JP3408835B2 - Method for producing E-type prostaglandins - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] The present invention relates to a carboxylic acid having a triple bond.
Of the lower alkyl alkyl of E-type prostaglandins conjugated with
Enzyme acts on stele and hydrolyzes, and carboxylic acid becomes 3
Producing E-type prostaglandins with conjugated heavy bonds
About the method. [0002] 2. Description of the Related Art A method for producing E-type prostaglandins and
In general, lower alkyl esters of prostanoic acid
Is used to obtain prostanoic acid from
Rostaglandins have β-hydroxyketo in their skeleton.
Is chemically unstable and causes dehydration reaction.
It is easy to rub, and it is not a little
With by-products. Therefore, using enzymes as the most powerful method
And a hydrolysis method is known [Japanese Patent Laid-Open No.
No. 1392 (lipase derived from porcine pancreas); A.
Porter et al. (Journal of American Chemi)
Cal Society, Vol. 101, Nos. 4319-432
Page 2, 1979) (Lipase from pig pancreas),
CH. Lin et al. (Journal of American Kei
Mical Society, Vol. 104, Nos. 1621-16
Page 28, 1982) (Lipper from porcine pancreas)
Ze), Atsuri Hari et al. (Journal of the Chemical Society of Japan, Vol. 9, No. 1390)
-1392, 1983) (S from pig liver)
Terase)]. [0003] SUMMARY OF THE INVENTION
In the method of hydrolysis using an enzyme, as a raw material,
E-type prostaglandins in which a triple bond is conjugated to boric acid
In the case of using an ester of the above, it is insufficient in terms of suppressing the formation of a dehydrated product.
Minutes. In addition, positions 13 and 14 are triple bond E-type
Staglandins (E-type prostag where B is an ethynylene group)
Isomers), by-products such as 8-β isomers
There was also a disadvantage of doing so. [0004] Means for Solving the Problems The present inventors have solved the above problems.
As a result of intensive research aimed at solving
This ester is added to the lower alkyl ester of taglandins.
With a specific enzyme that hydrolyzes
With good yield and extremely low generation of by-products
To produce the desired E-type prostaglandins.
We have found and completed the present invention. That is, the present invention provides [0006] [Wherein, R represents a lower alkyl group;¹
And R^TwoAre the same or different and represent a hydrogen atom or a hydroxyl group
A, R represents a protecting group^ThreeAnd R^FourIs not involved in the reaction
B represents a vinylene group or an ethynylene group.
You. ] The lower alkyl of the E-type prostaglandins represented by
Kill ester, Aspergillus or Pseudomona
Has the ability to hydrolyze esters produced by microorganisms belonging to the genus
Act on the enzyme [0008] (Where R¹, R^Two, A, R^Three, R^FourAnd B
Is as defined above. E type prostagland represented by)
This is a method for producing carrots. In the present invention, a lower alkyl group is a
Tyl group, ethyl group, n-propyl group, isopropyl group,
Linear or branched chain such as n-butyl group and isobutyl group
Refers to an alkyl group. A hydroxyl-protecting group is
It is usually used in the field of rostaglandins,
For example, t-butyldimethylsilyl group, triethylsilyl
Group, phenyldimethylsilyl group, t-butyldiphenyl
Silyl group, tetrahydropyranyl group, tetrahydrofuran
Nil group, methoxymethyl group, ethoxyethyl group, benzyl
And the like. A, R^ThreeAnd R^FourIs not involved in this reaction
If so, it doesn't matter. To give these examples
If A, for example, a straight chain having 1 to 6 carbon atoms
A chain alkylene group (eg, trimethylene group, tetrame
Tylene group, hexamethylene group, octamethylene group
), A linear alkenylene group having 4 to 7 carbon atoms
[For example, the expression [0012] (Wherein n₁Represents an integer of 1 to 4, and n_TwoIs
2 or 3 is indicated. ), One in the chain
From 2 to 2 carbon atoms via an oxygen or sulfur atom
6 linear alkylene groups [e.g. [0014] Wherein X represents an oxygen atom or a sulfur atom
Then n_ThreeRepresents an integer of 1 to 5, and n_FourRepresents an integer of 1 to 3
You. )), The formula [0016] (Wherein n_FiveAnd n₆Are the same or different
Represents an integer of 1 to 3. ) Group represented by the formula [0018] (Wherein n₇And n₈Are the same or different
Represents an integer of 1 to 3. )
Can be. In addition, R^ThreeAre, for example, a hydrogen atom,
Examples include tyl, ethyl, and vinyl groups.
You. R^FourIs, for example, a linear chain having 1 to 10 carbon atoms.
Or branched alkyl, alkenyl,
Rukinyl group (for example, pentyl group, hexyl group, 1-methyl
Lehexyl group, 2-methylhexyl group, 1,1-dimethyl
Ruhexyl group, 2-pentenyl group, 5-hexenyl group,
1-methyl-3-hexynyl group, 2-methyl-3-hexyl
A silyl group, a 2-pentynyl group, etc.), having 3 to 1 carbon atoms.
0 cycloalkyl groups (for example, cyclopentyl group,
Clohexyl, cycloheptyl, etc.), phenyl
Group, "phenyl group, phenoxy group or 5 to 5 carbon atoms
1 to 6 carbon atoms substituted with "six cycloalkyl groups"
4 alkyl groups (for example, cyclopentylmethyl group,
Clohexylmethyl group, 2-cyclopentylethyl group,
3-cyclopentylpropyl group, 3-cyclohexylp
Ropyl group, 4-cyclopentylbutyl group, benzyl group,
Phenoxymethyl group, 2-phenylethyl group, 4-phenyl
A butyl group). The enzyme used in the present invention is
Microorganisms belonging to the genus Lugilus or Pseudomonas grow
This is an enzyme that has the ability to hydrolyze esters. Product name
As a specific example, lipase PS [Pseudomonas sp.
Lipase from Pseudomonas cepacia],
Lipase MF [Pseudomonas sp.
Lipase], acylase 30,000 [Asper
Acylase from the genus Aspergillus sp.
Above, Amano Pharmaceutical Co., Ltd.). Next, the hydrolysis method of the present invention will be described in detail.
You. In the present invention, Aspergillus or Pseudomona
Has the ability to hydrolyze esters produced by microorganisms belonging to the genus
The enzyme and the compound of formula (I) are stirred or stirred in an aqueous solution.
This is done by shaking. At this time, phosphate buffer,
The use of a buffer such as squirrel-HCl buffer
Is preferable in keeping the pH of the solution constant, but without using a buffer solution.
The reaction can also be carried out. When using phosphate buffer
In the concentration of 100 mM or more, 8-β by-product
Increasing concentrations of 50 mM or less are desirable. Enzyme reaction base
Has low solubility in aqueous solution,
It is desirable to add an organic solvent such as acetone as
No. In the case of acetone, from 1% to 20% based on the reaction solution
It can be added, but preferably 3% to 8%.
You. The pH of the reaction solution should be set according to the enzyme used.
I just need. For example, various conditions when using Lipase PS
The condition is that the pH is between 5.0 and 8.5, most preferably
The pH is between 5.0 and 6.5. In addition, the reaction solution
Alternatively, an organic solvent can be used without using an aqueous solution. An example
For example, diethyl ether, diisopropyl ether,
Benzene, toluene, xylene, acetone, acetonitrile
Hexane, heptane and the like. Anti
The reaction can be performed at a reaction temperature of 5 ° C to 50 ° C.
35 ° C is desirable. The amount of enzyme used depends on the enzyme titer.
What is necessary is just to determine suitably according to and the amount of a substrate. Usually base
0.1 times to 20 times the mass, but 10 wt.
Use more than twice the amount of enzyme to complete the reaction in a short time
Is desirable in terms of suppressing by-products. Reaction time
Of the reaction by TLC analysis or HPLC analysis
It is sufficient to set while checking the progress. After the end of the reaction
If the reaction solution is acidic or neutral,
If the reaction mixture is
It can be extracted with any organic solvent. Also, the reaction solution is alkaline
In the case of, acidify with the addition of diluted hydrochloric acid, diluted phosphoric acid, etc.
Extract with an organic solvent such as ethyl or diethyl ether.
After distilling off the solvent, column chromatography if necessary
And the like, and a pure target product can be isolated. [0023] According to the present invention, a by-product of a dehydrated body and an 8-β form is obtained.
Of prostaglandins of high purity (Carbo
E-type prostaglandins in which a triple bond is conjugated to a carboxylic acid)
Can be produced in a short time and with good yield. [0024] EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples.
Although described, the present invention is limited only by the examples.
Not. The progress of the hydrolysis is shown below.
It was confirmed by TLC method and HPLC method. TLC-1: TLC plate (Merck, Art 57
15) Developing solvent: ethyl acetate or ethyl acetate / ethanol
= 1/1 HPLC: column; ODS 80TM (4.6φ ×
150mm) (Manufactured by Tosoh Corporation) Eluent; methanol / water = 7/3 Flow rate: 1 ml / min Temperature; 50 ° C Detection: UV 210 nm The amount of dehydrated and 8-β-forms produced was H
It was measured by the PLC method. HPLC: column; ODS 80TM (4.6φ ×
150mm) (Manufactured by Tosoh Corporation) Eluent: 6.6 mM phosphate buffer (pH 6.3) / medium
Tanol = 1/1 Flow rate: 1 ml / min Temperature; 50 ° C Detection: UV 210 nm Examples 1-4 Enzymes shown in Table 1 [Enzymes of Example 4 (porcine liver
Z (manufactured by Sigma) is used for comparison. ]
50 mg was added to 2.375 ml of phosphate buffer (10 mM,
pH 7.0) and 125 μl of acetone
(17R) -17,20-dimethyl-2,2,3
3,13,14-Hexadehydroprostaglandin E
₁  Add 5 mg of methyl ester, shake at 30 ° C.
Was. No spot corresponding to the raw material was found by TLC.
The reaction was continued until no more. Table 1 shows the time until the end of the reaction.
Showed a pause. After completion of the reaction, 15 ml of diethyl ether
, And the organic layer is washed with saturated saline and dried over anhydrous magnesium sulfate.
Dried and concentrated with nesium. Extract by HPLC method
The product was analyzed and (17R) -17,20-dimethyl-
2,2,3,3,13,14-hexadehydroprosta
Grangen E₁, 8-β form and dehydrated form in area%
(Table 1). [0026] [Table 1] Embodiment 5 3 g of lipase PS was added to 70 ml of a phosphate buffer (20 m
M, pH 7.0) and dissolved in 3.75 ml of acetone.
Dissolved (17R) -17,20-dimethyl-2,2,
3,3,13,14-hexadehydroprostaglandin
E₁  Add 150 mg of methyl ester, and at 30 ° C
Stir for 6 hours. The reaction solution is 100 ml of diethyl ether.
5 times, and the organic layer is washed twice with 100 ml of a saturated saline solution.
After washing, the extract was dried over anhydrous sodium sulfate and concentrated. Crude
The product is subjected to reverse phase silica gel column chromatography (Li
Chromprep RP18 (Merck), unfolded
Medium: methanol: water = 65: 35), 103 m
g of (17R) -17,20-dimethyl-2,2,3
3,13,14-Hexadehydroprostaglandin E
₁I got 8-β form as determined by HPLC analysis
Was not observed at all, and the content of dehydrated body was only 1.9%.
(Area%).¹ H-NMR (CDCl_Three, 300 Mz) δ (pp
m): 0.82 to 0.96 (m, 3H), 0.93
(D, J = 6.6 Hz, 3H), 1.10 to 1.88
(M, 15H), 2.22 to 2.32 (m, 1H),
2.26 (dd, J = 9.0 Hz, 18.6 Hz, 1
H), 2.37 to 2.44 (m, 2H), 2.68 (d
dd, J = 1.7 Hz, 8.2 Hz, 11.3 Hz, 1
H), 2.78 (ddd, J = 1.2 Hz, 7.4H
z, 18.6 Hz, 1H), 4.29-4.39 (m,
1H), 4.45-4.52 (m, 1H) IR (neat): 3391, 2930, 2861, 2
237, 1740, 1697, 1462, 1384, 1
259, 1154, 1071, 757 cm^-1 In the same manner as in the fifth embodiment, the corresponding
The following compounds were obtained by hydrolyzing the tyl ester. Implementation
Example 6 (17S) -2a-homo-17,20-dimethyl-2,
2,2a, 2a, 13,14-hexadehydroprosta
Grangen E₁ ¹ H-NMR (CDCl_Three, 300 Mz) δ (pp
m): 0.85 to 0.96 (m, 6H), 1.12
1.88 (m, 17H), 2.19 to 2.33 (m, 1
H), 2.25 (dd, J = 8.9 Hz, 18.5H)
z, 1H), 2.37 (t, J = 6.9 Hz, 2H),
2.67 (ddd, J = 1.7 Hz, 8.3 Hz, 1
1.2 Hz, 1 H), 2.77 (ddd, J = 1.3 H)
z, 7.1 Hz, 18.5 Hz, 1H), 4.29-
4.39 (m, 1H), 4.50 (dt, J = 1.7H)
z, 7.6Hz, 1H) IR (neat): 3392, 2930, 2860, 2
236,1707,1462,1384,1241,1
073,757cm^-1 Embodiment 7 (17S) -2-nor-17,20-dimethyl-3,
3,4,4,13,14-hexadehydroprostagland
Engine E₁ ¹ H-NMR (CDCl_Three, 300 Mz) δ (pp
m): 0.86-0.95 (m, 6H), 1.10
1.96 (m, 13H), 2.21-2.34 (m, 1
H), 2.26 (dd, J = 8.8 Hz, 18.7H)
z, 1H), 2.40 (t, J = 6.4 Hz, 2H),
2.68 (ddd, J = 1.5 Hz, 8.3 Hz, 1
1.3 Hz, 1 H), 2.78 (ddd, J = 1.1 H)
z, 7.3 Hz, 18.7 Hz, 1H), 4.30-
4.41 (m, 1H), 4.42 to 4.54 (m, 1
H) IR (neat): 3392, 2956, 2929, 2
872, 2237, 1739, 1698, 1458, 1
383, 1266, 1154, 1070, 757 cm^-1 Embodiment 8 (16RS) -16,20-dimethyl-2,2,3
3,13,14,18,18,19,19-decade
Loprostaglandin E₁ ¹ H-NMR (CDCl_Three, 300 Mz) δ (pp
m): 1.05 (d, J = 6.7 Hz, 3H), 1.1
1 (t, J = 7.5 Hz, 3H), 1.79 to 1.90
(M, 1H), 2.01-2.88 (m, 15H),
3.10 (q, J = 7.6 Hz, 1H), 3.71
(S, 3H), 4.24 to 4.33 (m, 1H), 4.
32 and 4.38 (d, J = 4.1 Hz and
  J = 2.9 Hz, 1H), 6.51 (br, 3H) Embodiment 9 (17S) -17,20-dimethyl-2,2,3,3
13,14-Hexadehydroprostaglandin E₁ ¹ H-NMR (CDCl_Three, 300 Mz) δ (pp
m): 0.80 to 0.92 (m, 6H), 1.02 to
1.88 (m, 15H), 2.20 to 2.46 (m, 2
H), 2.37 (t, J = 6.0 Hz, 2H), 2.5
9 to 2.66 (m, 1H), 2.75 (dd, J = 7.
4 Hz, 18.5 Hz, 1 H), 4.28 to 4.42
(M, 1H), 4.46 (t, J = 6.4 Hz, 1H) IR (neat): 3370, 2930, 2240, 1
700,1370,1240,1040,730cm
^-1

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:38) (72) Inventor Kazuki Hanada 3-24-1, Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Invention Person: Toru Tanami 3-24-1, Takada, Toshima-ku, Tokyo Taisho Pharmaceutical Co., Ltd. (72) Inventor: Fumie Sato 3-1-219, Kugenumahigashi, Fujisawa-shi, Kanagawa (56) References 21392 (JP, A) JP-A-63-254995 (JP, A) JP-A-1-202296 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 31/00 C07C 405 / 00 504 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] [Claim 1] Formula [Wherein, R represents a lower alkyl group, R ¹ and R ² are the same or different and represent a hydrogen atom or a hydroxyl-protecting group,
A, R ³ and R ⁴ represent any group not participating in the reaction,
B represents a vinylene group or an ethynylene group. A lower alkyl ester of an E-type prostaglandin represented by the formula (1), which is reacted with an enzyme having ester hydrolysis ability produced by a microorganism belonging to the genus Aspergillus or Pseudomonas, (Wherein, R ¹ , R ² , A, R ³ , R ⁴ and B have the same meanings as described above).