Note: Descriptions are shown in the official language in which they were submitted.
56;
BACKGROUND OF THE INVENTION
. ~ _
1. Field o~ 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 fer-
mentation of Streptomyces olivoreticul~ as disclosed in
__
various patents ~see Farmdoc o9548X) and the ~ollowing
publications:
` 1. Umezawa~ H.; T. Aoyagi, Ho Suda, M. Hamada &
T. Takeuchi: Bestatin an Inhibitor of Aminopeptidase
B, Produced by Actinomycetes. JO Antibiotics 22:97-99,
1976.
2. Suda, H.; T. Takita, T. Aoyagi ~ H. Umezawa:
The Structure o~ Bestatin. J. Antibiotics 29:100-101,
1976.
3. Nakamura, ~., H. Suda, T. Takita, T. Aoyagi,
H. Umezawa & Y. Iitaka: X-Ray Structure Determination
o~ (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoic Acid, a
New ~mino Acid Component of Bestatin. J. Antibiotics
29:102-103, 1976.
4. Suda~ H ; T. Takita, T. Aoyagi and H. Umezawa:
The Chemical Synthesis of Bestatin. J. Antibiotics
29:600-601, 1976.
Bestatin has the che-mical name [(2S,3R)-3-
amino-2-hydroxy-4-phenylbutanoyl]~L-leucine and the
~ollowing structure
-2-
5~
NH2 H H
CH2-1 - C-co-~H-l-cooH
H OH f H2
~C~
H3C CH~
SUMMARY_OF THE INVENTION
According to the present invention, there is
provided a compound represented by the ~ollowing ~or-
mula (I):
~ 2 1 2' 1'
Rl _ IH - 7H - CO - NH - IH - COOH (I)
NH2 OH R
where R is a group selected from a group consisting of
lower alkyl group, cycloalkanoalkyl group, phenyl groupg
benzyl group and substituted benzyl group; R2 is a
group selected from a group o~ ælkyl group having 1 to
6 carbon atoms, hydroxyalkyl group, mercaptoalkyl
group, carboxamidoalkyl group, alkoxyalkyl group, alkyl-
mercaptoalkyl group, carboxyalkyl group, aryl group,
aralkyl group and substituted aralkyl group. In the
case when Rl is benzyl group and R2 is isobutyl group,
the configuration of the compound is only (2S,3R,2'R),
(2S,~S,2'S) or (2S,3S,2'R).
Bestatin is (2S,3R)~3-amino-2-hydroxy-4-
phenylbutanoyl-(S)-leucine which is a compound repre-
sented by said Formula (I) wherein R is a benzyl group
and R an isobutyl group.
A preferred embodiment o~ the present invention
is a compound having the ~ormula
1 3 2 1 2' 1'
R -CH -CH CO-NH-CH-COOH
1 2
NH2 OH R
wherein Rl is (lower)alkyl, cyclopentyl, cyclohexyl,
cycloheptyl or
R3
~ (CH2)n- wherein R~ is hydro-
gen, chloro, methyl, nitro or amino and n is O or 1 and
R2 is (lower)alkyl having 1 to 6 carbon atoms,
hydroxy(lower)alkyl, alkylthioalkyl, carbox~mido(lower)-
alkyl, carboxy(lower)alkyl, phenyl or benzyl provided that
when Rl is benzyl and R2 is isobutyl the configuration o~
the compound is (2S,3R52'R)~ (2S,3S,2'S) or (2S,3S,2'R).
Another preferred embodiment of the present
invention is a compound having the formula
1 3 2 1 2' 1'
R -CH -CH-CO-NH-CH-COOH
N~2 OH R
wherein Rl is (lower)alkyl, cyclopentyl, cyclohexyl~
cycloheptyl or
R3 (CH2)n~ wherein R~ is hydro-
gen, chloro, methyl, nitro o~ amino and n is O or 1 and
R2 is (lower)alkyl havirlg 1 to 6 carbon atoms,
hydroxymethyl, methylthioethyl, -CH2CH2CONH2~ -CH2CH2COOH~
phenyl or benzyl provided that when Rl is benzyl and R2
is isobutyl the con~iguration o~ the compound is
(2S,3R,2'R), (2S~3S,2'S) or (2S,3S~2'R).
;i6
Another preferred embodiment o~ the present
invention is a-compound having the fo~nula
1 3 2 1 2' 1'
R -CH -CH-CO-NH-fH-COOH
NH2 OH R
wherein Rl is (lower)alkyl, cyclopentyl~ cyclohexyl or
cycloheptyl, and
R2 is (lower)alkyl having 1 to 6 carbon atoms,
hydroxymethyl~ methylthioethyl, ~CH2CH2CONH2~ -CH2CH2COOH~
phenyl or benzyl.
Another preferred embodiment of the present
invention is a compound having the formula
.
1 3 2 1 2' 1'
R ~CH ~CH-CO-NH-CH-COOH
1 ~
. NH2 OH R
wherein Rl is
R3
~ .
~ wherein R3 is hydro-
gen, chloro methyl, nitro or amino and
R ls (lower)alkyl having 1 to 6 carbon atoms,
hydroxymethyl, methylthioethyl, -CH2CH2CONH2, -CH2CH2COOH~
phenyl or benzyl.
Another preferred embodiment of the present
invention is a compound ha~ing the formula
1 3 2 1 2' 1'
R -fH -fH-CO~MH-IH-COOH
~2 OH R
--5--
wherein R is
R3
~ CH2- wherein R3 is
hydrogen, chloro, methyl, nitro or amino and
R is (lower)alkyl having 1 to 6 carbon atoms,
hydroxymethyl, methylthioethyl, -CH2CH2CONH2, -CH~CH2COOH,
phenyl or benzyl provided that when R is benzyl and R2
is isobutyl the configuration of the compound is
(2S~3R,21R), ~2S~3Sg2tS) or (2S,3S,2'R).
Another preferred embodiment of the present
invention is a compound having the formula
1 3 2 1 2' 1'
R -CH -CH-CO-NH-fH-COOH
NH2 R
wherein R is
R
~ CH2- wherein R3 is
chloro, methyl, nitro or amino and
R2 is (lower)alkyl having 1 to 6 carbon atoms,
hydroxymethyl, methylthioethyl, -CH2CH2CONH2~ -CH2CH2COOH,
phenyl or benzyl.
Another pre~erred embodiment of the present
invention is a compound having the formula
3 2 1 2' 11
Rl-CH ~CH-CO-NH-IH-COOH
NH2 R
~6--
wherein R is
R3
~ CH2- wherein R3 îs
chloro, methyl~ nitro or amino and
R is (lower)alkyl having 1 to 6 carbon atoms.
Another pre~erred embodiment of the present
invention is a compound having the formula
1 3 2
R -CH~ CH-CO-NH-CH-COOH
NH2 1H I 2
wherein Rl is
R3
~ H2 wherein R3 is
hydrogen, chloro, methyl, nitro or amino and
R2 is (lower)alkyl having 1 to 6 carbon atoms
provided that when Rl is benzyl and R2 is isobutyl the
configuration of the compound is (2S,3R,21R)~ (2S~3S,2'S)
or (2S,3S,2'R).
-7-
i6
Preferred species of the present invention in-
clude the following:
(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-
(X)-leucine,
(2S,3S)-3-amino-2-hydroxy-4-phenylbutanoyl-
(R)-leucine,
(2S,3S)-3-amino-2-hydroxy~4-phenylbutanoyl-
leucine,
(2S,3R)-3-amino-2-hydroxy-4-p-nitrophenyl-
butanoyl-(S)-leucine 3
(2$,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-
(S)-valine,
(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-
(S)-norvaline,
(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-
(S!-methionine~
(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-
(~)-isoleucine,
(2S,3R)-3-~mino-2-hydroxy-4-phenylbutanoyl-
(~)-norleucine,
(2RS,3RS)-3-~mino-2-hydroxy-4-p-chlorophenyl-
butanoyl-(S)-leucine,
(2RS,3RS)-3-amino-2-hydroxy-4-o-chlorophenyl-
butanoyl-(S)-leucine,
(2RS,3RS)-3-amino-2-hydroxy-4-p-methylphenyl-
butanoyl-(S)-leucine, and
(2S,3R)-3-amino~2-hydroxy-4-p-aminophenyl~
butanoyl-(S)-leucine
The invention also includes a process for the
preparation o~ the compounds represented by Formula (I)
in the following manner:
A nitrile derivative as represented by a ~ormula (II) 3
R -CH--CH-CN
IH2 OH (II)
(wherein R is a grou.p as defined abo~e) or such a
nitrile derivative whose amino group is protected is
hydrolyzed with an acid to prepare an amino acid as
represented by a formula (III),
Rl-CH--CH-COOH
l I (III)
NH2 OH
(wherein R1 is a group as defined above). After havlng
protected, i~ necessary, the functional groups which do
not take part in the reaction, this amino acid and an
amino acid represented by the ~ormula (IV)
NH2-CH-COOH
12 (IV)
(wherein R2 is a group as de~ined above) are condensed
in the ordinar~ peptide linking method. When said pro-
tected groups are removed, the compounds as represented
by Formula (I) are obtained.
There is also provided by the present in~ention
a process ~or producing a peptide represented by the
~ormula
1 ~ 2 1 2' 1'
R -CH--CH-CO-NH-CH-COOH
1 2
NH2 OH R
_g_
s~
(wherein R1 and R2 are groups as defined above) comprising
consecuti~e steps in which a nitrile represented by the
formula
Rl_fH- CH-CN
NH2 OH
(wherein R is a group as defined above) or a nitrile
derivative ~hose amino group is protected is
hydrolyzed with an acid to prepare an amino acid represented
by the ~ormula
R -IH~ 7H_~OOH
NH2 OH
(wherein Rl is a group as defined above), a~ter having
protected, i~ necessary, the functional groups which do
not participate in the reaction, the resulting amino acid
and an amino acid as represented by the ~ormula
NH2~fH-COOH
R2
(wherein R2 is a group as defined abo~e) are condensed
in an ordinary peptide linking process; and then said
protecting group is removed and, pre~erably~said process
wherein Rl is benzyl or substituted benzyl and R is
alkyl or hydroxymethyl having 3 or 4 carbon atoms and
also said process wherein Rl is benzyl and R2 is alkyl
having 4 carbon atoms and also said process wherein the
acid which is employed ~or hydrolysis is hydrochloric
acidl hydrobromic acid or sulfuric acid.
There is also provided by the present invention
a process for collecting an antipode comprising consecu-
tive steps in whîch an c~mino acid represented by the
-10-
~4~
formula
R -CH~ CH-COOH
NE~2 OH
(wherein Rl is a group as defined above) is protected
by benzyloxycarbonyl; the thusly protected amino acid
is allowed to react with brucine to prepare a diastexeo~
isomer of brucine salt; and said diastereoisomer is
optically resolved in an organic solvent and preferably
said process wherein Rl is benzyl~ and also said process
wherein ethyl acètate is used as the organic solvent.
There is further provided by the present inven-
tion a process for collecting an antipode comprising
consecutive steps in which an amino acid represented by
the formula
R -fH I E-COOH
~ NH2 ~
(wherein Rl is a group as deflned above) is protected by
benzyloxycarbonyl; the thusly protected amino acid is
dissolved in an organic solvent selected from a group of
alcohols, ethers, esters~ ketones, halogenated hydrocar-
bons, dimethylformamideg dimethylacetamide and acetoni-
trile; then to the resulting solution an organic solvent
selected from a group o~ petroleum hydrocarbons, aromatic
hydrocarbons, hexane and cyclohexane is added so as to
optically resolve the same and preferably said process
wherein Rl is benzyl and also said process wherein the
organic solvents are ethyl acetate and petroleum ether
There is also provided by the present invention
a cornpound represented by the formula
- 11-
Rl-CH CH-COOH
~12 o~
wherein R is lower alkyl, phenyl, benzyl and substituted
benzyl and particularly is
(2$,3R)-3-amino-2-hydroxy-4-phenylbutanoic acid,
(2RS~3RS)-3-amino-2-hydroxy-4-phenylbutanoic acid,
(2RS,3RS)-3-amino-2-hydroxy-4-p-chlorophenyl-
butanoic acid,
(2RS,3RS)-3-amino-2-hydroxy-4-o-chlorophenyl~
butanoic acid,
(2RS,3RS)-3-amino-2~hydroxy-4-p-methylphenyl-
butanoic acid or
(2RS,3RS)-3-amino-2-hydroxy-4-p-benzyloxyphenyl~
butanoic acid.
-12-
s~
Typical examples of nitrile derivatives as
represented by Formula (II) are 3-amino-2-hydroxy-
propionitrile, 3-amino 2-hydroxy-5-methylhexanonitrile,
~-amino-2-hydroxy-3-phenylpropionitrile, 3-amino-2-hydroxy-
4-p-chlorophenylbutyronitrile, 3-amino-2-hydroxy-4-phenyl-
butyronitrile~ 3-amino~2-hydroxy-1~-o-chlorophenylbutyro-
nitrile, 3-amino-2-hydroxy-1~-p-methylphenylbutyronitrile
and 3-amino-2-hydroxy-4-p-benzyloxyphenyl^butyronitrile.
Any known amino-protecting group such as those employed
in peptide chemistry may be used for protection of the
amino groups. Preferred examples of acyl type amino-
protecting groups are a formyl group, an acetyl group, a
trifluoroacetyl group, substi~u~ed or non-substituted
benzoyl group; examples of urethane type amino-protecting
groups are substituted or non-substituted benzylo~ycar-
bonyls, alkoxycarbonyl group having 1 to 6 carbon atoms
and cycloalkanoyxcarbonyl group, preferred examples of
other amino-protecting groups are substituted or non-
substituted arylsulfonyl group, phthalyl group, o-nitro-
phenylsulphenyl group or tri-tyl group. Any type of acid
usually employed for hydrolyzing nitriles may be used in
the present inven-tion for hydrolysis. Examples of inor~
ganic acids include hydrochloric acid, hydrobromic acid
and sulfuric acid; examples of organic acids include
alkylsulfonic acids such as methanesulfonic acid, ethane-
sulfonic acid, arylsulfonic acid such as benzenesul~onic
acid and toluenesulfonic acid. The concentration of
these acids may be at any level usable ~or hydrolyzing
ordinary nitriles. However~ a concentration of above lN
will be preferred.
To the compounds whose amino group is protected
~13-
and which are not readily dissolved in the aqueous solution
of acid, an organic sol~ent which is miscible with water
such as -tetrahydrofuran, dioxane, lower alcohols,
acetone, dimethylformamide, dimeth~lacetamide and dimethyl-
sulfoxide may be added to enhance the solubility of th
compounds in the aqueous solution
In the case when a protecting group is employed,
if the protecting group is remo~ed furing the hydrolysls to
form electrophilic bodies, a cation sca~enger 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 o~ the reaction mixture takes
place. To isolate the desired amino acid from the
r-eaction mixture, any ordinary method for isolating amino
acid may be applied. For example, when the hydrolysis is
effected with a volatile acid, the excessi~e acid may
be removed by concentrating the reaction mixture 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
is added to the reaction n~xture to crystallize the in~
tended compound as represented b~ ~lormula (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 ~rom the ion exchange resin
with a volatile alkali such as ammonia water and condensed
under reduced pressure. If necessary acetone, methanol or
ethanol is added to crystallize the intended compound as
4~
.~
represented by Formula (III), which is then separated by
filtration.
In the foregoing reaction~ when an (R)~nitrile
derivative is used as a starting material, (2RS~3R)-amlno
acid is obtained, when an (S)-nitrile derivative is used,
(2RS33S)-amino acid is obtained: when an (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 pro~
cess as described hereinbelow. For example, 3-amino-2-
hydroxy-4-phenylbutanoic acid (hereinafter, this amino
acid is abbr~viated as AHPA) which is a precursor of Bes~
tatin is in (2S,3R) form. There~oreg in order to obtain
this compound, it is necessary to resolve (2RS93R)-AHPA
to (2S,3R)- and (2R,3R)-AHPA.
(2RS,3R)-AHPA can be resolved optically in ethyl
acetate in the form of diastereoisomer which is obtained by
first benzyloxycarbonylation of (2RS,3R)-AHPA in the ordinary
manner to prepare benzyloxycarbonyl-(2RS,3R)-AHP~, which
is then allowed to react with brucine ~hereinafter, benzyl-
oxycarbonyl group is abbreviated as Z). More specifically,
Z~(2RS,3R)-AHPA and an equivalent or slightly excessive
quantity o~ brucine are dissolved in ethyl acetate under
heating and then filtered~ if necessary. When the filtrate
is cooled or allowed to cool, a crystal will be deposited.
This crystal is separated by filtration and, if necessary,
recrystallizing operations from ethyl acetate are repeated
to prepare the optically pure bruclne salt o~ Z-(2S~3R)~
AHPAo When this brucine salt is treated in the ordinary
way, optically pure Z-(2S,3R)-AHPA is obtained. Other-
wise optically pure Z (2R,3R)-AHPA may be prepared as
~14a-
.. . , . ... . . . . . . . .. . . ~ .. , . ~ . ... ..... ....... . ...
56
follows: A solu-tion of ethyl acetate containing a
brucine salt of Z~(2R,3R)-AHPA and a small quantity
of Z-(2S,~R)-AHPA is shaken with dilute hydrochloric
acid to remove brucine, the ethyl acetate layer is dried
with a dehydrating agent such as anhydrous sodium sul-
fate and concentrated under reduced pressure. Then tne
residue is dissolved into a small quantity of ethyl ace-
tate. Upon adding petroleum ether there is obtained
crystals which may then be separated by filtration to
prepare optically pure Z-(2R,3R)-A~IPA.
It was noted that the (2R33R)-form o~ Z-AHPA
had smaller solubilities into an organic solvent ~han
the (2S,~R)-form. Z-(2RS~R)-AHPA is dissol~ed in a
flrst soluble solvent at room te~perature or under heat-
ing. Then a second insoluble solvent is added so as to
precipitate crystals, which are then separated by fil-
tration. If necessary~ the foregoing operation is re-
peated until optically pure Z-(2R,3R)-AHPA 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 aceta-te and ethyl
acetate; ketones such as acetone and methyl ethyl ketone;
halogenated hydrocarbons such as methylene chloride and
chloroform; amides such as dimethylformamide and dimethyl-
acetamide; and nitriles such as acetonitrile, etc The
second insoluble sol~ent may include petroleum hydro-
carbons such a~ petroleum ether, petroleum benzene and
ligroin; aromatic hydrocarbons such as benzene and
toluene; alkanes such as pentane and hexane; and cyclo-
56
hexanes such as cyclopentane and cyclohexane. By useof the diastereoisomer method employing brucine, the
optically impure Z-(2S,3R)-A~IPA present in the filtra-te
may be purified to optically pure (2S,3R)-AHPA.
Z-AHPA as used in the abo~e-mentioned resolu-
tion may be synthesized by Schotte.n-Baumann method in
the presence of an alkali by reaction of (2RS,3R)-AHPA
and benzyloxycarbonyl chloride or, otherwise, in the
presence of an organic tertiary base such as triethyl-
amine and N-methylmorpholine by reaction with a benzyl-
oxycarbonylatlng reagent such as benzyloxycarbonyl-p-
nitrophenyl ester, benzyloxycarbonyl azide, benzyloxy-
carbonyl-N-hydroxysuccinimide ester and benzylox~car-
bonyl-4~6-dimethyl-2-mercaptopyrimidine in a solvent
prepared by mixing dioxane, tetrahydro~`uran, acetoni-
trile or dimethylformamide with water.
Although the foregoing optical resolution has
been described with respect to AHPA, a similar optical
resolution may be effected, if necessary, for the other
amino acids.
To prepare Bestatin and its related compounds
as represented by Formula (I), an amino acid as repre-
sented 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, leu-
cine, norleucine, isoleucine, tertiary-leucine, 2-amino-
-16-
S6
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, methionine, 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 1 ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide is used; an azide methocl ernploy-
ing nitrous acid or alkyl nitrite; a mixed anhydride
method employi~g ethyl chloroformate or isobutyl chloro-
~ormate; an active ester method employing cyanomethyl
ester, ~inyl ester, substituted and non-substituted
phenyl esters, thiophenyl ester o~ N-hydroxysuccinimide
ester; an O-acyl hydroxylamine derivative method employ-
ing O-acylacetoxime or O-acylcyclohexa~onneoxime; an.
N-acyl derivative method employing carbonyldiimidazole~
Organic solvents using in the above condensa-
tion reaction may be ethers such as diethyl ether,
tetrahydro~uran or dioxane; esters such as ethyl acetate
or methyl acetate; ketones such as acetone or methyl
ethyl ketone; halogenated hydrocarbons such as methylene
chloride or chloroform; amides such as dimethyl~ormamide
or dimethylacetamide, nitriles such as acet,onitrile.
In the case when an amino acid is represented
by Formula (III) and whose amino group is protected and
an amino acid as represented by Formula (IV) whose
17-
carbo~yl group is not pro-tected are condensed in an acti~e
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 remo~ed by the usual method in peptide chemistry such
as catalytic hydrogenation on palladium, sapo.ni~ication
with an alkali~ acidolysis with hydrogen bromide in
acetic acid, with tri~luoroacetic 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) is ob-
tained.
The nitrile derivatives as represented by
Formula (II) and used in this in~ention as the starting
material may be synthesized reducing an a~amino acid,
whose amino group is belng protected and carboxyl group
con~erted 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 tetrahydro~uran to
prepare an amino-aldehyde whose amino group is protected.
The resulting amino-aldehyde is then con~erted to the
adduct with sodium bisul~ite and then allowed to react
with an alkaline metal cyanide or directly allowed to
react with hydrogen cyanide to prepare cyanohydrin, or
3-amino-?-hydroxynitrile.
-Pre~erred examples o~ the secondary amine are
N,N-dimethylamine, aziridine, N-methylaniline, carbazole,
-18-
3,5-dimethylpyrazole and imidazole. Preferred examples
of me-tal hydride are lithium aluminum hydride, lithium
di- and tri-alkoxyaluminum hydride and sodium bis(2-
methoxyethoxy)aluminum hydride. Examples of the protect
ing group for the amino group are the protecting groups
usually employed in the peptide chemistry. Urethane
type protecting groups may be used preferably and,
above all, benzyloxycarbonyl group 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 gro~p similar to those mentioned pre-
viously is reduced at a temperature lower than -40 C.
in aromatic hydrocarbons such as tolue~e or benzene and
ethers such as diethyl ether or tetrahydrofuran with
metal hydrides such as sodium aluminum hydride, diiso-
butylaluminum hydride or sodium bis(2-me-thoxyethoxy)alu-
minum hydride thereby to prepare aminoaldehyde whose
amino group is protected When such an aminoaldehyde
has been trea~ed in the above-mentioned manner, 3-amino-
2-hydroxynitriles are prepared.
The physiological activities of Bestatin and
its related compounds represented by Formula (I) pre-
pared in the present invention were determined as fol-
lows: i
(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 ~ ml. of 1 mM arginine ~-naph-thylamide and 1~0 Ml. of
0.1 M Tris hydrochloride buffer (pH 7.0), 0.7 Ml. of distilled
water with or without a test
-19-
material is added and warmed at 37 C. for 3 mlnutes.
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. ~fter
30 minutes at 37 c., o.6 ml. of l.OM acetate buffer
(pH 4.2) containing diazonium salt of o-aminoazotoluene
at 1.0 mgO/ml~ and Tween 20 at 1.0~ is added. Fi~teen
minutes at room temperature thereafter, absorbance (a) at
530 nm is measured by spectrophotometer. As the control~
by similar means the absorbance (b) after the reaction
in the absence o~ a sample is measured. The inhibition
percent is calculated as follows: (b-a)/b x 100.
Inhibition percentages at various conc2ntrations
were measured and from the measured inhibition percen-
tages, 50~ inhibitions (ID50) were deduced. The results
are as listed in Table 1.
*Trade Marks
..~I
s~
Table 1. Inhibitory_Acti~ity of Bestatin and Its
Related Compounds Against Aminopeptidase B
No. Compound _ ID5o(mcg /ml )
1 (2S,3R) AHPA-(Sj-Leu 0.10
2 (2S,3S)-A~PA-(S)-Leu 1.25
3 (2S,3R)-AHPA-(R)-Leu o~56
4 (2S,~S)-AHPA-(R)-Leu 0~04
(2s~3R)-AHpA-Gly 21.5
6 (2S,3R)-AHPA-(S)-Val 0.55
7 (2S,3R)-AHPA-(S)-Ile 0.05
8 ~2S,~R)-AHPA-(S)-Met 0.22
9 (2S,3R)-AHPA-(S)-Gln 1.2
(2S,3R)-AHPA-(S)-Nva 0.17
11 (2S,3R)-AHPA-(S)-Nle 0.13
12 (2S,3R)-AHPA-(S)-Phe 4.2
13 (2S,3R)-AHPA-(S)-Ser 0.72
14 (2S,3R)-AHPA-(S)-Glu 25
(2S,3R)-AHPA-(RS)-Acc 3.1
16 (2RS~3R)-Me-Ise-(S)-Leu 16
17 (2RS,3R~-iso-Bu-Ise-(S)-Leu 12
18 (2RS,3R)-Ph-Ise-(S)-Leu 56
19 (2RS,3RS~-AHPA(p-Cl)-(S)-Leu 0.07
(2RS,3RS)-AHPA(o-Cl)-(S)-Leu o.48
21 (2RS,3RS)-AHP~(p-Me)-(S)-Leu 0.01
22 (2S,3R)-AHPA(p-N02)-(S)-Leu 0.01
23 (2S,3R)-AHPA(p-NH2)-(S)-Leu 0.10
24 (2S~3R)-AHPA(6H)-(S)-Leu 1.05
Note: In the ~able,
Leu~ leucine; ~ly, glycine~ Ser, serine; Gln, gluta~ine;
Glu, glutamic acid; ~al, ~aline; Nva, nor~aline; Met,
methioninej Ile, isoleucine; Nle, norleucine; Acc, 2-
aminooctanic acid; Phe, phenylalanine; Me-Ise, ~-methyl-
isoserine (3-amino-2-hydroxy-propionic acid); iso-Bu-Ise,
~- isobutylisoserine (3-amino-2~hydroxy-5-methylhexanoic
acid); Ph-Ise, ~-phenylisoserine (3-amino-2-hydroxy-3-
phenylpropionic acid), AHPA(p-Cl), 3-amino-2-hydroxy-4-
p-chlorophenylbutanoic acid; AHPA(o-Cl~ 3-amino-2-
hydroxy-4-o-chlorophenylbutanoic acid; AHPA(p-Me),
-21-
~-amino-2-hydroxy-4-p-methylphenylbutanoic acid;
AHPA(p-N02), ~-amino-2-hydroxy-4-p-nitrophenylbutanoic
acid; AHPA(p-NH2), 3-amino-2-hydroxy-4-p-aminophenyl-
butanoic acid; AHPA(6H), 3-amino~2-hydroxy-4-cyclohexyl-
butanoic acid.
(B) Organ distribution of Bleomycin in ~he presence
o~ Bestatin.
100 mg./kg. of Bleomycin B2 was subcutaneously
administered to each of three ICR male mlce (4 weeks of
age) and 100 mg~/kgO of Bleomycin B2 and .~L~O mg./kg. of
Bestatin were subcutaneously administered ~imultaneously
to each of three similar mice~ These 6 mice were killed
one hour after administration and the quantity of Bleo-
mycin B2 remaining in the serum an~ organs was determined
by a thin layer disc method employing Bacillus subtilis
PCI219 as the test microorganism. The results are a~
listed in Table 2a wherein the numerals show a~erage
~alues in mcg./ml,
Table 2~ The Effect o~ Simultaneous Administration of
Bestatin and Bleom~ B2 on Or~an Distribu-~ion
o~ Bleomycin in Mice.
Serum ~ Liver~~ y S leen Stomach Brain
P _ _
BLM lOOmg/kg 110 11.2 4.5 0 68.0 4~0 1~6 0
BLM lOOmg/kg t 140 21~2 42.0 0 84.0 5'~ 2.5 2'
RST lOOmg/kg ~
Note: BLM, Bleomycin B2; BST, Bestatin
(C) Contracepti~e property
Antifertility of Bestatin
Immediately after subcutaneously administer~ng
~22-
a56~
Bestatin (10 mg./kg.) to ICR JCL female mice at the age
of puberty, these mice were placed with male mice of
similar line. Female mice which had copulated were
killed two days after formulation of vaginal plug and the
ova were collected from their uterine tubes so as to
examine whether they had been fertilized or not The
fertilized ova were at 8-16 cell period.
Bestatin-administered Total
Mouse GrouPFemale No. 1 Female No.2 N=2
_ . _
Number of corpus luteum 10 19
Number of collected ovum 10 19 29
Fertilized ovum 4 (40~) 6 (3106~) 10 (34-5~)
Non-fertilized ovum _ 6 (60~) 13 (68 4~) 19 (65.5~)
Total
ControlFemale No. 1 Female No .2 N=2
Number of corpus luteum 13 13 26
Number of collected o~um 9 11 20
Fertilized ovum6 (66,7~) 8 (72.7%) 14 (7~)
Non-fertilized ovum _ 3 (33.3~)_ 3 (27.3~) 6 ~o~)
The experiments showed that the fertility of the
ova collected from the mice, which had copulated 4-16
hours after administration of Bestatin, was 3l~, 5~, whereas
the fertility of the ova collected from the control mouse
group was 70~, It is apparent from these results -that
Bestatin has an antifertility property.
Following is a discussion by way of example only
of methods of carrying the invention into effect. However~
it should be no-ted herein that any suitable functional
~2~
group-protecting methods, functional group-remo-~ing
methods and peptide lin~age-~orming methods other than
those described hereinbelow may be employed. In addi-
tion to the aforementioned abbreviations, the following
abbreviations are also used in the examples:
HOBt, N-hydroxybenzotriazole; DCCD, N,N'-dicyclohexyl-
carbodiimide; HOSu, N-hydroxysuccinimide; -OBzl-TosOH,
benzyl ester p-toluenesulfonate; -OSu, N-hydroxysuccini~
mide ester; -OMe, methyl ester; -ONb, p-nitrobenzyl
ester; Boc~ t-butoxycarbonyl; DCHA, dicyclohexylamine;
-OBut tert.-butyl ester.
Rf value is measured on a silica gel GF25l~ plate prepared
by Merck Corp. using n BuOH: AcOH: H20 (4:1:1) as
developing solvent.
A few 3~amino 2-hydroxycarboxylic acids and their
containing peptides possess two Rf values because they are
a mixture of threo-and erythro- configurations.
2~-
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Example 1
Step 1
35.4 g. of oily Z-(2RS,3R)-3-amino-2-hydroxy-4-
phenylbutyronitrile is dissolved in a mixture of 300 ml.
of concentrated hydrochloric acid and 300 ml. of dioxane.
After adding 21.2 g. of anisole, the reaction mixture is
re~luxed for 12 hours. Then dioxane is distilled away
under reduced pi essure, the resulting solution of hydro-
chloric acid is washed with ether and the water layer is
concentrated under reduced pressure and e~Taporated to
dryness. Subsequently, 200 ml. of water is added to the
residual substance and the insoluble substance is sepa-
rated by Iiltration. After adding an equal quantity o~
acetone, the mixture is adjusted to pH 5.5 with ammonia
water. The mixture is allowed to stand in a refrigerator.
The deposited crystals are separated by f`iltration,
13.66 g. of intended (2RS,3R)-AHPA is obtained.
C~]5778 ~ 16.8 (c 1.25, N HCl), Rf 0.23 and 0~26
CloH13N03, Found C: 61.06, H: 6 55
N: 6.80; ~alc'd. C: 61.52, H: 6.71, N: 7.18.
Step 2
13.66 g. of (2RS~3R)-AHP~ obtained in Step 1 is
dissolved in 70 ml~ of lN sodium hydroxide solution. While
vigorously agitating the solution under cooling with ice,
15 ml. of Z-Cl and 70 ml. of lN sodium hydroxide solution
are added in three portions o~Ter a period of 30 minutes.
Then the reaction mixture is ~igorously agitated for 1
hour under cooling with ice and foY 3 hours at room
-25-
temperature.
When the reaction has been completed, 6N hydro-
chloric acid is added to adjust the reaction mixture to
pH 1. As a result, oily materia] is separated which is
then extracted twice with 100 ml. of ethyl acetate. The
ethyl acetate layer is washed with water and dehydrated
to dryness by use of anhydrous magnesium sulfate. After
separating magnesium sulfate by filtration, the ~iltrate
is concentrated under reduced pressure and the residue
is crystallized in ethyl acetate-petroleum ether to pre-
pare 8.48 g. of optically impure Z-(2R,3R)~AHPA~ On the
other hand, when the mother liquor is concentrated under
reduced pressure and the residue is crystallized in petro-
leum ether, 8.o7 g. of optically impure Z-(2S,3R)-AHPA is
obtained.
m-p- 142~143 C., [a~5738 + 70.2 (c 1.44, AcOH)
8 00 g. of optically impure Z-(2S,3R)-AHPA and
10.35 g. of brucine dihydrate are dissolved under heating
in a mixture o~ 300 ml. o~ ethyl acetate and 10 ml~ of
methanol and filtered. When the filtrate is allowed -to
cool, the brucine salt of Z-(2S~3R)-AHPA i6 separated.
Then the crystal separated by filtration is recrystallized
twice from ethyl acetate to prepare 13.51 g. of pure bru--
cine salt of Z (2S~3R)-AHPA.
m.p. 144 C. [~]5728 ~ 3308 (c 1.12~ AcO~)
1~.5 g. of the obtained crystal is suspende~ in
150 ml. of ethyl acetate and brucine is removed with 30 ml.
of lN hydrochloric acid. Then the ethyl acetate layer is
washed with water and dehydra-ted to dryness by use of an-
hydrovs magnesium sulfate. Subsequently, magnesiwm sulfate
-26-
is separated bVV ~iltration and ethyl acetate is distilled
away under reduced pressure. When the residue is repreci-
pitated from ethyl acetate-petroleum ether, 6.21 g. of
Z-(2S,3R)-AHPA is obtained.
m.p. 154.5 C., [a]57~ ~ 83.5 (c 1.34, AcOH)
Step 3
2.25 g. of Z (2S~3R)-AHPA and 945 mg. of HOBt
are dissolved in 70 ml. of tetrahydrofuran. After adding
2.75 g. of Leu-OBzl TosOH, the mixture is neutralized with
o.98 ml. of triethylamine and cooled to -5 C. Then 1.40
g. of DCCD is added and the reaction mixture is allowed to
stand overnight for reaction. Tetrahydrofuran is distilled
away under reduced pressure and 200 ml. of ethyl acetate is
added. After filtering off the insoluble substances, the
filtrate is washed with lN sulfuric acid, water, 5%
aqueous sodium bicarbonate solution and water in that order
and then dehydrated to dryness wi-th c~nhydrous magnesium
sulfate. The residue obtained by concentrating the filtrate
under reduced pressure is solidified in ethyl acetate-
petroleum ether. Recrystallization from the same solvent
gives 3.26 g. of Z-(2S,3R)-A~IP~-(S)-Leu-OBzl.
m-p. 122-123 C., [a]5738 ~~ 14.2 (c 1, AcOEI),
Anal. for C31EI36N206, ~'ound C: 69.66, H: 7.01, N: 5.13,
Calc'd. C 69 90, H: 6.81, N: 5 26.
Step 4
3.22 g. of 2-(2S~3R)-AHPA-(S)-Leu-O~zl is dis-
solved in 100 ml. of methanol and hydrogenated ~or 3 hours
with 100 mg. of palladium black. The catalyst is -filtered
27-
off and the solvent is concentrated under reduced pres-
sure. When a recrystallization operation is carried out
with methanol-ethyl acetate, 889 mg. of (2S,3R)-3-amino-
2-hydroxy-4-phenylbutanoyl-(S)-leucine is obtained.
m.p. 208-213 C. (decomp.), L~]5728 - 23-5 (c o-48~ AcOH)~
Rf o.48, Anal. for C16H2~N204, Found C 62 61, H 7.86,
N: 8.83; Calc'd. C: 62 313 H: 7.85, N: 9.09.
Example 2
Step 1
11.0 g. of hydrochloride of (2RS,~R)-~~amino-2-
hydroxy-4-phenylbutyronitrile is refluxed for 4 hours in
200 ml. of 6N hydrochloric acid. Hydrochloric acid is
distilled away under reduced pressure. 200 ml. of water
is added and the reaction mixture is concentrated o~ce
more under reduced pressure. 100 ml. of water is added
to the solid matter and the insoluble substance is fil-
tered of~, 100 ml. of acetone is added and the reaction
mixture is adjusted to pH 5.5 with ammonia water and al-
lowed to stand o~ernight in a refrigerator. The separated
crystals are collected by filtration and rinsed with a
water/acetone (1:1) solution to prepare 6.19 g. of in-
tended (2RS,3R)-AHPA.
Step 2
In the similar manner to Example 1, Step 2,
(2RS,~R)-AHPA is benzyloxycarbonylated and resol~ed with
brucine to prepare Z-(2$,3R)-AHPA. When the resulting
Z-(2S,3R)-AHPA is allowed to react with an equ:imolar amoun-t
of HOSu and DC~D, Z-(2$,3R)-AHPA-OSu is obtained.
-28-
m.p. 111-112 C., [a]5758 + 87.7 (c 0 52, AcOH)
C2~H22N207, Found C: 61.27, H: 5~25 N:
6.47, Calc~d. C: 61.96, H: 5.20, N: 6.57.
Step 3
85 mg. o~ Z-(2S~ 3R)-AHPA-OSu and 86 mg. o~
(S)-Leu-OBzl TosOH are allowed to reac-t for 12 hours in
5 ml. of dioxane at room temperature in the presence o~
0.03 ml. of triethylamine. Then the reaction mixture is
concentrated under reduced pressure. Tne residue is ex-
tracted with ethyl acetate and washed wîth lN sul~uric
acid, water, 5~ aqueous sodium bicarbonate solution and
water in this order Then the residue is dried over
anhydrous magnesium sulfate and the sol~ent is distilled
away to prepare a solid matter. When the solid matter is
recrystallized from ethyl acetate-petroleum e-ther, 90 mg.
o~ intended Z-(2S,3R)-AHPA-(S)-Leu-OBzl is obtained.
m.p. 122 C-, ~a]578 ~ 15-1 (c 0 77, AcOH)
75 mg. of Z-(2S,3R)-AHPA-(S)-Leu-OBzl is then
treated in a similar manner to Example 1, Step 4 to thereby
prepare 33 mg. of (2S,3R)-3-amino-2-hydroxy-4-phenylbuta-
noyl-(S)-leucine.
[a]578 ~ 21.8 (c 0.45, AcOH), R~ o.48.
Example 3
Step 1
330 mg. of Z-(2S,3R)-AHPA prepared in the similar
manner to Example 1, Step 2, 162 mg~ o~ HOBt, 217 mg. o~
the hydrochloride of (S)-Leu-OMe~0.17 ml. of triethylamine
and 206 mg. o~ DCCD are reacted in the similar manner to
Example 1, Step 3 to prepare 293 mg. o~ Z-(2S,3R)-AHPA-
-29-
(S)-Leu-OMe.
m-p- 120 C , ~]578 ~ 22.1 (c 1.09, AcOH)
C25H32N206, Found C: 65 91, H: 7.16, N: 6 28;
Calc'd. C: 65.77, H: 7.07, N: 6.14
Step 2
250 mg. of Z-(2S,3R)-AHPA (S)-Leu-OMe is dissolved
in 10 ml. of methanol and 3.6 ml. of lN aqueous sodium hy-
droxide solution is added to the solution and the result-
ing mixture is agitated for 6 hours at room temperature.
Then methanol is distilled away under reduced pressure and
water is added to the remaining liquor~ which is then ad-
justed to pH 2 with lN sulfuric acid. The separated pre-
cipitates are extracted with ethyl acetate3 washed with
water, dehydrated to dryness over anhydrous magnesium sul-
fate and the sol~ent is removed. The resulting solid
matter is crystallized from ethyl acetate-petroleum ether.
~hen the crystal is recrystallized from the same solvents,
150 mg. of Z-(2S,3R)-AHPA-(S)-Leu is obtained.
m-p- 209 C. [~]578 ~ 27-2 (c 0.71, AcOH)
Anal. for C24H30N206, Found C: 65-29, H: 6-75j N: 6-33
Calc'd. C: 65.14, H: 6.75, N: 6.46
Step 3
120 mg. of Z-(2S,3R)-AHPA~(S)-I,eu is dissolved
in 30 ml. of methanol and hydrogenated for 3 hours with
50 mg. of palladium blac~. Then the catalyst is filtered
off and methanol is distilled away to crystallize 54 mg.
of (2S,3R)-3~amino-2-hydro~y-4-phenylbutanoyl (S)-leucine
from methanol-ethyl acetate.
-3
4~56;
Ca]578 -22.7 (c 0.95, AcOH), Rf o.48.
Example 4
85 mg. of Z-(2S,3R)-A~IPA-OSu prepared in the
similar manner to Example 2, Step 2 is allowed to react
with 54 mg. of (S)-Leu for 2 days at room temperature in
5 ml. of dioxane and 5 ml. of water in the presence of
0.03 ml. of triethylamine. The reaction mixture is con-
centrated under reduced pressure. 30 ml. of water is
added to the residue and the solution is then adjusted to
pH 2 with lN sulfuric acid and extraction is effected with
50 ml. of ethyl acetate. The ethyl acetate layer is
washed with lN sulfuric acid, water, 55~ aqueous sodium
bicarbonate solution and wa-ter in this order and dehy-
drated to dryness with anhydrous magnesium sulfate. Ethyl
acetate is removed and the resulting solid matter is crys-
tallized from ethyl acetate-petroleum e-ther. When the
crystal is reprecipitated from the same sol~rents, ~3 mg.
of intended Z-(2S,3R)-AHPA-(S)-Leu is obtained.
m.p. 209 C., [c~]5728 + 26.7 (c 0.55, AcO~I)
When 45 mg. of Z-(2S,3R)-AHPA~(S)-Leu is treated
in the similar manner to Example 3, Step 3, 15 mg. of
Bestatin is obtained.
~o~]572g -22.3 (c 0.52, AcOH), Rf o.48.
Example 5
330 mg. of Z-(2S,3R)-AHPA prepared in the similar
manner to Example 1, Step 2 is treated in the similar man-
ner to Example 1, Step 3 with 472 mg. of (R)-Leu-OBzl TosOH,
162 mg. of HOBt, 0.14 ml. of triethylamine and 206 mg. of
DCCD to prepare 450 mg. of Z-(2S,3R)-AHPA (R)-Leu-OBzl
m-p- 118-119 C., [~]5738 + 43- (c 1.23, AcOH).
When 430 mg. of resulting Z-(2S,3R)-AHPA-(R)-
Leu-OBzl is treated in the similar manner to Example 1,
Step 4, 144 mg. of (2S,3R)-3-amino-2-hydroxy-4-phenyl-
butanoyl-(S)-leucine is obtained
[~]570 + 0.9 (c 0.47, AcOH), Rf 0.49~ A~nal. for
C16H24N204, Found C: 62.47, H: 7.89; N: 9.09; Calcld.
C: 62.31, H: 7.85, N: 9~09.
Example 6
-
Step 1
Z-(2RS,3S)-AHPA is prepared by benzyloxycarbonyla-
tion of (2RS,3S)-AHPA which has been prepared in the simi-
lar manner to Example 1, Step 1. 7.75 g of Z-(2RS,3S)-
~HPA and 6.69 g. of dehydroabietylamine are allowed to
react to prepare a diastereoisomer, which is then dissol~ed
in ether so as to filter off the hardly soluble dehydro-
abietylamine salt of Z-(2R~3R)-AHPA. The filtered ether
solution is concentrated under reduced pressure and the
resultant oil~ material is treated with ethyl acetate-lN
hydrochloric acid to prepare crude Z-(2S,3S)-A~IPA when
the crystal is re-precipitated twice from ethyl acetate~
petroleum ether, 2.16 g. of pure Z-(2S,3S)-AHPA is ob-
tained.
m,p. 175-176 C- [~575 + 5-6 (c 1-39, AcOH)-
~` Step 2
660 mg. of Z-(2S,3S)-AHPA, 94~L mg. of (S)-Leu-
OBzl TosOH, 324 mg. of HOBt, 0.34 ml of triethylamine and
~32
56
412 mg. of DCCD are treated in the similar manner to
Example 1, Step 3 to prepare 710 mg of Z-(2S,3S)-AHPA-
(S)-Leu-OBzl.
m-p- 128 C. [~]57258 _ 38.o (c 0.81, AcOH).
500 mg. of Z-(2S,3S)-AHPA-(S)-Leu-OBzl is treated
in the similar manner to Example 1, Step 4 to prepare 270
mg. of (2S,3S)-3-amino-2-hydroxy-4 phenylbutan~yl (S)~
leucine.
[~]578 - 61.3(c 0 56, AcOH), Rf 0.37, Anal. for C~6H24N204,
Found C: 61.99, H: 7.71, N: 9.13; Calc'd. C: 62.31~ H:
7.85, N: 9.09
Example 7
165 mg. of Z-(2S,3S)-AHPA prepared in the similar
manner to Example 6, Step 1, 237 mg. of (R)-Leu-OBzl-TosOH~
81 mg. of HOBt, o.o8 ml. of triethylamine and 103 mg. of
DCCD are treated in the similar manner to Example 1, Step
3 to prepare 180 mg. of Z-(2S,3S)-AHPA-(R)-Leu-OBzl.
m.p. 128-219 C., [~]57238 _ 11.0 (c 1.02, AcOH).
155 mg. of Z-(2S,3S)-AHPA-(R)-Leu-OBzl is treated
in the similar manner to Example 1, Step 4 to prepare
72.1 mg. of (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoyl-
(R)-leUcine
[~]578 - 31.4 (c 0.47, AcOH, Rf 0.52,
Anal. for C16H24N204, Found C: 62.07, H: 7-63~ N. 8-80;
C~lc'd C: 62 31~ H: 7.85, N: 9.09.
Example 8
330 mg. of Z-(2S,3R)-AHPA prepared in ~he simi-
lar manner to Example 1, Step 2~ 405 mgO of Gly~OBzl TosOH,
162 mg. of HOBt, 0.14 ml. of triethylamine and 206 mg. of
-33-
s~;
DCCD are treated in the similar ma~ner to Example 1~ Step
to prepare oily Z-(2S,3R)-AHPA-Gly-OBzl. When examined
by a thin layer chromatography, this oily product was
proved to be pure.
When the oily product is treated in the similar
manner to Example 1, Step 4, 117 mg. of (2S,3R)-3-amino~
2-hydroxy-4-phenylbutanoyl-glycine is obtained.
[a]57238 - 7.4 (c ~49a AcOH), Rf 0.26 Anal. ~or
C12H1604N2'CH30H, Found C: 54.33, H: 6.87, N: 9.69;
Calc'd. C: 54.92, H: 7.09, N: 9.87.
Example 9
' 330 mg. o~ Z-(2Sg3R)~AHPA prepared in the simi~
lar manner to Exarnple 1, Step 2, 440 mg. of the hydro~
chloride of (S)-Ser(Bzl)~ONb 162 mg. of HOBt, 0.14 ml.
o~'triethylamine and 206 mg. o~ DCCD are treated in the
similar manner to Example 1, Step 3 to prepare 540 mg.
o~ Z-(2S,3R)-AHPA-(S)-Ser(Bzl)-ONb.
m.p. 121-5 C-, ~]578 ~ 17-7 (c 1~07, AcOH)
When 500 mg. o~ Z-(2S~3R)-AHPA(S)-Ser(Bzl)-ONb is treated
in the similar manner to Example 1~ Step 4, 147 mg. of
(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-serine
is obtained.
~a]576 _ g.o (c 0.50, AcOH), R~ 0.20,
Anal- for C13H1805N2 H20, Found C: 51-39, H: 6.7L~, N:
8.97; Calc'd. C: 51.99, H: 6.71, N: 9.33.
~;~ Exarn~
330 mg. of Z-(2S,3R)-AHPA prepared in the similar
manner to Example 1, Step 2~ 400 mg. o~ the hydrochloride
of (S)-Gln-OBzl~ 162 mg. o~ HOB-t~ 0.14 ml o~ triethylamine
S~i
and 206 mg. of DCCD are treated in the similar manner to
Example 1, Step 3 to prepare 200 mg. of Z-(2S,3R)-AHPA-
(S)-Gln-OBzl.
m-p- 155 C-~ [a]578 + 62.3 (c 0.75, AcOH)
When 180 mg. of Z-(2S,3R)-AHPA-(S)-Gln-OBzl is
treated in the similar manner to Example 1, Step 4, 61
~g. of (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-
glutamine is obtained.
~]57238 ~ 9.9 (c 0.37, AcOH), Rf 0.17,
Anal- for C15H215N3 2H2~ Found C: 50 64, M 6.~2, N
11.61; Calc'd. C: 50.13, H: 7.01, N: 11.69
Example 11
330 mg. of Z-(2S,3R)-AHPA prepared in the simi-
lar manner to Example 1, Step 2, 600 mg. of (S)-Glu(OBzl)-OBzl
TosOH, 162 mg. of HOBt, 0.14 ml. of triethylamine and
20~ mg. of DCCD are treated in the similar ma~ler to
Example 1, Step 3 to prepare 605 mg. of Z-(2$,3R)-AHPA-
~S)-Glu(OBzl)-OBzl.
m.p. 119-120 C., ~]5738 + 28.6 (c 1.04~ AcOH)
` When 250 mg. of Z-(2S,3R)-AHPA-(S)-Glu(OBzl)-
OBzl is treated in the similar manner to Example 1,
Step 4, 166 mg. of (2S,3R)-3-amino-2-hydroxy-4-phenyl-
butanoyl-(S)-glutamic acid is obtained.
[a]578 -18.4 (c 0 51, AcOH), Rf 0.27,
or C15~2006N2~ Found C: 55.20, H: 5-90, N: 8 20
Calc'd. C: 55.55, ~: 6.22, N: 8.64.
S~
Example 12
_
330 mg. of Z-(2S,3R) AHPA prepared in the simi-
lar manner to Example lg Step 2, 455 mg. of (S)~Val
OBzl TosOH, 162 mg. of HOBt~ 0.14 ml. of triethylamine
and 206 mg. of DCCD are treated in the similar manner
to Example 1, Step 3 to prepare 397 mgO of Z-(2S~3R)-
AHPA-(S)-Val-OBzl.
m-p- 96 5-97 C., [a]5738 + 19.2 (c 0.99, AcOH)
When 376 mg. of Z~(2S~3R)-AHPA-(S)~Val-OBzl
is treated in -the similar manner to Example 1, Step 4,
104 mg. of (2$,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-
(S)-valine is obtained
[~]578 ~ 1.4 (c 0.55, AcO'~), Rf 0-42~
~nal. for C15H2204N2g Found C: 60.98, H: 7 59 N: 9 28;
Calc'd. C: 61.20, H: 7.53, N- 9.52
Example 13
330 mg of Z-(2S,3R)-AHPA prepared in the simi-
lar manner to Example 1, Step 2~ 455 mg of (S)-Nva-
OBzl~TosOH, 162mgO of HOBt, 0.14 ml. of triethylamine
and 206 mg of DCCD are treated in the similar manner to
E~ample 1, Step 3 to prepare 310 mg of Z~(2S,3R)-AHPA-
(S)-Nva-OBzl
m pO 114-1~5 C-, [~578 + 22 0 (c 0-49~ AcO~I)
When 250 mg. of Z-(2S,3R)-~HPA-(S)-Nva-OBzl
i~ treated in the similar manner to Example 1, Step 4,
110 mg. of (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-
~S)-norvaline is obtained.
[a]578 ~ 14 5 (c 0.50, AcOH), Rf 0 41
Anal for C15H2204N2, Found C: 59.42, H: 7~58, N: 9 09;
Calc'd. C: 59033, H: 7.64~ N: 9.23
_36-
Example 14
When (2S,3R)-AHPA is t-butoxycarbonylated in
the usual manner by use of t-butyl S-4~6-dimethylpyri
midine-2-ylthiolcarbonate, DCH~ salt of Boc-(2S,3R)-
AHPA is obtained.
m.p- 158-159 C.g [a]5725g ~ 51.9 (c o.89, AcOH).
When 3CHA is removed from 952 mg. of this
product by treatment with lN sulfuric acid and ethyl
acetate the oily Boc-(2S,3R)~AHPA is obtained. The oily
material and 440 mg. of the hydrochloride of (S)-Met-
OMe are suspended in 20 ml. of tetrahydrofuran and
neutralized with o.~o8 ml. of triethylamine. The re-
sulting solution is cooled to -5 C. and after adding
~12 mg of DCCD~ agitated for 4 hours at room tempera-
ture.
The deposited crystal is filtered off~ the
mother liquor is concentrated under reduced pressure
and the residue is dissolved in 200 ml. of ethyl
acetate, washed wi-th lN sulfuric acid, water, 5~ aqueous
sodium bicarbonate solution and water in this order and
dehydrated to dryness over anhydrous magnesium sulfate.
The residue obtained by concentrating the
mother liquor under reduced pressure is dissolved in an
ethyl acetate-benzene mixture (2:5) and introduced into
a column chromatograph employing silica gel G (trade
name for an adsorbent for use in column chromatography
manufactured by Merck Inc.). The fraction containing
the intended material is collected and the solvent is
distilled away under reduced pressure to obtain a solid
matter. When the solid matter is recrystallized from
ether-petroleum ether~ 380 mg. of Boc-(2S,3R)-AHPA-
-~7~
(S)-Met-OMe is obtairled.
p 118 c ~ [ a] 578 + 514 (c 0.56, AcOH).
When 280 mg. of Boc-(2S,3R)-AHPA-(S)-Met-OMe
is treated in the similar manner to Example 3, Step 2,
200 mg. of Boc-(2S,3R)~AHPA-(S) Met) is obtained.
m.p. 116 C. (foaming, ~a~5728 + 44.1 (c 0 54, AcOH).
O.2 ml. of` thioglycolic acid and 50% aqueous
tri~luoroacetic acld solution are added to 150 mg. oî
Boc-(2S,3R)-AHPA-(S)-Met and the mixture is agitated ~or
one hour at room temperature.
An oily material obtained by concentrating the
mixture under reduced pressure is di~solved in water
and adsorbed to a column (5 ml.~ which is packed with
Dowex 50X4 (H type) (trade mark for an ion exchange
resin manufactured and sold by Dow Chemical Co.). men,
adsorbed material is eluted with a 2N aqueous ammonium
hydroxide solution and e~aporated to dryness to obtain
a solid material. When the solid material is recrystal-
lized in a water-acetone mixture (1:1), 87 mg. oî (2S,3R)-
3-amino-2-hydroxy-4-phenylbutanoyl-(S)-methionine is ob-
tained.
~a] 278 ~ 20.4 (c 0.49, AcOH), R~ 0.37,
C15H2204N2S 1/2H20, Found C: 53-64, H: 6 47
1~: 8.11; Calc'd. C: 53.67, H. 6 91, N: 8.35.
Example_15
330 mg. o~ Z-(2S,3R)-A~IPA prepared in the simi-
lar manner to Example 1, Step 2, 472 mgO o~ (S)-Ile-
03zl-TosOH, 162 mg. of HOBt, 0.14 ml. o~ triethylamine
and 206 mg. o~ I)CCD are treated in the similar manner
to Example 1, Step ~, to prepare ~66 mg o~ Z (2S93R)-
c~ ~38~
.. ..
AHPA~(S)-Ile-OBzl.
m.p. 100-101 C., [~]57238 + 23.6 (c 1.0, AcOH).
When 440 mg. of Z-(2S, 3R)-AHPA-(S)~Ile-OB~l
is treated in the similar manner to Example 1, Step 4,
191 mg. o~ (2S,3R)~3-amino~2~hydroxy~4~phenylbutanoyl-
(S~~isoleucine is obtained.
[a]578 ~ 6.4 (c 0.52l AcOH), Rf 0 48g
Anal- for C16H2404N2~ Found C: 62.31g H 7.70, N 8.99;
Calc'd. C: 62.31, H: 7.85, N: 9.09.
Example 16
330 mg. of Z~(2S, 3R)-AHPA prepared in the
similar manner to Example 1~ Step 2, 472 mg. of (S)~Nle-
OBzl-TosOH, 162 mg. of EIOBt, 0.14 ml. of triethylamine
and 206 mg. of DCCD are treated in the similar manner to
Example 1, Step 3 to prepare 437 mg. of Z-(2S,3R)~AHPA-
(S)~Nle~OBzl.
m.p. 114~115 C., [~]578 ~ 23.3 (c 0.94, AcOH)
350 mg. of Z~(2S,3R)~AHPA-(S)~Nle-OBzl is
treated in the similar manner to Example 1, Step 4, 160
mg. of (2S,3R)-3-amino~2-hydroxy-4~phenylbutanoyl~(S)-
norleucine is obtained.
[~]5378 ~8.9 (c 0.70, AcOH), Rf 0.47.
Anal. for C16H2404N2 H20, Found C: 58095~ H: 7-84~ N: 8-60;
Calc'd. C: 58.88~ H: 8.o3, N: 8.58.
Example 17
330 mg. of Z~(2S,3R)~AEIPA prepared in the
similar manner to Example 1, Step 2g 505 mg. of (RS)~
Aoc-OBzl~TosOH, 162 mg. of HOBt, 0~14 ml. of triethylamine
and 206 mg. of DCCD are treated in the similar manner to
-39-
Example 1, Step 3, 4~0 mg. of z-(2S7~R)-AHPA-(RS)-Aoc-
OBzl is obtained.
m.p. 90-92 C~ [a]573 ~ 32.9 (c 0.94, AcOH)
When 250 mg. of Z-(2S,3R)-AHPA-(RS)-Aoc-OBzl
is treated in the similar manner to Example 1, Step 4,
74 mg. of (2S,3R)-~-amino 2-hydroxy-4-phenylbutanoyl-
(RS)-2-aminooctanoic acid is obtained.
[~]578 ~ 34.9 (c 0.53, AcOH), Rf 0.58,
Anal. for C18H2804N2, Found C: 64 43, ~: 8-18, N: 8-02
Calc'd. C: 64 26, E: 8~9, ~: 8.~.
Example 18
330 mg. of Z-(2S,3R)-AHPA prepared in the simi-
lar manner to Example 1, Step 2, 51~ mg. of (S)-Phe-
OBzl TosOH, 162 mg. of HOBt, O.14 ml. of triethylamlne
and 206 mg. of DCCD are treated in the similar manner to
Example 1, Step 3 to prepare 378 mg. of Z-(2S,3R)-AHPA-
(S)-Phe-OBzl.
m p. 111-112 C., c~]5738 ~ 39 6 (c 0-97~ AcOH)
When 355 mg. of Z-(2S,3R)-AHPA-(S)~Phe-OBzl is
treated in the similar manner to Example 1, Step 4, 108
mg. of (2S,3R)-3-amino-2-hydroxy-4~phenylbutanoyl-(S)-
phenylalanine is obtained.
ca]5738 -~ 0.4 (c o.46, AcOH), Rf 0.44,
Anal. for ClgH2204N2 CH~OH, Found C: 63.58, H: 6-455 N: 7-57
Calc'd. C: 64.15, H: 7.00, N: 7.48.
Example 19
When 9.00 g. of benzyloxycarbonyl-(2RS,3R~-3-
amino-2-hydroxypropionitrile is treated in the similar
manner to Example 1, Step 1, 3.16 g. of (2RS73R)~Me-Ise
-40-
is obtained.
[a]578 ~ o~60 (c 1.10 N HCl) Rf 0,
Anal. ~or C4HgN03~ Found C: 40.53, H: 7.51, N: 11.95,
Calc'd C: 40~3~, H: 7.62, N: 11.76.
When 1.10 g. o~ oily Z-(2RS~3R)-Me-Ise prepared
by benzyloxycarbonylation of (2RS,~R)-Me Ise, 1.97 g. of
(S~-Leu-OBzl TosOH~ 675 mg~ o~ ~OBt, 0.70 ml. o~ tri-
ethylamine and 948 mg. o~ DCCD are treated in the similar
manner to Example 1I Step 3, oily Z-(2RSg3R)-Me-Ise-
(S)-Leu-OBzl is obtained. This oily product is proved to
be pure by thin-layer chromatography.
When this oily Z-(2RS,3R)-Me-Ise-(S)-Leu-OBzl
is treated in the similar manner to E~ample 1, Step 4,
765 mg. o~ (2RS,3R)-3 amino-2-hydroxypropionyl-(S)-
leucine is obtained.
[a]57~ - 28.7 (c 1.09, AcOH),
Anal. for CloH20N204, Found C: 51.49, H: 8.42, N: 11.96;
Calc'd. C: 51.70~ H: 8.58, N 12.06.
Example 20
2.76 g. o~ oily Z-(2RS,3R)-3-amino-2-hydroxy-5-
methylhexanonitrile is dissolved in 30 ml. of concentrated
hydrochloric acid and 30 ml. o~ dioxane. A~ter adding
2.16 g. of anisole, the solution is re~luxed for 4
hours. Dioxane is distilled away under reduced pressure
and the remaining hydrochloric acid solution is washed
with ether. The water layer is concentrated under re-
duced pressures to dryness.
Water is added to the residue and adsorbed on
a column packed with Dowex 50x4 (H type) ~ . A~ter
washing with water, the adsorbed material is eluted with
_L~l_
4N aqueous a~nonium hydroxide solution. Then the eluted
solution is concentrated under reduced p:ressure to dry-
ness.
When a water-acetone (1:1) mixture is aaded to
the residue and the resulting crystal is separated by f`il-
trat~on, 0.82 g. o~ intended (2RS,3R)-iso-Bu-Ise is ob-
tained.
~a]578 ~ 8.3 (c 1.0, AcOH), RI 0.18
1.32 g. of oily Z-(2RS,7R) iso-Bu Ise prepared
by benzyloxycarbonylation of (2RS,3R)-iso-Bu-Ise obtained
in the a~orementioned process, 1.76 g. o~ (S) Leu-OBzl
TosOH, o.60 g. of HOBt, o.63 ml. of triethylamine and
O.92 g. of DCCD are treated in the similar manner to
Example 1, Step 3 to obtain oily Z (2RS,3R) iso Bu-Ise-
(S)-Leu-OBzl. When this oily product is treated in the
similar manner to Example 1, Step 49 0.37 g. o~ (2RS,3R)-
30amlno-2-hydroxy-5-methylhexanoyl-(S)~leucine is ob-
tained.
[a]5728 ~ 26.6 (c 1.0, AcOH), R~ 0.43 and 0 49,
Anal- for C13H26N24~ F~und C: 57.01, H 9.80~ N 10.14;
Calc'd. C: 56.91, H: 9.55, N: 10~21.
E_ample ?l
When 3.00 g. of Z-(2RS,3R)-3-amino-2-hydroxy-
3-phenylpropionitrile is treated in the similar manner
to Example 1, Step 1, o.80 g. OI (2RS,3R)-Ph-Ise is ob-
tained.
[a]575 ~ 9.5 (c 1.0, N HCl), Rf 0~13 aMd 0.17,
Anal. ~or CgHllNO~ Found C: 59.18, H: 6 15, N: 7.67,
Calc'd. C: 59.66, H: 6 12, M: 7.73.
250 mg. of Z-(2RS,3R)-Ph-Ise ~m.p. 170-171 C.,
-~2-
~Q~
[a]5738 ~ 18.5 (c 0.47, ~cOH)] prepared by benzyloxy-
carbonylation of' (2RS,3R)~Ph-Ise, 394 mg. of (S)-Leu-
OBzl~TosOH, 130 mg. of HOBt, 0.14 ml. of triethylamine
and 165 mg. of DCCD are treated in the similar manner of
Example 1, Step 3 to prepare 285 mg. of Z-(2RS,3R)-Ph-
Ise-(S)-Leu-O'Bzl.
m-p. 93-95 C-~ [a]578 - 34-7 (c 0.49, AcOH).
When 200 mg. of' Z-(2RS,3R)-Ph-Isè-(S)-Leu-
OBzl is treated in the similar manner to Example 1,
Step 4, 45 mg. o~ (2RS,3R)-3-amino-2-hydroxy-~-phenyl-
propionyl-(S)-leucine is obtained.
[a]5758 ~ 2.4 (c 0.33, AcOH),
Anal- f'or C15H2204N2, Found C: 60.92, ~ 7-67, N 9-46
Calc'd. C: 61.20, H: 7.53, N: 9.52.
When 10 3 g. of Z (2RS,3RS)-3-amino-2-hydroxy-
4-p-chlorophenyl-butylonitrile is treated in the similar
manner to Example 1, Step 1, 3.10 g. of' (2RS,3RS)~AHPA
(p-Cl) is obtained.
R~ 0.25, Anal. for CloH12N03Cl, Found C: 52.11, H: 5.17,
N: 5.99; Calc'd. C: 52.29, H: 5.27, N: 6.10.
1.81 g. of Z-(2RS,3RS)-AHPA (p-Cl)(m.p. 147 C.)
prepared by benzyloxycarbonylation of (2RS~3RS)-AHPA(p-
Cl), 2.37 g. of (S)-Leu-OBzl TosO~, 810 mgO of HOBt,
o.84 ml. of triethylamine and 1.03 g. of DCCD are'treated
in the similar manner to Example 1, Step 3, 2.50 ~. of
Z-(2RS,~RS)-AHPA(p-Cl)-(S)-LeuYOBzl ls o'btained.
m.p. 122-124 C., [~5778 --12.3 (c 3.18, AcOH)
When 1.50 g. of' Z~(2RS,~RS)~AHPA(p-Cl)-(S)-
Leu-OBzl is treated in the similar manner to Example 1,
_1~3_
s~
Step 4, 650 mg. of (2RS,3RS)-3-amino-2-hydroxy-4-p-
chlorophenylbutanoyl-(S)-leucine is obtained.
[~]578 ~ 11.3 (c 1.33~ AcOH), Rf 0.42 and 0.52,
al for C16H23N204Cl~ Found C: 55.88, H 7-29~ N 8 11;
Calc'd. C: 56.o5, H: 6.76, N: 8.17.
When 10.3 g. of Z-(2RS,3RS)-3-amino-2-hydroxy
4-o-chlorophenyl~`butyronitrile i~ treated in the similar
manner to Example 1, Step 1, 4.82 g. of (2RS, ~RS)-AHPA
(o-Cl) is obtalned.
Rf 0.25 and 0029
Anal. for CloH12N03Cl, Found C: 52.01, H: 5.17, N: 5.91;
Calc'd. C: 52.29, H: 5.27, N: 6.10.
726 mg of Z-(2RS,3RS)-AHPA(o-C1) (m.p. 136.5 C.)
prepared by benzyloxycarbonylation of (2RS,3RS)-AHPA(o-Cl),
944 mg. of (S)-Leu-OBzl-TosOH, 324 mg. of HOBta 0.34 ml.
of triethylamine and 412 mg. of DCCD are treated in the
similar manner to Example 1, Step 3, 747 mg. of Z-(2RSa
3RS)-AHPA(o-Cl)-(S)-Leu-OBzl is obtained.
m-p~ 129-132 C., [a]572g ~ 29.1 (c 1.02, AcOH)
When 640 mg. of Z-(2RS,3RS)-~HPA(o-Cl)-(S)-Leu-
OBzl is treated in the similar manner to Example 1, Step
4 and 107 mg. of (2RS,3RS)-3-amino-2-hydroxy-4-o-chloro-
phen~lPutanoyl-(s)-leucine is obtained.
~a]578 ~ 20.8 (c o.48, AcOH) Rf o.46
r C16H23N204Cl, Found C: 56.21, H: 6 56 N: 7 90;
Calc'd. C: 56.o5, H: 6.76, N: 8.17.
When 11 2 g. of Z-(2RS,3RS)-3-amino 2-hydroxy-
4-p-methylphenylbutyronitrile is treated in the similar
manner to Example 1, Step 1, ~.41 g of (2RS~3RS)-AHPA(p-
Me) ls obtained.
R~ 0.20
Anal- for CllHl5NO33 Found C: 62.94, H 7.01 N 6 79;
Calc'd. C: 63.14, H: 7.23, N: 6.99.
1.57 g. of Z-(2RS,3RS)-AHPA~p-Me) (m p9 166.5-8
C.) prepared by benzyloxycarbonylation of (2RS,3RS)-AHPA (p-
Me), 1.42 g. o~ ~S)-Leu-OBzl-TosOH, 486 mg. o~ HOBt,
0.50 ml. OI triethylamine and 618 mg. of DCCD are treated
in the similar manner to Example 1, Step 3, oily Z (2RS,
3RS)-AHPA-(S)-Leu-OBzl is obtained.
When the oily product is treated in the similar
manner to Example 1, Step 4, 497 mg. of (2RS,3RS)-3-
amino-2-hydroxy-4-p-methylphenylbutanoyl-(S)-leucine is
obtained.
Rf o.58 and 0.53 [a]575 - 7.2 (c o.g6 AcOH),
Anal. for C17H26~204~ Found C: 6~ o8, H: 8.23, N: 8.49;
Calc'd. C: 63.33, H: 8,1~, N: 8.69.
10~ mg of Bestatin is dissolved in 5 ml. of
acetic acid and 5 ml. of ethanol and hydrogenated cata-
lytically on platinum oxide for 14 hours.
The catalyst is ~iltered o~ and the filtrate
is concentrated under reduced pressure to dryness.
When the res-ldue is triturated with ethyl
acetate and collected by filtration 6:L mg. of (2S,3R)
3-amino-2-hydroxy 4-cyclohexylbutanoyl-(S)-leucine is
obtained.
Rf 0-47 [~x]578 ~ 3.5 (c o.67, AcOH),
-~5-
for C16H30N204, Found Co 62.12, H: 9.52, N: 8 81;
Calc'd. G: 61 12~ H: 9.62, N: 8.91
Example 26
~ 00 mg of Bestatin ls suspended in 10 ml. of
concentrated sulfuric ac~d and 0.3 ml. of fuming nitric
acid at 0 C. Rçaction is continued for one hour at the
same temperature after the suspended materials disappear.
The reaction mixture is poured into 100 ml. of water and
adsorbed to a column packed with Dowex 50X4 (H type)~
Then adsorbed material is eluted with 2N aqueous ammonium
hydroxide solution and evaporated to dryness. The residue
is dissolved in 10 ml. of water and filtered and then the
filtrate is lyophilized to give 213 mg. o~ (2S,3R)-~-
amino-2-hydroxy-4-p-nitrophenylbutanoyl-(S)-leucine.
[~]578 + 5-2 (c 0.5, AcOH), Rf o.58,
r C16H23N306 Found C: 54.28, H: 6 58, N: 11 88;
Calc'd. C: 54038, H: 6.56, N: 11.89.
Example 27
100 mg. of (2S,3R)-AHPA(p-N02)~(S)-Leu obtained
in Example 26 is dissolved in 10 ml. of methanol and
catalytically hydrogenated on palladium black for 5
hours~ The catalyst -s filtered and the filtrate is
concentrated under reduced pressure. The residue is
dissolved in 5 ml. of water and filtered and then the
filtrate is lyophilized to give 81 mg. of (2S,~R) ~-
amino-2-hydroxy-4-p-aminophenylbutanoyl-(S)-leucine.
[a]578 - 23.2 (c 0.5, AcOH)g Rf 0.25,
Anal. for C16H25N~04 Found C: 59.01, H: 7.91, N: 12-81g
Calc'd. C: 59~42g H: 7.79, N: 13.00.
-~6-
Reference Example 1
Synthesis of Nitrile From Aldehyde
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 of~ and washed with water and ether in this
order to prepare 77 g. of crude ~-benzyloxycarbonylamino
2-hydroxy-4-phenylbutyronitrile. 68 G. of resulting adduct
is suspended in 250 ml. of water and cooled to 10-12 C.
After adding 500 ml. o~ 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
discarded. The ether layer is washed with an aqueous
solution o~ sodium chloride and dehydrated to dryness over
anhydrous magnesium sul~ate.
When magnesium sul~ate is filtered off and
ether is distilled away under reduced pressure, 49 g.
of oily benzyloxycarbonyl-(2RS~3R)-3-amino-2-hydroxy-4-
phenylbutyronitrile is obtained.
Other nitrile derivatives used in the present
invention are prepared in the same manner.
-~7-
