Note: Descriptions are shown in the official language in which they were submitted.
CA 02692549 2010-01-05
OPTICALLY ACTIVE
N-(ALPHA-MERCAPTOPROPIONYL)GLYCINE
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to an optically active N-(a-
mercaptopropionyl)glycine,
preparation method thereof, use of the same in preparation of detoxification
medicament for
improving metabolism, and pharmaceutical preparation containing the same.
Related Art
N-(a-mercaptopropionyl)glycine, generic name tiopronin, is used to treat, for
example,
acute and chronic hepatitis, and cirrhosis in clinics, with very excellent
therapeutic use in
treatment of liver diseases, and preparation method and pharmaceutical
preparation thereof
have been frequently disclosed. A tiopi-onin pharmaceutical preparation is
disclosed in
Chinese Patent Application No. CN02129300.7, and a process for synthesizing
tiopronin is
disclosed in Chinese Journal of Medicinal Chemistry (Vol. 7, No. 1, p. 55-56,
Mar. 1997).
However, there is not any report for the optical activity, i.e., levo and
dextro isomers of
N-(a-mercaptopropionyl)glycine. For the majority of the drugs, a single
enantiomer has
high therapeutic efficacy and low adverse effect, and thus is called as
eutomer, while the
inactive or low-active enantiomer is referred to as distomer; and in most
cases, the distomer
has no therapeutic efficacy, but can also partly neutralize the effect of the
enantiomer, and
even incur serious adverse effect. The enantiomeric drugs have very different
pharmacologic activity and metabolic process in the body, and thus exhibit
different
biological activity and efficacy. In clinical application, due to the lack of
awareness of the
differences in pharmacodynamic and pharmacokinetic behaviors of the individual
enantiomer of chiral drugs, the conclusions sometimes contradict the
therapeutic efficacy or
occurrence of adverse effect, and will even improperly direct the application
of drugs in
clinics. Therefore, researches on optical activities of drugs are very
necessary.
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CA 02692549 2010-01-05
SUMMARY OF THE INVENTION
The present invention is directed to an optically active
N-(a-mercaptopropionyl)glycine.
The present invention is further directed to a method for preparing the
optically active
N-(a-mercaptopropionyl)glycine.
The present invention is further directed to a use of the optically active
N-(a-mercaptopropionyl)glycine in preparation of detoxification medicament for
improving
metabolism.
The present invention is further directed to a pharmaceutical preparation
containing the
optically active N-(a-mercaptopropionyl)glycine.
As embodied and broadly described herein, the present invention provides an
optically
active N-(a-mercaptopropionyl)glycine, which is levo or dextro
N-(a-mercaptopropionyl)glycine.
The levo N- a-mercaptopropionyl)glycine is S-(-)-N-(a-
mercaptopropionyl)glycine
of S -configuration, or a pharmaceutically acceptable salt or ester
thereof, in which the salt can be an amino acid salt or a metal salt.
The amino acid salt has a general structural formula below:
, in which R can be an amino acid selected from, for example,
arginine, ysine, g ycine, aspartic acid, alanine, phenylalanine, leucine,
isoleucine, ornithine,
cystine, cysteine, tyrosine, valine, serine, histidine, threonine,
tryptoophan, methionine,
methionine, proline, glutamic acid, and hydroxyproline.
The metal salt has a general structural formula below:
in which Ro can be, for example, potassium or sodium.
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CA 02692549 2010-01-05
The ester has a general structural forrnula below:
, in which R, iis a linear CI-C5 alkyl group.
The dextro N-(a-mercaptopropionyl)glycine is R-(+)-N-(a-
mercaptopropionyl)glycine
of R-configuiration, or a pharmaceutically acceptable salt or
ester thereof, in which the salt can be an ainino acid salt or a metal salt.
The amino acid salt has a general structural formula below:
in which R can be an amino acid selected from, for example, arginine, lysine,
glycine,
aspartic acid, alanine, phenylalanine, leucine, isoleucine, ornithine,
cystine, cysteine,
tyrosine, valine, serine, histidine, threonine, tryptoophan, methionine,
methionine, proline,
glutamic acid, and hydroxyproline.
The metal salt has a general structural formula below:
, in which Ro, can be, for example, potassium or sodium.
The ester has a general structural forrnula below:
, in which R, is a linear C]-C5 alkyl group.
The present invention also provides a method for preparing the optically
active
N-(a-mercaptopropionyl)glycine, which includes a method for preparing
R-(-)-N-(a-mercaptopropionyl)glycine and a method for preparing
S-(-)-N-(a-mercaptopropionyl)glycine.
I. The method for preparing S-(-)-N-(a-mercaptopropionyl)glycine includes:
1. reacting R-(+)-2-chloropropionic acid with thionyl chloride to get
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CA 02692549 2010-01-05
R-(+)-2-chloropropionyl chloride;
2. reacting R-(+)-2-chloropropionyl chloride with glycine under a weak basic
condition
to get R-(+)-2-chloropropionyl glycine; and
3. reacting sodium sulfide with sublimed sulfur to get sodium disulfide, which
is then
acidified by reacting with R-(+)-2-chloropropionyl glycine, to get
S -(-)-N-(a-mercaptopropionyl)glycine;
where the resulting S-(-)-N-(a-mer(.aptopropionyl)glycine is further reacted
with an
acid, a base, or an alcohol to get a salt or an ester.
II. The method for preparing R-(+)-N-(a-mercaptopropionyl)glycine includes:
1. reacting S-(-)-2-chloropropioriic acid with thionyl chloride to get
S-(-)-2-chloropropionyl chloride;
2. reacting S-(-)-2-chloropropionyl chloride with glycine under a weak basic
condition
to get S-(-)-2-chloropropionyl glycine; and
3. reacting sodium sulfide with sublimed sulfur to get sodium disulfide, which
is then
acidified by reacting with S-(-)-2-chloropropionyl glycine, to get
R-(+)-N-(a-mercaptopropionyl)glycine;
where the resulting R-(+)-N-(a-mercaptopropionyl)glycine is further reacted
with an
acid, a base, or an alcohol to get a salt or an ester.
The present invention also provides a use of the optically active
N-(a-mercaptopropionyl)glycine, i.e., S-(-)-N-(a-mercaptopropionyl)glycine or
R-(+)-N-(a-mercaptopropionyl)glycine, or a pharmaceutically acceptable salt or
ester
thereof in preparation of detoxificatiori medicament for improving metabolism,
and
particularly a use in treatment of acute and chronic liver diseases and
improvement of liver
function, including protection of liver itissue cells, treatment of various
hepatitis, for
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CA 02692549 2010-01-05
example, acute and chronic hepatitis, viral hepatitis, alcoholic hepatitis,
drug induced
hepatitis, and heavy metal toxic hepatitisõ and treatment of fatty liver,
acute and chronic
liver injury, and cirrhosis; as well as in prevention and cure of peripheral
blood leukopenia
caused by chemoradiation therapy and accelerating of restoration of liver
cells, to reduce the
adverse effect of chemotherapy; in prevention and cure of early senile
cataract and vitreous
opacities; and in heavy metal detoxification.
It is found from pharmacodynamic experiment that both
S-(-)-N-(a-mercaptopropionyl)glycine and R-(+)-N-(a-mercaptopropionyl)glycine
have
good protection effect for liyer injury, which is better than that of
N-(a-mercaptopropionyl)glycine.
It is found from pharmacokinetic experiment that, conversion of
S-(-)-N-(a-mercaptopropionyl)glycine and R-(+)-N-(a-mercaptopropionyl)glycine
to each
other have not occurred in body. The specific experiment is a pharmacokinetic
test in the
body of a test animal, including determining the plasma drug concentration
with
high-performance liquid chromatogaphy-rnass spectrometry (HPLC-MS).
The process for treat a plasma sample to formulate an injected solution
includes plasma
acidification, extraction, and then derivatization, and is as follows:
1. plasma acidification, where the acid used for acidification can be, for
example,
hydrochloric acid, phosphoric acid, perchloric acid, or acetic acid, with
hydrochloric acid
being preferred, and hydrochloric acid of 1 mol/L being more preferred; and
where the
volume ratio of the acid to the plasma sample is (150 1-250 l):(2 ml-4 ml),
with 200 l:3
ml being preferred;
2. extraction of the acidified plasma, ivhere the organic solvent used for
extraction can
be, for example, ethyl acetate, chloroforrn, trichloromethane, diethyl ether,
or n-hexane,
with ethyl acetate being preferred; and
3. derivatization of the extract, whei-e the useful derivatization reagent can
be, for
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CA 02692549 2010-01-05
example, phenyl isothiocyanate and 2,3,4,6-tetra-O-acetyl-(3-D-pyranoglucose
isothiocyanate (GITC) solution, with GITC solution being preferred, GITC
solution of 2
mg/mi being more preferred, and solution of 2 mg/ml GITC in tetrahydrofuran
being most
preferred; where the derivatization temperature is 15-45 C, with 25-35 C being
preferred,
and 30 C being more preferred; and where the derivatization time is 10-30 min,
with 15-25
min being preferred, and 20 min being more preferred.
Chromatographic conditions include:
mobile phase A: methanol; mobile phase B: an aqueous solution, containing 0.05-
0.20
mmol/L of sodium chloride and 5.0-6.0 mmol/L of formic acid, with 0.10 mmol/L
of
sodium chloride and 5.3 mmol/L of formic acid being preferred, where the ratio
of A to B is
(40-50:50-60, with 44:56 being preferred).
Mass spectrometric conditions include:
ionization mode: electrospray ionization; selective ion detection; curved
desolvation
line (CDL); temperature: 200 C-300 C, with 250 C being preferred; heating
block
temperature: 150 C-250 C, with 200 C being preferred; CDL voltage: 20V-30V,
with 25V
being preferred; detection voltage: +1.2 k:V-+1.8 kV, with +1.50 kV being
preferred; flow
rate of atomizing gas: 1.2 L/min-1.8 L/min, with 1.5 L/min being preferred;
flow rate of
drying gas: 1.5 L/min-2.5 L/min, with 2.0 L/min being preferred; detected ion:
derivative of
test drug [M+Na]+(m/z): 575.20; internal standard: derivative of N-isobutanoyl-
D-cysteine
(NIDC) [M+Na]+(m/z): 603.05;
as long as the separation degree of the internal standard peak from the major
peak
meets the requirements of Chinese Pharmacopoeia.
The results indicate that, in spectrogram of S-(-)-N-(a-
mercaptopropionyl)glycine at
each time points after being administrated individually, the presence of
R-(+)-N-(a-mercaptopropionyl)glycine is not obviously detected; likewise, the
presence of
S-(-)-N-(a-mercaptopropionyl)glycine is not obviously detected either after
administrating
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CA 02692549 2010-01-05
R-(+)-N-(a-mercaptopropionyl)glycine, which suggest that conversions to each
other have
not occurred in body.
Therefore, it is practicable to make clinically useful pharmaceutical
preparation from
S-(-)-N-(a-mercaptopropionyl)glycine or R-(+)-N-(a-mercaptopropionyl)glycine.
In the present invention, the optically active N-(a-mercaptopropionyl)glycine
is made
into a clinically useful pharmaceutical preparation, which contains
S-(-)-N-(a-mercaptopropionyl)glycine oi- R-(+)-N-(a-mercaptopropionyl)glycine,
or a
pharmaceutically acceptable salt or ester thereof as active ingredient, and a
pharmaceutically acceptable adjuvant, and is an orally administrated
preparation or an
injection preparation.
The orally administrated preparation includes: a general oral preparation, for
example,
tablet, capsule, granule, chewable tablet, or effervescent tablet; a rapid
release preparation,
for example, dispersible tablet, or orally disintegrating tablet; or a slow
release preparation,
for example, slow release tablet, or slow release pellet. The injection
preparation
includes, for example, injectable solution, concentrated solution for
injection, or sterile
powder for injection.
The adjuvant in the oral pharmaceutical preparation includes a filler, a
binder, or a
disintegrant, in which the weight contents of the filler and the disintegrant
are 10-60%, and
2-30% respectively; and a glidant , a lubricant, and a surfactant can
optionally exist, in
which the weight contents of the glidant , the lubricant, and the surfactant
are 0.1-5%,
0.1-5%, and 0.005-1% respectively. 7'he filler can be starch, pregelatinized
starch,
carboxymethyl starch, microcrystalline cellulose, lactose, dextrin, sucrose,
glucose,
mannitol, sorbitol, calcium sulfate dihydrate, dibasic calcium phosphate,
tribasic calcium
phosphate, or calcium carbonate. The binder can be corn starch, pregelatinized
corn starch,
pregelatinized starch, gelatine, sucrose, arabic gum, povidone,
methylcellulose of various
viscosities, sodium carboxymethyl cellulose of low viscosity, ethylcellulose
of various
viscosities, polyvinyl alcohol of various viscosities, polyethylene glycol
6000, or solution of
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CA 02692549 2010-01-05
hydroxypropylmethyl cellulose in water or an alcohol. The disintegrant can be
starch,
pregelatinized starch, low-substituted hydroxypropyl cellulose,
microcrystalline cellulose,
absolute lignocellulose, alginic acid, sodium carboxymethyl starch,
croscarmellose sodium,
guar gum, crosslinked polyvinylpyrrolidone, ion exchange resin,
methylcellulose,
carboxymethylcellulose sodium, and effervescent disintegrant composed of an
organic acid
(e.g., citric acid, and tartaric acid) and carbonate (e.g., sodium carbonate
and sodium
bicarbonate). The lubricant can be tartaric acid, magnesium stearate, calcium
stearate,
zinc stearate, talc, polyethylene glycol 4000, polyethylene glycol 6000,
polyethylene glycol
8000, magnesium lauryl sulfate, sodium benzoate, sodium acetate, sodium
chloride, sodium
oleate, boric acid, leucine, adipic acid, fiimaric acid, glycerol triacetate,
polyoxyethylene
monostearate, monolauryl saccharate, magnesium lauryl sulfate, and sodium
lauryl sulfate.
The glidant can be gas phase micro-powder silica gel, synthetic micro-powder
silica gel,
and magnesia. The surfactant can be sodium dodecylsulfate, poloxamer, Tweens,
Spans,
hexadecyl trimethylamine bromide, sodium lauryl sulfate, sodium stearate
sulfonate,
polyoxyethylene castor oil, and polyoxyethylene monostearate. The prescription
can
optionally contain flavor, including stevioside, fructose, sucrose, glucose,
aspartame,
protein sugar, xylitol, mannitol, sorbitol, lactose, maltitol, glycyrrhizin,
sodium
cyclohexylaminosulfonate, banana flavoi-, orange flavor, pineapple flavor,
mint flavor,
fennel, vanillin, lemon flavor, cherry flavor, and rose flavor. The
prescription can also
optionally contain wetting agent, i.e., aqueous or ethanol solution of
different
concentrations. The coating material uiseful in the pharmaceutical preparation
of the
present invention can be, for example, cellulose and derivatives thereof,
acrylic resins, and
polymers of ethylene.
The injection preparation includes injectable solution, concentrated solution
for
injection, or sterile powder for injection. The adjuvant is an additive
meeting injection
requirements, including pH adjusting agent, isotonic adjustment agent, anti-
oxidant,
chelating agent, and excipient in the sterile powder for injection. The pH
adjusting agent
includes hydrochloric acid, lactic acid, methanesulfonic acid, sodium
hydroxide, sodium
bicarbonate, phosphoric acid and salts thereof, acetic acid and salts thereof,
citric acid and
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CA 02692549 2010-01-05
salts thereof, and amino acid and salts thereof, the isotonic adjustment agent
includes
glucose, sodium chloride, glycerol, and sodium sulfate; the excipient includes
sorbitol,
mannitol, dextran, lactose, sucrose, glucose, hydrolyzed gelatine, and sodium
chloride; the
anti-oxidant includes 0.01 %-0.1 % of sodium or potassium pyrosulfite, 0.01-
0.5% of sodium
sulfite, 0.01%-0.5% of sodium bisulphite, 0.01 %-0.5% of sodium thiosulfate,
0.1-0.2% of
sodium formaldehydesulfoxylate, 0.05%-0.1% of thiourea, 0.05%-0.2% of ascorbic
acid,
0.1%-0.5% of thioglycerol, 0.01-0.2% of glutathione, 0.01%-0.5% of alanine,
0.01%-0.5%
of cysteine, 0.01-0.1% of gallic acid and propyl or octyl ester thereof, 0.01-
0.1% of
tert-butyl p-hydroxyanisole, 0.01%-0.5% of di-tert-butyl p-cresol, 0.01 %-0.1
% of
tocopherol a, (3, and y, 0.01%-0.5% of nordihydroguaiaretic acid, or 0.01%-
0.5% of
palmityl ascorbate; and the chelating agent includes disodium ethylene diamine
tetraacetate,
and calcium sodium ethylene diamine tetraacetate.
The compound of the present invention is useful in preparation of
detoxification
medicament for improving metabolism.
The present invention further provicies a liquid chromatography for
determining an
optical purity of an optically active N-(a-mercaptopropionyl)glycine,
including:
1) chromatographic conditions: the chromatography column has a stationary
phase of a
chiral column with 3,5-dimethylphenyl-carbamate glycopeptides and a mobile
phase of
n-hexane/ethanol/glacial acetic acid (80-9:5:5-20:0.01-1.0), with (90:10:0.1)
being preferred;
the detection wavelength is 200 nm-230 nm, with 210 nm being preferred; and
flow rate of
the mobile phase is 0.2-3.0 ml/min, with 1.0 ml/min being preferred;
2) formulation of the sample solution: the sample
S-(-)-N-(a-mercaptopropionyl)glycine or R-(+)-N-(a-mercaptopropionyl)glycine
is
formulated into a solution of 0.1-20 mg/rnl (preferably 1 mg/ml) with an
organic solvent;
and the organic solvent is selected from, n-propanol, i-propanol, ethanol, i-
butanol, and
methanol, with ethanol being preferred; and;
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CA 02692549 2010-01-05
3) determination: the solution is injected into a high performance liquid
chromatograph
(HPLC), and the chromatogram is recorded and analyzed.
BRIEF DESCRIPTION OF THE DRAWINGS
No drawings.
DETAILED DESCRIPTION OF THE INVENTION
Embodiment 1: Preparation of S-(-)-N-(mercaptopropionyl)glycine
1) 54.3 g (0.5 mol) of R-(+)-2-chloropropionic acid, 60 g=(0.504 mol) of
thionyl
chloride were added to a 100 ml dry reaction flask, and stirred at reflux for
4 h with
moisture isolated. After the reaction was completed, excessive thionyl
chloride was
distilled out for reuse, and then the fraction having a bp of 95-105 C was
collected, to get
55 g of R-(+)-2-chloropropionyl chloride as a colorless liquid with a yield of
86.6%.
2) 29.9 g (0.40 mol) of glycine, 21.2 g (0.20 mol) of anhydrous sodium
carbonate, and
250 ml of water were added into a 1000 ml reaction flask, and stirred until
dissolved.
Cooling with an ice-salt bath, 50.6 g (0.40 mol) of R-(+)-2-chloropropionyl
chloride was
added dropwise with vigorously stirring., and a saturated solution of
anhydrous sodium
carbonate was added at the same time, to make the reaction solution weak
basic. After
addition, stirring was continued for additional 3-5 h till the reaction was
completed. The
reaction solution was acidified to pH = I with concentrated hydrochloric acid,
extracted
with ethyl acetate, and then dried oveir anhydrous magnesium sulfate. The
reaction
solution was filtered and concentrated und.er reduced pressure till a crystal
was precipitated.
Then, the reaction solution was stoocl still, filtered, and dried, to get 38.6
g of
R-(+)-2-chloropropionyl glycine as white small needle crystal. mp: 120-124 C,
a~ n=
+23.8 C (water).
3) 26.5 g(0.11 mol) of sodium sulfide (Na2S=9H2O), 3.52 g(0.11 mol) of
sublimed
sulfur, and 120 ml of water were added to a 250 ml beaker, and heated with
stirring until
CA 02692549 2010-01-05
dissolved, to get a red brown solution of sodium disulfide for later use. 16.4
g(0.10 mol)
of R-(+)-2-chloropropionyl glycine, and 5.6 g of anhydrous sodium carbonate
were added
into a 250 ml reaction flask, and then 100 ml of water was added slowly to
prevent the
generation of bubbles. After cooling to 0-10 C, the sodium disulfide solution
was added
dropwise, and then reacted for 10-15 h at 5-15 C. After the reaction was
completed, the
reaction solution was cooled to about 0 C', to which concentrated sulfuric
acid was added
dropwise, to adjust pH to approximately I. Then, the reaction solution was
filtered, and
then 17 g of zinc powder was added in portion to the filtrate with stirring,
and reacted for 3
h at normal temperature till the reaction was completed. The reaction solution
was filtered,
and then the filtrate was extracted with ethyl acetate, washed with saturated
sodium
chloride solution, and dried over anhydrous magnesium sulfate. The reaction
solution was
filtered, concentrated under reduced pressure, and stood still, to get a
solid. The solid was
filtered out and collected, and recrystallized with ethyl acetate, and then
dried under
vacuum, to get 8.4 g of S-(-)-N-(a-mercaptopropionyl)glycine as white
crystalline solid.
mp: 102-104 C, 1a1 , ~ =-36.5 C (water), content: 99.3% (titrated with 0.1
mol/L of iodine
titration solution ), related substance < 2% (thin layer chromatography:
silica gel G thin
plate, chloroform-acetone-glacial acetic acid (9:3:1), developed with iodine
vapor).
IHNMR (DMSO-D6) 8 ppm: 1.35 (d, 3H); 2.79 (d, 1H); 3.54 (m, 1H); 3.77 (m, 2H);
8.25(t, IH); 12.5(bs, 1H); MS (m/z) 163; element analysis C5H9NO3S (%): C
36.65, H 5.66,
N 8.50, S 19.68.
Embodiment 2: Preparation of R-(+)-N-(a-mercaptopropionyl)glycine
1) 54.3 g (0.5 mol) of S-(-)-2-chloropropionic acid, 60 g (0.504 mol) of
thionyl
chloride were added to a 100 ml dry reaction flask, and stirred at reflux for
4 h with
moisture isolated. After the reaction vvas completed, excessive thionyl
chloride was
distilled out for reuse, and then the fraction having a bp of 95-105 C was
collected, to get
56 g of S-(-)-2-chloropropionyl chloride as a colorless liquid, with a yield
of 88.2%.
2) 29.9 g (0.40 mol) of glycine, 21.2 g(0.20 mol) of anhydrous sodium
carbonate, and
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CA 02692549 2010-01-05
250 ml of water were added into a 1000 ml reaction flask, and stirred until
dissolved.
Cooling with an ice-salt bath, 50.6 g (0.40 mol) of S-(-)-2-chloropropionyl
chloride was
added dropwise with vigorously stirring, and a saturated solution of anhydrous
sodium
carbonate was added at the same time, to make the reaction solution weak
basic. After
addition, stirring was continued for additional 3-5 h till the reaction was
completed. The
reaction solution was acidified to pH = l with concentrated hydrochloric acid,
extracted
with ethyl acetate, and then dried ovei- anhydrous magnesium sulfate. The
reaction
solution was filtered and concentrated under reduced pressure till a crystal
was precipitated.
Then, the reaction solution was stood still, filtered, and dried, to get 37.8
g of
S-(-)-2-chloropropionyl glycine as wliite small needle crystal. ' mp: 120-124
C,
a n =-24.2 C (water).
3) 26.5 g(0.11 mol) of sodium sulfide (Na2S=9H2O), 3.52 g(0.11 mol) of
sublimed
sulfur, and 120 ml of water were added to a 250 ml beaker, and heated with
stirring until
dissolved, to get a red brown solution of sodium disulfide for later use. 16.4
g(0.10 mol)
15 of S-(-)-2-chloropropionyl glycine, and 5.6 g of anhydrous sodium carbonate
were added
into a 250 ml reaction flask, and then 100 ml of water was added slowly to
prevent the
generation of bubbles. After cooling to 0-10 C, the sodium disulfide solution
was added
dropwise, and then reacted for 10-15 h at 5-15 C. After the reaction was
completed, the
reaction solution was cooled to about 0 C, to which concentrated sulfuric acid
was added
20 dropwise, to adjust pH to approximately 1.. The reaction solution was
filtered, and then 17
g of zinc powder was added in portion to the filtrate with stirring, and
reacted for 3 h at
normal temperature till the reaction was completed. Then, the reaction
solution was
filtered, and then the filtrate was extracted with ethyl acetate, washed with
saturated sodium
chloride solution, and dried over anhydrous magnesium sulfate. The reaction
solution was
filtered and concentrated under reduced pressure, and stood still, to get a
solid. The solid
was filtered and collected, and recrystallized with ethyl acetate, and then
dried under
vacuum, to get 8.2 g of R-(+)-N-(a-mercaptopropionyl)glycine as white
crystalline solid.
0
mp: 102-104 C, n =+37.5 C (water), content: 99.1% (titrated with 0.1 mol/L of
iodine
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CA 02692549 2010-01-05
titration solution ), related substance < 2% (thin layer chromatography:
silica gel G thin
plate, chloroform-acetone-glacial acetic acid (9:3:1), developed with iodine
vapor).
1 HNMR (DMSO-D6) S ppm: 1.40(d, 3H): 2.80 (d, IH); 3.60 (m, IH): 3.81 (m, 2H);
8.28 (t, IH); 12.8 (bs, 1 H); MS (m/z) 163: element analysis C5H9N03S (%): C
36.68, H
5.65, N 8.53, S 19.65.
Embodiment 3: Preparation of R-(+)-N-(a-mercaptopropionyl)glycine
1) 54.3 g (0.5 mol) of S-(-)-2-chloropropionic acid, 60 g (0.504 mol) of
thionyl
chloride were added to a 100 ml dry reaction flask, and stirred at reflux for
4 h with
moisture isolated. After the reaction was completed, excessive thionyl
chloride was
distilled out for reuse, and then the fraction having a bp of 95-105 C was
collected to get
54.6 g of S-(-)-2-chloropropionyl chloride as a colorless liquid.
2) 29.9 g (0.40 mol) of glycine, 21.2 g (0.20 mol) of anhydrous sodium
carbonate, and
250 ml of water were added into a 1000 ml reaction flask, and stirred until
dissolved.
Cooling with an ice-salt bath, 50.6 g (0.40 mol) of S-(-)-2-chloropropionyl
chloride was
added dropwise with vigorously stirring, and a saturated solution of anhydrous
sodium
carbonate was added at the same time, to make the reaction solution weak
basic. After
addition, stirring was continued for additional 3-5 h till the reaction was
completed. The
reaction solution was acidified to pH = 1 with concentrated hydrochloric acid,
extracted
with ethyl acetate, and then dried ovei- anhydrous magnesium sulfate. The
reaction
solution was filtered and concentrated under reduced pressure till a crystal
was precipitated.
Then, the reaction solution was stood still, filtered, and dried, to get 38 g
of
S-(-)-2-chloropropionyl glycine as white sinall needle crystal.
3) 28.8 g (0.12 mol) of sodium sulfide (Na2S=9H2O), 3.84 g (0.12 mol) of
sublimed
sulfur, and 120 ml of ethanol were added to a 250 ml beaker, and heated with
stirring until
dissolved, to get a red brown solution of sodium disulfide for later use. 18.3
g of (0.11
mol) of S-(-)-2-chloropropionyl glycine, and 6.3 g of anhydrous sodium
carbonate were
13
,---------_ _ _ _---_ -
~~~ ,....... ~~
__ ___ _ - -------,
CA 02692549 2010-01-05
added into a 250 ml reaction flask, and then 100 ml of water was added slowly
to prevent
the generation of bubbles. After cooling to 0-10 C, the sodium disulfide
solution was
added dropwise, and then reacted for 12 h at 0-10 C. After the reaction was
completed,
the temperature was kept constant, and concentrated sulfuric acid was added
dropwise, to
adjust pH to approximately 1. The reaction solution was filtered, and then 17
g of zinc
powder was added in portion to the filtrate with stirring, and reacted for 3 h
at normal
temperature till the reaction was completed. The reaction solution was
filtered, and then
the filtrate was extracted with ethyl acetate, washed with saturated sodium
chloride solution,
and dried over anhydrous magnesium sulfate. The reaction solution was filtered
and
concentrated under reduced pressure, and stood still to get a solid. The solid
was filtered,
collected, recrystallized with ethyl acetate, and then dried under vacuum, to
get 8.1 g of
R-(+)-N-(a-mercaptopropionyl)glycine as white crystalline solid. Content:
99.3% (titrated
with 0.1 mol/L of iodine titration solution ), related substance < 2% (thin
layer
chromatography: silica gel G thin plate, chloroform-acetone-glacial acetic
acid (9:3:1),
developed with iodine vapor).
Embodiment 4: Determination of optical purity of
S-(-)-N-(a-mercaptopropionyl)glycine
Instrument: HPLC, SPD-10Avp UV detector, LC-lOAD pump
Mobile phase: n-hexane-ethanol-glac:ial acetic acid (90:10:0.1)
Flow rate: 1.0 ml/min
Chromatography column: CHIRALPAK AD-H (with 3,5-dimethylphenyl-carbamate
glycopeptide as stationary phase)
Column temperature: 25 C
Detection wavelength: 210 nm
Sample concentration: 0.5 mg/ml
14
!_ - ---
CA 02692549 2010-01-05
The optical activity of S-(-)-N-(a-mercaptopropionyl)glycine obtained in
Embodiment
I under the conditions above is determined to be 99.3%.
Embodiment 5: Determination of optical purity of
R-(+)-N-(a-mercaptopropionyl)glycine
Instrument: HPLC, SPD-10AvPUV detector, LC-lOAD pump
Mobile phase: n-hexane-ethanol-glacial acetic acid (90:10:0.1)
Flow rate: 1.0m1/min
Chromatography column: CHIRALPAKAD-H (with 3,5-dimethylphenyl-carbamate
glycopeptide as stationary phase)
Column temperature: 25 C
Detection wavelength: 210 nm
Sample concentration: 0.5 mg/ml
The optical activity of R-(+)-N-(a-mercaptopropionyl)glycine obtained in
Embodiment
1 under the conditions above is determined to be 99.2%.
Embodiment 6: Preparation of S-(-)-N-((x-mercaptopropionyl)glycine arginine
salt
10 g of S-(-)-N-(a-mercaptopropion),l)glycine obtained in Embodiment 1, 11.2 g
of
L-arginine, and 60 ml of 95% methanol were added into a reaction flask,
stirred, heated to
reflux, and reacted for 3 h. After the reaction was completed, the reaction
solution was
filtered while being hot, and then the filtrate was cooled to room temperature
and placed in
a freezer to precipitate a crystal by cooling. The crystal was filtered,
collected, and dried
to obtain 19.2 g of a white crystalline solid with a yield of 92.8%.
Embodiment 7: Preparation of R-(+)-T.T-(a-mercaptopropionyl)glycine lysine
salt
---
_--
.-.-
1
CA 02692549 2010-01-05
g of R-(+)-N-(a-mercaptopropionyl)glycine obtained in Embodiment 2, 9.4 g of
L-lysine, and 60 ml of 95% methanol were added into a reaction flask, stirred,
heated to
reflux, and reacted for 3 h. After the reaction was completed, the reaction
solution was
filtered while being hot, and then the filtrate was cooled to room temperature
and placed in
5 a freezer to precipitate a crystal by cooling. The crystal was filtered,
collected, and dried
to obtain 16.8 g of a white crystalline solicl with a yield of 88.6%.
Embodiment 8: Preparation of S-(-)-N-(a-mercaptopropionyl)glycine sodium
1) 16.3 g (0.10 mol) of S-(-)-N(a-m(.-rcaptopropionyl)glycine, and 45 ml of
methanol
were placed in a reaction flask, to which 0.15 g of dithiothreitol was added
at 15 C, and
10 stirred for 15 min, and then 5 g of molecular sieve was added.
2) 4.65 g of NaOH was slowly added into the methanol solution above in 6
portions.
3) After addition, the reactants were i-eacted for I h at 15 C with stirring,
and then for
another I h at 20-25 C with stirring. After the reaction was completed, the
molecular
sieve was filtered off, and then the filtrate was added into 130 mL of
acetone, and fully
agitated to be homogeneously mixed.
4) The large amount of white precipitate present in the reaction solution was
filtered by
suction, and then the filter cake was dried at 80 C, to get 12.8 g of
anhydrous
R-(-)-N-(a-mercaptopropionyl)glycine sodium.
Embodiment 9: Preparation of R-(+)-N-(a-mercaptopropionyl)glycine sodium
1) 16.3 g (0.10 mol) of R-(+)-N-(a-mercaptopropionyl)glycine, and 45 ml of
methanol
were placed in a reaction flask, to which 0.15 g of dithiothreitol was added
at 15 C, and
stirred for 15 min, and then 5 g of molecular sieve was added.
2) 4.65 g of NaOH was slowly added into the methanol solution above in 6
portions.
3) After addition, the reactants were reacted for 1 h at 15 C with stirring,
and then for
16
----
;_~. .
.. K_ ~
_ _. ,
CA 02692549 2010-01-05
another 1 h at 20-25 C with stirring. After the reaction was completed, the
molecular
sieve was filtered off, and then the filtrate was added into 130 mL of
acetone, and fully
agitated to be homogeneously mixed.
4) The large amount of white precipitate present in the reaction solution was
filtered by
suction, and then the filter cake was dried at 80 C, to get 13.1 g of
anhydrous
S-(+)-N-(a-mercaptopropionyl)glycine sodium.
Embodiment 10: Preparation of S-(-)-N-(ct-mercaptopropionyl)glycine tablet
Formula Composition:
S-(-)-N-(a-mercaptopropionyl)glycine 100 g
Microcrystalline cellulose 170 g
Pregelatinized starch 60 g
8% starch slurry suitable amount
Sodium carboxymethyl starch 7 g
Magnesium stearate 3 g
Preparation process: Taking the preparation of 1000
S-(-)-N-(a-mercaptopropionyl)glycine tablets as example, the raw and auxiliary
materials
were respectively pulverized and passed through a sieve of 100 mesh for later
use; next,
forrnula amounts of S-(-)-N-(a-mercaptopropionyl)glycine, microcrystalline
cellulose, and
pregelatinized starch were weighted, and homogeneously mixed to prepare a soft
material
with 8% starch slurry, which was granulated with a sieve of 20 mesh, dried,
and then sized
with a sieve of 18 mesh; afterwards, formula amounts of sodium carboxymethyl
starch and
magnesium stearate were added, homogeneously mixed, and then pressed into
tablets.
The weight of each tablet is about 345 mg.
17
. ..
, . ;
_ _ ------~
CA 02692549 2010-01-05
Embodiment 11: Preparation of film-coated S-(-)-N-(a-mercaptopropionyl)glycine
tablet
The naked tablet obtained in Embodiment 10 was coated with a formulated 8%
gastric
soluble OPADRY (OY-C-7000A) solution in ethanol in a high efficient coating
pan; and the
amount of the coating powder is 2.0-3.0% by weight of the naked tablet.
Embodiment 12: Preparation of enteric soluble S-(-)-N-(a-
mercaptopropionyl)glycine
tablet
The naked tablet obtained in Embodiment 10 was coated with a formulated 8%
enteric
soluble OPADRY solution in ethanol in a high efficient coating pan; and the
amount of the
coating powder is 4.0-5.0% by weight of the naked tablet.
Embodiment 13: Preparation of slow release S-(-)-N-(a-
mercaptopropionyl)glycine
tablet
Formula Composition:
S-(-)-N-(a-mercaptopropionyl)glycine 150 g
Hydroxypropylmethyl cellulose (K151V1) 150 g
Lactose 50 g
3% povidone solution in 80% ethanol suitable amount
Magnesium stearate 5 g
Preparation process: Taking the preparation of 1000 slow release
S-(-)-N-(a-mercaptopropionyl)glycine tablets as example, the raw and auxiliary
materials
were respectively pulverized and passed through a sieve of 100 mesh for later
use; next,
formula amounts of S-(-)-N-(a-mercaptopropionyl)glycine, hydroxypropylmethyl
cellulose,
and lactose were weighted, and homogeneously mixed to prepare a soft material
with 3%
18
CA 02692549 2010-01-05
povidone solution in 80% ethanol, which was granulated with a sieve of 20
mesh, dried,
and then sized with a sieve of 18 mesh; afterwards, formula amounts of
magnesium stearate
were added, homogeneously mixed, and then pressed into tablets. The weight of
each
tablet is about 360 mg.
Embodiment 14: Preparation of S-(-)-N-(a-mercaptopropionyl)glycine dispersible
tablet
Formula Composition:
S-(-)-N-(a-mercaptopropionyl)glycine: 100 g
Crosslinked polyvinylpyrrolidone 15 g
Microcrystalline cellulose 180 g
Mannitol 50 g
60% ethanol solution suitable amount
Sodium dodecyl sulfate 0.2 g
Micro-powder silica gel 5 g
Magnesium stearate 3 g
Stevioside 5 g
Preparation process: Taking the preparation of 1000
S-(-)-N-(a-mercaptopropionyl)glycine dispersible tablets as example, the raw
and auxiliary
materials were respectively pulverized and passed through a sieve of 100 mesh
for later use;
next, formula amounts of S-(-)-N-(a-mercaptopropionyl)glycine,
microcrystalline cellulose,
and mannitol were weighted, and homogeneously mixed to prepare a soft material
with
60% ethanol solution, which was granulated, dried, and then sized; afterwards,
formula
-----
19
CA 02692549 2010-01-05
amounts of crosslinked polyvinylpyrrolidone, sodium dodecyl sulfate, micro-
powder silica
gel, magnesium stearate, and stevioside were added, homogeneously mixed, and
then
pressed into tablets. The weight of each tablet is about 360 mg.
Embodiment 15: Preparation of S-(-)-N-(a-mercaptopropionyl)glycine capsule
Formula Composition:
S-(-)-N-(a-mercaptopropionyl)glycine 150 g
Starch 45 g
5% povidone solution in 60% ethanol suitable amount
Magnesium stearate 2 g
Preparation process: Taking the preparation of 1000
S-(-)-N-(a-mercaptopropionyl)glycine capsules as example, the raw and
auxiliary materials
were respectively pulverized and passed through a sieve of 100 mesh for later
use; next,
formula amounts of S-(-)-N-(a-mercaptopropionyl)glycine, and starch were
weighted, and
homogeneously mixed to prepare a soft material with 5% povidone solution in
60% ethanol,
which was granulated, dried, and then sized; afterwards, formula amounts of
magnesium
stearate was added, homogeneously mixecl, and then filled into a capsule.
Embodiment 16: Preparation of S-(-)-N-(a-mercaptopropionyl)glycine granule
Forrnula Composition:
S-(-)-N-(a-mercaptopropionyl)glycine: 100 g
Mannitol 350 g
Sucrose 350 g
Sodium carboxymethyl cellulose 50 g
~-- __
------
~
L_- _
CA 02692549 2010-01-05
Aspartame 10 g
Maltdextrin 140 g
5% povidone solution in 80% ethanol suitable amount
Flavor 1 g
Preparation process: Taking the preparation of 1000 packages of
S-(-)-N-(a-mercaptopropionyl)glycine granule as example, the raw and auxiliary
materials
were respectively pulverized and passed tlirough a sieve of 100 mesh for later
use; next, the
flavor was added into 5% povidone solution in 80% ethanol, and then formula
amounts of
S-(-)-N-(a-mercaptopropionyl)glycine, sucrose, mannitol, sodium carboxymethyl
cellulose,
aspartame, and maltdextrin were weighted to prepare a soft material with 5%
povidone
solution in 80% ethanol, which was grariulated with a sieve of 16 mesh, dried,
and then
sized with a sieve of 14 mesh; afterwards, the granulates were sieved to
remove the fine
powder with a sieve of 60 mesh, and packaged. The weight of each package is
about I g.
Embodiment 17: Preparation of injectable S-(-)-N-(a-mercaptopropionyl)glycine
solution
Formula Composition:
S-(-)-N-((x-mercaptopropionyl)glycine 100 g
Water for injection made up to 2000 ml
Preparation process: Taking the preparation of 1000 ampoules of injectable
S-(-)-N-(a-mercaptopropionyl)glycine solution as example, formula amount of
S-(-)-N-(a-mercaptopropionyl)glycine was added into 1600 ml of water for
injection, and
stirred until completely dissolved; then additional water for injection was
added to make up
to 2000 ml, and uniformly stirred; afterwards, 0.1% of activated carbon for
refinement of
injection was added, and uniformly stirred; the drug solution was processed
with titanium
21
;, ..
CA 02692549 2010-01-05
rod to remove active carbon, and then finely filtered with 0.45 m and 0.22 m
micro
membranes in sequence to a satisfactory clarification; then the solution was
detected for
intermediates, filled into 2 ml ampoules at 2 ml specification under nitrogen
flow if eligible,
and then sterilized.
Embodiment 18: Preparation of S-(-)-N-(a-mercaptopropionyl)glycine injection
Formula Composition:
S-(-)-N-(a-mercaptopropionyl)glycine 100 g
Dextran 40 50 g
Sodium bisulphate I g
Disodium ethylenediamine tetraacetate 0.2 g
Preparation process: Taking the preparation of 1000 vials of
S-(-)-N-(a-mercaptopropionyl)glycine foir injection as example, formula
amounts of
S-(-)-N-(a-mercaptopropionyl)glycine, dextran 40, sodium bisulphate, and
disodium
ethylenediamine tetraacetate were added into 1600 ml of water for injection,
and stirred
until completely dissolved; next, the pH of the solution was adjusted to 1.5-
2.5 with 1 mol/L
hydrochloric acid, and then additional water for injection was added to make
up to 2000 ml,
and uniformly stirred; afterwards, 0.1 % of activated carbon for refinement of
injection was
added, and uniforrnly stirred; the drug solution was processed with titanium
rod to remove
active carbon, and then finely filtered with 0.45 m and 0.22 m micro
membranes in
sequence and sterilized under aseptic conditions; the drug solution was
detected for the
presence of intermediates, pH, content, and clarification, and filled into a
penicillin bottle at
2 ml specification if eligible, partially stoppered, freeze dried, stoppered
and capped, and
then light inspected.
Embodiment 19: Preparation of R-(+)-N-(a-mercaptopropionyl)glycine tablet
22
~.. _..
CA 02692549 2010-01-05
Formula Composition:
R-(+)-N-(a-mercaptopropionyl)glycine 100 g
Microcrystalline cellulose 170 g
Pregelatinized starch 60 g
8% starch slurry suitable amount
Sodium carboxymethyl starch 7 g
Magnesium stearate 3 g
Preparation process: Taking the preparation of 1000
R-(+)-N-(a-mercaptopropionyl)glycine tablets as example, the raw and auxiliary
materials
were respectively pulverized and passed through a sieve of 100 mesh for later
use; next,
formula amounts of R-(+)-N-(a-mercaptopropionyl)glycine, microcrystalline
cellulose, and
pregelatinized starch were weighted, and homogeneously mixed to prepare a soft
material
with 8% starch slurry, which was granulated with a sieve of 20 mesh, dried,
and then sized
with a sieve of 18 mesh; afterwards, formula amounts of sodium carboxymethyl
starch and
magnesium stearate were added, homogeneously mixed, and then pressed into
tablets.
The weight of each tablet is about 345 mg.
Embodiment 20: Preparation of filrn-coated R-(+)-N-(a-
mercaptopropionyl)glycine
tablet
The naked tablet obtained in Embodiment 19 was coated with a formulated 8%
gastric
soluble OPADRY (OY-C-7000A) solution in ethanol in a high efficient coating
pan; and the
amount of the coating powder used is 2.0-3.0% by weight of the naked tablet.
Embodiment 21: Preparation of enteric soluble R-(+)-N-(a-
mercaptopropionyl)glycine
tablet
23
---
,
CA 02692549 2010-01-05
The naked tablet obtained in Embodiment 19 was coated with a formulated 8%
enteric
soluble OPADRY solution in ethanol in a high efficient coating pan; and the
amount of the
coating powder used is 4.0-5.0% by weight of the naked tablet.
Embodiment 22: Preparation of slow release R-(+)-N-(a-
mercaptopropionyl)glycine
tablet
Formula Composition:
R-(+)-N-(a-mercaptopropionyl)glycine 150 g
Hydroxypropylmethyl cellulose (K151VI) 150 g
Lactose 50g
3% povidone solution in 80% ethanol suitable amount
Magnesium stearate 5 g
Preparation process: Taking the preparation of 1000 slow release
R-(+)-N-(a-mercaptopropionyl)glycine tablets as example, the raw and auxiliary
materials
were respectively pulverized and passed through a sieve of 100 mesh for later
use; next,
formula amounts of R-(+)-N-(a-mercaptopropionyl)glycine, hydroxypropylmethyl
cellulose,
and lactose were weighted, and homogeneously mixed to prepare a soft material
with 3%
povidone solution in 80% ethanol, which was granulated with a sieve of 20
mesh, dried,
and then sized with a sieve of 18 mesh; afterwards, formula amount of
magnesium stearate
was added, homogeneously mixed, and then pressed into tablets. The weight of
each
tablet is about 360 mg.
Embodiment 23: Preparation of R.-(+)-N-(a-mercaptopropionyl)glycine
dispersible
tablet
Formula Composition:
24
CA 02692549 2010-01-05
R-(+)-N-(a-mercaptopropionyl)glycine 100 g
Crosslinked polyvinylpyrrolidone 15 g
Microcrystalline cellulose 80 g
Mannitol 50g
60% ethanol solution suitable amount
Sodium dodecyl sulfate 0.2 g
Micro-powder silica gel 5 g
Magnesium stearate 3 g
Stevioside 5 g
Preparation process: Takirig the preparation of 1000
R-(+)-N-((-t-mercaptopropionyl)glycine dispersible tablet as example, the raw
and auxiliary
materials were respectively pulverized and passed through a sieve of 100 mesh
for later use;
next, formula amounts of R-(+)-N-(mercaptopropionyl)glycine, microcrystalline
cellulose,
and mannitol were weighted, and homog;eneously mixed to prepare a soft
material with
60% ethanol solution, which was granulated, dried, and then sized; afterwards,
formula
amounts of crosslinked polyvinylpyrrolidone, sodium dodecyl sulfate, micro-
powder silica
gel, magnesium stearate, and stevioside were added, homogeneously mixed, and
then
pressed into tablets. The weight of each tablet is about 360 mg.
Embodiment 24: Preparation of R-(+)-N-(a-mercaptopropionyl)glycine capsule
Formula Composition:
R-(+)-N-(a-mercaptopropionyl)glycine 150 g
Starch 45 g
, _.
CA 02692549 2010-01-05
5% povidone solution in 60% ethanol suitable amount
Magnesium stearate 2 g
Preparation process: Taking the preparation of 1000
R-(+)-N-(a-mercaptopropionyl)glycine capsules as example, the raw and
auxiliary materials
were respectively pulverized and passed through a sieve of 100 mesh for later
use; next,
formula amounts of R-(+)-N-((x-mercaptopropionyl)glycine, and starch were
weighted, and
homogeneously mixed to prepare a soft material with 5% povidone solution in
60% ethanol,
which was granulated, dried, and then sized; afterwards, formula amounts of
magnesium
stearate was added, homogeneously mixed, and then filled into a capsule.
Embodiment 25: Preparation of R-(+).-N-(a-mercaptopropionyl)glycine granule
Formula Composition:
R-(+)-N-(a-mercaptopropionyl)glycin,e 100 g
Mannitol 350 g
Sucrose 350 g
Carboxymethylcellulose sodium 50 g
Aspartame l0 g
Maltdextrin 140 g
5% povidone solution in 80% ethanol suitable amount
Flavor I g
Preparation process: Taking the preparation of 1000 packages of
R-(+)-N-(a-mercaptopropionyl)glycine granule as example, the raw and auxiliary
materials
were respectively pulverized and passed through a sieve of 100 mesh for later
use; next, the
26
~ __ ;
CA 02692549 2010-01-05
flavor was added into 5% povidone solution in 80% ethanol, and then, formula
amounts of
R-(+)-N-(a-mercaptopropionyl)glycine, mannitol ,sucrosk', sodium carboxymethyl
cellulose,
aspartame, and maltdextrin were weighted to prepare a soft material with 5%
povidone
solution in 80% ethanol, which was granulated with a sieve of 16 mesh, dried,
and then
sized with a sieve of 14 mesh; afterwards, it was sieved to remove the fine
powder with a
sieve of 60 mesh, and packaged. The weight of each package is about I g.
Embodiment 26: Preparation of injectable R-(+)-N-(a-mercaptopropionyl)glycine
solution
Formula Composition:
R-(+)-N-(a-mercaptopropionyl)glycine 100 g
Sodium pyrosulfite I g
Disodium ethylene diamine tetraacetate 0.2 g
Water for injection made up to 2000 ml
Preparation process: Taking the preparation of 1000 ampoules of injectable
R-(+)-N-(a-mercaptopropionyl)glycine solution as example, formula amounts of
R-(+)-N-(a-mercaptopropionyl)glycine, sodium pyrosulfite and disodium
ethylenediamine
tetraacetate were added into 1600 ml of water for injection, and stirred until
completely
dissolved; then additional water for injection was added to make up to 2000
ml, and
uniformly stirred; afterwards, 0.1% of activated carbon for refinement of
injection was
added, and uniformly stirred; the drug solution was processed with titanium
rod to remove
active carbon, and then finely filtered wiith 0.45 m and 0.22 m micro
membranes in
sequence to a satisfactory clarification; then the solution was detected for
intermediates,
filled into 2 ml ampoules at 2 ml specification under nitrogen flow if
eligible, and then
sterilized.
Embodiment 27: Preparation of R-(+)-N-(a-mercaptopropionyl)glycine for
injection
27
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CA 02692549 2010-01-05
Formula Composition:
R-(+)-N-(a-mercaptopropionyl)glycine 100 g
Dextran 40 50 g
Preparation process: Taking the preparation of 1000 vials of
R-(+)-N-(a-mercaptopropionyl)glycine for injection as example, formula amounts
of
R-(+)-N-(a-mercaptopropionyl)glycine, and dextran 40 were added into 1600 ml
of water
for injection, and stirred until completely dissolved; next, the pH of the
solution was
adjusted to 1.5-2.5 with I mol/L hydrochloric acid or I mol/L sodium hydroxide
solution,
and then additional water for injection was added to make up to 2000 ml, and
uniformly
stirred; afterwards, 0.1% of activated carbon for refinement of injection was
added, and
uniformly stirred; the drug solution was processed with titanium rod to remove
active
carbon, and then finely filtered with 0.45 m and 0.22 m micro membranes in
sequence
and sterilized under aseptic conditions; the solution was detected for the
presence of
intermediates, pH, content, and clarification, and filled into a penicillin at
2 mi specification
if eligible, partially stoppered, freeze dried, stoppered and capped, and then
light inspected.
Embodiment 28: Effect of optically active N-(a-mercaptopropionyl)glycine
administrated by intravenous injection on serum biochemical indicators and
liver indexes of
mice with liver injury induced by acetamidophenol
The animals were randomly divided into the following 5 groups of 10 animals
according to weight: solvent control group, model group (acetamidophenol, 400
mg/kg, 10
mi/kg, intraperitoneal injection), tiopronin group (MPG),
S-(-)-N-(a-mercaptopropionyl)glycine group, and R-(+)-N-(a-
mercaptopropionyl)glycine
group. After fasting for 12 h, except the solvent control group and model
group were
given saline by intravenous injection, the other test groups were
intravenously injected
drugs, and then intraperitoneally injected 400/mg/kg of acetamidophenol
immediately, to
induce acute liver cell injury. The animals were fed with food at 6 hours
after modeling,
28
~ . 1
CA 02692549 2010-01-05
and then fasted for 16 hours; and at 24 hours after modeling, blood were
sampled by eye
ball removal, centrifuged, and collected for serum. ALT, and AST were
determined
following the method of the kit. 10% of iliver homogenate was prepared, to
measure GSH.
The liver and spleen were taken at the same time, weighted, and calculated for
organcoefficient. The results are shown in Tables I and 2.
Table 1. Effect of MPG and optical isomer thereof administrated by intravenous
injection on serum biochemical indicators of mice with liver injury induced by
acetamidophenol
Group Dosage Nurraber Liver ALT AST Serum
(mg/kg) of coefficient (Karl-Fischer (Karl-Fischer total
animals (mg/g) unit) unit) protein
(mg/ml)
Solvent control group - 12 5. 30.5 18.1 30.0f6.5 47.5f4.7
405 0.508
Model group - 10 6.139 0.68 5761.8 2761. 1682.8 1122. 42.9 8.1
3 8 0
Tiopronin group 800 8 5.804t0.59 648.1f412.1 Not detected Not
8 due to detected
insufficient due to
serum insufficient
seruin
R-(+)-N-(a-mercaptopropionyl)glycine 800 12 6.201f0.56 2880.2f2578.
1201.2+1123. 43.7 5.7
group 7 5 0
S-(-)-N-(a-mercaptopropionyl)glycine 800 12 5.816 0.61 1812.1+1780. 591.3
528.0 43.8 7.4
group 3 1
*P<005, **P<0.01 compared with the model group
It can be known from Table 1 that, compared with the solvent control group,
the liver
coefficient of the mice in the model group is significantly increased, and
inhibition of the
elevation of serum ALT is apparent in all the dosed groups, in which the
function in the
S-(-)-N-(a-mercaptopropionyl)glycine group is higher than that in the
R-(+)-N-(a-mercaptopropionyl)glycine group. The ALT value of tiopronin group
is lower
than that of S-(-)-N-(a-mercaptopropionyl)glycine group, but in the
experiment, after
modeling, 4 mouse dead in the tiopronin group die, 2 mouse dead in the model
group, and
no death occurs in the S-(-)-N-(a-mer(-,aptopropionyl)glycine group.
Therefore, the
29
CA 02692549 2010-01-05
protection against liver injury in mice induced by acetamidophenol in
S-(-)-N-(a-mercaptopropionyl)glycine group is superior to that in tiopronin
group.
Table 2. Effect of MPG and optical isomer thereof administrated by intravenous
injection on liver indexes of mice with liver injury induced by
acetamidophenol
Group Dosage Number Liver Liver GSH MDA
(mg/kg;) of total albumin (mo 1/L) (nmol/g wet
animal protein (mgJml) weight)
(mg/mi)
Solvent control group - 12 17.4 3.6 10.0 1.9 1.20 0.15 288. 9 84. 5
Model group - 10 13.0 1.5 8.3 0.7 0.93 0.12 452. 2t 132.
8
Tiopronin group 800 8 15.7 1.4 9. 6f1. 1 0.99f0.16 362.4 98.6
R-(+)-N-(a-mercaptopropionyl)glycine 800 12 16. 2 2. 9. 1t2. 0 0.99 0.16 440.0
group 9 134.6
S-(-)-N-(a-mercaptopropionyl)glycine 800 12 16. 5 1. 9.2 1.3 1.11 0.16 321.7
72.9
group 7
*P<0.05, **P<0.01 compared with the model group
It can be known from Table 2 that, cornpared with the model group, the content
of liver
total protein is significantly increased in all the dosed groups, liver GSH
content is
obviously increased and MDA content is decreased in the
S-(-)-N-(a-mercaptopropionyl)glycine group, but no such effects are present in
the
R-(+)-N-(a-mercaptopropionyl)glycine group and tiopronin group. The results
indicate
that S-(-)-N-(a-mercaptopropionyl)glycine can provide better protection
against liver injury.
Embodiment 29: Protection of N-(a-mercaptopropionyl)glycine and optical isomer
thereof against liver injury in rats induced by carbon tetrachloride
The animals were randomly divideci into the following 5 groups of 10 animals
according to weight: solvent control group, model group (50% CCl4, 2 ml/kg,
intraperitoneal injection), tiopronin group (MPG), S-(-)-N-(a-
mercaptopropionyl)glycine
group, and R-(+)-N-(a-mercaptopropionyl)glycine group. Except the solvent
control
group was given olive oil by intraperitoneal injection, the other test groups
were
intragastrically administrated drugs for 4 days, once daily, intraperitoneally
injected CCl4
CA 02692549 2010-01-05
once on the fifth day to induce acute liver cell injury, treated with drugs 30
min before
modeling (except for the model control group), and then administrated drugs
once 2 hours
after modeling. Then, the animals were fasted for 16 h, blood was sampled from
retrobulbar venous plexus 24 hours after injecting CCl4, centrifuged, and
collected for
serum, and ALT, AST, albumin were determined following the method of the kit.
The
results are shown in Tables 3, and 4. The left lobe of liver was taken to
prepare 10% of
liver homogenate and measure GSH and MDA contents. The results are shown in
Table 5.
Table 3. Effects of MPG and optical isomer thereof on ALT, and AST of rats
with liver
injury induced by CCl4
Group Dosage ALT (Karl-Fischer AST(Karl-Fischer
(mg/kg, ig) unit) unit)
Solvent control group - 327.6f11.4 117.6t46.5
Model group - 1020.1f275.0 1448.1f362.5
Tiopronin group 450 748.6 260.2 1126.0 159.2
R-(+)-N-(ct-mercaptopropionyl)glycine 450 532.8 352.7 818.3f347.0
group
S-(-)-N-(a-mercaptopropionyl)glycine 450 432.8t252.6 718.7+336.2
group
*P<0.05, **P<0.01 compared with the model group
It can be known from Table 3 that, all the three drugs can significantly
decrease the
ALT and AST level, in which the decrease in the R-(+)-N-(a-
mercaptopropionyl)glycine
group and the S-(-)-N-(a-mercaptopropionyl)glycine group is higher than that
in the
tiopronin group.
Table 4. Effects of MPG and optical isomer thereof on serum protein content of
rats
with liver injury induced by CC14
Group Dosage Total Albumin Globulin A/G
(mg/kg, protein (mg/mi) (mg/ml)
ig) (mg/mi)
Solvent control group - 76.2 5.14 30.1f2.2 46.1f5.1 0.660 0.08
3 3 7
Model group - 59.1t7.56 26.8f2.7 32.3f4.9 0.837 0.05
4 9 9
Tiopronin group 450 59.8f6.26 26.84:1.9 32.9 4.7 0.825f0.10
31
__ ;
CA 02692549 2010-01-05
8 9 1
R-(+)-N-(a-mercaptopropionyl)glycine 4510 64.7 6.40 27.5 1.4 37.2 5.0 0.747
0.07
group 4 6 3
S-(-)-N-(a-mercaptopropionyl)glycine 450 70.6 3.63 30.3 1.2 40.3f2.9
0.754f0.05
group 0 8 0
**P<0.01 compared with the model group
It can be known from Table 4 that, compared with the model group, in the
S-(-)-N-(a-mercaptopropionyl)glycine group, the serum total protein, albumin
and globulin
contents are obviously increased, and A/C'r value is decreased; A/G value is
also decreased
in the R-(+)-N-(a-mercaptopropionyl)glyc:ine group; but no obvious effect on
the indexes
are detected in the tiopronin group, which corresponds to the results obtained
in the model
group.
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