DE2406543C3 - Benzyloxycarbonyl-L-pyroglutamyl-L-histidyl-L-prolinamide or benzyloxycarbonyl-L-pyroglutamyl-L-3 methylhistidyl-L-prolinamide and TRF syntheses based therefrom - Google Patents

Description

In recent years theories have been put forward that the structure of the ihyrotropin releasing factor or hormone (hereinafter referred to simply as TRF) corresponds to that of pyroglutamyl-histidyl-prolinamide, in which all amino acids are in the L-form. TRF has now become a valuable drug and laboratory aid. It is special valuable in the treatment of depression and for the analysis of functional disorders of the

543

Pituitary gland. Recently it was found that the analogous compound, which has a methyl substituent in the 3-position the histidine imidazole side chain also has valuable biological activity. This compound is hereinafter referred to as 3-Me-TRF.

In the past few years, numerous methods have been used to synthetically produce TRF proposed and carried out. All of these methods have the same disadvantage as that of large-scale production is particularly disruptive: intermediate products and the end product require numerous cleaning stages, with which chromatography is always associated. Chromatography is an excellent tool for Cleaning of small amounts of product. However, when large-scale manufacture is required, it becomes almost prohibitive task. Synthetic routes that lead to 3-Me-TRF also have shortcomings on as the methods except the use of 3-methylhistidine as a building block in place of Histidine are chemically identical.

The invention therefore has the task of making a synthesis door TRF or 3-methyl-TRF available to make in which essentially no chromatography is required. The invention is also based on precursors for TRF and 3-Me-TRF as well as a precursor directed for TRF, which can be purified by crystallization. The invention also includes a process for the production of TRF or 3-Me-TRF in essentially quantitative yields from easy-to-clean preliminary stages.

The invention relates to compounds of the formula

O
CH ₁ O-CO

- CO-NH-CH-CO-N

j ^

CH,

Λ '

R-N-J CO-NH,

O)

in which R is a methyl radical or a hydrogen atom, the amino acid components all in the L-configuration and a process for the preparation of these compounds in an inert medium with gaseous hydrogen in the presence of finely divided palladium as a catalyst. This catalytic Hydrogenolysis takes place quickly at room temperature, but can also easily be done with a Temperature in the range between 15 and 55 "C. That used for this reaction Palladium can be used in any of a number of commercially available catalytic forms will. It can be used in a weight fraction of 2 to 50%, based on the compound (1). be used, amounts of 2 to 20% are preferred.

It has been found that the Catalylic Hydrogenolysc these compounds without racemization to the desired tripplide, d. H. to the connection, which as TRF is known and for which it already has numerous commercial and laboratory uses and leads to 3-Me-TRF.

In a general embodiment, the compound of structure (I) (R = H) is made by Conversion of benzyloxycarbonyl-pyroglutamyl-N-hydrosuccinimide with the sodium salt of histidine and subsequent neutralization with hydrochloric acid to form benzyloxycarbonylpyroglulamylhistidine, that in turn with prolinamide in the presence of hydroxysuccinimide and dicyclohexylcarbodiimide (hereinafter referred to as DCC) is converted to the compound of structure (1). The new connection has the advantage that it can be purified by recrystallization. She has a sharp melting point which makes it possible to determine the degree of purity in which the compound is obtained. The new Compound clearly differs from other Pcptidcn with a similar structure, in which the terminal nitrogen by one of the in the pcptidchemic common protective groups is protected. All other connections of this type must go through Be purified by chromatography. When R is methyl. is 3-methylhislidine in place of the The sodium salt of histidine is used with the aid of triethylamine. The N-protected tripeptide obtained is, however, an amorphous substance, which is cleaned and nonetheless nai 'known methods for making 3-Me-TRF in handy

theoretical yields can be used, with virtually no purification for that in this Way obtained 3-Me-TRF is required.

The process for the preparation of the above-mentioned novel compounds and their use is illustrated in the following examples. In all cases the connections made were by optical rotation (HHger-Watts polarimeter) identified. The infrared spectrum (Perkin-Elmer spectrophotometer) and the NMR spectrum (Varian spectrometer) were with the expected structures in harmony.

example 1

a) Benzyloxycarbonyl-L-pyroglutamic acid-N-hydroxysuccinimide ester

■

To a solution, cooled with an ice-acetone bath, of 15.79 g of benzyloxycarbonyl-L-pyroglutamic acid (described by G ibia et al. In Ann. Chem. 640 [1961], 145) and 7.59 g of N-hydroxysuccinimide in 60 ml 1,2-dimethoxyethane was added to 13.61 g of DCC with stirring. The mixture was stirred in the cold for 2 hours and then at room temperature overnight. The precipitated dicyclohexylurea was filtered off and washed with two small portions of 1,2-dimethoxyethane. The combined filirates were evaporated under reduced pressure to a viscous oil, which from elv. . 100 ml of isopropanol was crystallized. The crystals were filtered off, washed with isopropanol and ether and dried, whereby the desired compound with a melting point of 131 to 132 ° C. was obtained, [α] Γ = -23 " (c = 1.80 in DMF).

b) Bcnzyloxycarbonyl-L-pyroglutamyl-L-histidine

A solution of 13.51 g of the active ester prepared according to a) in 25 ml of dioxane was added, with stirring, to a solution of the sodium salt of L-histidine prepared from 6.42 g of L-histidine and 5.12 g of sodium carbonate penaldehyde in 50 ml of water. The reaction mixture was stirred for 4 hours at room temperature and concentrated under reduced pressure at a bath temperature below 40 ° C. to a volume of 30 ml. The solution was neutralized with 86.5 ml 0.957 N hydrochloric acid and then concentrated to about 20 ml. The precipitate obtained was filtered off. The solid was washed twice with 10 ml of ice water each time and recrystallized from 30% strength aqueous methanol, 9.40 g (62% of theory) of benzyloxycarbonyl-L-pyroglutamyl-L-histidine being obtained in the form of white needles after drying. The new compound does not melt, but begins to decompose at 149 ° C, [u]? ' = -1 U- = 1.08 in DMF). "

c) Benzyloxycarbonyl-L-pyroglutamyl-L-histidyl-i.-prolinamide

A solution of 1.005 g of the compound prepared according to b), 0.345 g of N-hydroxysuccinimide and 0.342 g of i.-prolinamide, prepared from L-prolinamide hydrochloride by treatment with "Rexyn No. 201" (OH form, manufacturer The Fisher Scientific Company, Fair Lawn, New Jersey) in 7 ml of DMF was chilled in an ice bath. After adding 0.578 g of DCC and 0.5 ml of Ν, Ν-Dirnelhylformarnid (referred to simply as DMF in this description), stirring was started and continued overnight while the reaction mixture was allowed to warm to room temperature. The precipitated dicyciohexyiharnsioff was separated off, filtered and washed three times with 1 ml of DMF each time. The combined filtrates were added dropwise to 200 ml of ether with stirring. The supernatant was decanted and the residue was dissolved in 10 ml of ethanol, which was added to 190 ml of ether. The white powder obtained was isolated and crystallized from ethanol to give 0.459 g of the desired peptide having a melting point of 184 to 186 ° C. The combined ethanolic mother liquors (from the precipitation and recrystallization with ether) were evaporated to dryness, a residue of 1.070 g being obtained, which was purified by chromatography on 40 g of silica gel. The non-polar impurities were removed by eluting the column twice with 100 ml of ethanol-chloroform (2: 3) each time. The desired peptide was then eluted three times with 100 ml of ethanol-chloroform (3: 2) each time. After evaporation of the solvent and crystallization of the residue from ethanol, a further 0.328 g of the desired compound with a melting point of 180 to 183 ° C. were obtained. The total yield of benzyloxycarbynyl-L-pyroglutamyl-L-histidyl-L-prounamide was thus 63% of theory. A further recrystallization in the manner described above increased the melting point to 186 to 187 ° C. ["] ?? = -43 ° (c = 1.02 in DMF).

d) L-pyroglutamyl-L-histidyl-L-prolinamide

A solution of 1.00 g of the compound prepared according to c) as Hcmihydrate in 50 ml of tetrahydrofuran and 50 ml of water was subjected to hydrogenolysis over 0.200 g of palladium black. To The catalyst was filtered off for 2 hours and washed several times with tetrahydrofuran-water (1: 1). The filtrate was concentrated to about 20 ml at a bath temperature below 35 ° C. The concentrated aqueous solution was filtered through a filter. The filter was rinsed twice with 10 ml of water each time. The resulting solution was frozen and freeze-dried, 0.704 g (98% of theory) of L-pyroglutamyl-L-histidyl-L-propynamide what remained was the same physical properties without further processing Properties like the best TRF material produced by the older process showed.

Example 2

a) Bcnzyloxycarbonyl-L-pyroglutamine-N'-norbornene-2,3-dicarboximidyl ester

A solution of 11.925 g of N-benzyloxycarbonyl-L-pyroglutamic acid and 8.95 g of N-hydroxy-5-norbornene-2,3-dicarboximide in 100 ml of dioxane and 100 ml of tetrahydrofuran was cooled in an ice bath. the 10.45 g of DCC were added to the solution with stirring. The mixture was left at ice bath temperature for 20 minutes and stirred for 40 minutes at room temperature, whereupon the precipitation separated and on the Filter was washed with the above-mentioned mixed solvent. The filtrate and the washing liquids were combined and the solvent was evaporated. The residue turned off

Recrystallized ethyl acetate heptane, with 18.743 g

(98% of theory) of the desired crystalline compound with a melting point of 145 to 146 "C. became.

b) N-Benzyloxycarbonyl-L-pyoglutamyl-

L-3-methylhistidine

A solution of 10.61 g of the product prepared according to a) and 4.23 g of 3-methylhistidine in 60 ml of dimethylformamide and 15 ml of water containing 3.5 ml of triethylamine as the reaction medium. was 4 hours stirred at room temperature. The solution was evaporated and the residue with 150 ml of ethyl acetate rubbed to form a gum. By crystallizing the gum with 75 ml of hot ethanol 8.33 g (77.5%) of the desired compound having a melting point of 197 ° to 198 ° C. were obtained. [«]? = -2.46 '- (C = 1.08 in DMF).

c) N-Benzyloxycarbonyl-L-pyroglutamyl-

i.-3-methylhistidyl-prolinamide

In the manner described in Example 1, c, a solution of 2.072 g of that prepared according to Example 6 was obtained Compound in 10 ml of DMF with 0.628 g of prolinamide in the presence of 1.135 α DCC and 0.986 ii N-Hydroxy-5-norbornene-2.3-dicarboxamide treated. The reaction was carried out in the manner described carried out, but the reaction mixture was diluted with 50 ml of water before filtration The oil obtained residue was applied to a column of 100 g of silica gel and treated with methanol-chloroform-acetic acid 35: 63: 2 eluted. The desired fractions were combined and evaporated to an oil. By precipitation of a methanol solution of the product with ether, a yield of 1.99 g (75%) obtained an amorphous powder whose analysis was eraab. that it is the 1.25 hydrate of the desired Connection acted. [, <] "= -50.6 (<· = 1.50 in DMF).

d) L-pyroglutamyl-L-3-methylhistidyl-L-prolinamide

A solution of 2.03 g of the product prepared according to c) in 100 ml of ethanol was subjected to hydrogenolysis with 2.0 g of palladium black in the manner described in Example 1. d). The desired compound was obtained as a hydrate in a yield of 92%. ['/ "K = -30.8 (c = 1.30 in DMF).

Claims (3)

Claims: 24 1. N-Benzyloxycarbonyl-L-pyroglutamyl-L-histidyl-L-prolinamide (I) and N-Benzyloxycarbonyl-L - pyrodutamyl - l - 3 - methylhistidyi - l - prolinamide (II). 2. A process for the preparation of v _O n TRF or 3-Me-TRF, characterized in that a solution of the compound 1 or 11 in an inert solvent with gaseous hydrogen at a temperature of 15 to 55 ° in a manner known per se C is hydrogenated in the presence of 2 to 50 percent by weight of finely divided palladium, based on the weight of the compound. 3. The method according to claim 1 and 2, characterized in that one is used as the inert solvent a mixture of 2 parts by volume of tetrahydrofuran and 1 to 3 parts by volume of water is used.