CS201033B2 - Method of producing derivatives of n-/3-amino-2-hydroxy-propionyl/-2-amino acetic acid - Google Patents

Abstract

For preparing the novel bestatin compounds of the formula <IMAGE> having R1 and R2 in accordance with the preceding Patent Claim 1, an alpha -hydroxybeta -amino acid of the formula <IMAGE> is used as the starting material. The latter is condensed, optionally after having protected the functional groups which are not to participate in the subsequent reaction, with a beta -amino acid of the formula <IMAGE> using a peptide-linking method. Protective groups which may optionally be present are subsequently removed. The optically pure diastereoisomers can be readily obtained from the compounds of the formula (III). The alpha -hydroxybeta -amino acids can be prepared by hydrolysing a corresponding cyanohydrin whose amino group is optionally protected. The novel compounds inhibit certain enzymes which decompose bleomycin and in this way amplify the anti-tumour activity of the bleomycins.

Description

The present invention relates to a process for the production of bestatin and related compounds which inhibit aminopeptidase B, leucine-aminopeptidase and bleomycin-1-ethylollase, which increase the antitumor activity of bleomysine as well as activity against fertilization.

An object of the invention is a process for the preparation of compounds of the formula I

2 1 2'T

R-CH-CH-CO-NH-CH-COOH ¹ III

NH 2 OHR 2 (I) wherein R 1 is alkyl of 1 to 4 carbon atoms, cyclopentyl methylC, cyclohexylmethyl, cyclohexylmethyl, phenyl and a substituent of the formula

R ³

(II) wherein R is hydrogen, chlorine, methyl, nitro, hydroxyl or and R8 is C1 -C6 alkyl, C1 -C2 hydroxyalkyl, C2 -C3 alkoxymethyl, C2 -C3 alkylthiomethyl carbon atoms, carboxyamidoalkyl of 2 to 3 carbon atoms, carboxyelkyl of 2 to 3 carbon atoms.

Bessatin is (2S, 3R) -3-phenoxy-2-hydroxy-4-phenylbutanoyl- (S)) leucine, the structure of which is represented by the above general formula I, wherein R 1 is benzyl and R 8 is isobutyl.

A group of compounds, including bestatin, which can be prepared from the culture medium of beetatin producing fungin and belonging to actinomycetes, are novel substances and are characterized by the aforementioned physiological activities.

The process for the preparation of compounds of the formula I is characterized by condensing an amino acid of the formula II

NH 2 OH (II) wherein R 1 is as defined above, optionally with protected functional groups that do not participate in the next reaction, and an amino acid of formula III

NHZ ~ Ooh CH ² I (III) wherein R is as defined above under general procedures poožití peptide chemistry. The compounds of formula I are obtained after removal of any protecting groups.

The aminocapsseins of formula II are obtained by acid tydrolysis from the nitriles of formula IV

NH 2 OH (IV) wherein R 1 is as defined above or from these protected amino nitriles.

The invention is illustrated in more detail in the following description, first of all the preparation of the aminookesein of formula II which serves as starting compounds in the process according to the invention.

Typical examples of nitriles in formula IV are nitrile of 3-amino-2-hydroxypropionic acid, nitrile of 3-amino-2-hydroxy-5-methylhexanoic acid, nitrile of 3-amino-2-hydroxy-3- flnylprkpiknkvé, nitrite ester 3-2-tmink tydroэχ-4-p-chlkrfenylmáselné acid nitrile, 3-amink-2-hydroxyethyl; _l iq r-4-phenylbutyric acid nitrile, 3-amnnk - hyddooχ-44-to-hlooflnnkmásslné , 3-amino-2-tyrosine-r-4-p-methylphenylbutyric acid nitrile, p-methocyclohexylbutyric acid nitrile, p-hydroxyphenylbutyric acid nitrile, and 3-amino-2-hydroxy-4-p-benzyloxyphenylbutyric acid nitrile.

For the protection of the amino group, a suitable amino-protecting group may be used, as is known in the art of chemistry.

Preferred examples of acyl groups for the protection of the amino group include phymyl, allyl, trifluoromethyl, benzyl, optionally substituted, and examples of the uglyethenyl-type protecting groups for Βΐηΐηοβ ^ ρ! ^ Include optionally substituted b ^ z ^ a!; a C 2 -C 7 alkyl group, an C 2 -C 7 alkyl group, and a C 1 -C 7 cycloalkyl group.

Preferred examples of other protecting groups useful for the protection of the amino group include those optionally substituted by a phosphorous group, a k-nitrophenyl group or a U-group.

Any of the acids commonly used in the hydrolysis of nitriles can be used in the hydrolysis. Examples of inorganic acids are hydrochloric, hydrobromic or sulfuric acids, and examples of organic acids include alkyl succinic acids such as mmone than sipphonic acid, ethanesulfonic acid, arylsulfonic acids such as benzenesulfonic acid and phosphonic acid. .

The concentration of these acids may be in the range if it is suitable for the hydrolysis of conventional nitriles. Concentrations above 1 N are preferred.

For amino-protected compounds that are reluctant to dissolve in aqueous acid solutions, a water miscible organic solvent such as tetrahydrofuran, dioxane, lower alcohols, acetone, dimethylformamide, dimethylacetimide and dimethyl sulfoxide may be added. This increases the solubility of the compounds in aqueous suspensions.

If a protecting group is used, and if the protecting group is removed by hydrolysis to form electrophilic materials, a cation-binding agent such as aii-sol is added. The hydrolysis temperature may range from room temperature to the boiling point of the reaction mixture. under reflux. Any of the usual procedures for such isolation can be used to isolate the expected amino acid from the reaction mixture.

For example, the volatile acid hydrolysis is carried out, then the excess acid used can be reduced by concentrating the reaction mixture under reduced pressure, and then the residue is dissolved in water and the solution is not naturalized with an aqueous solution of the base until isopropyl alcohol is obtained. point. Further, acetone, methanol or ethanol is added to the reaction mixture, if desired, thereby catalysing the compound of formula XI, which is then filtered off.

Alternatively, using a nonvolatile acid, the reaction mixture is diluted with water to a concentration of less than 1 N, the solution is poured over a strongly acidic ion exchange resin, and the desired amino acid is adsorbed onto the ion exchange resin. The adsorbed acid is then eluted from the ion exchange resin with a volatile base such as ammonia, optionally further diluted with water, and the solution te under reduced pressure. If necessary, acetone, methanol or ethanol is added to aid in the crystallization of the product, i.e. the compound of formula XI, which is then diluted.

The reaction is obtained by using the (R-nitrile) derivative as the starting compound (2RS, ZR) - β-nitrileine, when (S) - n-nitil is used, (2RS, 33) - γ-β) is obtained; when the derivative (RS-nitrile) is obtained (2RS-3RS.

The acids of the formula II obtained can be used in the crude state as starting materials in the following process. For example, 3-amino-2-hydroxy-4-phenylbutyric acid, which is the starting material for the preparation of bestatin, is in the form of (2S, 3R). Thus, in order to obtain this compound, (2RS, 3R) -3-taninion-2-oxo-4-phenylbenzylic acid must be cleaved to form (2S, 3R) - and (2R, 3R) -.

(2RS, 3R) -3-Amino-2-hydroxy} -4-benzenesulfonic acid is resolved into the optical isomers in ethyl acetate in the form of the Siaetherereomer, which is obtained first by benzyloxycarbonylation (2RS, ZR) -3-amino-2-hydroxys4 phenylbutylic acid to form benzyloxycarbonyl- (2RS, 3R) -3-aminio-2-hydroxysulf-phenylbutanoic acid, which is then reacted with brucine. More precisely, dissolving under heating in acetic acid, benzsloxycarbunyl (2RS, 3R) -3-amino-2- (4-phenylbutanoic), and brucine equivalent, or a small excess thereof, and the solution is dissolved. optionally filtered.

Upon cooling or cooling of the filtrate, crystals are precipitated which are filtered and, if necessary, repeated filtration from acetic acid until an optically pure salt of brucine with carbonic acid is obtained. (3R) -3-Amino-2-hydroxy-phenylbutanone. Conventional treatment of this brucine salt further provides optically pure benzyloxycarbonyl-1- (2S, 3R) -3-amino-2-hydroxy-4-phenylbutanoic acid.

Alternatively, the optically pure benzoic flux of carbon (2R, 3R) -3- and 4R-2-hexanoic acid can be prepared as follows:

Brucine salt solution benzxloxxkarbonxln acid (2R, 3R) -3-amino-2-hydroxy} χs4_f iylbutanové in _e - ^ χΙ ^ - ™ acetic obsaHnuící small application rates kxseliny benzχluxχkaгbonχl- (2S, 3R) -3-amino-2 The brucine was freed from the brucine by shaking with a dilute hydrochloric acid solution, then the acetic acid layer was dried with a dehydrating agent such as anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was dissolved in a small amount of ethyl acetate. Addition of petroleum ether precipitated crystals, and filtered to afford pure benzyloxycarbonyl- (2R, 3R) -3-amino-2-hydroxy-4-phenylbutanoic acid.

The (2R, 3R) -benzyloxycarbonyl-3-amino-2-hydroxy-4-phenylbutanoic acid form was found to be less soluble in organic solvents than the corresponding (2S, 3R) -form · Benzyloxycarbonyl- (2RS, 3R) - The 3-amino-2-hydroxy-4-phenylbutanoate is first dissolved in a solvent at room temperature or with heating and crystals are precipitated by the addition of a solvent which does not dissolve the solute, which can be separated by filtration. Alternatively, this process is repeated until optically pure benzyloxycarbonyl- (2R, 3R) -3-amino-2-hydroxy-4-phenylbutanoic acid is obtained.

The first solvent in which the substance dissolves may be one of the lower alcohols, such as methanol, ethanol and propanol, and one of the ethers, such as diethyl ether, diisopropyl ether, tetrahydrofuran and dioxane, followed by an ester group such as acetic acid methyl or ethyl ester, ketones such as acetone and methyl ethyl ketone, halogenated hydrocarbon derivatives such as methylene chloride and chloroform, amides such as dimethylformamide and dimethylacetamide, and finally one of the nitriles such as acetonitrile etc.

The second solvent which is diluted to precipitate the product is petroleum hydrocarbon fractions such as petroleum ether, gasoline and ligroin, aromatic hydrocarbons such as benzene and toluene, alkanes such as pentane and hexane, cycloalkanes such as cyclopentane and the like. cyclohexane. Using the diastereoisomer-brucine procedure, the filtrate 8 can be purified from the filtrate with impure benzyloxycarbonyl- (2S, 3R) -3-amino-2-hydroxy-4-phenylbutanoic acid to give optically pure (2S, 3R) -3-amino-2 acid. -hydroxy-4-phenylbutane.

The benzyloxycarbonyl-3-amino-2-hydroxy-4-phenylbutanoic acid used in the above-mentioned optical antipode cleavage can be synthesized using the Schotten and Baumann procedure by reaction of (2RS, 3R) -3-amino-2- hydroxy-4-phenylbutanoic with benzyloxycarbonyl chloride in the presence of a base or in the presence of a tertiary amine such as triethylamine or N-methylmorpholine, in which case benzyloxycarbonyl-p-nitrophenyl ester, benzyloxycarbonylazide, benzyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxycarbonyloxy 6-dimethyl-2-mercaptopyrimidine in a solvent which is a mixture of dioxane, tetrahydrofuran, acetonitrile or dimethylformamide with water.

Although the above-mentioned resolution to the optical antipodes is described 8 with respect to 3-amino-2-hydroxy-4-phenylbutanoic acid, a similar procedure may be used, if desired, for other amino acids.

In the preparation of bestatin and similar compounds of formula I, the amino acid of formula II is condensed with a compound of formula III using a procedure commonly used to rank amino acids in peptide chemistry, and then deprotecting any protecting groups which may have been used to protect them. functional groups that did not participate in the actual reaction.

Typical examples of amino acids of formula III include glycine, alanine, 2-aminobutyric acid, valine, norvaline, leucine, norleucine, isoleucine, tert.leucine, 2-aminoheptanoic acid, 2-amino-5-methylhexanoic acid, 2- aminoctanoic acid, 2-amino-6-methylheptanoic acid, serine, threonine, allothreonine, cysteine, homocysteine, asparagine, glutamine, O-methylserine, O-ethylserine, O-propylserine, methionine, ethionine, aspartic acid, glutamic acid, phenylglycine, p -methoxyphenylglycine, phenylalanine, tyrosine, p-methoxyphenylalanine and p-nitrophenylalanine.

Methods for coupling the amino acid of formula II with the amino acid of formula III include the carbodiimide procedure using either dicyclohexylcarbodiimide or 1-ethyl-3- (3-dimethylthiopropyl) carbodiimide.

Further, an azide process using nitrous acid or an alkyl ester thereof, a mixed anhydride process using ethyl chloroformate or isobutyl chloroformate, a process using active esters such as a cyanomethyl ester, a phenyl ester, optionally substituted thiophenyl ester may be used. or the N-hydroxy-8'-imido ester, the O-acyl hydroxylamine derivative, such as O-acylacetoxime or O-acylcyclohexanone oxime, and the N-acyl derivative procedure using carbohydrate. lů.

Among the organic solvents useful in the above condensation reaction are ethers such as diethyl ether, tris (hlsdrefuran or dioxane), esters such as ethyl or methylsulfate, ketones such as acetone or methyl ethyl ketone, halogenated hydrocarbon derivatives such as is or chloroform, amides such as dimethylfomamide or dimethylacetemide, and nitriles such as acetonitrile.

In the case of the condensation of an amino-protected amino group II with an amino protected formula and an amino acid III with an unprotected carboxyl group using active esters, a solvent may be a mixture of water and methanolic solvent with water in the presence of an inorganic base such as sodium hydrogen carbonate or magnesium oxide; or in the presence of an organic tertiary amine, such as or N-methylmoefficient.

Possible protecting groups can be removed from the resulting product using conventional techniques in peptide chemistry such as catalytic palladium hydrogenation, alkaline saponification, hydrogen bromide acid acetic acid, reaction with trifluoroacetic acid, with hydrogen chloride in dioxane, tetrahydrofuran or ethyl acetate, reaction with liquid in the form of hydrazinolysis by treatment with hydrazine or by reaction with sodium in water. liquid amoonac. This yields the expected compound of formula I.

The derivative of formula (IV) is prepared by reducing the alpha-amino acid, its amino group is protected, and the carboxyl group is transferred to the amide group, by reaction with a secondary amine at a temperature below 0 ° C and a metal hydride in ether such as 1β1Ьу1п11пг or tstrahsdгefuгau. amino-protected aminoaldehyde. The resulting amino aldehyde is converted into an addict with sodium hydrogen sulphite and then further reacted with alkali metal cyanide or directly with hydrogen cyanide to form cyanohydrin or 3-amino-2-hydroxynitrile.

Examples of preferred secondary amines are Ν, Ν-dimethyl amine, aziridine, carbazole, 3,5-dimethylpyrazole and imidazole. Examples of preferred metal hydrides are the hydride of hydride, l-trimethyl-oxyalminium-hydride, l-trimethyl-alkoxy-lithium-fluoride and uatrimbis- (2-methoxyethexyl) -amino-sulfide.

An example of an amino protecting group is: of protecting groups usually used in peptide chemistry. Nitrile-type protecting groups are preferably used, in particular a benzyloxycarbenyl group is preferred.

According to a second possible method, the amino group in the lower alkyl-alpha-1-amino acids is protected with a suitable protecting group, such as any of the above, to reduce the amino-protected product at a temperature below -40 ° C in a ruthenium hydrocarbon environment such as toluene or benzene or one of the ethers, such as dimethyl or tetrahydrofuran, by treatment with a metal hydride such as aluminum hydride (diisobutylaluminium hydride or sodium methacrylamide) with such amino amine hydride to form the aminoaldehyde with the aminoaldehyde formed. as mentioned above, 3-amino-2-hydro-sulfonyl is obtained.

б

The physiological activities of bestatin and the corresponding compounds of the formula I according to the invention are as follows: * (A) Inhibitory activity against aminopoeidase B

Test procedure ':

The method was described by K. Hopusu, K.K. Makinen, G.G. Glenner and Arch. Biochey. Biophys. 114, 557 (1966). To a mixture of 0.3 ml of a 1 mM arginine beta-naphthylmide solution and 1.0 ml of 0.01 M tris-buffer (pH 7.0), with or without the product to be tested, is added 0.7 ml of distilled water, and was heated at 37 ° C for 3 minutes.

The reaction is initiated by the addition of 0.2 ml aminopolyidase B solution, killing chromatosp'afil on sefadex G 100 as described by Hopus and spyl. (see above). After 30 minutes of storage at 37 ° C, 0.6 ml of 1.0 M acetate buffer (pH 4.2) containing 8 mg of the diazonium salt of o-aminoazotoluene at a concentration of 1.0 mg / ml and Tween 20 at a concentration of 1.0% are added. .

After 15 minutes at room temperature, the absorption (a) at 530 nm is measured by spectrophotometer. For control, the absorption (b) is measured in a similar manner after the reaction without the sample itself. The percentage 1у11Ь1о1 is calculated from the equation;

(b-a) / b.100.

Percentage data of the mushroom at various concentrations were measured, and a value of 50% of 1у11Ь1-1 (ΙΏ ^ θ) was derived from the observed% bUblce. The results are shown in Table 1.

Table 1

Inhibitory efficacy of bestatin. and similar compounds against aminopolpidasl B.

substance no. compound ^ID 50 (pg / ml) 1 (2S, 3R · 3-мno-2hlУdosy-4-felyУlbjaaoyl- (S J-loucin) 0.10 2 (2S, 3S) -3-Amino-2- (4-chloro-2-) - (S) -llucin 1,25 3 (2S, 3R) -3-Amino-22-hydroxy-1-amino-22-amino 0.56 4 (2S, 3S) -3-Amino-2-hy (1dosyy-4-phenylbenzylbutyl) - (R) -lljcin 0.04 5 (2S, 3S) -3-emino-2-hydroxy-4-flnylbutanoslglycine 21.5 б (2S, 3R) -3 “amnoo-2 ^ lrУaosУ“ 4-felyУbbjaaoySy (S) -vvlin 0.55 7 (2S, 3R) -3-Amino-2-amino: sy-У (f fθbbbjaloyoy ((S S S S S S S S S S S S) 0.05 8 (2S, 3R) -3-Amino-2-hydroxy-4-hydroxybenzyl) - ((S) -methylsnln) 0.22 9 (2S, 3R) -3-lminoo2-hydose-4-phenylethyl (S) -gl btemine 1,2 10 (2S, 3R) -3-lminys22hlУdooyy44phenyУbbjtnysl- 0.17 11 (2S, 3R) -3-Lminos2- (lrУaosyy44phenyУbbjtalУsУ- (S) -ysrlljcly 0.13 12 (2S, 3R) -3-Aminos-2-hexa-4-phenylbutyl-soyl (S ^^ πιΟ ^ ι ^ ι) 4.2 13 (2S, 3R) -3-Aminos-2hlУdooy-44feeyУbbjalУsУ- (S) -slaiy 0.72 14 (2S, 3R) -3-Aminos-2-γ-5-γ-5-γ-4-γ-butyutyl-γ- (S (-gluulic acid) 25 15 Dec (2S, 3R) -3-Amino-2-hydroxy-2-hydroxy-phenyl-2-amino-octanoic acid 3.1 16 (2RS, 3R) -3-Lminys2-llyido-10-oyypysiosy- 16 17 (2RS, 3R) y3-lmino-lly: dooyy 12 18 (2RS, 3R)) (3-lmiyys-2hlУdУsy-ЗзfelyУpoooOysyl- (S) -Уlbciy) 56 19 Dec (2RS, 3R) -3 ▼ m-22hlУdУo'-У-pochlysfenyУbb unnoS- (S) -Iouci 0.07

substance compound ΙΟ ^ θ (pg / ml)

20 May (2RS, 3R) -3-sclinO2-2-hydroxy-4-oociloorenol8C10J11 (S) - -ležcio 0.48 21 (2RS, 3RS) -3- ()Ilinlinožožožhhhhhhhhececececec) 0.01 22nd (2S, 3R) -3- (C) -in-2-yloxy-4-p-nitroenolbutanol (S) -leucine 0.01 23 (2S, 3R) -3-Amino-2-hydroxy-4-piperazin-1-ol (S) -leucine 0.10 24 (2S, 3R) -3-aminOž2- h ^ S ^{г г} Ooy ---- yУkožeeyllužsclOžУ- (S) -leucine 1.05 25 (2RS, 3R) | -leucine 0.10 26 (2S, 3R) -3-Amino-2-Hydrolines - Hydrolines (S). -J-eucine 0.007

(B) Distribution of bleomycin in organs in the presence of bestatin.

100 mg / kg bleomycin Bg is injected subcutaneously into each of the 3 male ICR δтё! (4 weeks of age), and the like, 100 mg / kg bleomycin Bg and 100 mg / kg bestatin are administered subcutaneously to each of 3 mice of the same age.

One hour after administration, all 6 mice were sacrificed and the amount of bleomycin remaining in serum and organs was determined using a thin layer on a Petri dish using Bacillus subttlis PCI219 as the test microorganism. The results are shown in Table 2; the figures are average values in pg / ml.

Table 2

Effect of concomitant administration of testctia and bleomycin Bg or bleomycin distribution in the. organs bleomycin 100 mg / kg lung serum skin liver kidney

110 11,2 4,5 0 68,0 spleen stomach brain

4.0 1.6 0 bleomrcin 100 mg / kg + piglets ^ 100 mg / kg 140

21,2 42,0

84.0

5.8 2.5 2.4 (C) Contraceptive properties.

Effect of fertility testing ·

Immediately after subcutaneous administration of 10 mg / kg testto females! The ICR JCL puberty in adolescence is placed together with males of similar age. The female mice are sacrificed after mating 2 days after the formation of the vial plug, and eggs are collected to determine whether they have been fertilized or not. Fertilized eggs were from 0 to 16 cells.

201033 8

The group has female N # 2 total N = 2 after beetatin administration

corpus luteim, count 10 19 Dec 29 eggs collected, number 10 19 Dec 29 fertilized eggs 4 (40%) 6 (31.6%) 10 (34.5 «) Unfertilized eggs 6 (60%) 13 (68.4 «) 19 Dec (65.5 ») Console corpus luteim, count 13 13 26 eggs collected, number 9 11 20 May fertilized eggs 6 (66.7 «) 8 (72.7 «) 14 (70 «) Unfertilized eggs 3 (33.3%) 3 (27.3 «) 6 (30 «)

From the experiment. it can be seen that the fertilization of eggs collected from mice and copulations 4 to 16 hours after the administration of bestatin corresponds to 34.5%, whereas the control group is 70. OF. These results show that bestatine is characterized by an action against fertilization.

(D) Effect. (2RS, 3IR:>) - 3-Amino-2-fluoro-4β-hykr ₀ -o-β-β-β-β-leucine for humoral production of antibodies to sheep red blood cells.

(2RS, 3R) - 3-Amino-2-tydrooχ-4-p-cyclooctylbenzanyl- (S) -benzin has also been tested for humalate antibody production in order to determine its efficacy as an immune cancer inhibitor. It has been found that the compound causes a significant increase in the number of hepatic pro-antibody cells. The results show that the compound can be a potent infectious cancer inhibitor. For humorous antibody formation in bsetatin, see J. A. J. Blotter et al., 29, 857-859 (1976).

^Ú red-handed ^{to the (2R, 3R)} -3-β] iinno ^- 2hy to ^{the kχ} - ^- 4 ^h ^ Droo; fknylbbžanny ^{χ o} - (S) NZ in -lβžc ^{i s h} UNOO protilá1teU the formation to sheep red blood cells um ^ he was studied as follows.

* 8

Mice (dd / y females) were immunized by intra-beta injection of 10 ° sheep red blood cells. Intraperitoneal injection (2RS, 3M>)) 3-α-innk - (hrooχ-fkenšlbžalnyl- (S) -llž) was administered immediately afterwards.

Beetatin, (2S, 3R) τЗ- (aninnk2-hyšrokX · w -fklnlbbžaanyk ^ (S) -loucin and (2RS, 3IR) -3-tninnk2 - yšdOkIk (-p-chiokfkeyšlbžannyl- (S S) -leucine.

After 4 days, the number of wild-type cells in the spleen was calculated by the JERNE method, (Jerne N.K, AA Nordin and - Henry. The Aga Plaque Technique Kor Reccoginzing Antibkdkpdjing Cells in Cellbboim Antibody (Wi ^ 1; ar Institute Press, Philadelphia, 1963) pp. 109 to 122.

The results are summarized in Table 3.

Table 3 ^{For the} red-handed ^{(2R, 3R>)} - 3 · ^{(aminck -} - ⁽ⁱ · _O ^Yd for ^χ - ^- 4pph ddOk ^{χ i n} FKE ^LBB Anna ^K - ^(S) on -llžcinž humor production by the Hátek on sheep red blood cells in mass.

antibody-forming cells

compound dose number Group-wiped group - - 12 500 ± 9 050 - (2RS, 3 ^ 0-3 ^ 1 ^ -2-Hydroxy-4-p- -hydro: χyeenУ1 · UutnιlooУ- (S ^ eeucin 1 mg 207 400 and 8,025 1.66 100 pg 261 000 and 11 700 2.09 10 pg 208 000 and 7 180 1.66 1. pg 175 800 tonnes 8 200 1.41 0.1 Ug 141 000 and 5 700 1.13 Bestatin 1 mg 190 000 tonnes 7 100 ⁵² 10 pg 136 250 ± 6 500 1.09 (2RS, 3R> S-3-Amino-2-Hydroxy-4-p-chloro-phenyl-butyno- (Sl-leucine) 1 mg 208,750 i - 8,000 1.67 10 pg 133 750 and 5 600 1.07

The number of antibody-forming cells in the group of m ^ S, which has been - received 10 pg bestatin or (2RS, 3R) ^-3-N- aninoo221h'yroo 4 "ppChhoofennyiuuanoyl- (S) -leucine is almost the same as u.neošetřené groups.

On the other hand, the number of antibody-forming cells to which 10 'pg (2RS, 3RЗ) -3' nninoo22hhУrooyy4-pp (hУ ^г o ooyУeeolbuUanoyl- (S) le leuhin) was applied is -1.66 times greater than that of untreated flies. even in those cases where only 1 pg is applied, it is 1.41 times higher.

As mentioned above, (2RS, 3R) -3-amino-2- (1-oxo-5-butanoyl (S) -leucine) has a remarkable effect on increasing the number of antibody-forming cells, and does not increase the spleen weight, or the number of non-speecic antibody-forming cells.

It will be understood, however, that in addition to the methods described, a method for introducing a protecting group may be used, any method for removing a protecting group and any method for forming a peetidic chain. '

The Rf values shown are based on GF ₂₅ silica gel plates of Merek Corp. eluent o-UutnloO-acetone-water (4: 1: 1).

Some of the 3-amino-2-cyclohexyloxy acids and also the peptides which contain them have 2 Rf values because they are mixtures - with threo- and erythro-cortfigination.

Example 1

Stage 1

13.66 g of (2RS, 3R) -3-amino-2-hydroxy-4 (eeenylbutyric acid) are dissolved in 70 ml of a 1 N sodium hydroxide solution, and the solution is stirred vigorously with ice-cooling and added over 30 minutes. 3 portions of 15 ml of Uenoyl: potassium chloride and 70 ml of 1 N sodium hydroxide solution. The reaction mixture was then stirred vigorously for 1 hour under ice-cooling and for a further 3 hours at room temperature.

After completion of the reaction, the pH of the reaction mixture was adjusted to 1 by addition of 6 N hydrochloric acid. This gave an oily product which was extracted twice into 100 ml of ethyl acetate.

The solution in the solvent is washed with water, then the solution in ethyl acetate is dried over anhydrous magnesium sulfate, and after filtration, the filtrate is concentrated under reduced printing. Crystallization of the residue from a mixture of ethyl acetate, acetic acid and petroleum ether gave 8.48 g of optically impure bennyloxycarbonylbenzyl (2R, 3R) -3-amino-2-hydroxy-4-phenylbutyric acid.

On the other hand, by concentrating the mother liquor under reduced pressure and crystallizing the residue from petroleum ether, 8.07 g of optically impure b < -minyloxyalkylalkyl- (2S, 3 3-3-amino-2-hyroo) -r-4-phenylbutyric acid can be obtained. .

^La TTA has mp ¹⁴² DEG C. ^F U3. [Al and ^f] |; g ⁰ + 7, 2 ° ^(c = ^{1 »44} acetic acid;).

A mixture of 8.00 g of optically impure benzylicoscarbonyl- (S, 3R) -3-trans-4-phenyl-4-phenylbutyric acid and 10.35 g of brucine hydrate is dissolved in 300 ml of ethyl ester with heating. of acetic acid and 10 ml of methanol. After filtration, the filtrate was cooled to precipitate the salt of brucine and oenzyloxy: trans-tert-butyl (2S, 3R) -3-amino-2-diolyl-phenyl-butyric acid.

The precipitated crystals were filtered off by co-crystallization from ethyl acetate to give 13.51 g of pure brucine and Oennyl-o-octylcarbonyl-1 (2S, 3R) -3- (110S-11) -11-oxo-2-ol. 4-Phylbutyrate.

The substance melts. · T. 144 ° C · ^[alpha] + 33.8 ° ^(c = ^{1, 12,} k ^y and ^YSE if acid).

13.5 g of the decanted crystals are suspended in 150 ml of ethyl acetate, and the brucine is removed by shaking the suspension into 30 ml of a 1 N hydrochloric acid solution. The ethyl acetate layer was washed with water and, after drying with anhydrous magnesium sulfate, the inorganic portion was washed with ethyl acetate, and the ethyl acetate was filtered off under vacuum. The residue from ethyl acetate and petroleum ether was precipitated to give 6.21 g of (2S, 3R) -3-cm-1-oxo-2-hydroxy-2-carboxylic acid. 4-Feryta-aaselyl acids.

^Substance and has a ^{TT-4, 5} ° C, [al ^ J ^ -y + 83.5 ° ^(c = 1.34 seliya ^alkyl acid).

Stage 2

2.25 g of benzyloxycarbonyl- (2S, 3R) -3-amino-2-hydroxybutylbutyric acid and 945 mg of N-hydroxynonylzazole are dissolved in 70 ml of tetrahyrrofuray. After adding 2.75 g · Sa-i bennylesteru leucine acid, p-toluene sulfonic neuuralizuje reaction mixture by adding ^0.98 ml trtottylmlnu, ochlafó ya is -5 DEG C. ^{EXAMPLE added 1.40 g} NYN ^ -cHcyklolux The reaction mixture was allowed to stand for one night.

The mixture was then removed under reduced pressure, 200 ml of ethyl acetate were added to the residue, the insoluble matter was washed with 1N sulfuric acid, water, 5% aqueous sodium hydrogen carbonate solution. and water (exactly in that order), after which the organic solution is dried over anhydrous magnesium sulfate.

The filtrate was concentrated under reduced pressure, and the residue solidified upon addition of a mixture of ethyl acetate and petoolether. Cyytalization from the same solvent mixture affords b (x) and z (1) - (0000-100-100 * 00-11- (3R) -3-amino- ² -ylamino ⁾ -4- ( ^4- methylbutyl) - (S) - leuciУU.

^And has an m. ^P _T ^122-12 ° С 3 [alpha] 2? ^{g + 14.2} ° ⁽ c = 1 acetic acid).

For C ^3iH3 6 ^N 2 ^O 6 Calculated: 69.90% C, 6.81% H, 5.26%. N;

Found:% C, 69.66;% H, 7.01;% N, 5.13.

Stage 3

In a 100 ml methrniolu was dissolved 3.22 g of the benzyl ester ^to benzyl and ^R bonnl- (2S, 3R) -3-amino-2-hydroa-4-fenylbutanoylleucinu, and after addition of 100 mg of palladium black was hydrogenated for 3 hours. The catalyst was filtered off and the filtrate was bent under reduced pressure. Crystallization of the residue from a mixture of methiwiol and ethyl acetate yielded 889 mg of (2S, 3R) -3-amino-2-hydroxy-4-phenyl-butenoeno- (S) -lescine.

Mp 208 DEG-213 DEG C. (decomposition), ([.alpha.] D @ 23 = 23.5 DEG (c = 0.48, acetic acid).

^P ro ^{C 1 -C} 6 ^H 24 ^N 2 ⁰ 4 calculated: 62.31% C, 7.85% H 9 _t 99% N;

found: 62.11% C, 7.86% Η, β, Β3% N.

The (2RS, 3R) -3-amino-2-hydroxy-4-phenyl-butyric acid used in Step 1 was prepared as follows:

35.4 g of benzyloxycarbonyl- (2RS, 3R) -3-amino-2-hydroxy-4-phenylbutyric acid are dissolved in 300 ml of concentrated hydrochloric acid and 300 ml of dioxane and, after addition of 21.2 g of anisole, the mixture was heated at reflux for 12 hours. Dioxane was distilled off under reduced pressure, the aqueous solution obtained was washed with ether, and the aqueous phase was brought to dryness under reduced pressure.

200 ml of water are added to the residue, the insoluble material is filtered, the same amount of acetone is added and the pH of the reaction mixture is adjusted to 5.4 by addition of aqueous ammonia solution. the mixture is left in the refrigerator, the precipitated crystals are filtered off to obtain.

13.66 g of the expected acid (2RS, 3 R) -3-a-2 R 1 (R 2 -) - (1 -) - - - - - - - - - - - - - - - - - - - - - - - <^ lné.

[α] D, + 16.8 ° (c = 1.25, N 3 H), R f = 0.23 and 0.26.

^{For C} 10 ^H 13 ^NO 3 calculated: ť ^ 1 / í 20 ° C, <5.1 1 50 H, 71.8 50 N;

Found:% C, 6.55% H, 6.80% N.

Example 2 '

Stage 1.

In a similar manner to that described in Step 1 of Example 1, (2RS, 1R-J-amino-4-hydroxy-4-phenylbutyric acid) was prepared from the acid which was cleaved with brucin to give benzyloxycarbonic acid. 3R) -3-amino-2-hydroiodo-4-phenylbutyric acid.

By treatment with an equimolar amount of N-N-dicyclo [beta] -carbodiimif, the yield is obtained. compound to give N-benzyloxy ^carbonyl hydroJχrltUcCnnmidfeter acid Boinou - (2S, 3R) -3-amino-2-hydroχr "4 fenllmmselné.

The substance has a melting point of 111-112 ° C, [alpha] ΙΥθ

2 ^{+ 8} 7 ^{7 (c} = ^0, 5-acetic tysellm

For ^C 22 ^H 22 ^N 2 ° 7 calculated:

61.96% C,

5.20% H found:

61.27% C,

H, 5.25

6.57% N;

6.47% N.

Stage 2

Smts of 85 mg of N-hydrosulphine cinnamic acid swabic acid: (2S, 3R) -3-amino-2-lysyl-4-feyl-maleate and 86 mg of salt (S-lancine with acetyl sulphate) The reaction mixture was reacted for 12 hours in an atmosphere of 5 ml of dioxane at room temperature and in the presence of 0.03 ml.

The mixture is concentrated under reduced pressure, the residue is extracted into acetic acid, the solution in this solvent is washed with 1 N sulfuric acid solution, then with water, 5% aqueous sodium hydroxide solution and water, respectively. .

After anhydrous magnesium hořlčyatýy next dream is distilled solvent, the coating is prepared in-solid residue which was ořlkrystlljjl from ethyl ^ te ^ acetic acid by adding ^θ trslltllru thereby give 90 mg Ь уУ ^^ ^^ ™ benyyls ^ χУkarbosyУ- (2S, 3R 13-lminos-2-lyosol-4-yl-butanosulfin.

Mp 122 ° C; [alpha]. + 15.1 (c = 0.77, acetic acid).

Similarly, as described in step 3 of Example 1, 33 mg of (2S, 3RR-3-amino-2-lysyloxycarbosyl) - (2S, 3R) -3-lmino-2-lysyl-butyryl-fluoride was obtained from 75 mg of 2-methyl-2-lysylcarbosyl. -flУylbutanyoУ '(S) -leuciУj.

The substance has [alpha] θθ θ - 21.8 ° (c = 0.45, acetic acid), Rf = 0.48.

(2RS, 3R) -3-Fluoro-2-lyarsxy-4-feyl-butyric acid ^is yellow in steps! . 1 is prepared as follows:

Smts 11.0 g. (2RS, 3R) -3-Dimethyl-2-ethylsilyl-4-phenyl-4-methyl-2-nitrile nitrile and 200 ml of a 6 N hydrochloric acid solution are heated under reflux for 4 hours; The hydrochloric acid was shared under reduced pressure, water (200 ml) was added to the residue, and the reaction mixture was concentrated under reduced pressure once.

100 ml of water are added to the solid residue, insoluble materials are filtered, and after addition of 100 ml of acetone, the pH of the reaction mixture is adjusted to 5.5 by addition of aqueous ammonia solution, and the mixture is left in the refrigerator overnight. The precipitated crystals were washed with water / acetone (1: 1) to give 6.19 g of (2RS, 3N) -3-1-oxo-2-hydroxy-4-butyric acid.

Example 3

Stage 1

Dissolve 250 mg of benzoic acid (10S) -butyl- (2S, 3R) -3-amino-2-cyclohexyl-4-fluorobutyl] -sulphane (S) in 10 ml of methanol and add 0.6 ml to the solution. 1 N aqueous sodium hydroxide solution, and the reaction was stirred at room temperature for 6 hours. The methanol was further distilled off under reduced pressure, water was added to the remaining liquid portion, and the pH was adjusted to 2 by acidification with 1 N sulfuric acid.

The precipitate was extracted into ethyl acetate, washed with water, then dried over anhydrous magnesium sulfate, and the solvent was distilled off after filtration. The residual solid was recrystallized from ethyl acetate / petroleum ether.

Repeated crystallization gave 150 mg of benzyloxycarbonyl- (2S, 3R) -3-amino-2-hydroxy-4-phenyl-butanol-CSI-leucine.

The substance has mp 209 DEG C. [alpha] D + 27.2 ° (c = ^0, 71 ýseHna acid).

C 24 ^H 30 ^N 2 ^O 6 requires C, 65.14; H, 6.75; N, 6.46.

Found: C, 65.29; H, 6.75; N, 6.33.

Stage 2

120 mg of benzyloxycarbonyl- (2S, 3R) -3-amino-2-hydroxy-4- (4-phenylbutanoyl- (S) -sucine) are dissolved in 30 ml of methanol and, after addition of 50 mg of palladium black, the reaction mixture is hydrogenated for 3 hours. clock. The catalyst was filtered off and the filtrate was stripped of methanol. · Are thus obtained 54 mg of (2S, 3R) -3-amine is - hydroxyethyl ^N-r -l ffnylbutanoyl- (S) -leucine (after crystallization from methmolu ethyl acetate).

[alfaJ ^ Q ^{- 22, 7} ° ^(c = 0.95, Acetic acid ^ ^f = 0, ^four eighth

The benzyloxycarbonyl-1- (2S, 3R) -3-amino-hyleone-4-phenyl-betanone (S) -nucine used in Step 1 was prepared as follows:

A mixture of 330 mg of benzyloxycarbonyl (2S, 3R) -3-amino-2-hydroxy-4-phenylbutyric acid, prepared similarly as described in step 1 of Example 1, followed by 162 mg of N-hydooenzooriazole, 217 mg of hydrochloride ( S) -leucine, 0.17 ml of triethyamine and 206 mg of N, N'-dicyclohexylcarbodiimide.

The reaction was carried out according to the procedure of Step 2 in Example 1 to prepare 293 mg of benzyl biphenyl-2-yl-(2S, 3R) -3-fluoro-2-haloo-1-phenylbutanoyl- (S) -. leucine.

The substance has a mp of 120 ° C, ^[af a] 278-22 ¹ DEG ^(c = ^ ^{0% to,} acetic acid).

^For ^ 5 ^ ² ° ^6: calculated: 65.77% C, 7.07% N, 6.14% N;

Found:% C, 65.91;% H, 7.16;% N, 6.28.

EXAMPLE 4 mg of benzyloxy / carbonyl-4S, 3R) -3-amino-2-hydroxy-4-phenylbutyric acid N-hydroxybenzimidazole is prepared in a similar manner to that described in Step 1 of Example 2, and is reacted with 54 mg. mg (S) -leucine in 5 ml dioxane and 5 ml water in the presence of 0.03 ml iriethyamine on standing for 2 days at room temperature.

The reaction mixture is concentrated under reduced pressure, 30 ml of water are added to the residue, and the pH of the solution is adjusted to 2 by addition of 1 N sulfuric acid solution. The occluded solution is extracted into acetic acid (50 ml), a solution in said solvent. Wash with 1N sulfuric acid solution, water, 5% aqueous sodium hydrogen carbonate solution and again with water, and then dry the solution with magnesium sulphate after filtration, after filtration.

The ethyl acetate is distilled off, and the remaining solid is recrystallized from ethyl acetate by addition of petroleum ether.

By precipitating crystals from the same solvent combination, 63 mg of the expected benzyloxycarbonyl- (2S, 3R) -3-amino-2-hydroxy-4-phenylbutanoyl (S) -leucine is obtained.

Mp 209 ° C, + 26.7 ° (c = 0.55, acetic acid). ,

Treatment with 45 mg of benzyoyloxycarbonyl- (2S, 3R) -3-amino-4-h-eroyl-4-phenoxy-(S) -leucine in a similar manner to that described in Step 2 of Example 3 affords 15 mg. best ^ tou.

The substance has a [alpha] D -22.3 ° (c = 0.52, acetic acid), Rf = 0.8.

ь

Example 5

Similar to Step 1 of Example 1, 330 mg of benzyloycarbonyl- (2S, 3R) -3-amino-2-4-hydroxy-4-: pheoylbutyric acid was prepared and a similar reaction as described in Step 2 of Example 1 by treatment with 472 g of the salt of R-leucine with p-lueotulfonic acid, 162 mg of N-hy (ldony-eeoziazole, 0.1 ml of triethymium and 206 mg of N, N-decyclohexylcarb bodiimide are prepared 450 mg). mg Benzz benzyloχlkarbonol- ester of (2S, 3R) -3-amino-2'4h eroyi - 4 ^L atka has i. ^{e 118} and 119 ° C, al and ^F | γ ^{4 8} + 3.0 ° ^(c = ^1, 23, acetic acid)

When 430 mg of the benzo-β-carbonyl- (2S, 3R) -3-amino-2-hydroxy-4-phenobutanone-1 (R) -leucine thus prepared benoz-ester benzyl-β-carbonyl is treated similarly to as described in Step 3 of Example 1 to give 144 mg of (2S, 3R) - 3-amino-4h-eooy-4-phenyl-1-quinone-1 (S) -leucine.

[al ^fa] ®g 0 + ⁹ ° ^(c = ^ ⁷ -ctová ^acid, HP = ^0, 49).

^p ro 0 H ²⁴ N ° ^4: calculated: 62.31% C, 7.55% H, 9.09% N; 'Found: 62.47% C 7.99% Η _9)} 9% 9 N.

Example 6

Stage 1

From 7.75 g of benzyl-ycarbonyl-4 (2RS, 3S) -3-amino-2-hydroxy-phen-butyric acid and 6.69 g of dehydroabielamine are prepared in ether the sparingly soluble eehydroabielamine amino acid salt. benzyloycarbol-1- (2R, 3R) -3-amino-2-heroxy-4-phenyl-methyl-deilirt was removed.

The clear ether solution was concentrated under reduced pressure after filtration, and a mixture of ethylene acetic acid and 1 N hydrochloric acid solution was added to the oily residue to give a crystalline crude benzylocybo-carboxylic acid (1S, 3S) -3. -amino-2-h-dr-χ1-4-phenylbutyric acid and from the obtained crystals by double precipitation from ethylene ™ acetic acid by addition of petmlether, 2.16 g of pure benzyl-ycarbonic acid - (2S, 3S) -34-amino-2 is obtained. -h-dr-χl --- fen-láselot.

T. í. 175 DEG-176 DEG C. [.alpha.] + 5.6 DEG (c = 1.39, acetic acid).

Stage 2

A mixture of 660 mg of benzyloxycarboryl- (2S, ZZ-Z-α-β-α-β-α-β-γ-β-β-β), 994 mg of (S) -leucine benzyl ester as p-toluenesulonic acid salt, 324 mg of N-hydroxybenzotriazole. 0.34 ml of triethylamine and 412 mg of Ν, Ν'-dicyclohexylcarbodiinide were treated in a similar manner to that described in step 2 of Example 1 to give 710 mg of benzyl oxycarboyl- (2S, 3S) -3-aminzo2 benzyl ester. -htlroyl-4-phenol-butazazyl- (S) -leucine.

The substance has a melting point of 128 [deg.] C., [[alpha]] D -38.0 [deg.] (C = 0.81, acetic acid).

270 mg of (2S, 3S) 434-amine was prepared from 500 mg of benzyloxyl / carboalone (S, 3S) -3-amino-2-hydroxy-4-benzylbutanoyl- (S) -leucine benzylestir. -2-Heterroyl-4-ylbutyl-11 (S) -leucine.

[alla] 24 ₆₁₃ ° ^{578-61 3} (c = 0.56, acetic acid). Rf = 0.37 For ^C 16 ^H 24 ^N 2 ^O 4 calculated: 62.11% C, H, 7.65; N, 9.09; found: 61.99% C, 7.71% H 9/3% N.

Example 7

A mixture of 165 mg of 0enzyloχyУalbo-NLL (2S, 3S) 2--34lmino'hl ^¢ droxyl4-lenylmááelné »237 mg salt Benzi you ^ ™ (R-leucine acid 81 mg of N-hyldooyloiozιOrrazolu»

0.08 ml and 103 mg triethyaaminu Ν, Ν ^/ 4dicyklohiyylkar0odiimidu was treated in a similar manner as described in Step 2 of Example 1 to give Oenzylester 0enzylo: yУckrbo-NLL _(t 2S 3S) 434amino 2-l4- hyddoэy ^r flnotallo-11 (R) -leucine.

Mp 128 DEG-129 DEG C., [.alpha.] D @ 20 = -18 DEG ( ^c = 1.02, acetic acid).

Similar to Example 1, Step 3, from 155 mg of benzyloyycarbonyl- (2S, 3S) - 434-amino-2,4-dihydro-4-lenphthalool-4R) -liucine Oenzylestir 72.1 mg of (2S, 3S) 434lio104 424-hydroxy-4-linylbutano- 1- (R) -liucine.

[α] D - 31.4 ° (c = 0.47, acetic acid), R = 0.52.

^p ro ^С1 бН ² ЛО ₄ calculated: 62.31% C,

H, 7.85; N, 9.09;

found:

H, 7.63; N, 8.80.

Example 8

A mixture of 330 mg of benzyloycarbonol- (2S → 3S) -34-amino-2-hydroxy-4-leolobutyric acid prepared according to the procedure of Step 1, Example 1, 405 mg of glycine benzyl ester of p-toluenesulfonic acid »162 mg N 0.14 ml of triethylamine and 206 mg of di-cyclohexylcarbodiimide were treated as described in Example 2, step 2 to give oily benzyl-benzyloxycarbonyl (2S, 3R) - 3-lminoo22hldr-yl441ennlbutanoylglycine.

Thin layer testing revealed that the product was uniform and pure.

Treatment of the oily product according to the procedure of Step 3 of Example 1 gave 117 mg of (2S, 3R) -3-amino-2-hydroxy-4-phenylbutylglycine.

[ay and ^f] _| γ β - ^{7, 4} ° ^(c = ^0, 49, ^ Selina acid). ^Rf = ^0.26

For C ₁₂ H 16 O ₄ N ₂ .CH ₃ OH Calculated: C 54.92, H 7.09, N 9.87;

Found: C 54.33, H 6.87, N 9.69.

Example 9

A mixture of 330 mg of bennylsulfonylcarbonyl- (2S, 3R) -3-amino-2-lysoxyl-4-fluorosulfate, prepared according to step 1 of Example 1, 440 mg of (5) -butylbenzyl ether (5) -benzyl ether. For example, 162 mg of N-hydroxybenzoleazole, 0.14 ml of triethyl ethyl and 206 mg of N, N-cyclicollol-4-carbonyl diimide were treated according to the procedure of Step 2 of Example 1 to give 540 mg OУit ^г sblnzyУesteru beУZyls: ιqrkkabbsyУl (2S, 3R)) (3-lminns2 - lrУdosχy 4fenyУbujta ⁴⁾ noУ- (S) -blУzyleeriУu.

^{The substance} has ^t. ^ T. 1 ² 1.5 ° C, [alpha] + 17.7 ° ^(c = 1, ^ tyseUn acid).

Treatment of 500 mg of o-nitrobenzyl ether with benzyl: (2S, 3R) -3-amino-2-lysyl- ^4- phenylbutayoyl- (S) -benzoyl -elly according to the procedure of Step 3 of Example 1 gives 147 mg (2S, 3R) -3. -lmiУO4 2-lydioxy ^{4 4} -γ-β-butadiene- (S-serine.

[alpha 2γθ - 9.0 ° (c = ^0, 50, kyselica мЮТ)). Rf = ⁰ 20th

^P ro ¢, ^, ⁸ ° ⁵ ¾.¾ ⁰ calculated: 51.99 * 0, <5.7 1% H, 9.33% Ni found: * ^ 1.39 «C ,, 6 ^ 4 5K H, 8.97 «N.

For example

A mixture of 330 mg of beynzls: ιχ rkarbbsyУ4 _l (2S, 3R) -3-2-amiУβ lydrsχy-4-feyylmáeelné prepared by analogy with the procedure of Step 1, Example 1, 400 mg boy ^ S ^^^ (S) -glutamine 162 mg of N, N-trimethylbenzyl, 0.14 ml of trimethyl amine and 206 mg of N, N-dicycloylcarbodiimide were treated similarly to step 2 of Example 1. This yielded 200 mg of benzyl ester of bennylsylcarbonyl- (2S, 3R) -1minos2. -llrУdboχy-4feyyУbutøn) sУ- (S) -glulιmiУu.

Mp 155 ° C [bIT] + 62.3 ° ( ^c = 0, acetic acid).

Treat with 180 mg of benzylsulfonyl (2S, 3R) -3-lminos-2-hydroxy-4-fluorobutan (S) -glutamine. Similarly, as described in Step 3 of Example 1, 61 mg of (2S, 3R) -3-lmino-2-lysyl- ^4- phenylbutyl (S) -glutamine was obtained.

Substances ^and ua [alpha] | Y ⁸ - 9.9 ° ^(c = 0 ^ ^l kyβeli.У acetate) ^{Rf 0,} 17th

For C 1 ₅ H ₂₁ O ₅ N3.2H ₂ ° Calculated: 50.13% С, 7.01% H, 11.69% N;

found: 50.64% c 6.32% H, 11.61% N.

Example 1 1

Similarly, as described in Step 1 of Example 1, benzyloxycarbooyl- (2S, 3R) -3-aeino-2-hydroxy-4-phenylenebutyric acid and a mixture of 330 mg of this material, 600 mg of benzyl ester salt (S) are prepared. p-tolunotulfonic acid, 162 mg of N-hydroxybesotriazole, 0.14 ml of triethylamine and 206 mg of Ν, Ν'-dicyclohexylcarbodiide were treated in a similar manner as described in Step 2 of Example 1, formation of 605 mg of benzyloxycarboyl- (2S, 3R) -3-amino-4-benzoxycarboyl- (S) -o-benzylglutmlic acid.

Mp 119-120 ° C, [alpha] + 28.6 ° (c = 1.04, acetic acid).

Treatment of 250 mg of benzyl ester with 3-methyl-3-methyl-2-carboxylic acid (2-carboxylic acid); Butylbutyloyl- (S) -Obenzylglutaminic acid according to the procedure of Example 1, Step 3, yielded 166 mg of (2S, 5R) -3- (α] -benzoic acid-phenobutyl-(S) (- glutamic acid). .

The substance has [alpha] and γθ - 18.4 ° (C R f = 0.27 For ^C 15 H 2 ^20OgN calculated: 55.55% C, 6.22% H, N, 8.64; found: 55.20% C, 5,90% H, 8.20% N.

Example 12

Similar to Step 1 of Example 1, benzyloic acid: - (- S, 3R) -3-amino-hydro-4-phenylethyl, and a mixture of 330 g of this material, 455 mg of benzyl ester salt are prepared. (S) -valyl with p -toluentulonic acid, 162 mg of N- (γr (lrooylbenzozriazole), 0.14 ml of triethylamine and 206 mg of β, β-dicyclohexyltrodiode were treated similarly as described in step 2 of Example 1. There was thus obtained 397 mg of benzylo [beta] -benzyl ester of O-benzyl ester - ((- S, 3R) -3-amino-hydroxy-N-phenylbutanoyl-S) valine.

Mp 96.5-97 ° C; ^{+ 9} > 2 ° (c = 0.99, acetic acid).

If 376 mg of benoyCester b-benzylcarbonyl-C - (- S, BR 4 -amino 2-hydroxy-oxo-4-phenylbuttooyC- (S) -propyl) was treated in a similar manner to that described in step 3 of Example 1, 104 mg of (2S) were obtained. (5R) -3-Amino-ydoxy-44-eneCbbutoyoyl- (S-valine.

[.alpha.] D @ 20 = 1.4 DEG (c = 0.55, acetic acid), Rf = 0.42.

H, 7.53; N, 9.52.

Found:% C, 60.98;% H, 7.59;% N, 6%.

Example 13

Similarly, as described in Step 1 of Example 1, benzylozycarbooyl- (2S, 5R) -5-amino-2-hydroxy-4-phenylbutyric acid was prepared, and a mixture of 330 mg of this material, 455 mg of beozylester salt (S) - oorvalium with p-toluenesulfonic acid, 162 mg of N-hydroxybenzazole, 0.14 ml of triethylamine and 206 mg of N, N ' -dicyclohexylcarbodiende were treated in a similar manner as described in step 2 of Example 1. 310 mg benzyloxy / carbonyl- (2S, 3R) -3-amino-2-hydroxy-4-fluorobutyl-thioyl- (S) -norvaline benzyl ester.

L ^and T ^k and И4 has mp 115 ° C, [alpha! | 4 ^g + 22 ⁰ ° ^(c = 0.49, Acetic acid).

Treatment of 250 mg of benzyloxycarbonyl- (2S, 3R) -3-amino-2-hydroxy-4-phenylbutanool- (S) -norvaline benzyl ester according to the procedure of Example 1, Step 3, afforded 110 mg of (2S, 3R) -3-amino- 2-Hydroxy-4-phenyl-1-butyoyl- (S) -norvalint.

-14.5 ° (c = 0.50, acetic acid), R f = 0.41.

For C ₁₅ H ₂₂ O ⁴ N ₂ calculated: 59.33% C,

H, 7.64; N, 9.23;

found:

% C, 59.42;% H, 7.58;

Example 14

When (2S, 3R) -3-amino-2-hydroxy-4-phenylbutyric acid is reacted in a conventional manner after the ingestion of tert-butyl-S-4, -dimethylplasimide-2. yl-lhtoaaгbtУátu then added dicykloheyilamin give a salt of tert-butoyikarbonyl- ^ s, 3R) -3-amino-2- ^HID royy- ⁴ -phenyl ^and numerical · s klohejqrliminem di ^ tt 15 ^{8 and} 159 DEG C. [ ^.alpha .] And ^.alpha .] 29 ^g + 51.9 DEG (c = 0.89, acetic acid).

The dicycloheyl-Emin of 952 mg of the above salt was reacted with 1 N sulfuric acid and ethyl acetate to give the oleic acid tertiary-butylacetic acid ^and (2S, 3R) -3-amino-2-hydroxy-2-carboxylic acid. 4-phenylbutyric.

This oily portion and 440 mg of methyl ester tydaochloaid (S-methionine was dried in 20 ml of tetraldehyde), and the reaction mixture was neutralized by the addition of 0.308 ml of triethylamine. . the solution was cooled to -5 DEG C., 412 mg of _N> N * -diclkloltyyL.kaгbtdiimiSt reaction mixture was stirred for 4 hours at místnoosi, after which the crystals of (šutrují and the mother liquor was concentrated under reduce pressure Dissolve the residue in 200 ml of acetic acid, wash this solution with 1N sulfuric acid, water, 5% aqueous sodium bicarbonate solution and then again with water as indicated. · Dry the organic solution with anhydrous magnesium sulphate.

The residue obtained by concentrating the mother liquor under reduced printing is dissolved in a mixture of ethyl acetate and benzene (2: 5), and the solution is applied to a column of chromatographic silica G (Merck). The fractions containing the expected material were collected, the solvent was distilled off under reduced printing, and the solid residue was recrystallized from a mixture of ether and petroleum ether to obtain 380 mg of methanol ^and tert-butoxy- (2S, 3R) -3-amino-2-lydino. Y-14-phthylbutayoyl- (S) -methyl.

^Lát k has ^m. P. ¹¹⁸ ° C, [Al and ^{f] |} θ ⁴ + 51.4 ° ^(c = ^{0 5б,} acetic acid a).

When treated with 280 mg of tert-butyutyl- (2S, 3R) -3-amino-2-lyddaoyl-4-naphthylbutalol (S) -mithlityl methyl ester, 280 mg of the target was obtained as described in Example 3, step 1. -butons! (2S, 3R) -3-Amino-2-hydroxysoyl-4-naphthylbutyl-(S) -mithlitol.

^Fabric and has ^t . t. ¹¹⁶ ° ^{C (pěnёn s). [Alpha]} | γ 44J + ₈ ° ^(c = ⁰ 54, acetic acid).

To 150 mg of tert-butyl (S, 3R) -3-amino-2-iodo-4-phenylbutyl-(S) -mithlitic acid was added 0.2 ml of tert-glycolic acid and a 50% aqueous solution of trifluoroacetic acid, and the reaction The mixture was stirred at room temperature for 1 hour. The oily fraction obtained by concentrating the reaction mixture under reduced pressure was dissolved in water and aisorbed on a 5 ml column packed with Dowex 50X4 resin (in a hydrogen cycle).

The adsorbed material was eluted with a 2 N aqueous ammonia solution, and then concentrated to dryness to give a solid material from the eluate. Crystallization of this sub-portion from water / acetone (1: 1) afforded 87 mg of (2S, 3R) -3-amino-22-hydroxy-4-phenyl-4-methoxy-(S) -methionine.

[alpha] Θ ⁷ - 20, ⁴ ° ^(c = ^{0 49;} acetic acid). ^R = ^0, the 37th

For O ^ 2204 ^ 1/2 ^ 0 calculated: 53.67% C,

H, 6.01; N, 8.35.

found:

% C, 53.64;% H, 6.47;

Example 15.

A mixture of 330 mg of benzyloxycarbonyl- (2S, 3R) -3-amino-2-hydroxy-γ-4-phenylbutanoic acid, prepared similarly as described in step 1 of Example 1, 472 mg of benzyl ester salt (S) by acid-leuce with an acid p-luentuf: 162 mg of N-trienoxy-thiazole, 0.14 ml of triethylamine and 206 mg of Ν, Ν-cyclic-hexylcarbodiimide were treated as described in Example 2 step 2 to give 466 mg of benzyl-xycarbonylbenzyl ester (2S, 3R-1-amino-2-hydroxy-4-benzylbutyl) -1- (S) -it-leucine.

The substance has a mp 100-101 ° C, [alpha] | γ ₈ + 23.6 ° (c = 1.0, kzselioa acid).

Treatment of 440 mg of the title compound according to the procedure of Step 3 of Example 1 afforded 191 mg of 2S, 3R) -3-amino-2- (4-yloxy) -1-4-azobutanoz-1 (S) -oleucine. [.alpha.] D @ 20 = 6.4 DEG (c = 0.52, acetic acid), Rf = 0.48.

^{for C} 6 ^H 24 ₁ ° 4 ^N 2 Calculated: 62.1% C 1, 7.85% H, 9, 09% N;

Found:% C, 62.7;% H, 7.70;% N, 8.99.

Example 16

A mixture of 330 mg of benzyl-4-carbonitrile (2S, 3R) -3-aminoo-2-yl-4-yl] -4-butyric acid, prepared analogously to step 1 of Example 1, 472 mg of benzyl ester salt (S-nodleucine with p-luentulnonic acid, 162 mg). N-hydroxybenzodiazole, 0.14 ml of triethylamine and 206 mg of N, N'-cyclic-hexylcarbodiimide were treated similarly to the procedure described in Example 2, step 2 to give 437 mg of benzyl ester of benzyl ester (2S). 3R) -3-amino-2-hydroxy-4-phenylbutanoyl- (S) -o-resolution.

Mp 114-115 ° C, [alpha] 24g + 23.3 ° (o = 0.04, acetic acid):

From 350 mg of the just mentioned compound were obtained poyie procedure of Example 1, Step 3, 160 mg of (2S, 3R) -3- (2hhУdony-amine is L-ffeo lbutanoyl- ^r (S) -nonleucinu.

^{L p} has atka MICK Company ec ^ [^ alfaj g ^{- 8 0 (c} = ^0, 7 ^0, tyseltoa ^- C ^{t in} A), ^Rf = ^0.4 7th Pra эд ^^ ⁰ ^ ² ^ ²⁰ vzpočteno: 58.88% C 8.03% H 8.58% N found: 5β, 95% C, 7 ^84% H 8 00% N

Example

A mixture of 330 mg of benzyloxycarbonyl- (2S, 3R) -3-amino-2-hydroxy-4-phenylbutyric acid, prepared according to Example 1, step 1, 505 mg of (RS) -2-amino-octaonic acid bennylester salt with p-toluenesulfonic acid, 162 mg of N-hyllghoxyleno-Oriazole, 0.14 ml of triethyamine and 206 mg of N, N'-dicyclohexylcarbodiimide were treated in a similar manner to that described in step 2 of Example 1; 430 mg of benzyloxycarbonyl-4S, 3R-3-amino-4-methyl-4-ylene-1-yl-benzyl ester are obtained. butanoyl-OS-minoctane. ¹

Substances ^and m mp 90-92 ° C (alpha ») @ + 32.9 ° (c = ^{0 94;} acetic acid).

If 250 mg of the title compound were obtained according to the procedure of Step 3 of Example 1, 74 mg of (2S, 3R) -3-amino-2-1H-pyrrolidinyl-butyno- (RS) -2-amino-octanoic acid was obtained.

The substance has a [alpha] θθ - 34.9 ° (c = 0.53, acetic acid), R1 = 0.58.

^Pr 18 ^{° C} 8 ° 4 ^{Hg, N} 2 ': calculated: 64.26% C, 8.39% H, 8.33% N;

Found:% C, 64.43;% H, 8.18;% N, 8.02.

Example 18

A mixture of 330 mg of benzyloxycarbonyl-4S, 3R) -3'-amino-2-] hydroxy-4-fumaric acid, prepared as described in Example 1, step 1, 513 mg of (S) -leoylalcoin acid ester salt p-toluenesuioone, 162 mg of N-hylroo-pyrrolo-o-triazole, 0.14 ml of triethyamine and 206 mg of N, N'-dicyclohexylcarbodiimide were treated similarly as described in step 2 of Example 1;

378 mg of benzyloxycarbonyl-4S, 3R) -3-amino-4-amino-4-phenylbenzoyl- (S) -lenylcyclohexane are obtained.

Latte and has mp 111 ¹¹² ° C [alpha] | γθ ⁺ 39.6 ° ^(c = ^{0, 97, to,} acetic acid).

Treatment with 355 mg of the title compound according to the procedure of Step 3 of Example 1 afforded 108 mg of (2S, 3R) -3-amino-1H-pyrrolidin-4-ylbutanoyl- (S) -phenylalanine.

And let ^{K L} has a ^p ECI ^{f ic} ou rotation ^[01 ^ q] + ^0, 4 ° ^(c = ⁰ 4-acetic acid, ^ ^R = ^0, the 44th

For C ₁₉ H ₂₀ O ₄ N ₂ .CH ₃ OH calculated: 64.15% C, 7.00% H, 7.48% M;

found: 63.58% С, 6 »55% H,% N.

Example 19

Z dacováváv ^p-i with 1.10 g of oily mixture of Z- (2RS, 3R) -miihhlisošeřinu prepared benzyloxykarbonylací (2RS, 3R) -mithhlisošeřinu, 1.97 g salt BE-zy ester of (S) -leucine acid p toluensuioonovou, 675 mg of N ^ ^ц г lгooχlenoyOriazolu 0.70 mL tr ^ tty! amine and 948 mg N, N'-diclklohexylkcrbodimidu similarly as described in Example 1, step 3, yielding the oily Ъenoyles'te Benzyloxycarbonyl- (2RS, 3R) -lithlissosulfonyl- (S) -fluoric acid. According to the Thin Layer Chromatography, this oily θ is sold clean.

From ^the process, the oily oil was sold as described in Example 1, step 3, to give 765 mg of (2RS, 3R) -3-amino-3-amino-3-aminooylropiooyl- (S) -leuce.

The substance has a specific rotation [alpha] - 28.7 ° (c = 1.09, acetic acid).

For ^{C 10 H} 20 ^N 2 O 4 Calculated: C 51.70, H 8.68, N 12.06.

Found:% C, 51.49;% H, 8.42;% N, 11.96.

The benzyloxycarbonyl- (2RS, 3R) -methylisoserine used in this example was prepared as follows:

9.00 g of benzyloxycarbonyl- (2RS, 3R) -3-amino-2-hydroxypropionitrile was treated as in Example 1 to give 3.16 g of (2RS, 3R) -methylisoserine.

[alpha] - -0.6 For ₉ and ₃ years (c = 1.10, N hydrochloric acid solution), Rf = 0, calculated: 40.33% C, 6.72% H, 11.76% N; found: 40.53% C, 7.51% H, 11.95% N.

Example 20

1.32 g of oily benzyloxycarbonyl- (2RS, 3R) -isobutylisoserine prepared by benzyloxycarbonyllation of the (2RS, 3R) -isobutylisoserine obtained in the above process, 1.76 g of (S) -leucine benzyl ester salt with p-toluenesulfonic acid, 0 60 g of N-hydroxybenzotriazole, 0.63 ml of triethylamine and 0.92 g of N, N'-dicyclohexylcarbodiimide were treated as described in Step 2 of Example 1 to give oily benzyloxycarbonyl- (2RS, 3R) -isobutylisoseryl- (S) - leucine.

Working up in a similar manner to that described in Step 3 of Example 1 gave 0.37 g of (2RS, 3R) -3-amino-2-hydroxy-5-methylhexanoyl- (S) -leucine.

The substance has a specific rotation of [alpha] - 26.6 ° (c = 1.0, acetic acid), Rf = 0.43 and 0.49.

For C ₁₃ H ₂₆ N ₂ O ₄ Calculated: C 56.91, H 9.55, N 10.21.

Found: C, 57.01; H, 9.80; N, 10.14.

The (2RS, 3R) -isobutylisoserine used in this example was prepared as follows.

2.76 g of oily benzyloxycarbonyl- (2RS, 3R) -3-amino-2-hydroxy-5-methylhexanoic acid nitrile are dissolved in 30 ml of concentrated hydrochloric acid and 30 ml of dioxane. After the addition of 2.16 g of anisole, the solution was refluxed for 4 hours, the dioxane was distilled off under reduced pressure, and the remaining solution in hydrochloric acid was washed with ether. The aqueous portion was concentrated to dryness under reduced pressure.

Water is added to the residue and the active ingredient is adsorbed on the Dowex 50X4 resin column (in a hydrogen cycle) from the solution. After washing with water, the adsorbed material is eluted with 4 N aqueous ammonium hydroxide solution and then the eluted solution is concentrated to dryness under reduced pressure.

A 1: 1 mixture of water and acetone was added to the residue, and the precipitated crystals were filtered to give 0.82 g of the desired (2RS, 3R) -istyl ester. Substances exhibit Speccy-flckou rotation [alpha] | γθ ^{- 8,} 3 ° ^(c = 00 kyselka acid), ^R = ^0, 1 · ⁸

EXAMPLE 21

Benzyloχlkarbonyl- mixture of 250 mg of (2RS, 3R) -ftyyliststгiУU _J mp 170-171 ° C, alpha 2 · · + 105 ° (c = 0.47, Acetic acid) připraveyého beyzyloyykarboynlací (2RS, 3R) -ftylli8tseřiyu 394 mg The salt of benzyl ester (S-leucine with p-toluenesulfonic acid), 130 mg of N-lydroethyl ether, 1.14 ml of tert-butyl amine and 165 mg of N, N'-Syclyclolylcarbonyl imide was treated as described in step 2 of Example 1. 285 mg of cyanobotyl- (2RS, 3R) -eyl -isteeayl-4 (S) -leuilyl are obtained.

^Fabric and has ^t . ^t . ⁹³ and ⁹⁵ DEG C., ^{[alpha] - 3} 4 ⁷ ° ^(c = ⁰ · ^{4 9,} acetic acid).

Treatment of 200 mg of (2R, 3R) -phenylcarbonyl- (2RS, 3R) -phenylcarbonyl- (S) -EleuUU according to the procedure of Step 3 of Example 1 afforded 45 mg of (2RS, 3R) -3-amino-2-indolyl-2-indolyl-2- phthylprtp ions - (S) -1 eucine.

e [Al and ^f] | ^γ8 - ^{2, 4 (c} = ^{0, 33, to,} acetic acid).

H, 7.33; N, 9.52.

Found:% C, 60.92;% H, 67;% N, 9.6%.

The (2RS, 3R)) feyylistseries used in this example were prepared as follows:

Jessiiže 3.00 g of the nitro-acid in ^the beyzyltyy aabtnyl (2RS, 3R) -3-amino-2-Lys R YY-3-feyylproptoyová treated as in Example 1, yielding 0.80 g of (2RS, 3R)) · Ftyylist sednu.

^Substances and ^y Kazuo EPEC ^f ic ^to the rotation of ^{[al fa] 2γ8 +} 9.5 ° ^(c = 00 N HC1), ^Rf = ^0.13 and ^0.17.

calculated: 59.66% C, 6.12% H, 7.73% N; found: 59.18% C, 6.15% H, 7.67% N. For example 22nd

A mixture of 1.81 g beuz ^y Lo ^to aгbonyl- yl (2RS, 3RS-3-Amiya-2-y (Sroэyrl ⁴ -p-cllorfeyylmáetlné kyseLízi, mp 147 ° C ¢ í beyyyltχlkarboynla obtained (2RS, 3RS) -3- -amino-2-indolyl- ⁴ -β-chloromethylbutyric acid, 2.37 g of the salt of silyl ester (S-leucine with p-tolueneultonic acid, 810 mg of N-1-hydroxyethyltriazole), 0.84 m treelhylamine and 1.03 g of N, N The cycloltylkabodimide was worked up as in Example 1 and Step 2.

There was thus obtained 2.50 g of cyanoethyl (2RS, 3RS) -3-amino-2-lydroyl ⁴ -.beta.

^Lát ka M ^{& T. t} . 122 to ^{ž 1} 2 ⁴ ° 0 [al ^f a] ² ? ⁸ - ^{12, 30 (} c = ^3.1 8 acetylene).

Treatment of 1.50 g of the title compound as described in Step 3 of Example 1 gives 650 mg of (2RS, 2RS) -3-amino-2-hydroxy-4-chlorophenyl-4-chloro (S) -nucine. .

The substance has a specific rotation [alpha] µg - 11.3 ° (c = 1.33, acetic acid), R f = 0.42 = = 0.52.

For C 6 ^H 23 ^N 2 ^O 4 ^Cl calculated: 56.05% C, 6.76% Η, Θ17% NT;

Found: C, 55.88; H, 7.29; N, 7.1%. The (2RS, 3RS) -3-amino-22-hydroxy-44-chlorophenylbutyric acid used in this Example was prepared as follows:

10.3 g of the nitrile ester of Z- (2SS, 3R) -3- <cmnn ^{p-22hhУdrxχl4-} --Uhorfnnχlmmselná was treated as in Example 1 to obtain 3.10 g of (2RS, 3SSS-amino-2-hydroxy -4 ~ Phenylphenyl.

Rf = 0.25.

For c ₁₆ h ₁₂ n ° ₃ C1 calculated: 52.29% C, 5.27% H, 6.10% N; Found: C 52.11, H 5.17, N 5.99.

Example 23

A mixture of 726 mg of benzyloxycarbonyl-1- (2RR, 3RR) -3-amino-2-hydroxy-4-chlorophenylbutyric acid (mp 136.5 ° C), prepared by benzyloxycarbonylation of the acid (2RS, 3RSS-4-amino-4-hydroxy-4). 944 mg of benzyl ester salt (Slleucine with p-toluenesulfonic acid, 324 mg of N-hydroxytrennenetrrazole, 0.34 ml of triethylamine and 412 mg of dicyclohexylcarbodiimide was treated similar to that described in step 2 of Example 1).

There was thus obtained 747 mg of methylbenzyl-oxybutylbenzyl (2SR, 3R) -3-amino-2-hydroxy-4-o-carbonaphenylleutanol (S) -leucine.

The substance has a melting point of 129 to 132 ° C, [alpha] γγθ - 29.1 ° (u = 1.02, -ut-acid).

Treatment with 640 mg of enzyl-xylcarbonyl-enyl ester (2SS, 3RS-3-amino-2-hydroxy-4-o-chloro-phenyl-butanoyl- (S) -leucine) similar to that described in step 3 of Example 1 yields with 107 mg <2RS, 3RS) -3-ε-cmnn-2-nitro: χ <χl-o-hloophenine-betaine-(S) -nucine.

^{L p} has atka ecifickou rot-ation ^ 1 ^ 273 - 20.8 ° ^(u = ^0, 48, tyselina alpha.-VA) ^Rf = ⁰ 46th

For ^C 16 ^H 23 ⁿ 2 ° ₄ ^C1 calculated: '56> 55% C, 6% -6% Η, 81Π% N; · Found: 56 _s 1,% C, 6.66% H, 7.90 N.' ( 2RS, 3] R-3-Amino-2-hydroxy-1,440-Chlorophenylbutyric acid used in this example was prepared as follows:

10.3 g of benzyl-xylcarbonyl-nitrile- (2SS, 3R) -3-amino-2-hydroxy-4-oxo-phenyl-phenyl-butyric acid nitrile was treated as described in Example 1. This gave 4.82 g of (2RS, 3R) -3. 4-Chloro-2-hydroxy-4-chlorophenyl-butyric acid.

201033 24

Rf = 0.25 and 0.29.

For C ₁₀ H ₁₂ NO ₃ Cl calculated: 52.29% C, 5.27% H, 6.10% N; Found:% C, 52.01;% H, 5.17;% N, 5.91.

Example 24

A mixture of 1.57 g of benzyloxycarbonyl- (2RS, 3RS) -3-amino-2-hydroxy-4-p-methylphenylbutyric acid. 166.5-168 ° C), prepared by benzyloxycarbonylation of (2RS, 3RS) -3-amino-2-hydroxy-4-p-methylphenylbutyric acid, 1.42 g of (S) -leucine benzyl ester salt with p-toluenesulfonic acid 486 mg of N-hydroxybenzotriazole, 0.50 ml of triethylamine and 618 mg of dicyclohexylcarbodiimide were treated analogously to Example 2 step 2. Benzyloxycarbonyl- (2RS, 3RS) -3-amino-2-hydroxy-4-phenylbutanoyl- (S) -leucine benzyl ester is thus obtained in oily form.

Treatment of the oily product similarly to that described in Example 1, Step 3 gives 497 mg of (2RS, 3RS) -3-amino-2-hydroxy-4-p-methylphenylbutanoyl- (S) -leucine.

The substance has Rf = 0.58 and 0.53, [.alpha.] D @ 20 = 7.2 DEG (c = 0.96, acetic acid).

For C ₁₇ H ₂₆ N ₂ O ₄

calculated: % C, 63.33;% H, 8.13; found: % H, 8.23;% N, 8.49.

The (2RS, 3RS) -3-amino-2-hydroxy-4-p-methylphenyl butyric acid used in this example was prepared as follows:

11.2 g of benzyloxycarbonyl- (2RS, 3RS) -3-amino-2-hydroxy-4-p-methylphenylbutyric acid nitrile was reacted as in Example 1 to give 3.41 g of (2RS, 3RS) -3- amino-2-hydroxy-4-p-methylphenylbutyrate.

Rf = 0.20.

For C _H H ₁₅ NO ₃

calculated: % C, 63.14;% H, 7.23; found: % C, 62.94;% H, 7.01;% N, 6.79.

Example 25

103 mg of bestatin are dissolved in 5 ml of acetic acid and 5 ml of ethanol. The reaction mixture was catalytically hydrogenated on platinum oxide for 14 hours. After filtration, the filtrate is concentrated to dryness under reduced pressure and trituration of the residue in ethyl acetate yields after filtration 61 mg of (2S, 3R) -3-amino-2-hydroxy-4-cyclohexylbutanoyl- (S) -leucine.

The substance has an Rf = 0.47, [alpha] - 30.5 ° (c = 0.67, acetic acid) For C ₁₆ H ₃₀ N ₂ O ₄ calculated: 61.12% C, 9.62% H, 6.91 % N; found: C, 62.12; 9.52% H, 8.81 % N.

Example 26

300 mg of bestatin are suspended in 10 ml of concentrated sulfuric acid and, while cooling to 0 ° C, 0.3 ml of fuming nitric acid is added. The reaction is allowed to proceed for one hour at the indicated temperature; during this time, the suspended product dissolves, the reaction mixture is diluted by pouring into 100 ml of water, and adsorption is carried out on a Dowex 50X4 resin column (hydrogen cycle).

The adsorbed material was eluted into a 2 N aqueous ammonia solution, the solution was concentrated to dryness, the residue was dissolved in 10 ml of water, filtered and lyophilized to give 213 mg of (2S, 3R) -3-amino-2-hydroxy-4. -p-nitrophenylbutanoyl- (S) -leucine.

The substance had [α] D + 5.2 ° (c = 0.5, acetic acid). Rf = 0.58.

For C ₁₆ H ₂₃ N ₃ 0 ₆ calculated: 54.38% C, 6.56% H, 11.89% N;

Found:% C, 54.28;% H, 6.58;% N, 11.88.

Example 27

100 mg of (2S, 3R) -3-amino-2-hydroxy-4-p-nitrophenylbutanoyl- (S) -leucine as obtained in Example 26 were dissolved in 10 ml of methanol, and the solution was hydrogenated on palladium for 5 hours. blacks. The catalyst is filtered, the filtrate is concentrated under reduced pressure, the residue is dissolved in 5 ml of water, and after filtration, by filtration of the filtrate by lyophilization, 81 mg of (2S, 3R) -3-amino-2-hydroxy-4-p-aminophenylbutanoyl- ) -leucine.

The substance has [alpha] 2θ _β - 23.2 ° (c = 0.5, acetic acid), Rf = 0.25 For C ₁₆ H ₂₅ N ₃ O ₄ calculated: 59.42% C, 7.79% H, 13.00% N; found: 59.01% C, 7.91% H, 12.81% N.

Example 28

Stage 1

2.11 g of (2RS, 3RS) -3-amino-2-hydroxy-4-p-hydroxyphenylbutyric acid are dissolved in 10 ml of 1 N sodium hydroxide solution. With vigorous stirring of the solution and ice cooling, 4.5 ml of benzyloxycarbonyl chloride is added in three portions over 30 minutes.

The reaction mixture was then vigorously stirred for 1 hour under ice-cooling and then for 3 hours at room temperature. During the reaction, the pH is adjusted to 8-9 by addition of 1 N sodium hydroxide solution.

After the addition was complete, the pH of the reaction mixture was adjusted to 2 with 6 N hydrochloric acid. The resulting oily product was then extracted twice with 100 ml of ethyl acetate. The ethyl acetate phase was washed with water and dried over anhydrous magnesium sulfate.

After separating the magnesium sulfate, the filtrate was concentrated under reduced pressure and the residue was crystallized from a mixture of ethyl acetate and petroleum ether. There was thus obtained 3.64 g of benzyloxycarbonyl- (2RS, 3RS) -3-amino-2-hydroxy-4-p-benzyloxycarbonylphenyl butyric acid, m.p. 138-140 ° C.

Stage 2

479 mg of benzyloxycarbonyl- (2RS, 3RS) -3-amino-2-hydroxy-4-p-benzyloxycarbonylphenyl butyric acid and 162 mg of N-hydroxybenzotriazole are dissolved in 10 ml of tetrahydrofuran. After addition of 472 mg of benzyl ester of (S) -leucine with toluenesulfonic acid, the mixture is neutralized with 0.168 ml of triethylamine and cooled to -5 ° C.

206 mg of N, N ^from dicyclohexylcarbodiimide are then added and the reaction mixture is allowed to stand overnight. Tetrahydrofuran was distilled off under reduced pressure and 30 ml of ethyl acetate were added. After filtering off the insoluble matter, the filtrate was washed with 1 N sulfuric acid, water, 5% sodium bicarbonate and water, and then dried over anhydrous magnesium sulfate.

The residue obtained by concentrating the filtrate under reduced pressure solidified in a mixture of ethyl acetate and petroleum ether. Crystallization from the same solvent gave 450 mg of benzoylcarbonyl- (2 H, 3RS) -3-amino-2-hydroxy-4-p-benzyloxycarbonylphenylbutanoyl- (S) -leucine benzyl ester.

M.p. 98 DEG-99 DEG C., [.alpha.] D @ 14.0 DEG (c = 0.58, acetic acid).

Stage 3

400 g of benzyloxycarbonyl- (2RS, 3RS) -3-amino-2-hydroxy-4-p-benzyloxycarbonylphenylbutanoyl- (S) -leucine benzyl ester is dissolved in 10 ml of methanol and hydrogenated to 10 mg of palladium black for 3 hours.

The catalyst was filtered off and the solvent was concentrated under reduced pressure. Crystallization from methanol / ethyl acetate gave 219 mg of (2RS, 3RS) -3-amino-2-hydroxy-4-p-hydroxyphenylbutanoyl- (S) -leucine.

[.alpha.] D @ 20 = 8.8 DEG (c = 0.90, acetic acid), Rf = 0.48 and 0.51.

For C ₁₆ H ₂₄ N ₂ O ₅ calculated: 59.24% C, 7.46% H, 8.64% N;

Found:% C, 59.38;% H, 7.23;% N, 8.95.

The (2RS, 3RS) -3-amino-2-hydroxy-4-p-hydroxyphenylbutyric acid used in Step 1 was prepared as follows:

22.0 g of oily benzyloxycarbonyl- (2RS, 3RS) -3-amino-2-hydroxy-4-p-methoxyphenyl butyric acid are dissolved in 200 ml of concentrated hydrochloric acid and 200 ml of dioxane. After the addition of 13.2 g of anisole, the reaction mixture is heated at reflux for 12 hours. The dioxane was then distilled off under reduced pressure and the remaining aqueous solution was washed with ether and the aqueous phase was concentrated under reduced pressure and evaporated to dryness.

100 ml of water are then added to the residue and the insoluble material is filtered off. After the same amount of acetone was added, the mixture was adjusted to pH 5.5 by adding a solution of ammonia in water. The precipitated crystals were collected by filtration to give 6.93 g of the desired (2RS, 3RS) -3-amino-2-hydroxy-4-p-hydroxyphenylbutyric acid.

Example 29

When (2RS, 3RS) -3-amino-2-hydroxy-4-p-hydroxyphenylbutyric acid is benzyloxycarbonyl using benzyl S-4,6-dimethylpyrimidine-2-lythiocarbonate and dicyclohexylamine, benzyloxycarbonyl- (2RS, 3R) is obtained. -3-amino-2-hydroxy-4-p-hydroxyphenylbutyric acid in the form of the dicyclohexylammonium salt.

85.22 g of crude dicyclohexylammonium salt was crystallized from methanol, ethyl acetate and petroleum ether to give 3.2 g of optically impure dicyclohexylammonium salt of benzyloxycarbonyl- (2RS, 3R) -3-amino-2-hydroxy-4-p-hydroxyphenylbutyric acid in first crystalline fraction.

If the mother liquors were evaporated to dryness and the residue was crystallized three times from ethyl acetate and ether, 5.02 g of optically pure benzyloxycarbonyl- (2S, 3R) -3-amino-2-hydroxy-4-p-hydroxyphenylbutyric acid dicyclohexylammonium salt was obtained.

Mp 121-122 ° C, [alpha] + 55.3 (c = 4.22, CH 3 COOH).

For ^C 3 H 42 N ² O ⁶ calculated: 69.46% C, 8.16% H, 6.17% N;

Found: C, 69.81; H, 8.55; N, 6.42.

Benzyloxyxycabooyl- (2S, 3R) -3-amino-2-hydroxy-4-p-hydroxyphenylbutyric acid (1.05 g) in ethyl acetate is reacted with dilute sulfuric acid to give benzyloxycarbonyl- (2S, 3S, 3R) 3R) -3-Amino-2-hydroxy-4-p-hydroxyphenol-butyric acid is reacted with 866 mg of the benzyl ester salt (S-leucine with p-toluenesulfonic acid, 405 mg of N-hydroxy-zriazole, 0.308 ml of tree amine and 412 mg). Ν ^/ -dicyklohexllkarboíiimiíu procedure of Example 28, step 3. quant yield itatiwiím bennzyester gave Z- (2S, 3R) -3 'aminoo2 hhldooχl4-p-hhldooχ: leeolbbtaaool- (S) -leucine.

If the benzyloxy ester of benzyloxycarbonyl-4S, 3S) -3-amino-2-hydroxy-4-p-hydroxy-phenoxy-butanol (S) -leule was reacted according to the procedure of Example 28, step 3, 630 mg were obtained. (2S, 3R) -3-Amino-hydroxy-1β-hydroxy-butanoyl (S) -leucine.

[α] D - 89.9 ° (c = 1.19, CH 3 COOH), R f = 0.48.

^P ro calculated: 60.55% C, 7.00% И, 10.08% N;

Found:% C, 59.97;% H, 10.2%;

The (2RS, 3R) -3-amino-2-hydroxy-3-p-hydroxyphenyloybutyric acid used in this example was prepared as follows:

When 30 g of benzyloxycarbonyl- (2RS, 3R) -3-amino-2-hydroxy-4-p-hydrophenyl-butyric acid nitrile was reacted according to the procedure of Example 28, 12.61 g of (2RS, 3R) -3-amino were obtained. -2-hydroxy-4-p-hydroxyphenylbutyric acid.

Rf = 0.20.

Why 10Hi ₂ N ° 4 calculated: 58.81% C, 5.92% H, N, 7.82; found: 58.63% C, 5.99% H, 7.43% N. Reference Example 8

Synthesis of nitrile from aldehyde

A solution of 20.8 g of sodium hydrogen sulphite in 50 ml of water is added to 52.7 g of oily benzyloxycarbonyl (R) -feeyyaaaninal, the editorial product is filtered off and washed with water and ether to give 77 g of crude sodium salt. Benzyloxycarboylamino-1-hydroxy-1'-phenylphenoxyan-sulfonic acid, 68 g of the resulting adduct is suspended in 250 ml of water. and cooled to 10-12 ° C. After addition of 500 ml of ether, a solution of 13 g of potassium cyanide in 100 ml of water was added over 30 minutes.

The mixture is then reacted for 3 hours at a temperature and the aqueous phase is discarded. The ethereal layer was washed with brine and dried over anhydrous magnesium sulfate.

After separation of the magnesium sulfate and evaporation of the ether under reduced pressure, 49 g of benzyloxycarbonyl-4R, 3R) -3-amino-2-lyloxy-4-phenyl-phenylallyl nitrile are obtained.

The other nitrile used in the process of the invention is prepared in an analogous manner.

Claims (4)

SUBJECT OF THE INVENTION 1. A process for the preparation of N- (3-lmino-2-lydro) -pyrrolidone) -2 (lactic acid cyanoborohydrates). Joint of formula I. 3 2 1 2 'T R — CH — CH — CO — NH — CH — COOH li ^{1 (} d NH' OH R ₂ ' where ^R ! is C1 -C4 alkyl, cyclopentylmol, cycloalkyl, cyclopentyl, phenyl. or eubbeitjlyt of formula ХУ ^сн '~ r3 where R ³ R ^{2 is} ^ thanks ^ cllsrj ^{MEL th} r ^y yitroe ^y u ^{p iУl,} Tydra .mu.l aminos ^y u ^{p INA} is alkyl of 1 to 6 carbon atoms, tydrojxyakyl having 1-2 carbon atoms, Al ^ xy ^ U ^! C 2 -C 3 alkyl, C 2 -C 3 alkylthomyl. C3 Vilik, klrboχy lnidísl ^y ^ yl having 2-3 carbon atoms У1гО: ^ з ^ 8 ^) ^ 1 ^; ^ 1 with 2-3 carbon atoms; characterized in that the amino amine of the formula II is condensed RI — CH — CH — COOH ⁽ II) NH 2 OH where R1 is as defined above, optionally with protected functionalities which may be reacted in the reaction and amino amino compounds of formula III H'N-CH-COOH ¹² (III) wherein R ₂ is as defined above, after which the protecting groups are triturated. O 2. A method from point ooujijl to a compound wherein R, 11, characterized in that, as in general wooc II is a group of the general formula • 5 where R. is hydrogen, chlorine, hydrogen, nitroelectric, hydrogen. or amino. 3. The process of claim 1 wherein the starting compound is a compound of formula (II) wherein R (is bennzS) and a compound of formula (III) wherein R8 is alkyl of 4 carbon atoms. 4. A process according to claim 1, wherein the starting compound is a compound of formula (II) wherein R @ 1 is p-hydroxybenzyl, and a compound of formula (III) wherein R8 is alkyl of 4 carbon atoms.