Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
SPECI~IC~TION 2 0 ~ 0 2 0 0
lTîtle o~ th~ Invention3
Antihepatopathic composition
[Technical Ficld]
The present invention relates to an
antihepatopathic composition containing a novel and
useful glutathione-S-lower fatty acid derivative.
[Background Art]
There are known several glutathione-S-lower fatty
acid derivatives. Among them, S-(2-
carboxypropyl)glutathione has been isolated from onion
and garlic (Virtanen and Matikkala, 1960; Suzuki et al,
1961) but there is little information on its
pharmacologic activity.
The inventors of the present invention previously
found that glutathione-S-succinic acid derivatives have
platelet aggregation-inhibitory, antiinflammatory,
antiallergic, antitumoral and hepatic impairment-
protective activities (Japanese Kokai Patent
Application No. 63-8337 and Japanese Patent Application
No. 1-79956, No~ 1-183484, No. 1-251534, No. 1-256370
and No. 2-36745).
In search of still more pharmacologically active
compounds, the inventors of the present invention
synthesized a variety of novel glutathione derivatives
and screened them, as well as said S-(2-
carboxypropyl)~lutathione, for their pharmacologic
activities. As a consequence, they found that S-(2-
carboxypropyl)glutathione and a series o~ compou~which can be synthesized by reacting glutathione or an
ester thereof with an a , ~ -unsaturated -fatty acid,
such as acrylic acid, methacrylic acid, crotonic acid,
cinnamic acid, etc., or an ~ (or ~ )-halogenated
organic monocarboxYlic acid, such as monochloroacetic
acid, or an ester or amide thereof have excellent
antihepatopathic ef~icacy. The present invention has
been attained based on this finding.
[Disclosure of thc I~vention]
The present invention relates to an
antihepatopathic composition comprising a compound of
the formula
Hooc-cH-c~2cH2co - NH
2 CH2-S-(CH)n-CH-COR~
R R
wherein R1 and R3 are the same or di~-~erent and
respectivelY mean a hYdrogen atom or a lower alkyl
group which may be substituted: R~ is a hydroxyl group,
a lower alkoxy group which may be substituted or an
amino group which may be substituted; n means O or 1
and when n-1, R2 is a hydrogen atom, a lower alkyl
group which may be substituted or a phenyl group which
may be substituted or a salt thereo~ as an active
ingredient.
Where Rl, R3 in the above ~ormula means a lower
alkyl group, such alkYl group preferably has 1 to 102
carbon atoms. This alkyl group may be straight-chain,
branched or cyclic or contain a cyclic moiety. Thus,
methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-
butyl, tert-butYl, sec-butyl, n-pentYl. 1-ethylpropyl,
isopentyl and ben~yl may be mentioned by way o~
example.
Referring, further, to the above formula, where n
is equal to 1, R2 is a hydrogen atom, a lower alkyl
group or a phenyl group. The lower alkyl group is as
previously defined, and the phenyl group may be
substituted with an alkyl group and so on.
Further in the formula, R~ is a hydroxyl group, a
lower alkoxy group or an amino group. The lower alkoxy
group includes, among others, methoxy, ethoxy, propoxy,
isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-
butoxy, n-pentyloxy, isopentyloxy, tert-pentyloxy,
neopentyloxy, 2-methylbutoxy, 1,2-dimethylpropoxy, 1-
ethylpropoxy and so on. The lower alkoxy group may
have a hydroxyl group or a cyclic group such as phenyl.
The hYdrogen atom or atoms of said amino group may be
substituted by, for example, alkyl.
In the antihepatopathic composition of the
invention, the above compound can occur as the free
acid or as a pharmaceutically acceptable salt thereof,
for instance an alkali metal salt thereof, e.g. the
sodium salt, potassium salt, etc., or an alkaline earth
metal salt, e.~. the calcium salt, magnesium salt and
, . :, . . . . :, ::, :, . : : :: ,.. :, :.:. ,. . ., -
. :.:.. . : . . ~ . .: ~ :: .: . ., .; .,
so on. Regarding the salt, any or all of the carboxYl 2 ~ o
functions available in the compound may have been
converted to such salt or salts. Any of these salts
can be used advantageously in the manufacture of the
antihepatopathic composition of the present invention.
This antihepatopathic composition effectively
inhibits the onset of acute and chronic hepatic
disorders, suppresses elevation of GOT and GPT values
and, as such, is not only useful for the prevention and
treatment of acute or chronic hepatitis but also
effective in the prevention and treatment of
hepatocirrhosis. It can also be used with advantage in
cases of hepatic impairment induced by drugs such as
acetaminophen.
The antihepatopathic composition of the present
invention can be administered orallY or parenterally.
With regard to dosage form, it can be provided, for
example, in various solid dosage forms such as tablets,
granules, powders, capsules, etc. or in liquid dosage
forms such as injectable preparations. These
preparations can be manu~actured by the established
pharmaceutical procedures and according to the type of
disease to be controlled. In such preparations, there
may be incorporated conventional additives such as the
binder, disintegrating agent, thickener, dispersing
agent, reabsorption promoter, corrigent, buffer,
surfactant, cosolvent, preservative, emulsifier,
isotonizing agent, stabilizer, pH adjusting agent and
: ., ~ ., :,, -, ;; .. , ~.
'~ so on. 2~2
; The dosage of the active ingredient according to
the present invention is dependent on the particular
species of compound used, type o~ disease, patient's
age and body weight, dosage form, indication and so on.
In the case of an injectable preparation, for instance,
about 1 to 500 mg per day per adult is administered
once a day, and in the case of an oral preparation,
about 10 to 2000 mg per dose per adult is administered
a few times a day.
Depending on the objective and necessity of
treatment, the antihepatopathic composition of the
present invention may contain two or more species o~
the active compounds in suitable proportions.
Unless contrary to the object of the invention,
the antihepatopathic composition of the present
invention may additionally contain other active
ingredients having similar efficacy or different
efficacies in suitable proportions.
Among the compounds which can be used in the
antihepatopathic composition of the invention; the
compound wherein n=l, Rl and R2 are hydrogen, R3 is
methyl and R~ is hydroxy is a known compound as
mentioned hereinbefore and can be extracted from onion
or garlic or chemically synthesized by, or in
accordance with, the method described in Journal of
Agricultural and Food Chemistry 37, 611 (1989). The
compound described in Synthesis Example 1 which appears
hereinafter is also a known compound. Thus, either 2 ~ ~ ~ 2
glutathione or a glutathione monoester (~ -
glutamylcysteinylglycine ester) which is obtainable by
reacting glutathione with the corresponding alcohol in
the presence of an acid, e.g. sulfuric acid, is reacted
in water or aqueous medium with an a , ~ -unsaturated
acid, e.g. acrylic acid, methacrylic acid, crotonic
acid, cinnamic acid, etc., or an ~ (or ~ )-halogenated
organic monocarboxylic acid, e.g. monochloroacetic
acid, or an ester or amide thereof, preferably at pH
about 4 to 8, at room temperature or under mild heating
with stirring. This reaction readily goes to
completion. This reaction mixture is purified by
column chromatography or recrystallization from a
solvent to give the object compound. Almost all of the
compounds wherein Rl is hydrogen can be purified by way
of the copper salt.
Since most of the compounds synthesized as above
have asymmetric carbon within the molecule, they may
occur as optical isomers but all such optically active
compounds as well as mixtures thereof can be used for
purposes of the present invention.
The following synthesis examples, test example and
preparation examples are intended to illustrate the
invention ln further detail.
[Synthesis Example 1]
S-(2-Carboxyethyl)glutathione
[R~=R2=R3=H, R~=OH, n=1]
- , : : :................... ,. :, . ,
'. ~ ' ' . ., ' ~ ' . .` '. ! . , . .
~- In 100 ml of water are dissolved 6.2 g of ~ 9 ~ ~ 2
; glutathione and 4 ml of acrylic acid and the solu~ion
is adjusted to pH 6.5 with sodium hydroxide and stirred
at room temperature -for 12 hours. Then, 5 ml of acetic
acid and 4.4 g of copper acetate.are added and
dissolved, following by addition of 150 ml of ethanol.
The precipitated copper salt is recovered by filtration
and washed with 50~ ethanol. From this salt, copper is
removed using hydrogen sulfide as described in
Synthesis Example 2 and the filtrate is concentrated.
To the residue is added ethanol and the resulting white
crystalline precipitate is recovered by filtration and
recrystallized from water-ethanol. Yield 5.4 g.
TLC, silica gel Rf=0.15 tn-butanol-acetic acid-water=
4:1:1)
Elemental analysis, for C13H2108N3S
Calcd. (~): C, 41.16; H, 5.58; N, 11.08
Found (~): C, 40.97; H, 5.46; N, 11.12
[Synthesis Example 2]
S-(2-Methyl-2-carbethoxyethyl)glutathione
[Rl=R2-H, R3=CH~, R~=OC2H~, n=l]
[S-(2-carbethoxypropyl)glutathione]
In lOO ml of water is dissolved 6.2 g of
glutathione and the solution is adjusted to pH 7 with
2N-sodium hydroxide. To this solution is added 4 ml of
ethyl methacrylate and the mixture is stirred at room
temperature for 48 hours. Thereafter, 4.4 g of copper
acetate is added and dissolved and the precipitated
., . : , :: :: , .~ ~ ::"
: ::, : :. .: :,:. : : . , ~ . .
, . : : ,: ., .,. :: ~ .: . . ;: :.
copper salt is collected by filtration and rinsed with 2
water. This copper salt is suspended in 150 ml oe
water and hydrogen sulfide is bubbled through the
suspension with stirring to precipitate the copper
sulfide. This copper sulfide is filtered off and the
filtrate is concentrated. The resulting white crystals
are recovered by filtration and recrystallized from
water to give 4.5 g of needlets melting at 193-194C
(decompn.)
TLC, silica gel Rf=0.28 (n-butanol-acetic acid-water =
4:1:1)
Elemental analysis, for C1~H270~N3S
Calcd. (%): C, 45.60; H, 6.46; N, 9.97
Found (%): C~ 45.33; H, 6.65: N, 9.97
tSYnthesis E~ample 3]
S-[(2-Methyl-2-carbethoxyhydroxy)ethyl]glutathione
[R1=R2=H, R3=CH3, R~=OCzH~OH, n=l)
Using 6.2 g of glutathione and 3.3 g of 2-
hydroxyethyl methacrylate, the procedure of Synthesis
Example 2 i9 followed and the resulting crop of
crystals is recrystallized from water-ethanol to give
4.5 g o-f white powdery crystals melting at 173-175C
(decompn.)
TLC, silica gel Rf=0.19 (n-butanol-acetic acid-water =
4:1:1)
Elemental analysis, for Cl~H2~0aN3S
Calcd. (%): C, 43.93; H, 6.22; N, 9.60
Found (%): C, 43.64; H, 6.09; N, 9.72
[Synthesis E~ample 4]
S-(2-Methyl-2-carbamoylethyl)glutathione 2 ~ ~ ~ 2 ~ a
[R =R2=H. R~=CH9, ~=NHz, n=1]
Using 6.2 g of glutathione and 4.0 g of
methacrylamide, the procedure of Synthesis Example 2 is
followed and the resulting crop o~ crystals is
recrystallized from water-ethanol to give 5.6 g of
white crystals melting at 165-167C (decompn.)
TLC, silica gel Rf=O.l~ (n-butanol-acetic acid-water=
4~
Elemental analysis, for C1~H2~07N~Sl/2H20
Calcd. (%): C, 41.89; H, 6.28; N, 13.96
Found (~): C, 41.65; H, 6.11; N, 13.84
[Sy~thesis E~ample 5]
S-(2-Carbethoxyethyl)glutathione
[Rl=R2=R3=H, R~=OC2Hs, n=1]
Using 6.2 g of glutathione and 5 ml of
ethylacrylate, the procedure of Synthesis Example 2 is
followed and the resulting crop of crystals is
recrystallized from water to gi~e 6.0 g of white
crystals melting at 194-195~C (decompn.)
TLC, silica gel Rf=0.24 (n-butanol-acetic acid-water =
4:1:1)
[a ]20_ 22.80 (c=l, H20)
Elemental analysis, for C1~H2~08N3S
Calcd. (%): C, 44.22; H, 6.18; N, 10.31
Found (%): C, 44.08; H, 6.36: N, 10.~6
Synthesis Example 6]
': ' ' ' ' :" ' ' "' . ' . " . . ' ' '. ' . . :: . ' ' . ~ . . ' : , ! ` "
:. ' . '', -: :-: ':. . ;: .. ; . . .~: . ;. : r, ~.. " -
S-(2-MethYl-2-carbobenzoxyethyl]glu-tathione 2 ~ ~ 0 2
[Rl=Rz=H, R~=CH3, R~=OCHaC~H~, n=1]
A mixture of 6.2 g of glutathione and 3.8 g o~
benzyl methacrylate is stirred in a hydrous solvent (80
ml water, 80 ml ethanol) at room temperature for 48
hours and the reaction mixture is concentrated to about
40 ml. The residue is acidified with acetic acid and
the resulting white crystalline precipitate is
recovered by filtration. The crystals are dissolved in
2% sodium hydrogen carbonate, acidified with acetic
acid and the resulting crop of white crystals is
collected by filtration, washed with water and ethanol
and dried.
Yield 4.5 g; m.p. 191-192C (decompn.)
TLC, silica gel Rf=0.34 (n-butanol-acetic acid-water
=4:1:1)
Elemental analysis, for C21Hz~08N3S
Calcd. (%): C, 52.16; H, 6.04: N, 8.69
Found (%): C, 51.98; H, 6.02; N, 8.72
[Synthesis Example 7]
S-(2-Methyl-2-carboisobutoxyethyl)glutathione
[R1=Rz=H, R9=CH9, R~=OC~H~, n=1]
Using 6.2 g of glutathione, 3.0 g of isobutyl
methacrylate and, as a solvent, 100 ml water-50 ml
ethanol, the procedure of Synthesis Example 2 is
followed and the resulting crop of crystals is
recrystallized from water to give 4.0 g of white ;
crystals melting at 195-196C (decompn.)
.~
TLC, silica gel Rf=0.34 (n-butanol-aCetlC acid-water
4:1:1)
Elemental analysis, for C1BH9l08N~S
Calcd. (~0): C, 48.10; Il, 6.95: N, 9.35
Found (~0): C, 47.96; H, 6.82; N, 9.37
[Synthesis E~mple 8~
S-(2-Methyl-2-carbethoxyethyl)glutathione iso-
propyl ester
[Rl=C3H7, Rz=H, R3=CHs, R~=OC2H5, n=l]
In 50 ml of water is suspended 4.0 g of
glutathione isopropyl ester sulfate and the solution is
adjusted to pH 6.5 with 2N-sodium hydroxide. After 2
ml of ethyl methacrylate is added, the mixture is
stirred at room temperature for 3 hours and
concentrated. To the residue is added ethanol and the
precipitated inorganic salt is filtered off. To the
filtrate is added acetone and the resulting crop of
colloidal crystals is recovered by filtration and
purified by Sephadex G-10 column chromatography
(eluent: water-ethanol = 1:1). Recrystallization from
ethanol-acetone giv0s 2.4 g of amorphous crystals.
TLG, silica gel Rf=0.48 (n-butanol-acetic acid-water =
4:1:1)
Elemental analysis, for Cl~H3908N3S
Calcd. (~0): C, 49.23: H, 7.18; N, 9.06
Found (%): C, 49.09; H, 7.10; N, 9.16
[Synthesis E~ample 9~
S-(2-Methyl-2-carboisobutoxyethyl)glutathione
.. : . . . - . . ; . i
.. . . , , :, ,: ,.,., ,:,, - ~,: .: : ~. . . . ::
''' ' "' '
isopropyl ester 2
[R1=C~H7, R2=H, R3=CH3, R~=OC~H~, n=1]
Using 4.0 g of glutathione isopropyl ester sulfate
and 1.6 g of isobutyl methacrylate, the procedure o~
Synthesis Example 8 is followed (stirring time: 48
hours). The resultin~ crop of crystals is
recrystallized from ethanol-ethyl acetate-petroleum
benzin to give 2.0 g of colorless amorphous crystals.
TLC, silica gel Rf=0.54 (n-butanol-acetic acid-~Yater =
4:1:1)
Elemental analysis, for C21~3708N3S
Calcd. (%): C, 51.31; H, 7.59; N, 8.55
Found (%): C, 51.13: H, 7.48; N, 8.57
tSYnthesls E~ample 10]
S-(l-Methyl-2-carboisopropoxyethyl)glutathione
[Rl=R3=H, R2=CH3, R~=OC3H7, n=1]
Using 6.2 g of glutathione and 3.7 g of isoprop~l
crotonate, the procedure of Synthesis Example 7 is
followed and the resulting crop of crystals is
recrystallized from water to give 3.2 g of white
crystals melting at 189-190C (decompn.)
TLC, silica gel Rf=0.27 (n-butanol-acetic acid-water =
4:1:1) .
Elemental analysis, for C17H2~08N9S
Calcd. (%): C, 46.89; H, 6.71; N, 9.65
Found (/0): C, 46.66; H, 6.53; N, 9.68
Synthesis E~ample lll
S-(1-Phenyl-2-carbethoxyethyl)glutathione
[Rl=R3=H, R2=C~H~, R~=OCzH5~ n=1] 2 ~ 3
Using 6.2 g of glutathione and ~.0 g of ethyl
cinnamate, the procedure of Synthesis Example 7 is
followed (stirring at room temperature for about 7
days) and the resulting crop of crystals is
recrystallized from water to give 2.7 g of white
needles melting at 185-186C (decompn.)
TLC, silica gel Rf=0.29 (n-butanol-acetic acid-water =
4:1:1)
Elemental analysis, for C21H2~08N3S- 1/2H20
Calcd. (%): C, S1.21; H, 6.14; N, 8.53
- Found (%): C, 51.14; H, 5.89; N, 8.42
~Synthesis Example 12]
S-(Carboisopropoxymethyl)glutathione
~Rl=R3=H, R~=OC3H7, n=O]
In 80 ml of water is dissolved 6.2 g of
glutathione and the solution is adjusted to pH 6.5 with
2 N-sodium hydroxide. Following addition of 5 g of
isopropyl monochloroacetate, the mixture is stirred at
room temperature. The pH of the mixture fell with
progress of the reaction. Therefore, the reaction
mixture is read~usted to pH 6.5 with 2 N-sodium
hydroxide. This procedure is repeated and when the pH
has almost ceased to fall, 2 ml of acetic acid and a
sufficient amount of water are added to make a total of
200 ml. Then, 4.4 g of copper acetate is added and
dissolved and the precipitated copper salt is recovered
by filtration, washed with water and methanol and
13
. . ,: .: :: . .: , ::: , :.,., . ~ . ::, :: .:, - . :
., , ,. ~: , . :.. ., ~; ; . : ., . : :
suspended in 200 ml of water. Then, hydrogen sulfide
is bubbled through the suspen~ion with stirrin~ and th~ 0
resulting copper sulfide is filtered off, the filtrate
is concentrated and the resulting crop of white
crystals is harvested by ~iltration and recrys~allized
from water to give 5.2 g of white crystals melting at
194-195C (decompn.)
TLC, silica gel Rf=0.21 (n-butanol-acetic acid-water =
4:1:1)
[ a ]2Do-29.oo (c=l.O, HzO)
Elemental analysis, for ClsH2~O8N3S
Calcd. (%): C, 44.22; H, 6.18; N, 10.31
Found (%): C, 44.10; H, 6.24; N, 10.26
tTest Example 1]
Effect on acetaminoPhen-induced hePatic impairment
Method: Male SD rats (body weights ca. 180 g)
purchased from Japan SLC were used. The test substance
was orally administered (0.5 mmole/kg) and one hour
later 300 mg/kg of acetaminophen was intraperitoneally
administered. After 24 hours, blood was drawn from ths
abdominal aorta and the serum was separated. Using
this serum, s-GOT and GPT were determined.
Result: Nine different glutàthione derivatives
were tested for inhibitory effect on acetaminophen-
lnduced hepatlc lmpairment. As shown in Table 1,
compound Nos. 1, 2, 3, 4, 7, 8 and 9 (corresponding to
the structures indicated in the table) showed
slgnificant antihepatopathic effects.
14
., : . . , ~ . ,. .. ~, , .. ~ . ,
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~ 2~2~g
~ 7 o a~
,~ ~ ~ cr t~ r
a) a~ r r ~rl ~ r
,~ 1 ~ ~
t~ o r ~ O N u~ ~r
~7 O ~
~al ~ ô ~ --
~ ~ r a~ r r ai a~
~ C~ ~
o O ~ u7 o
r ~D r ~D r o o o o
O ~ ~ ~ . .
E ~ . E E
u O c c c E
u E ~ ~ N IY ~ ~r E E
¦, ~ ~ c ~ e.~
E~ ~ r J
~ ~ ~r~ ~r~ r~ r~ ~ .
,~.;, . ~: ~ X P~ PC K ~ PC ~
æ I ~ r~
[Best Modes o~ Working the Invention]
[Preparation E~ample 1] Oral tablet 2 ~ fi ~ 2 9
S-(2-Methyl-2-carboisobutoxyethyl)glutathione
isopropyl ester 100 mg
Lactose 80 mg
Starch 17 mg
Magnesium stearate 3 mg
Using the above materials per tablet, tablets for
oral administration were manufactured by the
established pharmaceutical procedure. Where necessary,
the tablets may be sugar-coated.
tPreparation Example 2] Injection
S-(1-Methyl-2-carboisopropoxyethyl)glutathione
1.0 g
Sodium chloride 0.6 g
Distilled water for injection 100 ml
The above materials are admixed, adjusted to pH
6.5 with 2N-sodium hydroxide and sterilized by
filtration. The filtrate is aseptically distributed in
2 ml portions into glass ampules and sealed by fusion
of the glass to provide a batch of injections.
16
- . . . ,. : :. . . ~:
; . - . ,: .,...,, ,~
... . - ~ . .
