DE2760007C2 - - Google Patents

Description

The invention relates to a further embodiment of the 3-amino 2-hydroxy-4-phenylbutanoic acid derivatives of those given below general formula (I) according to patent 26 60 626 and a process for their manufacture, the valuable intermediates for the production of peptides, in particular To represent bestatin derivatives of the general formula:

Subject of the patent 26 60 626 are 3-amino-2-hydroxy-4- phenyl-butanoic acid derivatives of the general formula

with the following configuration

C-2C-3

RSRS RSS RSR SS SR

and the meanings:

R1 hydrogen, 2-chloro, 4-chloro or 4-methyl and R2 hydrogen, benzyloxycarbonyl or t-butoxycarbonyl.

The other described in this additional application Formation of 3-amino-2-hydroxy-4-phenyl-butanoic acid Derivatives of the general formula (I) given above is characterized in that R¹ is hydroxy or known per se Protected hydroxy and R² signify hydrogen and the configuration at the 2-C atom (S) or (RS), the amino group in can be protected in a manner known per se.

A particularly preferred 3-amino-2-hydroxy-4-phenyl-butanoic acid Derivative of the general formula given above (I) is the compound (2S, 3R) -3-amino-2-hydroxy-4-p- hydroxyphenylbutanoic acid.

The compounds of the above specified according to the invention general formula (I) represent valuable intermediates for the production of peptides, especially bestatin and related compounds, the aminopeptidase B, Inhibit leucine aminopeptidase and bleomycin hydrolase, which Anti-tumor effects of bleomycin intensify and anti-fertility effects have.  

Bestatin is (2S, 3R) -3-amino-2-hydroxy- 4-phenyl-butanoyl- (S) -leucine of the general given above Formula (I '), wherein R₁ is benzyl and R₂ isobutyl. This class of compounds including bestatin itself can from a bred medium from a bestatin forming mushroom, which belongs to the Actinomycetes will.

The invention further relates to a method for the production the 3-amino-2-hydroxy-4-phenyl-butanoic acid derivatives of the general formula (I) given above, which thereby is characterized in that a nitrile of the general formula

wherein R¹ and R² have the meanings given for the formula (I) have, in a manner known per se with an acid hydrolyzed and, if desired, the amino and / or Protects hydroxy groups in a manner known per se.

In the implementation of the method according to the invention used nitrile of the general formula given above (II) it is preferably 3-amino-2-hydroxy-4- p-hydroxyphenyl-butyronitrile.

To protect the amino groups, any amino protective groups, as usually used in peptide chemistry will be used. Preferred examples of amino protecting groups of the acyl type are a formyl group, an acetyl group, a trifluoroacetyl group and a substituted or unsubstituted benzoyl group. Examples of amino protecting groups Urethane type are substituted or unsubstituted  Benzyloxycarbonyl, cycloalkanoxycarbonyl and Alkoxycarbonyl groups with 1 to 6 carbon atoms, in particular the t-butoxy carbonyl group. Preferred examples for other amino protecting groups are substituted or unsubstituted Arylsulfonyl groups, a phthalyl group, a o-nitrophenylsulfenyl group and a trityl group.

In the method according to the invention can be carried out any acids are used in hydrolysis, as usually used for the hydrolysis of nitriles will. Examples of usable inorganic acids are hydrochloric acid, hydrobromic acid and sulfuric acid; Examples of usable organic acids are Alkyl sulfonic acids, such as methanesulfonic acid and ethanesulfonic acid, Aryl sulfonic acids such as benzenesulfonic acid and toluenesulfonic acid. These acids can be used in any concentration as usual for the hydrolysis of ordinary nitriles is. However, a concentration above 1n is preferred.

The compounds whose amino group is protected and the can not easily dissolve in aqueous acid solution organic solvent that is miscible with water, such as Tetrahydrofuran, dioxane, lower alcohols, acetone-dimethylformamide, Dimethylacetamide or dimethyl sulfoxide, added are used to improve the solubility of the compounds in the aqueous solution.

If a protective group is used and becomes the protective group during hydrolysis to form electrophilic species removed, a cation capture agent such as anisole can be added will. The hydrolysis temperature can be within the Range from room temperature to reflux temperature of the reaction mixture.

To isolate the desired amino acid from the reaction mixture can be a common method for isolating  Amino acids can be applied. For example, if Hydrolysis can be carried out with a volatile acid excess acid by concentration of the reaction mixture be removed at reduced pressure. The residue can be dissolved in water and with an aqueous alkali solution be neutralized until the isoelectric point is reached is. Optionally, acetone, methanol or ethanol to crystallize the desired compound of the formula (I) are added to the reaction mixture. These The connection is then filtered off. On the other hand, becomes a non-volatile acid is used, so the reaction mixture diluted with water to an acidity of less than 1n and are passed through a strongly acidic ion exchange resin be so that the desired amino acid on the Ion exchange resin is adsorbed. The adsorbed amino acid is from the ion exchange resin with a volatile Alkali such as ammoniacal water eluted and reduced Pressure condensed. If necessary, acetone, Methanol or ethanol to crystallize the desired one Compound of formula (I) added. This connection will then filtered off.

If an (R) -nitrile derivative is used in the process according to the invention is used as the starting material, a (2RS, 3R) - Received amino acid; an (S) -nitrile derivative is used, a (2RS, 3S) amino acid is thus obtained; becomes an (RS) - Nitrile derivative is used, then a (2RS, 3RS) amino acid receive.

The compounds of formula (I) obtained can be used as Starting material in the process described below be used. For example, 3-amino-2-hydroxy-4- phenylbutanoic acid (hereinafter abbreviated as AHPA) is a precursor by Bestatin in the (2S, 3R) form. To make this Connection is therefore required to (2RS, 3R) -AHPA in Split (2S, 3R) - and (2R, 3R) -AHPA.  

(2RS, 3R) -AHPA in diastereoisomeric form can be found in ethyl acetate split into the optical antipodes. These will obtained by first (2RS, 3R) -AHPA in the usual way producing benzyloxycarbonyl- (2RS, 3R) -AHPS the Subjects to benzyloxycarbonylation and the latter compound then reacted with brucine (hereinafter the benzyloxycarbonyl group abbreviated as Z).

Z- (2RS, 3R) -AHPA and an equivalent or slightly larger Amount of brucine is dissolved in ethyl acetate with warming and then optionally filtered. When the filtrate cools down or is cooled, crystals are deposited. These crystals are filtered off and, if necessary, a or several times from ethyl acetate for the preparation of the optically pure brucine salt of Z- (2S, 3R) -AHPA recrystallized. If this brucine salt is treated in the usual way, then you get optically pure Z- (2S, 3R) -AHPA.

Otherwise optically pure Z- (2R, 3R) -AHPA can be produced as follows be: a solution of ethyl acetate containing a brucine salt of Z- (2R, 3R) -AHPA and a small amount of Z- (2S, 3R) - AHPA contains is diluted with hydrochloric acid Removal of brucine shaken. The ethyl acetate layer with a drainage agent, such as anhydrous sodium sulfate, dried and concentrated under reduced pressure. The residue is then in a small amount of ethyl acetate solved. After adding petroleum ether, crystals are obtained, which filters to produce optically pure Z- (2R, 3R) -AHPA can be.

The (2R, 3R) form of the Z-AHPA has a lower solubility in an organic solvent as the (2S, 3R) form. Z- (2RS, 3R) -AHPA is first in a solvent in which it is soluble, dissolved at room temperature or with heating. Then a second solvent in which it is insoluble is added to precipitate the crystals. These will  filtered off. If necessary, the previously described Repeat the process until optically pure Z- (2R, 3R) -AHPA is obtained becomes.

As the first solvent in which the compounds are soluble, can be used: lower alcohols, such as methanol, ethanol or propanol; Ether, such as diethyl ether, diisopropyl ether, Tetrahydrofuran or dioxane; Esters, such as methyl acetate or Ethyl acetate; Ketones such as acetone or methyl ethyl ketone; halogenated Hydrocarbons such as methylene chloride or chloroform; Amides such as dimethylformamide or dimethylacetamide; and Nitriles such as acetonitrile.

As a second solvent in which the compounds are insoluble you can use: petroleum hydrocarbons like Petroleum ether, petrolbenzene or ligroin; aromatic hydrocarbons, such as benzene or toluene; Alkanes, such as pentane or Hexane; and cycloalkanes such as cyclopentane or cyclohexane. At Application of this diastereoisomer method in which brucine used, the optically impure Z- (2S, 3R) -AHPA, which in the filtrate is too optically pure (2S, 3R) -AHPA getting cleaned.

Z-AHPA, which is used in the splitting process described above can be used in accordance with the Schotten-Baumann method Presence of an alkali by reacting (2RS, 3R) -AHPA and benzyloxycarbonyl chloride or otherwise in the presence an organic tertiary base such as triethylamine and N-methylmorpholine, by reaction with a benzyloxycarbonylation reagent, such as benzyloxycarbonyl-p-nitrophenyl ester, Benzyloxycarbonyl azide, benzyloxycarbonyl-N-hydroxysuccinimide ester and benzyloxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine, in a solvent made by mixing dioxane, Tetrahydrofuran, acetonitrile or dimethylformamide with water is produced, obtained.  

Although the optical splitting method described above with regard to AHPA can also be described with the other amino acids performed a similar optical splitting will.

For the production of bestatin and its related compounds, represented by the formula (I ′) becomes a Amino acid as represented by formula (I) with a compound of formula (IV) H₂N-CHR₂-COOH, wherein R₂ has the meaning given above, condensed. To one uses a common peptide condensation method, and then the protecting groups, if any to protect functional groups that are not involved in the reaction participate, have been used, removed again.

The process for the condensation of an amino acid of formula (I) with an amino acid of formula (IV) can be a carbodiimide method be in which dicyclohexylcarbodiimide or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide is used; one can also use the azide process, in which nitrous acid or alkyl nitrite is used; you can also use a mixed one Anhydride process in which ethyl chloroformate or isobutyl chloroformate is used, apply; it can be an active one Ester process in which cyanomethyl esters, vinyl esters, are substituted and unsubstituted phenyl esters, thiophenyl esters or N-hydroxysuccinimide esters can be used; an O-acylhydroxylamine derivative Process in which O-acylacetoxime or O-acylcyclohexanone oxime is used, or an N-acyl derivative Process using carbonyldiimidazole applied will.

Organic solvents used in the above condensation reaction can be used ether, such as diethyl ether, Tetrahydrofuran or dioxane; Esters, such as ethyl acetate or methyl acetate; Ketones such as acetone or methyl ethyl ketone; halogenated hydrocarbons, such as methylene chloride or  Chloroform; Amides such as dimethylformamide or dimethylacetamide; Nitriles such as acetonitrile.

When an amino acid of formula (I), its amino group is protected and an amino acid of formula (IV) whose Carboxyl group is not protected according to the active ester process can be condensed, a solvent mixture Water with a water-miscible organic solvent in the presence of an inorganic base such as sodium carbonate or magnesium oxide, an organic tertiary base, such as triethylamine or N-methylmorpholine, can be used.

Protecting groups in the resulting compounds can usual methods of peptide chemistry are removed, e.g. B. by catalytic hydrogenation with palladium, saponification with an alkali, acid splitting with hydrogen bromide in acetic acid, with trifluoroacetic acid, with hydrogen chloride in dioxane, tetrahydrofuran or ethyl acetate, with liquid hydrogen fluoride, Hydrazinolysis with hydrazine or treatment with sodium and liquid ammonia. The desired connection is thus obtained of the formula (I ').

The nitrile derivatives represented by the formula (II) and which are used as starting material in the process according to the invention can be obtained by reducing an α- amino acid whose amino group is protected and whose carboxyl group has been converted into the amide by reaction with a secondary amine at low temperatures below 0 ° C with a metal hydride in ethers, such as diethyl ether or tetrahydrofuran, to produce an amino aldehyde, the amino group of which is protected. The resulting amino aldehyde is then converted to the adduct with sodium bisulfite and then allowed to react with an alkali metal cyanide, or it can react directly with hydrogen cyanide to give cyanohydrin or 3-amino-2-hydroxynitrile.

Preferred examples of the secondary amine to be used are N, N-dimethylamine, aziridine, N-methylaniline, carbazole, 3,5-dimethylpyrazole and imidazole. Preferred examples of the metal hydride that can be used are lithium aluminum hydride, Lithium di and tri alkoxy aluminum hydride and sodium bis (2-methoxyethoxy) aluminum hydride. Examples of protection Groups for the amino group are the protecting groups that commonly used in peptide chemistry. Prefers protecting groups of the urethane type are used, and most preferred are benzyloxycarbonyl groups.

According to a second method, a lower alkyl ester of an α- amino acid, the amino group of which is protected by an amino protecting group, is similar, as mentioned above, at a temperature below -40 ° C. in aromatic hydrocarbons, such as benzene or toluene, and ethers, diethyl ether or tetrahydrofuran, with metal hydrides, such as sodium aluminum hydride, diisobutyl aluminum hydride or sodium bis (2-methoxyethoxy) aluminum hydride. An amino aldehyde with a protected amino group is obtained. If such an amino aldehyde is treated as described above, 3-amino-2-hydroxynitrile is obtained.

The invention is illustrated by the following examples. The abbreviations used therein have the following Meanings: HOBt = N-hydroxybenzotriazole; DCCD = N, N'-dicyclohexylcarbodiimide; -OBzl · TosOH = benzyl ester- p-toluenesulfonate; DCHA = dicyclohexylamine; -OBut = t-butyl ester; AHPA = 3-amino-2-hydroxy-4-phenylbutanoic acid, Z = benzyloxycarbonyl; Ac = acetyl. The Rf values were with silica gel plates using n-BuOH: AcOH: H₂O (4: 1: 1) determined as a developing solvent.  

Example 1 Step 1

22.0 g of oily Z- (2RS, 3RS) amino-2-hydroxy-4-p-methoxyphenylbutyronitrile were in a mixture of 200 ml concentrated hydrochloric acid and 200 ml of dioxane. After the addition of 13.2 g of anisole, the reaction mixture Heated under reflux for 12 hours. Then was the dioxane distilled off under reduced pressure, and the resulting hydrochloric acid solution was washed with ether. The Water layer was concentrated under reduced pressure and evaporated to dryness. Then 100 ml of water added to the remaining substance, and the insoluble Substance was separated by filtration. To Adding an equal amount of acetone to the pH the mixture was adjusted to 5.5 with ammonia water. The Mixture was left in a refrigerator. The deposited crystals were by filtration separated. 6.73 g of the desired (2RS, 3RS) - AHPA (p-OH) obtained.

Level 2

2.11 g (2RS, 3RS) -AHPA (p-OH) obtained in Step 1 were obtained dissolved in 10 ml of 1N sodium hydroxide solution. Under vigorous stirring of the solution and cooling with ice 4.5 ml of Z-Cl were added in three portions over 30 minutes. The reaction mixture was then under ice cooling for 1 hour and then stirred vigorously for 3 h at room temperature. During the reaction the pH was adjusted with a 1n Sodium hydroxide solution adjusted to 8 to 9.

After the reaction had ended, 6N hydrochloric acid was added, to adjust the pH of the reaction mixture to 2. As a result, an oily material separated out twice  was extracted with 100 ml of ethyl acetate. The ethyl acetate layer was washed with water and using anhydrous Magnesium sulfate dried. After filtering off of the magnesium sulfate, the filtrate was reduced Pressure was reduced and the residue was removed Ethyl acetate / petroleum ether crystallized, giving 3.64 g Z- (2RS, 3RS) -AHPA (p-OZ), mp 138 to 140 ° C.

Level 3

479 mg Z- (2RS, 3RS) -AHPA (p-OZ) 162 mg HOBt were dissolved in 10 ml tetrahydrofuran. After the addition of 472 mg of Leu-OBzl-TosOH, the mixture was neutralized with 0.168 ml of triethylamine and cooled to -5 ° C. Then 206 mg of DCCD was added and the reaction mixture was left to react overnight. Tetrahydrofuran was distilled off under reduced pressure and 30 ml of ethyl acetate was added. After filtering off insoluble substances, the filtrate was washed with 1N sulfuric acid, water, 5% aqueous sodium bicarbonate solution and water in that order, and then dried with anhydrous magnesium sulfate. The residue, obtained by concentrating the filtrate under reduced pressure, solidified in ethyl acetate / petroleum ether. Recrystallization from the same solvent gave 450 mg of Z- (2RS, 3RS) -AHPA (OZ) - (S) -Leu-OBzl, mp 98 to 99 ° C [ α ] -14.0 ° ( c = 0, 58, AcOH).

Level 4

400 mg of Z- (2RS, 3RS) -AHPA (p-OZ) - (S) -Leu-OBzl was added in 10 ml Dissolved methanol and with about 10 mg of palladium black for 3 hours hydrated. The catalyst was filtered off and the solvent was concentrated under reduced pressure. At Carry out a recrystallization with methanol / ethyl acetate 219 mg (2RS, 3RS) -3-amino-2-hydroxy-4-p-hydroxyphenylbutanoyl-  (S) -leucine obtained.

[ α ] -8.8 ° ( c = 0.90, AcOH), Rf 0.48 and 0.51.

Analysis for C₁₆H₂₄N₂O₅:
Found: C 59.38 H 7.23 N 8.95%; calc .: C 59.24 H 7.46 N 8.64%.

Example 2

When treating 30 g of Z- (2RS, 3R) -3-amino-2-hydroxy- 4-p-hydroxyphenylbutyronitrile in a similar manner as in Example 1, stage 1, 12.61 g (2RS, 3R) -AHPA (p-OH) receive.

Rf 0.20.

Analysis for C₁₀H₁₂NO₄:
Found: C 58.63 H 5.92 N 7.43%; calc .: C 58.81 H 5.92 N 7.82%.

If (2RS, 3R) -AHPA (p-OH) using benzyl- S-4,6-dimethylpyrimidin-2-ylthiol carbonate, benzyloxycarbonylated then Z-AHPA (p-OH) was obtained as the DCHA salt.

15.22 g of crude DCHA salt was extracted from methanol, ethyl acetate and Petroleum ether crystallizes. 3.2 g optically impure Z- (2R, 3R) -AHPA (p-OH) -DCHA salt were the first yields receive.

When the mother liquor was evaporated to dryness and the residue was precipitated three times from ethyl acetate and ether, 5.02 g of optically pure Z- (2S, 3R) - AHPA (p-OH) -DCHA salt were obtained.
M. 121 to 122 ° C [ a ] + 49.9 ° ( c = 0.87, AcOH).

Analysis for C₃₀H₄₂N₂O₆:
Found: C 69.81 H 8.35 N 6.42%; calc .: C 69.46 H 8.16 N 6.17%.

After treatment with Z- (2S, 3R) -AHPA (p-OH) -DCHA salt (1.05 g) with ethyl acetate and dilute H₂SO₄ the Z- (2S, 3R) -AHPA) p-OH) obtained, 866 mg (S) -Leu-OBzl. TosOH, 405 mg HOBt, 0.308 ml triethylamine and 412 mg DCCD treated in a manner similar to Example 1, Level 3. Oily Z- (2S, 3R) -AHPA (p-OH) - (S) -Leu-OBzl was obtained quantitatively.

If the oily Z- (2S, 3R) -AHPA (p-OH) - (S) -Leu- OBzl treated in a similar manner as in Example 1, Level 4 then 630 mg (2S, 3R) -3-amino-2-hydroxy- 4-p-hydroxyphenylbutanol- (S) -leucine obtained.

[ α ] -19.9 ° (c = 1.19, AcOH); Rf 0.48.

Analysis for C₁₆H₂₃N₂O₅:
Found: C 59.98 H 7.42 N10.42%; calc .: C 60.55 H 7.30 N 10.08%.

Claims (3)

1. Further training of the 3-amino-2-hydroxy-4-phenyl-butanoic acid derivatives of the general formula with the following configuration anC-2C-3RSRS RSS RSR SS SR and the meanings: R¹hydrogen, 2-chlorine, 4-chlorine or 4-methyl, R²hydrogen, benzyloxycarbonyl or t-butoxycarbonyl, according to patent 26 60 626, characterized in that R¹ for hydroxy or in a manner known per se protected hydroxy and R 2 are hydrogen and the configuration on the 2-C atom is (S) or (RS), where the amino group can be protected in a manner known per se. 2. (2S, 3R) -3-amino-2-hydroxy-4-p-hydroxyphenylbutanoic acid. 3. A process for the preparation of the 3-amino-2-hydroxy-4-phenyl-butanoic acid derivatives of the general formula (I) given in claim 1, characterized in that a nitrile of the general formula wherein R¹ and R² have the meanings given in claim 1, hydrolyzed in a manner known per se with an acid and, if desired, protects the amino and / or hydroxyl groups in a manner known per se.