DE2406543B2 - 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 thyrotropin releasing factor or hormones (hereinafter referred to simply as TRF) those of pyroglutamyl-histidyl-prolinamide corresponds, in which all amino acids are in the L-form. TRF is now a valuable drug and become laboratory tools. It is especially valuable in treating depression and for the analysis of dysfunction of the pituitary gland. Recently it was found that the analog Compound which has a methyl substituent in the 3-position the histidineimidazole side chain contains, also has valuable biological activity. This compound is hereinafter referred to as 3-Me-TRF.

In the past few years, numerous methods of synthetically producing TRF have been proposed and practiced. All d> -c processes have the same disadvantage, which is particularly troublesome in large-scale production: intermediate products and the end product require numerous purification stages, which are always associated with chromatography. Chromatography is an excellent means of purifying small amounts of product. However, when large-scale manufacturing is required, it becomes an almost prohibitive task. Synthetic routes that lead to 3-Me-TRF are equally deficient, since the methods are chemically identical with the exception of the use of 3-methylhistidine as a building block instead of histidine.

The object of the invention is therefore to provide a synthesis for TRF or 3-methyl-TRF which requires essentially no chromatography. The invention is also based on Precursors for TRF and 3-Me-TRF as well as a precursor for TRF which is purified by crystallization can be addressed. The invention also encompasses a process for the production of TRF or 3-Me-TRF in essentially quantitative yields from easy-to-clean precursors.

The invention relates to compounds of the formula

CO - NH - CH - CO - N

CO— NH,

in which R is a methyl radical or a hydrogen atom, wherein the amino acid components are all in the L-configuration and a method of preparation these compounds in an inert medium with gaseous hydrogen in the presence of finely divided palladium as a catalyst. This catalytic Hydrogenoiysc takes place quickly at room temperature, however, a temperature in the range between 15 and 55 ° C. can also be used without further ado will. The palladium used for this reaction can be in any of the various im Commercially available catalylic forms can be used. It can range in weight from 2 to 50%, based on the compound (I), can be used, amounts of from 2 to 20% being preferred.

It has been found that thick catalytic hydrogenolysis these compounds without racemization to the desired tripcpiid, 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) prepared by converting 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 (I). 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 is protected by one of the protective groups customary in Pcptidchemic. All other compounds of this type must be purified by chromatography. If R is a Is methyl radical, 3-methylhistidine is used in place of the sodium salt of histidine with the help of triethylamine used. The N-schülzlc tripcptide obtained is, however, an amorphous substance, which is nevertheless cleaned and cleaned according to known methods for the production of 3-Mc-TRF in practical

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 (Hilger-Watts polarimeter) identified. The infrared spectrum (Perkin-Elmer spectrophotometer) and the NMR spectrum (Varian spectrometer) were consistent with the expected structures.

example 1

a) Benzyloxycarbonyl-L-pyroglutaminsUuie-N-hydroxysuccinimideater

To one: solution cooled with an ice-acetone bath from 15.79 g of benzyloxycarbonyl-L-pyroglutamic acid (described by G i b i a η and co-workers in Ann. Chem., 640 [1961], 145) and 7.59 gN-hydroxysuccinimide 13.61 g of DCC were added to 60 ml of 1,2-dimethoxyethane 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 filtrates were reduced under Pressure evaporated to a viscous oil which was crystallized from about 100 ml of isopropanol. the Crystals were filtered off, washed with isopropanol and ether and dried to give the desired Compound of melting point 131 to 132 C was obtained, [r,] "= -23 (c = 1.80 in DMF).

b) Benzyloxycarbonyl-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 pentahydrate 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 resulting precipitate 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. [<0? = -Γ " ic = 1.08 in DMF).

c) Bcnzyloxycarbonyl-L-pyroglutamyli.-hislidyl-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 L-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-dimethylformamide (referred to simply as DMF in this description), stirring was started, which continued overnight while the reaction mixture was allowed to warm to room temperature. The precipitated dicyclohexylurea 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, whereby 0.459 g of the desired peptide having a melting point of 184 ° to 186 ° C. was obtained. 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 benzyloxycarbonyl-L-pyroglutamyl-L-histidyl-L-prolinamide was thus 63% of theory. The melting point was increased to 186 to 187 ° C. by a further recrystallization in the manner described above. [ _fi ] f = -43 ° (c = 1.02 in DMF).

d) l-pyroglutamyl-i.-hislidyl-i.-prolinamide

A solution of 1.00 g of the compound prepared according to c) as a hemihydrate 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 with 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-i.-histidyl-L-propynamide remained, the same physical properties as those produced by the older process without further processing showed best TRF material.

Example 2

a) Benzyloxycarbonyl-i.-pyroglutamic acid c-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. Of 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 with the solvent mixture mentioned above was washed. The fillrat 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-pyroglutamyl-L-3-methylhistidine

A solution of 10.61 g of the product prepared in accordance with 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 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).

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c) N-Benzyloxycarbonyl-L-pyroglutamyl-L-3-methylhistidyl-prolinam H.

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 DMF with 0.628 g pmlinamide in the presence of 1.135 g DCC and 0.986 g

N-Hydroxy-5-norbornene-2,3-dicarboxamide treated. The reaction was carried out in the manner described, but the reaction mixture was diluted with 50 ml of water before filtration. The oil obtained as residue was applied to a column of 100 g of silica gel and eluted with methanol-chloroform-acetic acid 35:63: 2. The desired fractions were combined and evaporated to an oil. By precipitation of a methanol solution of the product with ether, an amorphous powder was obtained in a yield of 1.99 g (75%), the analysis of which showed that it was the 1,25 hydrate of the desired compound. [«]? = -50.6 ' (c = 1.50 in DMF).

d) L-pyroglutamyl-L.-3-methyIhistidyl-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 palladium black on the in example 1. d) subject to the manner described. Hereby, the desired compound was obtained as a hydrate in one yield received by 92%. [«]? = -30.8 ° (c = 1.30 in DMF).

Claims (3)

Patent claims: 1. N-Benzyloxycarbonyl-L-pyroglutamyl-L-hisUdyl-L-prolinamide (1) and N-benzyloxycarbonylu - pyroglutamyl - L - 3 - methylhistidyl - L - prolinamide (II). 2. Process for the preparation of TRF or 3-Me-TRF, characterized in that one in a manner known per se, a solution of the compound I or II in an inert solvent with gaseous hydrogen at a temperature of 15 to 55 ° C in the presence of 2 to 50 percent by weight finely divided palladium, based on the weight of the compound, hydrogenated. 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.