PT99654A - PREPARATION PROCEDURE OF THE L-HYSTIDINE-D-TRYPTOPHANE-L-ALANINE-L-TRYPTOPHANE-D-PHENYLALANINE-L-LYSINE-AMIDE PEPTIDE AND ITS INTERMEDIARIES - Google Patents

Description

73 404 SB CASE 14549 -2

DESCRIPTIVE MEMORY

This invention relates to a process for the preparation of 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 which are used in the process of the invention.

BACKGROUND OF THE INVENTION

In U.S. Patent 4,411,890, peptide L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH 2 is described as having pituitary growth hormone releasing activity. The peptide is useful for treating symptoms related to deficiencies in growth hormones. The method of preparing this peptide, which is exemplified in U.S. Patent 4,411,890, is a solid phase process in which the starting material, amino acid, is attached to a resin, and 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 hydrofluoric acid. While it is mentioned in U.S. Patent 4,411,890 that solution methods for the preparation of peptides known in the art can be used, no methods in solution are exemplified. The only intermediates disclosed for solution phase processes are the protected hexapeptides.

DESCRIPTION OF THE INVENTION

This invention provides an advantageous process for the preparation of L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2. The process -3- 73 404 SB CASE 14549 is a solution phase method which provides solid, recrystallizable intermediates which are readily isolated and purified. These solid intermediates are generally crystalline and can be purified by recrystallization. Solid, recrystallizable intermediates are rare in peptide chemistry, particularly at all steps of a process and offer an advantage in the purification.

Thus, this invention is a process for the preparation of a compound of formula: L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2 which comprises: a) coupling of L -Lys (BOC) -NH 2 with ZD-Phe; b) removal of the Z group and coupling of the resulting D-Phe-L-Lys (B0C) -NH2 with Z-L-Trp-NH2; c) removal of the Z group and coupling of the resulting L-Trp-D-Phe-L-Lys (BOC) -NH2 with Z-L-Ala; d) removal of the Z group and coupling of the resulting L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 with Z-D-Trp; e) removal of the Z group and coupling of the resulting D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 with (BOC) 2-L-His; and f) removal of the BOC groups to give L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH 2.

The intermediates are preferably recrystallized at each step in the process to prevent the appearance of impurities. When each intermediate is purified prior to use in the next step of the process, purification of the final product is facilitated.

73 404 SB CASE 14549 -4-

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 resin product. The solution phase process of the invention requires only an acid treatment (at the end of the reaction process to remove the amino protecting groups), thereby minimizing the decomposition of tryptophan residues; while the solid phase method involves repetitive acid treatments.

The protected peptide intermediates in the process of this invention are also a feature of this invention. Intermediates are particularly useful in the process of this invention because they are solid, generally crystalline and are easily purified by recrystallization. The process of this invention may be represented as follows: Z-L-Lys (BOC) -NH 2 (I) 1) Recryst. 2) Cat. H2 3) Z-D-Phe Z-D-Phe-L-Lys (BOC) -NH2 (II) 1) Recryst. 2) Cat. H2 3) Z-L-Trp Z-L-Trp-D-Phe-L-Lys (BOC) -NH 2 (III) 1) Recryst. 2) Cat. H2 3) Z-L-Ala

(IV) 73 404 SB CASE 14549 Z-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 1) Recryst. 2) Cat. H2 3) Z-D-Trp Z -D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH 2 (V) 1) Recryst. BOC-L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH 2 and BOC-L- His (BOC) -D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 (VI) 1) Recryst. 2) L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH 2 (VII)

The intermediates of this invention are represented by structures (I) - (VI) above.

The abbreviations of the amino acids / residues used herein follow the standard nomenclature of the peptides. The following are abbreviations and other terms used herein: L-His D-Trp L-Trp L-Ala L-histidine D-tryptophan L-tryptophan L-alanine

73 404 SB CASE 14549 D-Phe = D-phenylalanine L-Lys = L-lysine Z = benzyloxycarbonyl BOC = t-butyloxycarbonyl TFA = trifluoroacetic acid WSC = water soluble carbodiimide [1- (3- dimethylaminopropyl) -3-ethylcarbodiimide] DMF = dimethylformamide

In the above-described process steps, the intermediates (I) - (VI) are recrystallized before being used in the subsequent steps. Intermediates (II) - (VI) are prepared by removal of the benzyloxycarbonyl (Z) group by catalytic hydrogenolysis and then coupled with the appropriate amino acid. The starting material, N (a) -benzyloxycarbonyl-N- (e) -t-butyloxycarbonyl-L-lysine amide (I), is prepared from N - (a) -benzyloxycarbonyl-N- (e) t-butyloxycarbonyl-L-lysine hydrochloride by reaction with ammonia, water-soluble carbodiimide [i.e., 1- (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 deprotection sequence (removal of the Z group) and coupling is repeated with benzyloxycarbonyl-L-tryptophan, benzyloxycarbonyl-L-alanine, benzyloxycarbonyl-D-tryptophan and N (a), N (im) -di-t -butyloxycarbonyl-L-histidine to give a mixture of di-t-butyloxycarbonyl hexapeptide and tri-t-butyloxycarbonyl hexapeptide (VI). Removal of the t-butyloxycarbonyl groups with acid gives the desired peptide product (VII). The peptide product (VII) is purified, for example, by the use of reverse-phase liquid chromatography.

The t-butyloxycarbonyl groups are removed using acid, and optionally a carbonic ion scavenger. The acids

Suitable for removal of the t-butyloxycarbonyl group are well known in the art, and include mineral acids or strong organic acids, such as trifluoroacetic acid. Carbon-ion scavengers are also well known in the art, and include electrophilic aryl compounds and mercaptans, such as n-propylmercaptan.

The hydrogenolysis to remove the Z protecting groups is carried out 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 hydrogenolysis is performed is not critical, and can be carried out from atmospheric pressure to a few hundred psi. Typically, it is performed at atmospheric pressure up to 6.9 x 105 Pa (100 psi). Increasing the pressure of the hydrogen increases the rate of the reaction. It is preferred to carry out the reaction at about 6.9 x 105 Pa (100 psi). Vigorous stirring, such as at about 10 Hz (600 rpm), in the autoclave, is very advantageous for increasing the speed of the reaction.

J

Coupling reactions are well known in the art and can be carried out by activating the carboxyl group of the intermediate, for example by forming an activated acyl halide, activated ester or anhydride, or by coupling with a coupling reagent such as carbodiimide, for example dicyclohexylcarbodiimide or water soluble carbodiimide, PPA (cyclic anhydride of 1-propanophosphonic acid), DPPA (diphenylphosphorylazide) 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 the carbodiimides, such as N-hydroxybenzotriazole.

After completion, the coupling reaction solutions are poured into an aqueous base, for example in the preparation of the intermediates (II) - (V), the coupling reaction solutions are poured into aqueous solutions of potassium carbonate or potassium bicarbonate with vigorous shaking. 73 404 SB CASE 14549 -8-

In the preparation of the di-BOC and tri-BOC (VI) intermediates, the coupling reaction solution is preferably poured into aqueous potassium bicarbonate solution. During addition of the coupling solution, the aqueous solution of potassium bicarbonate is vigorously stirred. The addition rate should preferably be about 500 ml-1 1 / minute.

When the intermediates (VI) are collected by filtration, they must be thoroughly washed until the filtrate is neutral. The wet product should be dried to constant weight prior to recrystallization and / or removal of the BOC protecting groups.

The BOC protecting groups are removed with acid, optionally in the presence of a carbonic ion scrubber. Trifluoroacetic acid is a suitable acid for the removal of the BOC group. The use of a carbonic ion scrubber is preferred. Typical carbonic ion scavengers are anisole, dimethoxybenzene and mercaptans. The n-propylmercaptan is suitable.

The following examples illustrate the specific practice of this invention but are not intended to limit its scope.

Example 1

Preparation of Z-L-LysfBOO-NHo

ZL-Lys (BOC) -OH (682.0 g, 1.79 mol) and 1-hydroxybenzotriazole hydrate (330.4 g, 2.15 mol) were dissolved sequentially in DMF (1.06 L) in a flask of Round bottom equipped with a head stirrer. The resulting solution was cooled in an ice bath to 0-5øC and wsc (377.0 g, 1.98 mol) was added through a funnel to powders at such a rate that the temperature did not exceed 12øC. After addition, the ice bath was removed and the mixture was stirred for thirty minutes at room temperature. The solution was then cooled again in an ice bath at 0-5øC and a solution of concentrated ammonium hydroxide (225 ml, 3.59 moles of NH 3) in -9-73 404 SB CASE 14549 DMF (500 ml) 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 to an aqueous solution of potassium carbonate (5.6% w / v, 361). The white solid, which separated, was collected in the pump, washed with deionized water (approximately 25 L) until the washings were neutral to the 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 give white crystals which were collected on the pump, washed with isopropanol-water (2: 3 v / v, 2 1) and dried to a constant weight in vacuo at 45øC (436 g, 64.1%): 1 H NMR (DMSO-d6, 360 MHz) δ 7.4-7.15 (m, 6H) , 6.93 (s, 2H), 6.72 (broad t, 1H), 5.02 (S, 2H), 3.93-3.83 (m, 1H), 2.91-2.80 m, 2H), 1.65-1.20 (m, 6H), 1.36 (s, 9H); MS (FAB) m / e 380.3 [M + H] +; TLC Rf 0.57 (silica, chloroform: methanol: acetic acid 90: 8: 2); HPLC TA 7.0 min (5μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml / min, gradient, A: 0.3 M ammonium acetonitrile / water-dihydrogenphosphate, 1/9 v / v, B: 0.3 M ammonium acetonitrile / water-dihydrogenphosphate, 1/1 v / v, 0% -100% B for 10 minutes, 100% B 35 minutes, UV detection at 220 nm).

Example 2

Preparation of Z-D-Phe-L-Lys (BOC) -γ2

Z-L-Lys (BOC) -NH2 (20.0 g, 53 mmol) was dissolved in DMF (100 mL). This solution was transferred to a suitable autoclave for hydrogenation reactions (300 ml capacity). To this solution was added a suspension of 10% palladium on carbon catalyst (0.67 g) in DMF (20 ml). The autoclave was charged with 6.9 x 10 Pa Pa (100 psi) hydrogen and the turbine agitator was connected at approximately 10 Hz (600 rpm). The temperature was maintained at 22-24 ° C. After 2 hours the stirring was stopped and the autoclave was vented. HPLC indicated the absence of starting material. The reaction mixture was filtered through a bed of

73 404 SB CASE 14549 -10-

Washed with DMF. In turn, the solids were washed with DMF (35 mL) and the filtrate and washings were combined.

This product, as a whole consisting of a solution of L-Lys (BOC) -NH 2 (53 mmol) and toluene in DMF (155 ml) was placed in a round bottom flask equipped with a head 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 a rate maintaining the temperature below 10øC. When the WSC dissolved, the reaction mixture was stirred at room temperature for one hour. The reaction mixture was filtered through a bed of Celite® washed with DMF, and then added slowly and with vigorous stirring to an aqueous solution of potassium carbonate (5% w / v, 1.6 L). The precipitated solid was collected on a Buchner funnel and the filter cake washed with deionized water (about 1.2 L) until the eluent remained neutral to the pH paper. The solid was dissolved in boiling isopropanol (240 ml). Deionized water (approximately 240 ml) was added to the boiling solution to the cloud point; the resulting solution was cooled to 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 under vacuum at 40øC (26.3 g, 95%): NMR (DMSO-d6, 360 MHz) δ 8.09 (d, 1H, J = 7Hz), 7.54 J = 7Hz), 7.4-7.15 (m, 10H), 7.04 (s, 2H), 6.70 (broad t, 1H), 4.95 (m, 2H), 4.3-4.15 (m, 2H), 3.0-2.7 (m, 4H), 1.7-1.0 (m, 6H), 1.36 (s, 9H); MS (FAB) m / e 527.4 [M + H] +, 525.3 [M-H]; TLC Rf 0.60 (silica, chloroform: methanol: acetic acid 90: 8: 2) HPLC 16 min (5 μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml / min, gradient, A: acetonitrile / water 0.3% ammonium dihydrogen phosphate, 1/9 v / v, 75% B over 5 minutes, 75% -100% B for 7.5 minutes, 100% B 35 minutes, UV detection at 220 nm).

73 404 SB CASE 14549 -11-

Example 3

Preparation of Z-L-Trp-D-Phe-L-Lys / BOCy-NHU

Z-D-Phe-L-Lys (BOC) -NH2 (26.0 g, 49 mmol) was dissolved in DMF (100 mL). This solution was transferred to a suitable autoclave for hydrogenation reactions (300 ml capacity). To this solution was added a suspension of 10% palladium on carbon catalyst (1.3 g) in DMF (20 ml). The autoclave was sealed, charged and vented several times with hydrogen at 6.9 x 105 Pa (100 psi). The autoclave was charged with hydrogen at 100 psi and the turbine agitator was connected at approximately 10 Hz (600 rpm). The temperature was maintained at 22-24 ° C. After approximately 2 hours, the stirring was stopped and the autoclave was vented. TLC and HPLC indicated the absence of starting material. The reaction mixture was filtered through a bed of Celite® washed with DMF. In turn, the solids were washed with DMF (50 mL) and the filtrate and washings were combined. The product as a whole consisting of a solution of D-Phe-L-Lys (BOC) -NH 2 (49 mmol) and toluene in DMF (approximately 170 ml) was placed in a round bottom flask equipped with a head 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 a rate maintaining the temperature below 10øC. When the WSC dissolved, the reaction mixture was stirred at room temperature for 2.5 hours. At this point, TLC indicated the absence of D-Phe-L-Lys (BOC) -NH2. The reaction mixture was filtered through a pad of Celite ® washed with DMF, and then slowly added and with vigorous stirring to an aqueous solution of potassium carbonate (5% w / v, 1.6 L). The product, separated as a solid, was collected on the pump and washed with deionized water (1.2 L) until the washes were no longer basic. The wet cake was dissolved in boiling methanol (500 - 12 73 404 SB CASE 14549 ml); deionized water (150 ml) was added to the cloud point. The solution was cooled to 0-5 ° C overnight. The solid which separated was collected on a Buchner funnel and washed with aqueous methanol (50% v / v, 100 ml). The solid was dried under vacuum at 40øC (33.3 g, 95%): NMR (DMSO-d 6, 360 MHz) δ 10.73 (d, 1H, J = 1Hz), 8.45 (d J = 7Hz), 8.05 (d, 1H, J = 7Hz), 7.6 (d, 1H, J = 7Hz), 7.4-6.9 (m, 17H), 6.70 (broad t, 1H), 4.92 (m, 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, chloroform: methanol: acetic acid 90: 8: 2) HPLC TA 21 min (5μ Altex Ultrasphere® ODS, 4.6 x 250mm, 1ml / min, gradient, A: acetonitrile / water 0.3 M ammonium hydrogen phosphate, 1/9 v / v, B: 0.3 M ammonium acetonitrile / water-dihydrogen phosphate, 1/1 v / v, 75% B over 5 minutes, 75% -100% B for 7.5 minutes, 100% B 35 minutes, XJV detection at 220 nm).

Example 4

Preparation of Z-L-Ala-L-Trp-D-Phe-L-LyssBOCT-NHn

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 a suitable autoclave for hydrogenation reactions (300 ml capacity). To this solution was added a suspension of 10% palladium on carbon catalyst (2.2 g) in DMF (20 ml). The autoclave was sealed, charged and vented several times with hydrogen at 6.9 x 105 Pa (100 psi). The autoclave was charged with hydrogen at 100 psi and the turbine agitator was connected at approximately 10 Hz (600 rpm). The temperature was maintained at 20-28Â ° C. After approximately 3 hours, the stirring was stopped and the autoclave was vented. TLC indicated the absence of starting material; HPLC showed that traces remained. The reaction mixture was filtered through a pad of Celite® washed with DMF. In turn, the solids were washed with DMF (50 mL) and the filtrate and washings were combined. 73 404 SB CASE 14549 -13-

The solution of L-Trp-D-Phe-L-Lys (BOC) -NH2 (46 mmol) in DMF and toluene (ca. 20 mL) was placed in a round bottom flask equipped with a head stirrer and cooled to 0 ° C. -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 a rate maintaining the temperature below 10øC. When the WSC dissolved, the reaction mixture was stirred at room temperature for 2.5 hours. The reaction mixture was filtered through a bed of Celite® washed with DMF, and then slowly added and with vigorous stirring to an aqueous solution of potassium carbonate (5% w / v, 1.6 L). The product, separated as a solid, was collected in the pump and washed with deionized water (1.6 L) until the washings became neutral. After suctioning to dry as much as possible, the wet cake was dissolved in boiling methanol (700 ml); deionized water (300 ml) was added to the cloud point. The resulting solution was cooled to 0-5 ° C overnight. 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%): 1 H NMR (DMSO- (d, 1H, J = 7Hz), 7.5 (d, 1H, J = 1Hz), 8.27, 8.15, 1H, J = 7Hz), 7.4-6.9 (m, 17H), 6.69 (bt, 1H), 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, chloroform: methanol: acetic acid 90: 8: 2) HPLC TA 19 min (5μ Altex Ultrasphere® ODS, 4.6 x 250mm, 1ml / min, gradient, A: acetonitrile / water 0.3 M ammonium hydrogen phosphate, 1/9 v / v, B: 0.3 M ammonium acetonitrile / water-dihydrogenphosphate, 1/9 v / v, 75% B over 5 minutes, 75% -100% B for 7.5 minutes, 100% B 35 minutes, UV detection at 220 nm).

Example 5

Preparation of Z-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOCl -NH 2

Z-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH 2 (34.5 g, 43 mmol) was dissolved in DMF (100 mL). This solution was transferred to a

73 404 SB CASE 14549 -14-autoclave suitable for hydrogenation reactions (300 ml capacity). To this solution was added a suspension of 10% palladium on carbon catalyst (3.45 g) in DMF (20 ml). The autoclave was sealed, charged and vented several times with hydrogen at 6.9 x 105 Pa (100 psi). The autoclave was charged with 6.9 x 105 Pa (100 psi) hydrogen and the turbine agitator was connected at approximately 10 Hz (600 rpm). The temperature was maintained at 20-28Â ° C. After approximately 3.5 hours, the stirring was stopped and the autoclave was vented. TLC indicated the absence of starting material; HPLC showed that traces remained. The reaction mixture was filtered through a bed of Celite® washed with DMF. In turn, the solids were washed 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 a stirrer and cooled to 0-5 ° C. Z-D-Trp solution (16.0 g, 48 mmol) and 1-hydroxybenzotriazole 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 a rate maintaining the temperature below 10øC. When the WSC dissolved, the reaction mixture was stirred at room temperature for 3 hours. At this point, TLC indicated the absence of L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2. The solution was filtered through a pad of Celite®. The reaction mixture was added slowly and with vigorous stirring to an aqueous solution of potassium carbonate (5% w / v, 1.6 L). The product, separated as a solid, was collected in the pump and washed with deionized water (1.6 L) until the washings became neutral. After suction to dry as much as possible, the wet 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 to 0-5 ° C overnight. 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%): 1 H NMR (DMSO-d 6, 360 MHz) δ 10.8, 10.7 each signal (d, 1H, J = 1Hz), 8.20-7.85 (m, 4H), 7.65-6.9 (m, 23H), 6.70 (bt, 1H), 5.0 (m, 2H), 4.6-4.0 (m, 5H), 3.15-2 , 6 (m, 8H), 1.7-0.9 (m, 6H), 1.37 (s, 9H), 1.04 (d, 3H, J = 8 Hz); MS (FAB) m / e 970.7 [M + H] +, 968.6 [M-H] +; TLC Rf 0.30 (silica, chloroform: methanol: acetic acid 90: 8: 2) HPLC TA 25 min (5μ Altex Ultrasphere ™ ODS, 4.6 x 250 mm, 1 ml / min, gradient, A: acetonitrile 0.3 M ammonium water-dihydrogen phosphate, 1/9 v / v, B: 0.3 M, 1/4 / v / v, 75% B ammonium acetonitrile / water-dihydrogenphosphate over 5 minutes , 75% -100% B for 7.5 minutes, 100% B 35 minutes, UV detection at 220 nm).

Example 6

Preparation of BOC-L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-LyssBOC) -NH2 and BOC-L-His (BOC) -D-Trp-L-Ala-L -Trp-D-Phe-L-I-VS (BOCl-NH2.

Z-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys- (CHO) -NH2 (39.0 g, 40 mmol) was dissolved in DMF (100 mL). This solution was transferred to a suitable autoclave for hydrogenation reactions (300 ml capacity). To this solution was added a suspension of 10% palladium on carbon catalyst (5 g) in DMF (30 ml). The autoclave was sealed, charged and vented several times with hydrogen at 6.9 x 105 Pa (100 psi). The autoclave was charged with hydrogen at 100 psi and the turbine agitator was connected at approximately 10 Hz (600 rpm). The temperature was maintained at 20-28Â ° C. After approximately 5 hours, the stirring was stopped and the autoclave was charged and vented three times with nitrogen at 6.9 x 10 Pa Pa (100psi). TLC and HPLC indicated the absence of starting material. The reaction mixture was filtered through a bed of Celite® washed with DMF. In turn, the solids were washed with DMF (50 mL) and the filtrate and washings were combined.

This solution of D-Trp-L-Ala-L-Trp-D-Phe-Lys- (BOC) -NH2 (40 mmol) in DMF and toluene (approximately 200 ml) was placed in a round bottom flask equipped with a head stirrer and cooled to 0-5 ° C in an ice bath. Were dissolved

(15.7 g, 44 mmol), 1-hydroxybenzotriazole hydrate (7.39 g, 48 mmol), and WSC (9.63 g, 50 mmol) were sequentially sequenced in this solution (BOC). There were less than five minutes between each addition. When the WSC dissolved, the reaction mixture was stirred at 0-5 ° C for 1 hour and then at room temperature overnight. The disappearance of D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 was monitored by HPLC. The reaction mixture was filtered through a pad of Celite® S /. The resulting filtrate was added slowly and with vigorous stirring to an aqueous solution of potassium bicarbonate (10% w / v, 31). The product, separated as a solid, was collected in the pump and washed with deionized water (approximately 5 L) until the washings became neutral. After suction until no more filtrate appeared, the wet cake was dried under vacuum at 35 ° C to give 45.4 g of solid (95%). HPLC showed that the major species were tri-BOC-hexapeptide (88.3%) and the next was di-BOC-hexapeptide (9.5%). The crude mixture of 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 charcoal (25 g). The mixture was boiled for two minutes and filtered while hot through a pad 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 on the pump, washed with methanol (200 ml) and dried in vacuo overnight at ambient temperature (37.1 g, 74.2%): 1 H NMR (DMSO-d6) , 400 MHz) δ 10.8, 10.7 each signal (d, 1H, J = 1Hz), 8.25-7.9 (m, 5H), 8.1 (s, 1H), 7.55 (M, 6H), 3.1-2.6 (m, 10H), 1,7-dioxo- 1.0 (m, 6), 1.51 (s, 9H), 1.36 (s, 9H), 1.30 (d, 9H), 1.0 (d, 3H, J = 8 Hz); 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, chloroform: methanol: 90: 8: 2 acetic acid), 0.94 (tri-BOC), 0.47 BOC) (silica, butanol: acetic acid: water 4: 1: 1); HPLC TA 31 min (tri-BOC, 90.2%), 8 min (di-BOC, 8.4%) (5 μ Altex Ultrasphere® ODS, 4.6 x 250 mm, 1 ml / min, gradient, acetonitrile / water-0.3 M ammonium dihydrogen phosphate, 1/9 v / v, B: acetonitrile / water-dihydrogenphosphate

73 404 SB CASE 14549 ammonium 0.3 M, 1/1 v / v, 75% B for 5 minutes, 75% -100% B over 7.5 minutes, 100% B 35 minutes, UV detection at 220 nm ).

Example 7

Preparation of L-Hys-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH 2

Trifluoroacetic acid (960 ml) and n-propanethiol (150 ml) were stirred and stirred for one minute. After stirring the mixture of BOC-L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH 2 · and BOC-L-His (BOC) -D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH 2 (60.0 g). When the dissolution was complete, the solution was kept at ambient temperature for two hours.

At this point, a sample was analyzed by HPLC to confirm the presence of the desired product and absence of the starting material. The reaction mixture was evaporated to a gum, which was dissolved in distilled water (300 ml) and re-evaporated twice. The mixture was then dissolved in distilled water (11) and the pH was adjusted to approximately 3 by the dropwise addition of a dilute aqueous ammonium hydroxide solution.

This material was purified by reverse-phase liquid chromatography using spherical, YMC AS-5055, styled octadecyl silica having a particle size of 50 μ and a pore size of 120 μm. The bed size was 5 cm d.i. x 50 cm in length, corresponding to a bed volume of 981 ml, placed on a stainless steel HPLC column. There was also a 5 cm d.i. x 50 cm in length, containing the same filler (196 ml). This was equilibrated with a 0.1 M aqueous ammonium acetate solution pH 4.5 (4 L) using a Beckman Prep-350 HPLC system, controlling the eluent at 254 nm. The pH-adjusted reaction mixture referred to above was 73 404 SB CASE 14549-18

diluted further with distilled water (300 ml) and then applied to the column at a flow rate of 100 ml / minute. The column was washed with 1.0 M ammonium acetate, pH 8 (6.01), followed by 0.1 M ammonium acetate, pH 4.5 (2.5 L). The product was removed by stepwise elution with acetonitrile solutions in 0.1 M aqueous ammonium acetate pH 4.5 as described below: 10% V / V (11), 15% v / v ( 900 ml), 17% v / v (11), 20% v / v (2.5 l), 25% v / v (2.5 l). The fractions were collected, ranging in size from 125 ml to 11. 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 product were pooled. The acetonitrile was evaporated and the aqueous residue lyophilized. The resulting solid was redissolved twice in distilled water and reliofilified to give L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH 2 (47.67 g). The product was dissolved in distilled water, combined, and filtered through a 0.2 μm membrane (cellulose nitrate). The filtrate was lyophilized to give L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH2 (powder): Amino acid analysis: His (0.99), Ala (1, 0), Phe (1.0), Lys (1.01); Analysis for Trp by Ultraviolet Absorption (1.93); TA 17 minutes (5μ Altex Ultrasphere® ODS, 4.6x250mm, 1ml / min, gradient, A: acetonitrile / water-0.1M ammonium dihydrogen phosphate, 0.8M phosphoric acid, 1/4 v / v (adjusted to pH 3.0 with triethylamine), B: acetonitrile / water-0.1 M ammonium dihydrogen phosphate-0.8 M phosphoric acid, 3/7 v / v (adjusted to pH 3 , 0% with triethylamine), 0% B over 23 minutes, 0% -100% B for 37 minutes, UV detection at 210 nm).

Variations of this process and of these intermediates will be apparent to those skilled in the art. The scope of this invention is not limited to the specific examples disclosed, but is intended to encompass all that is within the claims set forth.

Claims (15)

A process for the preparation of a compound of the formula: ## STR2 ## in which the compound of the formula: ## STR3 ## a) the coupling of L-Lys (BOC) -NH2 with ZD-Phe; b) removal of the Z group and coupling of the resulting D-Phe-L-Lys (BOC) -NH2 with Z-L-Trp-NH2; c) removal of the Z group and coupling of the resulting L-Trp-D-Phe-L-Lys (BOC) -NH2 with Z-L-Ala; d) removal of the Z group and coupling of the resulting L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 with Z-D-Trp; e) removal of the Z group and coupling of the resulting D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 with (BOC) 2-L-His; and f) removal of the BOC groups to give L-His-D-Trp-L-Ala-L-Trp-D-Phe-L-Lys-NH 2. Process according to claim 1, characterized in that the intermediate is recrystallized after each coupling. A process according to claim 1, characterized in that the Z protecting groups are removed by catalytic hydrogenolysis. A process according to claim 3, characterized in that the catalyst is 5-10% palladium on carbon. A process according to claim 3, characterized in that the catalytic hydrogenolysis is carried out at a pressure from atmospheric pressure to about 6.9x105 Pa. A process according to claim 4, characterized in that the catalytic hydrogenolysis is carried out at about 6,9x105 A process according to claim 1, characterized in that the BOC protecting groups are removed using acid and a carbonic ion scrubber. 8. A process according to claim 7, wherein the acid is trifluoroacetic acid and the carbonic ion scavenger is n-propylmercaptan. 9. A process according to claim 1, wherein the coupling reaction is carried out using water-soluble diisocyanate and 1-hydroxybenzotriazole. A process for the preparation of a compound of the formula: ZL-Lys (BOC) -NH2 where Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl, which comprises reacting ZL-Lys (BOC) with aqueous NH 3 and a coupling reagent . (BOC) -D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH 2 where BOC is t-butyloxycarbonyl , characterized in that it comprises the reaction of: D-Trp-L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH 2 with BOC-L-His (BOC) and a coupling reagent. A process for the preparation of a compound of the formula: wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl, characterized in that it comprises reaction of: L-Ala-L-Trp-D-Phe-L-Lys (BOC) -NH2 with ZD-Trp and a coupling reagent. A process for the preparation of a compound of the formula: wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl, Z is Al-L-Trp-D-Phe-L-Lys (BOC) -NH2 characterized in that it comprises the reaction of: L-Trp-D-Phe-L-Lys (BOC) -NH 2 with ZL-Ala and a coupling reagent. A process for the preparation of a compound of the formula: wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl, characterized in that it comprises the reaction of: D-Phe-L (Z) -D-Phe-L-Lys (BOC) -Lys (BOC) -NH 2 with ZL-Trp and a coupling reagent. A process for the preparation of a compound of the formula: ZD-Phe-L-Lys (BOC) -NH2 wherein Z is benzyloxycarbonyl and BOC is t-butyloxycarbonyl, which comprises reacting L-Lys (BOC) ZD-Phe and a coupling reagent. Lisbon 1991 By SMITHKLINE BEECHAM CORPORATION = 0FACIAL AGENT =