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Description  |
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This invention relates to a novel group of ML-236B carboxylic acid
derivatives and their use as antihyperlipemic agents.
More particularly, it is concerned with an ML-236B carboxylic acid
derivative having the formula
##STR2##
wherein R represents an alkyl group, a benzyl group optionally substituted
with alkyl, alkoxy or halogen or a phenacyl group optionally substituted
with alkyl, alkoxy or halogen; a group of 1/n M in which M represents a
metal and n represents a valency of said metal; or a group of AH.sup.+ in
which A represents an amino acid. Also, it relates to a new use of the
ML-236B carboxylic acid derivatives (I) as an agent for the treatment of
hyperlipemia.
Heretofore, it was disclosed and claimed in Japanese Patent Application
Provisional Publication No. 155690/1975 (which was laid open to public on
Dec. 16, 1975 and corresponds to U.S. Pat. No. 3,983,140; British Pat. No.
1453425; Belgian Pat. No. 830033; and West German Laying Open
Specification No. 2524355) that the compound having the following chemical
structure, i.e., "ML-236B" itself can be prepared by cultivation of
Penicillium citrinum SANK 18767 and exhibits a pharmacological activity as
hypocholesteremic and hypolipemic medicaments.
##STR3##
As a result of further studies made by the present inventors, it has been
found that the ML-236B carboxylic acid derivative (I) can be easily
derived from ML-236B itself and show a unexpectedly higher biological
activity against hyperlipemia as compared with ML-236B and, therefore, the
present invention has been completed upon this finding.
It is a principal object of this invention to provide the new ML-236B
carboxylic acid derivatives (I) which possess a valuable activity as
antihyperlipemic agents.
Another object of this invention is to provide a composition for the
treatment of hyperlipemia which comprises the ML-236B carboxylic acid
derivative (I) as an active ingredient.
These and other objects of this invention will be apparent to those skilled
in the art from the following description.
According to one aspect of this invention, there is provided a new class of
the ML-236B carboxylic acid derivatives having the above formula (I). The
ML-236B carboxylic acid derivatives (I) of this invention include, more
specifically, (a) esters of ML-236B carboxylic acid of the formula (I)
wherein R represents an alkyl group, a benzyl group optionally substituted
with alkyl, alkoxy or halogen or a phenacyl group optionally substituted
with alkyl, alkoxy or halogen; (b) metal salts of ML-236B carboxylic acid
of the formula (I) wherein R represents a group of 1/n M in which M
represents a metal and n represents a valency of said metal; and (c) amino
acid salts of ML-236B carboxylic acid of the formula (I) wherein R
represents a group of AH.sup.+ in which A represents an amino acid.
In the esters of ML-236B carboxylic acid, R may be exemplified by an alkyl
group, preferably of 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, n-hexyl etc; a benzyl group optionally substituted
with an alkyl group, an alkoxy group or a halogen atom such as benzyl,
2-methylbenzyl, 3-methylbenzyl, 4-methylbenzyl, 2-ethylbenzyl,
3-ethylbenzyl, 4-ethylbenzyl, 2-methoxybenzyl, 3-methoxybenzyl,
4-methoxybenzyl, 2-ethoxybenzyl, 3-ethoxybenzyl, 4-ethoxybenzyl,
2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 2-bromobenzyl,
3-bromobenzyl, 4-bromobenzyl etc; or a phenacyl group optionally
substituted with an alkyl group, an alkoxy group or a halogen atom such as
phenacyl, 2-methylphenacyl, 3-methylphenacyl, 4-methylphenacyl,
2-ethylphenacyl, 3-ethylphenacyl, 4-ethylphenacyl, 2-methoxyphenacyl,
3-methoxyphenacyl, 4-methoxyphenacyl, 2-chlorophenacyl, 3-chlorophenacyl,
4-chlorophenacyl, 2-bromophenacyl, 3-bromophenacyl, 4-bromophenacyl etc.
The ML-236B carboxylic acid esters are new substances and can be easily
prepared according to any conventional methods, for example, by one of the
following processes.
(1) Reaction of ML-236B with an alcohol wherein an inorganic acid such as
hydrochloric acid, sulfuric acid etc, a boron fluoride or an acidic ion
exchange resin may be employed as a catalyst and the same alcohol or any
other solvents that do not participate in the reaction, e.g., chloroform,
benzene and ether can be employed as a solvent.
(2) Reaction of a metal salt of ML-236B carboxylic acid with an alkyl
halide (the metal salt of ML-236B carboxylic acid can be prepared by
saponification of ML-236B with a weak alkali) wherein dimethylformamide,
tetrahydrofuran, dimethylsulfoxide or acetone may be employed as a
solvent.
(3) Reaction of ML-236B carboxylic acid with an alcohol in the same manner
as the above Process (1). (ML-236B carboxylic acid can be prepared by
neutralization of a metal salt of ML-236B carboxylic acid).
The ML-236B carboxylic acid metal salts are also new substances and can be
prepared according to any conventional methods, for example, by
saponification of ML-236B with a weak alkali.
As the ML-236B metal salts, there may be mentioned alkali metal salts,
e.g., sodium or potassium salt, alkaline earth metal salts, e.g., calcium
or magnesium salt, aluminum salt, iron salt, zinc salt, copper salt,
nickel salt, cobalt salt and the like. Among others, alkali metal salts,
alkaline earth metal salts and aluminum salt are preferable and sodium
salt, calcium salt and aluminum salt are most preferable.
The ML-236B carboxylic acid.amino acid salts are novel compounds and can be
prepared, for example, by contacting ML-236B with an amino acid.
As the ML-236B carboxylic acid.amino acid salt, there may be preferably
mentioned those salts with a basic amino acid such as arginine, lysine,
histidine, .alpha.,.gamma.-diaminobutyric acid, ornithine and the like.
The ML-236B carboxylic acid derivatives (I) can inhibit biosynthesis of
cholesterol in liver similarly as ML-236B itself does. However, a much
more potent activity is available with the said derivatives as compared
with that of ML-236B. Such a potent inhibitory action of cholesterol
biosynthesis of ML-236B carboxylic acid derivatives is not expected upon
the action of ML-236B itself. Accordingly, ML-236B carboxylic acid
derivatives are very effective as an antihyperlipemia.
An inhibition activity of cholesterol biosynthesis, a lowering activity of
serum cholesterol, an effective dose and a toxicity of the ML-236B
carboxylic acid derivative (I) are illustrated hereinbelow.
(1) Inhibition activity of cholesterol biosynthesis
The ML-236B carboxylic acid derivatives have been found to specifically
inhibit 3-hydroxy-3-methylglutaryl-CoA reductase, which is known as a
rate-limiting enzyme during the biosynthesis of cholesterol. Inhibition
activity of cholesterol biosynthesis [determined according to the method
disclosed in J. Biol. Chem., 234, 2835 (1959)] and inhibition activity on
3-hydroxy-3-methylglutaryl-CoA reductase [determined according to the
method disclosed in Anal. Biochem., 31, 383 (1969)] of these compounds are
summarized in Table 1.
Table 1
______________________________________
Inhibition of cholesterol biosynthesis and
3-hydroxy-3-methylglutaryl-CoA reductase
Concentration
to inhibit
50% of Concentration to inhibit
Test cholesterol 50% of 3-hydroxy-3-
Compound biosynthesis
methylglutaryl-CoA
R (.mu.g/ml) reductase (.mu.g/ml)*
______________________________________
methyl 0.01 0.15
ethyl 0.01 0.16
isopropyl 0.04 0.16
n-butyl 0.02 0.16
n-hexyl 0.02 0.18
benzyl 0.01 0.2
4-methylbenzyl
0.01 0.2
4-methoxybenzyl
0.01 0.2
4-chlorobenzyl
0.01 0.2
phenacyl 0.01 0.2
4-methylphenacyl
0.01 0.2
4-methoxyphenacyl
0.01 0.2
4-bromophenacyl
0.01 0.2
Na 0.006 0.16
Ca 0.006 0.16
Al 0.006 0.16
Arginine 0.007 0.18
Lysine 0.006 0.16
ML-236B 0.01 4.8
______________________________________
*Employed as an enzyme microsome of rat liver.
Namely, ML-236B carboxylic acid esters have an approximately equivalent
inhibition effect of cholesterol biosynthesis as compared with ML-236B,
while ML-236B metal salts show about 2 times stronger inhibition activity
of cholesterol biosynthesis and 30 times stronger inhibition activity of
3-hydroxy-3-methylglutaryl-CoA reductase as compared with those of
ML-236B.
(2) Dose and administration
A lowering activity of blood cholesterol in beagle dogs (a body weight of
about 10 kg) was assayed on ML-236B carboxylic acid derivatives (I). The
test was to orally administer an encapsulated test sample twice per day,
in the morning (9 o'clock) and evening (16 o'clock), for continuous 5
days, collect a blood sample in the morning after 5 days and determine a
total cholesterol level in serum by a conventional method. The results are
shown in Table 2.
Table 2
______________________________________
Lowering activity of blood
cholesterol in beagle dogs
Test Lowering rate of
Compound Dose total serum cholesterol
R (mg/kg/day)
(%)
______________________________________
methyl 25 12
100 26
ethyl 25 13
100 19
isopropyl 25 19
100 30
n-butyl 25 32
100 46
n-hexyl 25 17
100 29
benzyl 25 24
100 38
4-methylbenzyl
25 30
100 41
4-methoxybenzyl
25 26
100 36
4-chlorobenzyl
25 20
100 36
phenacyl 25 32
100 42
4-methylphenacyl
25 20
100 33
4-methoxyphenacyl
25 26
100 39
4-bromophenacyl
25 21
100 30
Na 25 24
100 47
Ca 25 28
100 44
Al 25 26
100 41
Arginine 25 26
100 45
Lysine 25 24
100 44
ML-236B 25 <5
100 19
______________________________________
It is apparent that the ML-236B carboxylic acid derivatives (I) have a
several times stronger activity in lowering of blood cholesterol as
compared with ML-236B.
A lowering activity of blood cholesterol in rats was assayed on rats. The
test was to orally administer a test sample suspended in water, collect a
blood sample after 18 hours and determine a cholesterol level in serum by
a conventional method.
The results are shown in Table 3.
Table 3
______________________________________
Lowering activity of blood
cholesterol in rats
Test Lowering rate
Compound Dose of serum cholesterol
R (mg/kg) (%)
______________________________________
methyl 5 18
20 23
ethyl 5 19
20 25
isopropyl 5 16
20 25
n-butyl 5 20
20 26
n-hexyl 5 14
20 23
n-benzyl 5 14
20 24
4-methylbenzyl
5 13
20 26
4-methoxybenzyl
5 17
20 20
4-chlorobenzyl
5 15
20 21
phenacyl 5 14
20 21
4-methylphenacyl
5 14
20 19
4-methoxyphenacyl
5 14
20 23
4-bromophenacyl
5 17
20 24
Na 5 20.6
20 23.1
Ca 5 16.5
20 20.9
Al 5 18.3
20 22.1
Arginine 5 19.6
20 24.1
Lysine 5 16.9
20 23.8
ML-236B 5 11.2
20 17.1
______________________________________
It is apparent that the ML-236B carboxylic acid derivatives (I) have a very
satisfactory activity in lowering of blood cholesterol as compared with
ML-236B.
(3) Acute toxicity
Acute toxicity of ML-236B carboxylic acid derivatives (I) was determined in
oral and intraperitoneal administration (both as an aqueous suspension).
The results are shown in Table 4.
Table 4-(1)
______________________________________
Acute toxicity
LD.sub.50 (mg/kg)
Test Compound Mouse Rat
R i.p. P.O.
______________________________________
methyl >500 >2,000
ethyl >500 >2,000
isopropyl >500 >2,000
n-butyl >500 >2,000
n-hexyl >500 >2,000
n-benzyl >500 >2,000
4-methylbenzyl >500 >2,000
4-methoxybenzyl >500 >2,000
4-chlorobenzyl >500 >2,000
phenacyl >500 >2,000
4-methylphenacyl >500 >2,000
4-methoxyphenacyl >500 >2,000
4-bromophenacyl >500 >2,000
______________________________________
Table 4-(2)
______________________________________
Acute toxicity
Admini-
Compound Animal stration LD.sub.50 (mg/kg)
______________________________________
p.o. >2,000
Mouse
ML-236B Na salt i.p. > 500
p.o. >2,000
Rat
i.p. > 500
p.o. >2,000
Mouse
i.p. > 500
"Ca salt
p.o. >2,000
Rat
i.p. > 500
p.o. >2,000
Mouse
"Al salt i.p. > 500
p.o. >2,000
Rat
i.p. > 500
p.o. >5,000
Mouse
Arginine salt i.p. > 500
p.o. >5,000
Rat
i.p. > 500
p.o. >5,000
Mouse
i.p. > 500
Lysine salt p.o. >5,000
Rat
i.p. > 500
______________________________________
As apparent from the above-recited experiments, ML-236B carboxylic acid
derivatives can be administered orally, through intravenous injection or
the like manner and their effects are distinctly far more superior to
ML-236B. A dose for the treatment of an adult may vary depending upon
administration route and frequency, but it is usually within the range of
100-3000 mg per day, preferably 1500 mg per day.
The ML-236B carboxylic acid derivatives (I) may be formulated to various
preparations for administration by any conventional methods in analogy
with other known antihyperlipemic agents such as Clofibrate, Sinfibrate
and the like.
Then, in another aspect of this invention, there is provided a
pharmaceutical preparation which comprises as an active ingredient at
least one of the ML-236B carboxylic acid derivatives (I). This preparation
may naturally include any pharmaceutically acceptable carrier or
excipient.
The preparation is desirably provided in the form preferable for absorption
in gastrointestinal tracts. Tablets and capsules for oral administration
are of a unit dosage form and may comprise conventional vehicles such as a
binding agent such as syrup, gum arabic, gelatin, sorbit, tragacanth gum
or polyvinylpyrrolidone; an excipient such as lactose, sucrose, corn
starch, calcium phosphate, sorbitol or glycine; a glidant such as
magnesium stearate, talc, polyethylene glycol or silica; a disintegrating
agent such as potato starch; or a wetting agent such as sodium lauryl
sulfate. Tablets may be coated by any methods well-known in the art.
Liquid preparations for oral administration may be an aqueous or oily
suspension, a solution, a syrup, an elixir and the like or they may be of
any dried forms which may be re-dissolved in water or other suitable
vehicles when applied. Such liquid preparations may comprise conventional
additives such as a suspending agent such as sorbit syrup, methyl
cellulose, glucose/sugar syrup, gelatin, hydroxyethyl cellulose,
carboxymethyl cellulose, aluminum stearate gel or a hydrogenated edible
fat; an emulsifying agent such as lecitin, sorbitan monooleate or gum
arabic; a non-aqueous vehicle such as almond oil, fractionated coconut
oil, oily ester, propylene glycol or ethanol; a preservative such as
methyl p-hydroxybenzoate, propyl p-hydroxybenzoate or sorbic acid.
Injectable preparations are provided in a unit dosage ampoule or multiple
dosage vessel with added preservatives. The preparations may be in the
form of a suspension, a solution or an emulsion in oily or aqueous
vehicles and also comprise formulating agents such as a suspending agent,
a stabilizer and/or a dispersing agent. Alternatively, the active
ingredient may be in the form of a powder which may be re-dissolved in a
suitable vehicle, e.g., pyrogen-free, sterilized water when applied.
These preparations may contain not less than 0.1%, preferably 10-60% of the
active ingredient, depending upon administration route. A unit dosage form
of the preparation may preferably contain 50-500 mg of the active
ingredient. The ML-236B carboxylic acid amino acid or metal salts when in
an aqueous solution have a property of being lactonized to ML-236B in an
acidic pH range. Therefore, an aqueous solution of the salts is preferably
kept in a neutral to weakly alkaline pH range.
This invention will be more fully explained by way of the non-limiting
Examples and Preparation Examples as shown hereinbelow.
EXAMPLE 1
Ethyl ML-236B carboxylate
10 g of ML-236B were dissolved with heating in 150 ml of ethanol and to the
resulting solution were added 20 g of an acidic ion exchange resin (dry
Dowex 50 W - H.sup.+ form) and then stirring was effected at
60.degree.-70.degree. C. for 3 hours. After completion of the reaction,
the resin was filtered off, the solvent was distilled off under reduced
pressure and the residue was isolated and purified by silica gel (100 g)
chromatography to give 6.1 g of the desired product and 3.1 g of the
starting ML-236B.
Analysis for C.sub.25 H.sub.40 O.sub.6 -- Calcd.: C; 68.77%, H; 9.24%.
Found: C; 68.82%, H; 9.31%.
NMR spectrum: .delta. ppm (CDCl.sub.3), 4.20 (2H, quartet), 1.25 (3H,
triplet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1730.
EXAMPLE 2
Propyl ML-236B carboxylate
The same procedure as in Example 1 was repeated except that 2.5 g of
ML-236B, 50 ml of n-propanol and 5 g of the acidic ion exchange resin were
employed, thereby yielding 1.5 g of the desired product.
Analysis for C.sub.26 H.sub.42 O.sub.6 -- Calcd.: C; 69.30%, H; 9.40%.
Found: C; 69.52%, H; 9.45%.
NMR spectrum: .delta. ppm (CDCl.sub.3), 3.6-4.4 (4H, multiplet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1732.
EXAMPLE 3
Isopropyl ML-236B carboxylate
The same procedure as in Example 1 was repeated except that 10 g of
ML-236B, 200 ml of isopropanol and 20 g of the acidic ion exchange resin
were employed, thereby yielding 6.5 g of the desired product and 3.6 g of
the starting ML-236B.
Analysis for C.sub.26 H.sub.42 O.sub.6 -- Calcd.: C; 69.30%, H; 9.40%.
Found: C; 69.83%, H; 9.30%.
NMR spectrum: .delta. ppm (CDCl.sub.3), 5.10 (1H, multiplet), 1.25 (6H,
doublet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1725.
EXAMPLE 4
Hexyl ML-236B carboxylate
To a solution of 10 g of ML-236B carboxylic acid in 50 ml of n-hexylalcohol
and 20 ml of benzene was added 0.5 ml of conc. sulfuric acid and the
resulting mixture was stirred at room temperature for 16 hours. Then, the
reaction mixture was neutralized with an aqueous solution of sodium
hydrogencarbonate, washed with water, dried over anhydrous sodium sulfate
and then the solvent was distilled off at a temperature of not more than
60.degree. C. under reduced pressure. The residue was isolated and
purified by a silica gel (100 g) chromatography to give 7.4 g of the
desired product and 2.3 g of the starting ML-236B.
Analysis for C.sub.29 H.sub.48 O.sub.6 -- Calcd.: C; 70.69, H; 9.82. Found:
C; 70.54, H; 10.12.
NMR spectrum: .delta. ppm (CDCl.sub.3), 3.5-4.5 (4H, multiplet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1735.
EXAMPLE 5
Benzyl ML-236B carboxylate
To a solution of 2.1 g of sodium ML-236B carboxylate in 10 ml of
dimethylformamide were 10 mg of sodium iodide and 2 ml of benzyl chloride
and the resulting mixture was left for 16 hours. Then, water was added to
the reaction mixture and extraction was effected with ethyl acetate. The
extract was washed with water and dried over anhydrous sodium sulfate and
then the solvent was distilled off. The redidue was isolated and purified
by silica gel (60 g) chromatography to give 2.2 g of the desired product.
Analysis for C.sub.30 H.sub.42 O.sub.6 -- Calcd.: C; 72.26, H; 8.49. Found:
C; 72.45, H; 8.46.
NMR spectrum: .delta. ppm (CDCl.sub.3), 5.18 (2H, singlet), 7.40 (5H,
singlet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1730.
EXAMPLE 6
4-Methylbenzyl ML-236B carboxylate
The same procedure as in Example 5 was repeated except that 2.0 g of sodium
ML-236B carboxylate, 2 ml of 4-methylbenzyl chloride and 10 ml of
dimethylformamide were employed, thereby yielding 2.3 g of the desired
product.
Analysis for C.sub.31 H.sub.44 O.sub.6 -- Calcd.: C; 72.62, H; 8.65. Found:
C; 72.50, H; 8.32.
NMR spectrum: .delta. ppm (CDCl.sub.3), 2.30 (3H, singlet), 5.10 (2H,
singlet), 7.40 (5H, singlet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1730.
EXAMPLE 7
4-Methoxybenzyl ML-236B carboxylate
The same procedure as in Example 5 was repeated except that 2.0 g of sodium
ML-236B carboxylate, 2.5 g of 4-methoxybenzyl chloride and 10 ml of
dimethylformamide were employed, thereby yielding 2.2 g of the desired
product.
Analysis for C.sub.31 H.sub.44 O.sub.7 -- Calcd.: C; 70.43, H; 8.39. Found:
C; 70.81, H; 8.52.
NMR spectrum: .delta. ppm (CDCl.sub.3), 3.75 (3H, singlet), 5.12 (2H,
singlet), 6.80, 7.05 (4H, quartet),
IR spectrum: .nu. cm.sup.-1 (liquid film) 1735.
EXAMPLE 8
4-Chlorobenzyl ML-236B carboxylate
The same procedure as in Example 5 was repeated exept that 1.0 g of sodium
ML-236B carboxylate, 1.5 g of 4-chlorobenzyl chloride and 7 ml of
dimethylformamide were employed, thereby yielding 1.3 g of the desired
product.
Analysis for C.sub.30 H.sub.41 O.sub.6 Cl -- Calcd.: C; 67.58, H; 7.75.
Found: C; 67.71, H; 7.77.
NMR spectrum: .delta. ppm (CDCl.sub.3), 5.20 (2H, singlet), 7.38 (4H,
singlet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1730.
EXAMPLE 9
Phenacyl ML-236B carboxylate
A solution of 10.5 g of sodium ML-236B carboxylate and 6.3 g of phenacyl
bromide in 50 ml of dimethylformamide was left at room temperature for 3
hours. Then, water was added to the reaction mixture and the resulting
mixture was extracted with ethyl acetate. The extract was washed with
water, dried over anhydrous sodium sulfate and then the solvent was
distilled off. The residue was isolated and purified by silica gel (150 g)
chromatography to give 11.3 g of the desired product as a colorless oily
substance.
Analysis for C.sub.31 H.sub.42 O.sub.7 -- Calcd.: C; 70.69, H; 8.04. Found:
C; 70.45, H; 8.21.
NMR spectrum: .delta. ppm (CDCl.sub.3), 5.44 (2H, singlet), 7.4-8.2 (5H,
multiplet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1745, 1725, 1710.
EXAMPLE 10
4-Bromophenacyl ML-236B carboxylate
The same procedure as in Example 9 was repeated except that 1.0 g of sodium
ML-236B carboxylate, 1 g of p-bromophenacyl bromide and 10 ml of
dimethylformamide were employed to give 1.4 g of the desired product.
Analysis for C.sub.31 H.sub.41 O.sub.7 Br -- Calcd.: C; 61.48, H; 6.82.
Found: C; 61.20, H; 7.01.
NMR spectrum: .delta. ppm (CDCl.sub.3), 5.40 (2H, singlet), 7.45-8.0 (4H,
multiplet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1730, 1710.
EXAMPLE 11
4-Methylphenacyl ML-236B carboxylate
The same procedure as in Example 9 was repeated except that 1.0 g of sodium
ML-236B carboxylate, 1.0 g of 4-methylphenacyl bromide and 10 ml of
dimethylformamide were employed to give 1.1 g of the desired product.
Analysis for C.sub.32 H.sub.44 O.sub.7 -- Calcd.: C; 71.08, H; 8.20. Found:
C; 71.38, H; 8.46.
NMR spectrum: .delta. ppm (CDCl.sub.3), 2.46 (3H, singlet), 5.42 (2H,
singlet), 7.5-8.2 (4H, multiplet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1745, 1730, 1710.
EXAMPLE 12
4-Methoxyphenacyl ML-236B carboxylate
The same procedure as in Example 9 was repeated except that 1.0 g of sodium
ML-236B carboxylate, 1.2 g of 4-methoxyphenacyl bromide and 10 ml of
dimethylformamide were employed to give 0.9 g of the desired pruduct.
Analysis of C.sub.32 H.sub.44 O.sub.8 -- Calcd. C; 69.04, H; 7.97. Found:
C; 69.20, H; 8.19.
NMR spectrum: .delta. ppm (CDCl.sub.3), 3.81 (3H, singlet), 5.45 (2H,
singlet), 6.6-8.0 (4H, multiplet).
IR spectrum: .nu. cm.sup.-2 (liquid film) 1740, 1725, 1710.
EXAMPLE 13
Methyl ML-236B carboxylate
A mixture of 9.75 g of ML-236B and 225 ml of a 0.1 N aqueous NaOH solution
was heated on a water bath at 80.degree.-90.degree. C. for 1 hour while
occasionally shaken. After 1 hour, undissolved ML-236B was filtered off,
the filtrate was made acidic with HCl under ice-cooling and extracted with
ethyl acetate. The extract was well washed with a saturated aqueous NaCl
solution and dried over Na.sub.2 SO.sub.4. The dried ethyl acetate extract
was treated with an etheral solution of diazomethane. The solvent was
distilled off and the residue was purified by silica gel chromatography to
give 6.1 g of the desired product as colorless oily substance. Yield: 58%.
[.alpha.].sub.D.sup.25 = + 202.55 (c = 1% ethanol).
NMR spectrum: .delta. ppm (CDCl.sub.3), 3.7 (3H, singlet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1725, 3450.
Mass spectrum (molecular weight 422.54): M.sup.+ 422.
EXAMPLE 14
Ethyl ML-236B carboxylate
A mixture of 30.9 g of ML-236B and 775 ml of a 0.1 N aqueous NaOH solution
was heated on a water bath at 80.degree.-90.degree. C. for 1 hour, while
occasionally shaken. The ML-236B gradually dissolved. After 1 hour,
undissolved ML-236B was filtered off, the filtrate was freeze-dried to
give 27.0 g of sodium ML-236B carboxylate as white powders.
5.0 g (0.01 mol) of the substance thus obtained were added to 30 ml of
absolute ethanol to form a suspension. Dry hydrogen chloride gas was
bubbled into the suspension under ice-cooling, whereupon NaCl was
separated out. The solvent was dissolved off at a low temperature and the
residue was dissolved in benzene. The resulting solution was washed with
an aqueous sodium hydrogencarbonate solution and then with a saturated
aqueous sodium chloride solution and dried over Na.sub.2 SO.sub.4. The
solvent was distilled off and the residue was purified by silica gel
column chromatography to give 2.0 g of the desired product as colorless
oily substance. Yield: 45.87%
[.alpha.].sub.D.sup.25 = + 188.25 (c = 1%, ethanol).
NMR spectrum: .delta. ppm (CDCl.sub.3), 4.14 (2H, quartet), 1.25 (3H,
triplet).
IR spectrum: .nu. cm.sup.-1 (liquid film): 1725, 3450.
Mass spectrum (molecular weight 436.57): M.sup.+ 436.
EXAMPLE 15
Ethyl ML-236B carboxylate
To a mixture of 1.0 g (0.0025 mol) of ML-236B and 15 ml of absolute ethanol
were added several drops of acetyl chloride and stirring was continued at
room temperature overnight. The solvent was distilled off and the residue
was purified by silica gel column chromatography to give 0.3 g of the
desired product as colorless oily substance. Yield: 26.85%
[.alpha.].sub.D.sup.25 = + 188.25 (c = 1%, ethanol).
NMR spectrum: .delta. ppm (CDCl.sub.3), 4.14 (2H, quartet), 1.25 (3H,
triplet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1725, 3450.
Mass spectrum (molecular weight 436.57): M.sup.+ 436.
EXAMPLE 16
Isopropyl ML-236B carboxylate
To 20 ml of absolute isopropanol were added 3.0 g (0.0069 mol) of sodium
ML-236B carboxylate to form a suspension. Dry hydrogen chloride gas was
bubbled into the suspension under ice-cooling and stirring. NaCl was
separated. The solvent was distilled off at a low temperature and the
residue was dissolved in benzene. The solution was washed with an aqueous
sodium hydrogen-carbonate solution and dried over Na.sub.2 SO.sub.4. The
solvent was distilled off and the residue was purified by silica gel
column chromatography to give 1.6 g of the desired product as colorless
oily substance. Yield: 51.02%
[.alpha.].sub.D.sup.25 = + 178.24 (c = 1%, ethanol).
NMR spectrum: .delta. ppm (CDCl.sub.3), 5.08 (1H, multiplet), 1.25 (6H,
doublet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1725, 3450.
Mass spectrum (molecular weight 450.60): M.sup.+ 450.
EXAMPLE 17
Isopropyl ML-236B carboxylate
To 30 ml of absolute isopropanol were added 3.9g (0.01 mol) of ML-236B and
several drops of acetyl chloride and the resulting mixture was heated to
50.degree. C. for several minutes. Then, the solvent was distilled off and
the residue was purified by silica gel column chromatography to give 2.6 g
of the desired product as colorless oily substance. Yield: 57.78%
[.alpha.].sub.D.sup.25 = + 178.24 (c = 1%, ethanol).
NMR spectrum: .delta. ppm (CDCl.sub.3), 5.08 (1H, multiplet), 1.25 (6H,
doublet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1725, 3450.
Mass spectrum (molecular weight 450.60): M.sup.+ 450.
EXAMPLE 18
n-Butyl ML-236B carboxylate
To 50 ml of a 1% aqueous sodium hydroxide solution were added 10.0 g (0.026
mol) of ML-236B and a complete solution was formed with heating and
shaking. Then, the solution was adjusted to pH 4 with 1 N hydrochloric
acid under ice-cooling. Immediately after the adjustment, the solution was
extract twice with ethyl acetate and the combined extracts were dried over
sodium sulfate. The ethyl acetate was distilled off and the residue
dissolved in n-butanol. To the solution was bubbled dry hydrogen chloride
gas under ice-cooling and stirring for 10 minutes. The reaction mixture
was washed with an aqueous sodium hydrogencarbonate solution and a
saturated aqueous sodium chloride solution. The n-butanol was distilled
off under reduced pressure and the residue was dissolved in benzene and
then subjected to silica gel column chromatography to give 4.9 g of the
desired product as colorless oily substance. Yield: 41.21%
Analysis for C.sub.27 H.sub.44 O.sub.6 -- Calcd.: C; 69.79, H; 9.55. Found:
C; 69.40, H; 9.45.
[.alpha.].sub.D.sup.25 = + 207.44 (c = 1%, ethanol).
NMR spectrum: .delta. ppm (CDCl.sub.3), 4.13 (5H, triplet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1725, 3450.
Mass spectrum (molecular weight 464.62): M.sup.+ 464.
EXAMPLE 19
n-Butyl ML-236B carboxylate
To 100 ml of n-butanol were added 13 g (0.033 mol) of ML-236B and a little
amount of acetyl chloride and the resulting mixture was stirred at room
temperature. Formation of the corresponding n-butyl ester was investigated
occasionally by a thin layer chromatography and, whenever a poor formation
was found, a further little amount of acetyl chloride was added and
stirring was continued. When the starting material was scarcely observed,
the reaction was ceased. A total amount of acetyl chloride was 1 g. The
reaction mixture was washed with an aqueous sodium hydrogen-carbonate
solution and a saturated aqueous sodium chloride solution and the
n-butanol was distilled off under reduced pressure and the residue was
subjected to silica gel column chromatography by the use of benzene as a
developing agent to give 10.9 g of the desired product. Yield: 70.4%
[.alpha.].sub.D.sup.25 = + 207.44 (c = 1%, ethanol).
NMR spectrum: .delta. ppm (CDCl.sub.3), 4.13 (3H, triplet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1725, 3450.
Mass spectrum (molecular weight 464.62): M.sup.+ 464.
EXAMPLE 20
Benzyl ML-236B carboxylate
A mixture of 39.0 g (0.1 mol) of ML-236B and 1 l. of a 0.1 N aqueous NaOH
solution was heated on a water bath at 80.degree.-90.degree. C. for 1 hour
with occasional shaking. The ML-236B gradually dissolved. After 1 hour,
undissolved ML-236B was filtered off and the filtrate was made acidic with
HCl under ice-cooling and extracted with ethyl acetate. The extract was
well washed with a saturated aqueous sodium chloride solution and dried
over sodium sulfate. This ethyl acetate extract was mixed with a
separately prepared etheral solution of phenyl diazomethane. The solvent
was distilled off and the residue was purified by silica gel column
chromatography to give 8.6 g of the desired product as colorless oily
substance. Yield: 17.27%
NMR spectrum: .delta. ppm (CDCl.sub.3), 5.08 (2H, singlet), 7.34 (5H,
singlet).
IR spectrum: .nu. cm.sup.-1 (liquid film) 1725, 3450.
Mass spectrum (molecular weight 498.64): M.sup.+ 498.
EXAMPLE 21
Sodium ML-236B carboxylate
A suspension of 39 g of ML-236B in an aqueous solution of sodium hydroxide
(0.2 N, 500 ml) was stirred at 50.degree. C. for 2 hours to form a
substantially clear aqueous solution. A minor amount of the residual
insolubles was filtered off and the filtrate freeze-dried to afford 40.2 g
of ML-236B sodium salt.
UV spectrum (aqueous solution):
______________________________________
.lambda. max (m.mu.)
D.sub.1cm.sup.1%
______________________________________
229.0 509
236.2 580
245.0 386
______________________________________
EXAMPLE 22
Calcium ML-236B carboxylate
To 1 l. of an aqueous solution containing 4.7 g of calcium hydroxide were
added 50 g of ML-236B and the mixture was stirred at 50.degree. C. for 2
hours. A whole solution containing the precipitates formed in situ was
freeze-dried. The dried specimen (about 55 g) was suspended in 500 ml of
ethyl acetate. The suspension was stirred at room temperature for 1 hour
and precipitates were collected by filtration. The precipitates were
further washed with 1 l. of ethyl acetate and dried under vacuum to afford
49.5 g of ML-236B calcium salt.
UV spectrum (aqueous solution):
______________________________________
.lambda. max (m.mu.)
E.sub.1cm.sup.1%
______________________________________
229.0 516
236.2 596
245.0 393
______________________________________
EXAMPLE 23
Aluminum ML-236B carboxylate
A solution of 10 g of the ML-236B sodium salt produced in Production
Example 1 in 100 ml of water was adjusted to pH 2 with hydrochloric acid
and 100 ml of benzene was added thereto. After shaking, the benzene layer
was recovered and concentrated to dryness. 8 g of the residue and 1.3 g of
aluminum isopropoxide were dissolved in 200 ml of anhydrous benzene and
the resulting solution was boiled under reflux for 2.5 hours. Then, the
solvent was distilled off to leave 9.0 g of ML-236B aluminum salt.
UV spectrum (aqueous solution):
______________________________________
.lambda. max (m.mu.)
E.sub.1cm.sup.1%
______________________________________
229.0 506
236.2 580
245.0 389
______________________________________
EXAMPLE 24
Lysine salt of ML-236B carboxylic acid
To a solution of 4.10 g of ML-236B carboxylic acid in 50 ml of ethanol was
added an aqueous solution (5 ml) of 1.46 g of lysine. The resulting
mixture was stirred at 60.degree. C. for 30 minutes. Then, the solvent was
distilled off under reduced pressure. To the residue were added 100 ml of
ethyl acetate and stirring was effected at 70.degree. C. Crystalline
substance thus separated was recovered by filtration. Yield 4.7 g, m.p.
182.degree. C.
Analysis for C.sub.29 H.sub.48 N.sub.2 O.sub.7 -- Calcd.: C, 71.58; H,
6.99; N, 2.89%. Found: C, 71.53; H, 7.08; N, 2.82%.
IR spectrum cm.sup.-1 (Nujol): 3150, 2100, 1730, 1610, 1585, 1510, 1410,
1180, 1080, 545.
EXAMPLE 25
Arginine salt of ML-236B carboxylic acid
To a solution of 4.05 g of ML-236B carboxylic acid in 40 ml of
tetrahydrofuran was added an aqueous solution (10 ml) of 1.75 g of
arginine. Then, the same treatment as in Example 24 was conducted to yield
4.71 g of the desired product. m.p. 147.degree. C.
Analysis for C.sub.29 H.sub.48 N.sub.4 O.sub.7 -- Calcd.: C, 59.77; H,
8.64; N, 9.62%. Found: C, 59.63; H, 8.72; N, 9.56%.
IR spectrum cm.sup.-1 (Nujol): 3350, 3150, 1730, 1640, 1560, 1400, 1260,
1180, 1080, 830, 530.
Some preparation examples of this invention will be given below for
illustration purpose only.
PREPARATION EXAMPLE 1
Capsule for Oral Administration
______________________________________
Methyl ML-236B carboxylate
250 mg
Lactose 75 mg
Magnesium stearate 15 mg
Total 340 mg
______________________________________
According to the above-defined formulation, ingredient powders were admixed
and passed through a sieve of 60 mesh. Then, the sieved powder (340 mg)
was packed into a No. 1 gelatin capsule to produce a capsule.
PREPARATION EXAMPLE 2
Injectable Preparation
ML-236B sodium salt was asceptically placed into a vial so that 250 mg of
the sterilized salt was contained therein, asceptically freeze-dried and
then sealed. It was admixed with 2 ml of physiological saline when
applied, thereby forming an injectable preparation.
PREPARATION EXAMPLE 3
Capsule for Oral Administration
______________________________________
ML-236B calcium salt 250 mg
Lactose 75 mg
Magnesium stearate 15 mg
Total 340 mg
______________________________________
According to the above-defined formulation, ingredient powders were admixed
and passed through a sieve of 60 mesh. Then, the sieved powder (340 mg)
was packed into a No. 1 gelatin capsule to produce a capsule.
PREPARATION EXAMPLE 4
Injectable Preparation
ML-236B carboxylic acid.arginine salt was asceptically placed into a vial
so that 100 mg of the sterilized salt was contained therein, asceptically
freeze-dried and then sealed. It was admixed with 2 ml of physiological
saline when applied, thereby forming an injectable preparation.
PREPARATION EXAMPLE 5
Capsule for Oral Administration
______________________________________
Lysine salt of ML-236B
carboxylic acid 100 mg
Lactose 75 mg
Magnesium stearate 15 mg
Total 190 mg
______________________________________
According to the above-defined formulation, ingredient powders were admixed
and passed through a sieve of 60 mesh. Then, the sieved powder (190 mg)
was packed into a gelatin capsule to produce a capsule.
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