4- 7.15 (m,lQH), 7.04(s,2H), 6.70 (br t, 1H) , 4.95(m,2H), 4.3-4.15 (m,2H), 3.0-2.7 (m, H) , 1.7-1.0 (m, 6H) , 1.36 (s,9H); MS (FAB) m/e 527.4 [M+H]+, 525.3 [M-H]"; TLC R-' 0.60 (silica, 90:8:2 chloroformrmethanol:acetic acid) ; HPLC RT 16 min (5μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml/min, gradient, A: 1/9 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate B: 1/1 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate, 75% B for 5 min, 75%-100% B over 7.5 min, 100% B 35 min, UV detection at 220 n ) . EXAMPLE 3
Preparation of Z-L-Trp-D-Phe-L-Lys (BOC)-NH Z-D-Phe-L-Lys (BOC-NH2 (26.0 g, 49 mmol) was dissolved in
DMF (100 mL) . This solution was transferred to an autoclave suitable for hydrogenation reactions (capacity 300 mL) . To this was added a suspension of 10% palladium on carbon catalyst (1.3 g) in DMF (20 L) . The autoclave was sealed, then filled and vented several times with hydrogen to 100 psi. The autoclave was filled with hydrogen at 100 psi and the turbine agitator turned on at approximately 600 rpm. The temperature was maintained at 22-24°C. After approximately 2 -hours, stirring was stopped and the autoclave was vented. TLC and HPLC' indicated the absence of starting material.
The reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (50 mL) and the filtrate and washings were combined.
The entire product, consisting of a solution of D-Phe-L- Lys(BOC)-NH (49 mmol) and toluene in DMF (approximately 170 mL) , was placed in a round-bottomed flask equipped with an overhead stirrer and cooled to 0-5°C. Z-L-Trp (18.6 g, 55 mmol) and 1-hydroxybenzotriazole hydrate (9.02 g, 59 mmol) - were dissolved sequentially in this solution. WSC (12 g, 62 mmol) was added with vigorous stirring through a powder addition funnel at such a rate as to keep the temperature below 10°C. When the WSC had dissolved, the reaction mixture was allowed to stir at ambient temperature for 2.5 hours. At this time, TLC indicated the absence of D-Phe-L-Lys (BOC)-NH2. The reaction mixture was filtered through a DMF-washed bed of Celite® and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) . The product separated as a solid which was collected at the pump and washed with deionized water (1.2 L) until the washings were no longer basic. The damp cake was dissolved in boiling methanol (500 mL) ; deionized water (150 mL) was added to cloud point. The solution was allowed to cool at 0- 5°C overnight . The solid that separated was collected on a Buchner funnel and washed with aqueous methanol (50% v/v, 100 mL) . The solid was dried in vacuo at 40°C (33.3 g, 95%) : λE NMR(DMSO-d6, 360 MHz) δ 10.73 (d, 1H,J=lHz) , 8.45
(d,lH,J=7Hz) , 8.05 (d,lH,J=7Hz) , 7.6 (d, 1H,J=7Hz) , 7.4-6.9 (m,17H), 6.70(br t, 1H) , 4.92 ( ,2H) , 4.6, 4.25, 4.15 each signal(m,1H) , 3.0-2.6 (m, 6H) , 1.7-1.0 (m, 6H) , 1.36 (s,9H); MS (FAB) m/e 713.6 [M+H]+, 711.4 [M-H]-; TLC Rf 0.46 (silica,
90:8:2 chloroform:methanol:acetic acid); HPLC RT 21 min (5μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml/min, gradient. A: 1/9 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate B: 1/1 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate, 75% B for 5 min, 75%-100% B over 7.5 min, 100% B 35 min, UV detection at 220 nm) .
EXAMPLE 4
Preparation of Z-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2
Z-L-Trp-D-Phe-L-Lys(BOC)-NH2 (33.0 g, 46 mmol) was dissolved in DMF (100 mL) . This solution was transferred to an autoclave suitable for hydrogenation reactions (capacity 300 mL) . To this was added a suspension of 10% palladium on carbon catalyst (2.2 g) in DMF (20 mL) . The autoclave was sealed, then filled and vented several times with hydrogen to 100 psi. The autoclave was filled with hydrogen at 100 psi and the turbine agitator turned on at approximately 600 rpm." The temperature was maintained at 20-28°C. After approximately 3 hours, stirring was stopped and the autoclave was vented. TLC indicated the absence of starting material; HPLC showed a trace to be remaining. The reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were -rinsed with DMF (50-mL) and the filtrate and washings were combined.
The solution of L-Trp-D-Phe-L-Lys (BOC)-NH2 (46 mmol) in
DMF and toluene (approximately 20 mL) was placed in a round- bottom flask equipped with an overhead stirrer and cooled to 0-5°C, Z-L-Ala (11.3 g, 50 mmol) and 1-hydroxybenzotriazole hydrate (8.47 g, 50 mmol) were dissolved sequentially in this solution. WSC (11.1 g, 58 mmol) was added with vigorous stirring through a powder addition funnel at such a rate as to keep the temperature below 10°C. When the WSC had dissolved, the reaction mixture was allowed to stir at ambient temperature for 2.5 hours. The reaction mixture was filtered through a DMF-washed bed of Celite® and then was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) . The product separated as a solid, which was collected at the pump and washed with deionized water (1.6 L) until the washings were neutral. After sucking as dry as possible, the damp filter cake was dissolved in boiling methanol (700 mL) ; deionized water (300 mL) was added to cloud point. The resulting solution was cooled overnight at 0-5°C. The solid was collected on a Buchner funnel, washed with methanol-water (1:1, v/v, 200 mL) and dried in vacuo at 40°C (34.8 g, 95%) : -R NMR(DMSO-d6, 360 MHz) 5 10.73 (d, 1H,J=lHz) , 8.27, 8.15,
7.88 each signal (d, 1H, =7Hz) , 7.5 (d, 1H, J=7Hz) , 7.4-6.9
(m, 17H), 6.69 (br t,lH), 5.0 (m,2H), 4.6-4.0 (m, 4H) , 3.0-2.7
(m,6H), 1.7-1.0 (m, 6H) , 1.36 (s, 9H) , 1.12 (d, 3H, J=8Hz) ; MS (FAB) m/e 784.6 [M+H] +, 782.5 [M-H]~; TLC Rf 0.46 (silica, 90:8:2 chloroform:methanol:acetic acid); HPLC RT 19 min (5μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml/min, gradient. A: 1/9 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate B: 1/1 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate, 75% B for 5 min, 75%-100% B over 7.5 min, 100% B 35 min, UV detection at 220 nm) .
EXAMPLE 5
Preparation of Z-D-Trp-L-Ala-L-Tro-D-Phe-L-Lys (BOC) -NH2 ■
Z-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 (34.5 g, 43 mmol) was dissolved in DMF (100 mL) . This solution was transferred to an autoclave suitable for hydrogenation reactions (capacity 300 mL) . To this was added a suspension of 10% palladium on carbon catalyst (3.45 g) in DMF (20 mL) . The autoclave was sealed, then filled and vented several times with hydrogen to 100 psi. The autoclave was filled with hydrogen at 100 psi and the turbine agitator turned on at approximately 600 rpm. The temperature was maintained at 20-28°C. After approximately 3.5 hours, stirring was stopped and the autoclave was vented. TLC indicated the absence of starting material; HPLC showed a trace to be remaining. The reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (50 mL) and the filtrate and washings were combined.
This solution of L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 (43 mmol) in DMF and toluene (approximately 200 mL) was placed in a round-bottom flask equipped with an overhead stirrer and cooled to 0-5°C. Z-D-Trp (16.0 g, 48 mmol) and 1-hydroxy- benzotriazole hydrate (7.99 g, 52 mmol) were dissolved sequentially in this solution. WSC (10.4 g, 54 mmol) was added with vigorous stirring through a powder addition funnel at such a rate as to keep the temperature below 10°C. When the WSC had dissolved, the reaction mixture was allowed to stir at ambient temperature for 3 hours. At this time, TLC indicated the absence of L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2.
The solution was filtered through a bed of Celite®. The reaction mixture was added slowly and with vigorous stirring into aqueous potassium carbonate solution (5% w/v, 1.6 L) . The product separated as a solid, which was collected at the pump and washed with deionized water (1.6 L) until the washings were neutral. After sucking as dry as possible, the damp filter cake was dissolved in boiling ethanol (1.4 L) and DMF (100 mL) at 60°C. Deionized water (400 mL) was added to the cloud point. The resulting solution was cooled overnight at 0-5°C. The solid was collected on a Buchner funnel, washed with ethanol-water (1:1 v/v, 200 mL) and dried in vacuo at 40°C (39.3 g, 93.6%) : 1H NMR(DMSO-d6, 360 MHz) δ
10.8, 10.7 each signal (d,1H, J=lHz) , 8.20-7.85 (m, 4H) , 7.65- 6.9 (m,23H), 6.70 (br t,lH), 5.0 (m,2H), 4.6-4.0 ( ,5H) , 3.15-2.6 (m,8H), 1.7-0.9 (m, 6H) , 1.37 (s,9H), 1.04 (d,3H,J=8Hz) ; MS (FAB) m/e 970.7 [M+H]+, 968.6 [M-H]"; TLC Rf 0.30 (silica, 90:8:2 chloroform:methanol:acetic acid); HPLC
RT 25 min (5μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml/min, gradient. A: 1/9 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate B: 1/1 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate, 75% B for 5 min, 75%-100% B over 7.5 min, 100% B 35 min, UV detection at 220 nm) .
EXAMPLE $
Preparation of BOC-L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 and BOC-L-His (BOC)-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2.
Z-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys(BOC)-NH2 (39.0 g, 40 mmol) was dissolved in DMF (100 mL) . This solution was transferred to an autoclave suitable for hydrogenation reactions (capacity 300 mL) . To this was added a suspension of 10% palladium on carbon catalyst (5 g) in DMF (30 mL) . The autoclave was sealed, then filled and vented several times with hydrogen to 100 psi. The autoclave was filled with hydrogen at 100 psi and the turbine agitator turned on at approximately 600 rpm. The temperature was maintained at 20-28°C. After approximately 5 hours, stirring was stopped and the autoclave was filled and vented three times with nitrogen to 100 psi. TLC and HPLC indicated the absence of starting material.
The reaction mixture was filtered through a DMF-washed bed of Celite®. In turn, the solids were rinsed with DMF (50 mL) and the filtrate and washings were combined.
This solution of D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) - H2 (40 mmoles) in DMF and toluene (approximately 200 mL) was placed in a round-bottom flask equipped with an overhead stirrer and cooled to 0-5°C in an ice bath. (BOC) 2-L-His
(15.7 g, 44 mmol), 1-hydroxybenzotriazole hydrate (7.39 g, 48 mmol), and WSC (9.63 g, 50 mmol) were dissolved sequentially in this solution. Less than five minutes occurred between each addition. When the WSC had dissolved, the reaction mixture was allowed to stir at 0-5°C for 1 hour and then at ambient temperature overnight . The disappearance of D-Trp-L- Ala-L-Trp-D-Phe-L-Lys (BOO-NH2 was monitored by HPLC. The reaction mixture was filtered through a bed of Celite®. The resulting filtrate was added slowly and with vigorous stirring into aqueous potassium bicarbonate solution (10% w/v, 3 L) . The product separated as a solid, which was collected at the pump and washed with deionized water (approximately 5 L) until the washings were neutral. After sucking until no more filtrate appeared, the damp cake was dried in vacuo at 35°C to afford 45.4 g of solid (95%) . HPLC showed that the major species was the tri-BOC-hexapeptide (88.3%) and next was the di-BOC-hexapeptide (9.5%) .
The crude mixture of the tri-BOC and di-BOC hexapeptide (50 g) was dissolved at 60°C in DMF (130 mL) . Methanol (1.3 L) was added, followed by activated carbon (25 g) . The mixture was boiled for two minutes and filtered, while hot, through a bed of Celite®. The cake was washed with hot methanol (500 mL) . The combined filtrate and washings were refrigerated overnight at -15°C. The white solid, which separated, was collected at the pump, washed with methanol (200 mL) and dried, in vacuo, overnight at room temperature (37.1 g, 74.2%) : -E NMR(DMSO-d6f 400 MHz) δ 10.8, 10.7 each signal (d,1H,J=lHz) , 8.25-7.9 (m,5H) , 8.1 (s,lH), 7.55-6.9 (m,20H), 6.75 (br t,lH), 4.6-4.1 (m,6H) , 3.1-2.6 (m,10H) , 1.7-1.0 (m,6H), 1.51 (s,9H), 1.36 (s, 9H) , 1.30 (d, 9H) , 1.0(d,3H,J=8Hz) ; MS (FAB) m/e 1173.6 [M+H]+ (tri-BOC), 1073.5 [M+H]+ (di-BOC); TLC Rf 0.44 (tri-BOC), 0.02 (di-BOC) (silica, 90:8:2 chloroform:methanol:acetic acid), Rf 0.94 (tri-BOC), 0.47 (di-BOC) (silica, 4:1:1 butanol:acetic acid:water); HPLC RT 31 min (tri-BOC, 90.2%), 8 min (di-BOC, 8.4%) (5μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml/min, gradient. A: 1/9 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate B: 1/1 v/v acetonitrile/water-0.3M ammonium dihydrogen phosphate, 75% B for 5 min, 75%-100% B over 7.5 min, 100% B 35 min, UV detection at 220 nm) .
EXAMPLE 7
Preparation of L-His-D-Trp-L-Ala-L-Ti' -D-Phe-L-Lys-NH2. Trifluoroacetic acid (960 mL) and n-propanethiol (150 mL) were mixed and stirred for one minute. The mixture of BOC-L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys(BOC)-NH2 and BOC-
L-His (BOC)-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 (60.0 g) was added with stirring. When dissolution was complete, the solution was kept at room temperature for two hours.
At this time, a sample was analyzed by HPLC to confirm the presence of the desired product and absence of starting material.
The reaction mixture was evaporated to a gum which was dissolved in distilled water (300 mL) and re-evaporated twice. Then, the mixture was dissolved in distilled water (1 L) and the pH was adjusted to approximately 3 by the dropwise addition of dilute aqueous ammonium hydroxide solution.
This material was purified by reverse phase liquid chromatography using YMC-type AS-5055, end-capped octadecyl silica, spherical in shape, with a particle size of 50 μ and a pore size of 120 A0. The bed size was 5 cm i.d. x 50 cm length, corresponding to a bed volume of 981 mL, packed in a stainless steel HPLC column. There was also a guard column of 5 cm i.d. x 10 cm length containing the same packing material (196 mL) . This was equilibrated with 0.1 M aqueous ammonium acetate solution pH 4.5 (4 L) , utilizing a Beckman Prep-350 HPLC system, monitoring the eluant at 254 nm.
The above pH adjusted reaction mixture was further diluted with distilled water (300 mL) and then applied to the column at a flow rate of 100 mL/min. The column was washed with 1.0 M ammonium acetate pH 8 (6.0 L), followed by 0.1 M ammonium acetate pH 4.5 (2.5 L) . The product was removed by step-wise elution with solutions of acetonitrile in 0.1 M aqueous ammonium acetate pH 4.5 as follows: 10% v/v (1 L) , 15% v/v (900 mL) , 17% v/v (1 L) , 20% v/v (2.5 L) , 25% v/v (2.5 L) . Fractions were collected, varying in size from 125 mL to 1 L. Finally, the column was washed with 50% v/v acetonitrile in 0.1 M aqueous ammonium acetate pH 4.5 (3.5 L) .
The fractions were analyzed by HPLC and those containing the product were pooled. The acetonitrile was evaporated and the aqueous residue lyophilized. The resulting solid was twice redissolved in distilled water and relyophilized to afford L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2 (47.67 g) . The product was dissolved in distilled water, combined, and filtered through a 0.2 μ membrane (cellulose nitrate) .
The filtrate was lyophilized to afford L-His-D-Trp-L-Ala-L- Trp-D-Phe-L-Lys-NH2 (powder) : Amino Acid Analysis: His (0.99), Ala(1.0), Phe(l.O), Lys(l.Ol); Analysis for Trp by Ultraviolet Absorption (1.93); RT 17 min (5μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml/min, gradient,. A: 1/4 v/v acetonitrile/water-0.1M ammonium dihydrogen phosphate- 0.8M phosphoric acid (adjusted to pH 3.0 with triethylamine) B: 3/7 v/v acetonitrile/water-0.1M ammonium dihydrogen phosphate-0.8M phosphoric acid (adjusted to pH 3.0 with triethylamine), 0% B for 23 min, 0%-100% B over 37 min, UV detection at 210 nm) .
Variations of this process and these intermediates will be apparent to those skilled in the art. This scope of this invention is not limited to the specific examples disclosed, but is intended to encompass all within the claims that follow.

Claims

What is claimed is :
1. A process for preparing a compound of the formula:
L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2
which comprises: a) coupling L-Lys (BOC) -NH2 with Z-D-Phe; b) removing the Z group and coupling the resulting D-Phe-L-Lys(BOC)-NH2 with Z-L-Trp-NH2; c) removing the Z group and coupling the resulting L-Trp-D-Phe-L-Lys (BOC)-NH2 with Z-L-Ala; d) removing the Z group and coupling the resulting L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 with Z-D-Trp; e) removing the Z group and coupling the resulting
D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH with (BOC) 2-L-His; and f) removing the BOC groups to give L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2.
2. A process according to Claim 1 wherein the intermediate is recrystallized after each coupling.
3. A process according to Claim 1 in which the protecting groups Z are removed by catalytic hydrogenolysis.
4. A process according to Claim 3 in which the catalyst is 5-10% palladium on carbon.
5. A process according to Claim 3 in which the catalytic hydrogenolysis is carried out at atmospheric pressure to about 100 psi.
6. A process according to Claim 4 in which the catalytic hydrogenolysis is carried out at about 100 psi.
7. A process according to Claim 1 in which the protecting groups BOC are removed using acid and a carbonium ion scavenger.
8. A process according to Claim 7 in which the acid is trifluoroacetic acid and the carbonium ion scavenger is n-propylmercapta .
9. A process according to Claim 1 in which the coupling reaction is carried out using water soluble carbodiimide and 1-hydroxy-benzotriazole.
10. A solid, recrystallizable compound of the formula:
Z-L-Lys(BOC)-NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl.
11. A solid, recrystallizable compound of the formula: Z-D-Phe-L-Lys (BOC)-NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl.
12. A solid, recrystallizable compound of the formula:
Z-L-Trp-D-Phe-L-Lys (BOC) -NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl.
13. A solid, recrystallizable compound of the formula:
Z-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl.
14. A solid, recrystallizable compound of the formula:
Z-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl.
15. A solid recrystallizable compound of the formula:
Boc-L-His (BOC)-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH in which BOC is t-butyloxycarbonyl.
16. A process for preparing a compound of the formula: BOC-L-His (BOC)-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 wherein BOC is t-butyloxycarbonyl, which comprises reacting:
D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH with BOC-L-His (BOC) and a coupling reagent .
17. A process for preparing a compound of the formula: Z-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl, which comprises reacting:
L-Ala-L-Trp-D-Phe-L-Lys (BOC)-NH2 with Z-D-Trp and a coupling reagent .
18. A process for preparing a compound of the formula:
Z-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl, which comprises reacting:
L-Trp-D-Phe-L-Lys (BOC) -NH2 with Z-L-Ala and a coupling reagent.
19. A process for preparing a compound of the formula:
Z-L-Trp-D-Phe-L-Lys (BOC)-NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl, which comprises reacting:
D-Phe-L-Lys (BOC) -NH2 with Z-L-Trp and a coupling reagent .
20. A process for preparing a compound of the formula: Z-D-Phe-L-Lys(BOC)-NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl, which comprises reacting:
L-Lys(BOC)-NH with Z-D-Phe and a coupling reagent.
EP9292903706A 1990-11-30 1991-11-25 Solution phase process for synthesis of peptide Withdrawn EP0564587A4 (en)

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