CA1118441A - Analogs of bestatin - Google Patents

Abstract

IMPROVEMENTS IN AND RELATING TO
ANALOG OF BESTATIN
ABSTRACT
p-Hydroxy-bestatin, which is [(2S,3R)-3-amino-2-hydroxy-4-p-hydroxyphenylbutanoyl]-L-leucine, and related compounds which inhibit aminopeptidase B were synthesized and tested.

Description

~844~

~ACKGROUND OF THE INVENTION
1. Field of the Invention This invention relates to chemical compounds of the peptide type produced by synthetic methods and includes various processes.

2. Description of the Prior Art Bestatin is a chemical active as an inhibitor of certain enzymes which was originally produced by ~er-me~tation of Streptomyces olivoreticuli as disclosed in various patents (see Farmdoc 09548X) and the following publications:
1. Umezawa, H.; T. Aoyagi, H. Suda, M. Hamada &
T. Takeuchi: Bestatin an Inhibitor of Aminopeptidase B, Produced by Actinomycetes. J. Antibiotics 29:97-99, 1976.
2. Suda, H.; T. Takita, T. Aoyagi & H. Umezawa:
The Structure of Bestatin. J. Antibiotics 29:100-101, 1976.

3. Nakamura, H.; H. Suda, T. Takita, T. Aoyagi, H. Umezawa & Y Iitaka: X-Ray Structure Determlnation of (2S,3R)-3-amino-2-h~droxy-4-phenylbutanoic Acid, a New Amino Acid Component of Bestatin. J. Antibiotics 29:lG2-103, 1976.

4. Suda, H.; T. Takita, T. Aoyagi and H. Umezawa:
The Chemical Synthesis of Bestatin. J. Antibiotics 2 :600-601, 1976.

5. Umezawa, H.; M. Ishizuka, T. Aoyagi and T. Takeuchi, Enhancement of Delayed-Type Hypersensitivity by Bestatin, an Inhibitor of Aminopeptidase B and Leucine Aminopeptidase, J.

Antibiotics 29, 857-859, 1976.
Bestatin has the chemical name [(2S,3R)-3-amino-2~
hydroxy-4-phenylbutanoyl[-L-leucine and the following structure -2- ~

CH2-~_C-CO--NH--C-COOH
H OH fH2 CH

SU~MARY OF THE INVENTION
According to the present invention, there is provided a compound represented by the following formula (I):
1 3 2 1 2' 1' R -- CH - jH - CO - NH -- CH - COOH ( I ) where Rl is hydroxybenzyl and R2 is a member selected from the group consisting of alkyl having 1 to 6 carbon atoms, hydroxy-alkyl, mercaptoalkyl, carboxamidoalkyl, alkoxyalkyl, alkyl-mercaptoalkyl, carboxyalkyl, aryl, aralkyl and substituted aralkyl.
Bestatin is (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucine which is a compound represented by said Formula (I) wherein Rl is benzyl and R2 is isobutyl.

~.~

11 1 8 4~

A preferred embodiment of the present invention is a compound having the formula l 3 2 l 2' l' R -CH -CH-CO-NH-IH_COOH

wherein R is nd ~ i8 (lower)alkyl ha~ing 1 to 6 carbon atoms, hydroxy(lower~alkyl, alkylthioalkyl, carboxamido(lower)-alkyl, carboxy(lower)alkyl, phenyl or benzyl.
Another pre~erred embod~ent of the present invention iB a compound ha~lng the formula 3 2 l 2' 1' Rl-fH--CIH-CO-NH-CIH-COOH

~herein Rl i~
OH

~CH2 a n ~ R2 18 (lower)alkyl having 1 to 6 carbon atoms, hydroxymethyl, methylthioethylJ C~ C~2CO ~ , -C~ C~ COOH, phenyl or benzyl.

34~1 Another preferred embodiment of the present invention is a compound naving the formula 3 2 1 2' 1' Rl-CH -CH~CO NH-CH-COOH

wherein Rl is and R2 ~ (lower)alkyl ha~lng 1 to 6 car~on atom~ 3 hgdroxymethgl, methylthioethyl, -C ~ C ~ CO ~ , -C~ C~ COOH, phenyl or ~enzyl.
Another preferred embodiment of the present lnvention i8 a compound having the formula 1 3 2 1 2' 1' R -CH -CH-CO-NH-CH-COOH

whereln Rl i~

HO ~ C ~ -and R2 i~ (lower)alkyl having 1 to 6 carbon a~oms.
Preferred specie~ of the pre~ent invention in-clude the ~ollowlng:
(2R~3RS)-~-amino-2-hydroxy-4-p-hydroxyphenyl-butanoyl-(S)-leu~ine.
(2~,3R)-3-amino-2-hydroxy-4-p-hydroxyphenyl-butanoyl-(5)-leucine.

~, ,,~

``` 111~441 The invention also includes a process for the preparation of the compounds rePresented by Formula (I) in the following manner:
A nitrile derivative as represented by a formula (II), R -CH CH-CN
NH2 OH (II) (wherein Rl is a group as defined above) or such a nitrile der-ivative whose amino group is protected is hydrolyzed with an acid to prepare an amino acid as represented by a formula (III), Rl -IH - CH-COOH
NH2 OH (III) (wherein Rl is a group as defined above). After having protected, if necessary, the functional groups which do not take part in the reaction, this amino acid and an amino acid represented by the formula (IV) NH2 -fH-COOH
R2 (IV) (wherein R2 is a group as defined above) are condensed in the ordinary peptide linking method. When said protected groups are removed, the compounds as represented by Formula (I) are obtained.
There is also provided by the present invention a process for producing a peptide represented by the formula 1 3 2 1 2' 1' R -~H CH-CO-NH-CH-COOH

(wherein Rl and R2 are groups as defined above)com~rising con-secutive steps in which a nitrile represented by the formula R1 _ ~H I H- CN

(wherein Rl is a group as defined above) or a nitrile derivative whose amino group is protected is hydrolyzed with an acid to lllB4'~

repare an amino acid represented by the formula R _CH--CH_COOH

(wherein Rl is a group as defined above); after having pro-tected, if necessary, the functional groups which do not participate in the reaction, the resulting amino acid and an amino acid as represented by the formula (wherein R2 is a group as defined above) are condensed in an ordinary pe tide linking process; and then said protecting group is removed and, preferably, said process wherein R is p-hydroxybenzyl or su~stituted benzyl and R2 is alkyl or hydroxymethyl having 3 or 4 carbon atoms and also said process wherein Rl is p-hydroxybenzyl and R2 is alkyl having 4 carbon atoms and also said process wherein the acid which is employed for hydrolysis is hydrochloric acid, hydrobromic acid or sulfuric acid.
There is also provided by the present invention a process for collecting an antipode comprising consecutive steps in which an amino acid represented by the formula R1 - fH--CH_COOH

(wherein R is a group as defined above) is protected by benzyloxycarbonyl; and said diasteroisomer is selectively crygtallized in organic solvents in the form of dicyclohexyl-amine salt.

4~1 mere is further provided bv the present invention a pro-cess for collecting an antipode comprising consecutive steps in which an amino acid re~resented by the formula R -~CH ~ H-COOH

(wherein R is a group as defined above) is protected by benzy-loxycarbonyl; the thusly protected amino acid is dissolved in an organic solvent selected from a grou~ of alcohols, ethers, esters, ketones, halogenated hydrocarbons, dimethylformamide, dimethylacetamide and acetonitrile; then to the resulting sol-ution an organic solvent selected from a group of ~etroleum hydrocarbons, aromatic hydrocarbons, hexane and cyclohexane is added so as to optically resolve the same and preferably said process wherein Rl is p-hydroxybenzyl and also said process wherein the organic solvents are ethyl acetate and petroleum ether.
There is also provided by the present invention a com~ound represented by the formula ~; -8-' ,~.~';;~

R -fH - CH-COOH

wherein Rl is hydroxybenzyl and particularly is (2S,3R)-3-amino-2-hydroxy-4-~-hydroxy~henyl-butanoic acid or (2RS,3RS)-3-amino-2-hydroxy-4-p-hydroxy~henyl-butanoic acid.

_ g Typical examples of nitrile derivatives as represented by Formula (II~ are 3-amino-2-hydroxy~4-p-hydroxyphenylbutyronitrile and 3-amino-2-hydroxy-4-o-hydroxyphenylbutronitrile. Any known amino-protecting group such as those el~Dloyed in peptide chem-istry may be used for protection of the amino groups. Preferred exam~les of acyl type amino-~rotecting groups are a formyl group, an acetyl group, a trifluoroacetyl group, substituted or non substituted benzoyl group; examples of urethane type amino-protecting groups are substituted or non-substituted benzyloxy-carbonyls, alkoxycarbonyl group having 1 to 6 carbon atoms andcycloalkanoxycarbonyl group; preferred examples of other amino-protecting groups are substituted or non-substituted arylsulfonyl group, phthalyl group, o-nitrophenylsulphenyl group or trityl group. Any type of acid usually employed for hydrolyzing nitriles may be used in the present invention for hydrolysis.
Examples of inorganic acids include hydrochloric acid, hydro-bromic acid and sulfuric acid; examples of organic acids include alkylsulfonic acids such as methanesulfonic acid, ethanesulfonic acid, arylsulfonic acid such as benzenesulfonic acid and toluene-sulfonic acid. The concentration of these acids may be at anylevel usable for hydrolyzing ordinary nitriles. However, a concentration of above lN will be preferred.
To the compounds whose amino group is protected S ~ , 10--and which are not readily dissolved in the aqueous solution of acid, an organic solvent which is miscible with water such as tetrahydrofuran, dioxane, lower alcohols, acetone, dimethylformamide, dimethylacetamide and dimethyl-sulfoxide may be added to enhance the solubility of the compounds in the aqueous solution.
In the case when a protecting group is employed~
if the protecting group is removed during the hydrolysls to form electrophilic bodies, a cation scavenger such as anisole may be added. The hydrolytic temperature may be at any level within the range from room temperature to the temperature at which reflux of the reaction mixture takes place. To isolate the desired amino acid from the reaction mixture, any ordinary method for isolating amino acid may be applied. For example, when the hydrolysis is effected with a volatile acid, the exce~sive acid may be removed by concentrating the reaction rnixture under reduced pressure and the residue dissolved in water and neutralized with aqueous alkali solution to the isoelectric point. Then, if necessary, acetone, methanol or ethanol i~ added to the reaction mixture to crystallize the in-tended compound as represented by Formula (III), which is then separated by filtration. Or otherwise, when a non-volatile acid is used the reaction mixture is diluted with water to less than lN acid and allowed to pass through a strong acidic ion exchange resin so that the desired amino acid can be adsorbed to the ion exchange resin. The ad-sorbed amino acid is eluted from the ion exchange resin with a volatlle alkali such as ammonia water and condensed under reduced pressure. If necessary acetone, methanol or ethanol is added to crystallize the intended compound as ~B~
represented byFormula (III)~ which is then se~arated by filtra-tion.
In the foxegoing reaction, when an (~)-nitrile derivative is used as a starting material, (2RS,3R)-amino acid is obtained;
when a (S)-nitrile derivative is used, (2RS,3S)-amino acid is obtained: whenan (RS)-nitrile derivative is used, (2RS,3RS)-amino acid is obtained.
The comPounds obtained as represented by Formula (III) can be used as a raw material in the process as described herein-below. For example, 3-amino-2-hvdroxy-4-p-hydroxyphenylbutanoic acid (hereinafter, this amino acid is abbreviated as AHPA(p-OH)) i5 in (2S,3R) form. Therefore, in order to obtain this compound, it is necessary to resolve (2RS,3R)-AHPA(p-OH) to (2S,3R)- and (2R,3R)-AHPA(p-OH).
(2RS,3R)-AHPA(p-OH) can be resolved optically in ethyl acetate in the form of diasteroisomer which is obtained by first benzyloxycarbonylation of (2RS,3R)-AHPA(p-OH) in the ordinary manner to prepare benzyloxycarbonyl-(2RS,3R)-AHPA(P-OH), which is then allowed to react with dicyclohexylamine (hereinafter, benzyloxycarbonyl group is abbreviated as Z).
(2R,3R)-AHPA(p-OH) is benzyloxycarbonylated by benzyl S-4,

6-dimethylpyrimidin-2-yl-t'niocarbonate in the ordinary procedure to give benzyloxycarbonyl- (2RS,3P~)-AHPA(p-OH), which is then crystallized as its dicyclohexylamine salt (hereinafter, benzvl-oxycarbonyl group is abbreviated as Z).
When the salt is selectively recrystallized in organic sol-vents Z-(2R,3R)-AHPA(p-OH) is obtained. When the filtrate is concentrated under reduced pressure and the residue is recrystal-lized from ethyl acetate and ether, the dicyclohexylamine salt 84~1 of optically pure Z-( 2S,3R)-AHPA(p-OH) is obtained. When this dicyclohexylamine salt is selectively crvstallized the dicyclo-hexylamine salt of optically pure Z-( 2S,3R)-AHPAtp-OH) is obtained. The dicyclohexylamine salt of Z-( 2S,3R~-AHPA~p-OH) is obtained by concentration of the filtrate under reduced pressure and recrystallization from ethyl acetate and ether.
It was noted that he ( 2P~,3R) -form of Z-AHPA(p-OH) had smaller solubilities into an organic solvent than the (2S,3R)-form. Z-(2RS,3R)-AHPA(p-OH) is dissolved in a first soluble solvent at room temperature or under heating. Then a second in-soluble solvent is added so as to precipitate crystals, which are then separated by ~iltration. If necessary, the foregoing operation is repeated until optically pure Z-( 2P~,3R)-AHPA(p-OH) is obtained.
The first soluble solvent may include lower alcohols such as methanol, ethanol and propanol; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran and dioxane; esters such as methyl acetate and ethyl acetate; ketones such as acetone and methyl ethyl ketone; halogenated hydrocarbons such as methylene chloride and chloroform; amides such as dimethylformamide and dimethylacetamide; and nitriles such as acetonitrile, etc. The second insoluble solvent may include petroleum hydrocarbons such as petroleum ether, petroleum benzene and ligroin; aromatic hydrocarbons such as benzene and toluene; alkanes such as pen-tane and hexane; and cyclohexanes such as cyclopentane and 34~

cyclohexane. By use of the afore-mentioned method em~loying dicyclohexylamine, the opticall~ im~ure Z-(2S,3R)-AHPA(p-OH) present in the filtrate may be purified to optically pure Z-(2S, 3R)-AHPA(p-OH).
Z-AHPA(p-OH) as used in the above-mentioned resolution may be synthesized by Schotten-Baumann method in the presence of an alkali by reaction of (2RS,3R)-AHPA(p-OH) and benzyloxycarbonyl chloride or, otherwise, in the presence of an organic tertiary base such as triethylamine and N-methylmorpholine by reaction with a benzyloxycarbonylating reagent such as benzyloxycarbonyl-p-nitrophenyl ester, benzyloxycarbonyl azide, benzyloxycarbonyl-N-hydroxysuccinimide ester and benzyloxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine in a solvent ~repared by mixing dioxane, tetrahydrofuran, acetonitrile or dimethylformamide with water.
Although the foregoing optical resolution has been described with respect to AHPA(p-OH), a similar optical resolu-tion may be effected, if necessary, for the other amino acids.
To prepare compounds as represented by Formula (I), an amino acid as represented by Formula (III) is condensed with a compound as represented by Formula (IV) in the ordinary pePtide linking method and then the protecting groups, which, if necessary have been used for protecting the functional groups which do not take part in the reaction, are removed.
Typical examples of amino acids are glycine, alanine, 2-amino-butanoic acid, valine, norvaline, leucine, norleucine, isoleucine, tertiary-leucine, 2-amino-3~

34~1 heptanoic acid~ 2-amino-5-methylhexanoic acid, 2-amino-octanoic acid, 2-amino-6-methylheptanoic acid, serine, threonine, allothreonine, cysteine, homocysteine, as-paragine, glutamine, O-methylserine, O-ethylserine, O-propylserine, methionine3 ethionine, aspartic acid, glutamic acid, phenylglycine, p-methoxyphenylglycine, phenylalanine, tyrosine, p-methoxyphenylalanine and p-nitrophenylalanine.
The method for condensing an amino acid as represented by Formula (III) and an amino acid as repre-sented by Formula (IV) includes a carbodiimide method wherein dicyclohexylcarbodiimide or l-ethyl-~-(3-dimethyl-aminopropyl)carbodiimide is used; an azide method employ-ing nitrous acid or alkyl nitrite; a mixed anhydride method employing ethyl chloroformate or isobutyl chlor)-~^rmate; an active ester method employing cyanomethyl ester, vinyl ester, substituted and non-substituted phenyl esters, thiophenyl ester of N-hydroxysuccinimide ester; an O-acyl hydroxylamine derivative method employ-ing O-acylacetoxime or O-acylcyclohexanonneoxime; an N-acyl derivative method employing carbonyldiimidazole.
Organic solvents using in the above ~onderlsa-tion reaction may be ethers such as diethyl ether, tetrahydrofu-ran or dioxane; esters such as ethyl acetate or methyl acetate; ketones such as acetone or methyl ethyl ke~tone; halogenated hydrocarbons such as methylene chloride or chloroform; amides such as dimethylformamide or dimethylacetamide; nitriles such as acetonitrile.
In the case when an amino acid is represented by Form~la (III) and whose amino group is protected and an amino acid as represented by Formula (IV) whose 4~

carboxyl group is not protected are condensed in an active ester method, a mixed solvent of water and water miscible organic solvent may be used in the presence of an inor-ganic base such as sodium bicarbonate, magnesium oxide or an organic tertiary base such as triethylamine or N-methylmorpholine Protecting groups in the resulting compound may be removed by the usual method in peptide chemistry such as catalytic hydrogenation on palladium, saponification with an alkali, acidolysis with hydrogen bromide in acetic acid, with trifluoroacetic acid~ with hydrogen chloride in dioxane, tetrahydrofuran or ethyl acetate~
with liquid hydrogen fluoride, hydrazinolysis with hydra-zine or treatment with sodium in liquid ammonia. Thus, the intended substance as represented by Formula (I) ~s ob-ained.
The nitrile derivatives as represented byFormula (II) and used in this invention as the starting material may be synthesized reducing an a-amino acid, whose amino group is belng protected and c,arboxyl group converted to an amide by reaction with a secondary amine, at low temperature less than 0 C. with a metal hydride in ethers such as diethyl ether or tetrahydrofuran to prepare an amino-aldehyde whose amino group is protected.
The resulting amino-aldehyde is then converted to the adduct with sodium bisulfite and then allowed to react with an alkaline metal cyanide or directly allowed to react with hydrogen cyanide to prepare cyanohydrin, or 3-amino-2-hydroxynitrile.
Preferred examples of the secondary amine are N,N-dimethylamine, aziridine, N-methylaniline, carbazole, 3,5-dimethylpyrazole and imidazole~ Preferred exa~les of metal hydride are lithium aluminum hydride, lithium di- and tri-alkoxyaluminum hydride and sodium bis~2-methoxyethoxy)aluminum hydride. Examples of the protecting group for the amino group are the protecting groups usually employed in the peptide chem-istry. Urethane type protecting groups may be used preferably and above all, benzyloxycarbonyl grou3 may be preferred.
According to a second method a lower alkyl ester of a_ amino acid whose amino group is protected by an amino-protecting group similar to those mentioned previously is reduced at a temperature lower than -40 C. in aromatic hydrocarbons such as toluene or benzene and ethers such as diethyl ether or tetra-hydrofuran with metal hydrides such as sodium aluminum hydride, diisobutylaluminum or sodium bis(2-methoxyethoxy)aluminum hydride thereby to prepare aminoaldehyde whose amino group is protected.
When such an aminoaldehyde has been treated in the above-mentioned manner, 3-amino-2-hydroxynitriles are pre~ared.
Following is a discussion by way of example only of methods of carrying the invention into effect. However, it should be noted herein that any suitable functional group-protecting methods, functional group-removing methods and peptide linkage-forming methods other than those described hereinbelow may be employed. In addition to the aforementioned abbreviations, the following abbreviations may be used in the examples:
HOBt, N-hydroxybenzotriazole; DCCD, N,N'-dicyclohexylcarbodi-imide; HOSu, N-hydroxysuccinimide; -OBzl.TosOH, benzyl ester p-toluenesulfonate; -OSu, N-hydroxysuccinimide ester; -OMe, 34~

methyl ester; -ONb, p-nitrobenzyl ester; Boc, t-butoxycarbonyl;
DCHA, dicyclohexylamine, -Osut tert.-butyl ester.
Rf value is measured on a silica gel GF254 ~late prepared by Merck Corp. using n BuOH: AcOH: H2O (4:1:1) as developing solvent.
A few 3-amino-2-hydroxYcarboxylic acids and their contain-ing peptides possess two Rf values because they are a mixture of threo- and erythro- configurations.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Example 1 Step 1 22.0 g. of oily Z-(2RS,3RS)-3-amino-2-hydroxy-4-p-methoxy-phenylbutyronitrile was dissolved in a mixture of 200 ml. of concentrated hydrochloric acid and 200 ml. of dioxane. After adding 13.2 g. of anisole the reaction mixture was refluxed for 12 hours. Then the dioxane was distilled away under reduced pressure, the resulting solution of hydrochloric acid washed with ether and the water layer concentrated under reduced pressure and evaporated to drvness. Subsequently, 100 ml. of water was added to the residual substance and the insoluble sub-stance was separated by filtration. After adding an equal quantity of acetone the mixture was adjusted to pH 5.5 with am~onia water. The mixture was allowed to stand in a refriger-ator. The deposited crystals were separated by filtration to obtain 6.73 g. of intended (2RS,3RS)AHPA(p-OH).

1~184~1 Step 2 2.11 g. of (2~S,3RS)-AHPAtp-OH) obtained in Ste~ 1 was dissolved in 10 ml. of lN sodium hydroxide solution. While vigorously agitating the solution under coolin~ with ice, 4.5 ml. of Z-Cl was added in three portions over a period of 30 minutes. Then the reaction mixture was vigorously agitated for 1 hour under cooling with ice and for 3 hours at room temper'a-ture. During the reaction the pH was adjusted to 8-9 with lN
sodium hydroxide solution.
T~hen the reaction had been com~leted, 6N hydrochloric acid was added to adjust the reaction mixture to pH 2. As a result oily material separated which was then extracted twice with 100 ml. of ethyl acetate. The ethyl acetate layer was washed with water and dehydrated to dryness by the use of anhydrous magnes-ium sulfate. After separating magnesium sulfate by filtration the filtrate was concentrated under reduced pressure and the residue crystallized in ethyl acetate-petroleum ether to provide 3.64 g. of Z-(2RS,3RS)-AHPA(P-OZ). M.p. 138-140 C.

,~. . .

11184 ~1 Step 3 479 mg. of Z-(2RS,3RS)-AHPA(P-OZ) and 162 mg.
of HOBt were dissolved in 10 ml. of tetrahydrofuran.
After adding 472 mg. of Leu-OBzl-TosOH the mlxture was neutralized with 0.168 ml. of triethylamine and cooled to -5 C. Then 206 mg. of DCCD was added and the reac-tion mixture was allowed to stand overnight for reaction.
~-~rahydrofuran was distilled away under reduced pressure and ~0 ml. of ethyl acetate was added. After filtering off the insoluble substances the filtrate was washed with lN sulfuric acid, water, 5% aqueous sodium bicarbonate solution and water in that order and then dehydrated to dryness with anhydrous magnesium sulfate. The residue obtained by concentrating the filtrate under reduced pressure was solidified in ethyl acetate-petroleum ether.
Recrystallization from the same solvent gave 450 mg. of Z-(2RS,3RS)-AHPA(P-OZ)-(S)-Leu-OBzl. M.p. 98-99 C., [a]5788 -14-0 (c o.58, AcOH).
Step 4 400 mg. of Z-(2RS,3RS)-AHPA(P-OZ)-(S)-Leu-OBzl was dissolved in 10 ml. of methanol and hydrogenated for 3 hours with about 10 mg. of palladium black. The catalyst was filtered off and the solvent was concentrated under reduced pressure. When a recrystallization opera-tion was carried oùt with methanol-ethyl acetate, 219 mg.
of (2RS,~RS)-3-amino-2-hydroxy-4-p-hydroxyphenylbutanoyl-(S) leucine were obtained.
[a]3578 -8~8(c 0.90, AcOH), Rf o.48 and 0.51, An~l. for C16H24N205, Found: C~ 59.38; H, 7.23; N, 8.95. Calc'd.: C, 59.24;

H, 7.46; N, 8.64.

xample 2 When 30 g. of Z-~2RS,3R~3~amino-2-hydroxy-4-p-hydroxyphenylbutyronitrile was treated in similar manner to Example 1, Step 1, 12.61 g. of (2RS,3R)-AHPA(p-OH) was obtained.
Rf 0.20 Anal. for CloH12NO4, H, 5.99; N, 7.43. Calc'd.: C, 58.81; H, 5.92; N, 7.82.
When (2RS,3R~-AHPA(p-OH) was benzyloxycarbonylated using benzyl-S-4,6-dimethylpyrimidin-2-ylthiocarbonate, Z-AHPA(p-OH) was obtained as DCHA salt. 15.22 g. of that crude DCHA salt was crystallized from methanol, ethyl acetate and petroleum ether and 3.2 g. of optically impure Z-(2R,3R)-AHPA(p-OH) DCHA salt was obtained as a first crop.
When the mother liquor was evaporated to dryness and the residue precipitated three times from ethyl acetate and ether, 5.02 g. of optically pure Z-(2S,3R)-AHPA(p-OH)DCHA
salt was obtained.
M.p. 121-122C., [~]578 + 49 9 (c 0.87, AcOH);
Anal. for C30H42N2O6, Found: C, 69.81; H, 8-35; N, 6-42-Calc'd.: C, 69.46; H, 8.16; N, 6.17.
After a treatment of Z-(2S,3R)-AHPA(p-OH) DCHA
salt (1.05 g.) with ethyl acetate and dilute H2SO4, the obtained Z-(2S,3R)-AHPA(p-OH), 866 mg. of (S)-Leu-OBzl TosOH, 405 my. of HOBt, 0.308 ml. of triethylamine and 412 mg. of DCCD were treated in similar manner to Example 1, Step 3.
Oily Z-(2S,3R)-AHPA(p-OH)-(S)-Leu-OBzl was obtainea quan-titatively~
When the obtained oily Z-(2S,3R)-AHPA(p-OH)-(S)-Leu-OBzl was treated in similar manner to Example 1, Step 4, 630 mg. of (2S,3R)-3-amino-2-hydroxy-4-p-hydroxy-11184~1 phenylbutanol-(S)-leucine was obtained.
~ ~-7578 -19-9 (c~ 1.19, AcOH) Rf 0.48, Anal. for C16H23N2O5, Found C, 59.98; H, 7.42;
N, 10.42. Calc'd: C, 60.55; H, 7.30; N, 10.08.
The physiological activities o~ Bestatin and two compounds represented by Formula (I) prepared in the present invention were determined as follows:
(A) Inhibitory activity against aminopeptidase B Testing method:
The method described by V. K. Hopusu, K. K. Makinen, G. G. Glenner in Archives of Biochemistry and Biophysics 114, 557, (1966) was modified. To the mixture of 0.3 ml. of 1 mM
arginine ~ -naphthylamide and 1.0 ml. of 0.1 M Tris hydro-chioride buffer (pH 7.0), 0.7 ml. of distilled water with or without a test material is added and warmed at 37 C. for 3 minutes. The reaction is started by addition of 0.2 ml. of aminopeptidase B solution which is prepared by Sephadex* 100 chromatography as described by Hopusu et al. After 30 minutes at 37 C., 0.6 ml. of l.OM acetate buffer (pH 4.2) containing diazonium salt of o-aminoazotoluene at 1.0 m~./ml. and Tween* 20 at 1.0% is added. Fifteen minutes at room temperature there-after, absorbance (a) at 530 nm is measured by spectrophotometer.
As the control, by similar means the absorbance (b) after the reaction in the absence of a sample is measured. me inhibition percent is calculated as follows: (b-a)/b x 100.
Inhibition percentages at various concentrations were measured and from the measured inhibition percentages, 50%
inhibitions (ID50) were deduced. The results are as listed in Table 1.

1~184 ~1 Table 1 Compounds 50 (mcg./ml.) Example 1 0.10 Example 2 0.007 Bestatin [(2S,3R~-AHPA-(S~-Leu~ 0.10 As you will see from the table the compound of Example 1 has substantially the same inhibitory effects as Bestatin but the compound of Example 2 can attain the same effect in a far less amount, one-fourteenth of Bestatin. Gathering from these results, it is expected that the new compounds, especially the compound of Example 2 which is an optically active form of the com-pound of Example 1, can be an extremely useful physio logically active substance.
(B~ The compound of Example 1 was also tested for its humoral antibody formation to find its efficaGy as an immunizing cancer inhibitor. As a result, it was found that the compound has an effect of increasing the number of humoral antibody cells to a considerable degree. The results suggest that the compound can serve as an excel-lent immunizing cancer inhibitor. For the humoral anti-body formation of Bestatin per se see J. Antibiotics, 29(8), 857-859 (August, 1976~.
The effect of (2RS,3RS)-3-amino-2-hydroxy-4-p-hydroxyphenylbutanoyl-(S)-leucine on humoral antibody formation to Sheep Red Blood Cell (SRBC~ in mice was studied as follows.
Mice (dd/y female) were immunized by intravenous injection of 10 SRBC. Intraperitoneal injection of 44~

(2RS,3RS)-3-amino-2-hydroxy-4-p~hydroxyphenylbutanoyl-(S)-leucine was made soon afterwards.
Bestatin [(2S,3R~-3-amino-2-hydroxy-4-phenyl-butanoyl-(S)-leucine] and (2RS,3RS)-3-amino-~-hydroxy-4-p-chlorophenylbutanoyl-(S~-leucine were used as control.
Four days thereafter the number of plaque forming cells in the spleen were enumerated by JERNE's hemo-lytic plaque technique [Jerne, N.K.; A. A. Nordin & C.
Henry: "The Agar Plaque Technique for Recognizing Anti-body-producing Cells" in cell-bound antibodies (Wistar Institute Press, Philadelphia, 1963~, pp. 109-122].
The results are as listed in Table 2.
Table 2 Effect of (2RS,3RS~-3-amino-2=hydroxy-4-p-hydroxyphenyl-butanoyl-(S)-Leucine on Humoral Antibody Formation to SRBC in Mice Antibody Forming CeIls Treated Group Non-treated Name of Compound Dose Number Group - - 12,500+ 9,050 (2RS,3RS)-3-amino- 1 mg. 207,400_ 8,025 1.66 2-hydroxy-4-p- 100 mcg 261,000+11,700 2.09 hydroxyphenylbuta-noyl-(S)-leucine10 mcg. 208,000+ 7,180 1.66 1 mcg. 175,800+ 8,200 1.41 0.1 mcg. 141,000+ 5,700 1.13 Bestatin 1 mg. 190,000+ 7,100 1.52 10 mcg. 136,250+ 6,500 1.09 (2RS,3RS)-3-amino- 1 mg. 208,750+ 8,000 1.67 2-hydroxy-4-p-10 mcg. 133,750+ 5,600 1.07 chlorophenyl-butanoyl-(S)-Leucine ~1~84'~1 The number of antibody forming cells in mice group given 10 mcg. of Bestatin or ~2RS,3RS~-3-amino-2-hydroxy-4-p-chlorophenylbutanoyl-(S~-leucine was nearly equal to that of non-treated group.
On the other hand the number of antibody forming cells in mice given 10 mcg. of (2RS,3RS~-3-amino-2-hydroxy-4-p-hydroxyphenylbutanoyl-(S~-leucine was 1.66 times larger than that of the non-treated mice and even when given only 1 mcg. it showed 1.41 times.
As mentioned above (2RS,3RS~-3-amino-2 hydroxy-4-p-hydroxybutanoyl-(S)-leucine has the excellent effect of increasing the number of antibody forming cells and did not increase the weight of the spleen or the number of nonspecific antibody forming cell.s.

1~18441 'Re'fe'renc'e Example 1 Synthesis of Nitr le'From A'l'dehyde A solution consisting of 20.8 g. of sodium hydrogen sulfite and 50 ml. of water is added to about 52.7 g. of oily Z-(R)-phenylalaninal and separated adduct is filtered off and washed with water and ether in this order to prepare 77 g . of crude 3-benzyloxycarbonylamino-2-hydroxy-4-phenylbutyronitrile. 68 G. of resulting adduct is suspended in 250 ml. of water and cooled to 10-12C. After adding 500 ml. of ether, a solution consisting of 13 g. of potassium cyanide and 100 ml. of water is added dropwise over 30 minutes.
Then the reaction mixture is allowed to react for 3 hours at room temperature and the water layer is dis-carded. The ether layer is washed with an aqueous solution of sodium chloride and dehydrated to dryness over anhydrous magnesium sulfate.
When magnesium sulfate is filtered off and ether is distilled away under reduced pressure, 49 g. of oily benzyloxycarbonyl-(2RS,3R)-3-amino-2-hydroxy-4-phenylbutyro-nitrile is obtained.
Nitrile derivatives used in the present inventionare prepared in the same manner.

Claims (10)

Div.
RD-9203C Div.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for producing an amino acid represented by the formula in which R1 is hydroxybenzyl wherein a nitrile derivative represented by the formula (wherein R1 is a group as defined above), or such a nitrile whose amino group is protected, is hydrolyzed with an acid and then removing said protecting group if one is present.

2. A process according to Claim 1, wherein the acid employed for hydrolysis is hydrochloric acid, hydrobromic acid or sulfuric acid.

3. A process according to Claim 1 wherein R1 is hydroxybenzyl.

4. A process according to Claim 2 wherein R1 is hydroxybenzyl.

5. A compound represented by the formula wherein R1 is hydroxybenzyl whenever prepared or produced by the process of Claim 3 or 4 or by an obvious chemical equivalent thereof.

6. A process for producing an amino acid represented by the formula in which R1 is para-hydroxybenzyl wherein a nitrile derivative represented by the formula (wherein R1 is a group as defined above), or such a nitrile whose amino group is protected, is hydrolyzed with an acid and then removing said protecting group if one is present.

7. A process according to Claim 6, wherein the acid employed for hydrolysis is hydrochloric acid, hydrobromic acid or sulfuric acid.

8. A process according to Claim 6 wherein R1 is para-hydroxybenzyl.

9. A process according to Claim 7 wherein R1 is para-hydroxybenzyl.

10. A compound represented by the formula wherein R1 is para-hydroxybenzyl whenever prepared or produced by the process of Claim 8 or 9 or by an obvious chemical equivalent thereof.