JPH06319588A - Production of optically active norstatin derivative - Google Patents

Abstract

PURPOSE:To stereoselectively produce an optically active norstatin derivative useful as an intermediate for various renin inhibitors, HIV protease inhibitors and carcinostatic agents from a cyanohydrin derivative or an aldehyde derivative using an enzyme. CONSTITUTION:An optically active norstatin derivative is stereoselectively produced from a cyanohydrin derivative or an aldehyde derivative using a nitrile hydrolase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a stereoselective method for producing an optically active norstatin derivative useful as an intermediate for pharmaceuticals. More specifically, the present invention provides a method for stereoselectively producing an optically active norstatin derivative, which is an intermediate of various renin inhibitors, HIV protease inhibitors, and carcinostatics, from a cyanohydrin derivative or an aldehyde derivative using an enzyme. Regarding

[0002]

2. Description of the Related Art As a method for producing a norstatin derivative, JP-A-62-33141 and JP-A-1-172 are known.
Japanese Patent No. 365 and Japanese Patent Laid-Open No. 4-208257 are known. As a method using an enzyme, Japanese Patent Laid-Open No. 60595/1990 is already known.

[0003]

However, in the above method, the diastereoselectivity of the cyanohydrin derivative produced as an intermediate is low, and therefore complicated purification is required to obtain the objective optically active norstatin derivative. There are drawbacks such as the necessity, and it cannot be said that the manufacturing method is satisfactory.

[0004]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a method for stereoselectively producing a norstatin derivative from a cyanohydrin derivative or an aldehyde derivative using an enzyme. The present invention has been completed and the present invention has been completed. That is, the present invention is

The following formula (1);

[Chemical 7] [In the formula, R ₁ represents hydrogen or an amino group-protecting group, and R ₂ represents a substituted or unsubstituted alkyl group, aryl group, or aralkyl group. Further, the carbon atom marked with (S) has the S configuration. ]

An enzyme acts on the cyanohydrin derivative represented by the following formula (2);

[Chemical 8] [In the formula, R ₁ , R ₂ and (S) have the same meanings as described above. ]
A method for stereoselectively producing an optically active norstatin derivative represented by further

The following formula (3):

[Chemical 9] IN FORMULA, R < ₁ >, R < ₂ > REPRESENTS THE SAME MEANING AS THE ABOVE-MENTIONED, AND THE CARBON ATOM SHOWING (R) IS R CONFIGURATION. ] The following formula (4) which makes an enzyme act on the cyanohydrin derivative shown by these;

[0008]

[Chemical 10] [In the formula, R ₁ , R ₂ , (R), and (S) have the same meanings as described above. ] The stereoselective manufacturing method of the optically active norstatin derivative shown by these is provided. further

Formula (5) below;

[Chemical 11] [In the formula, R ₁ , R ₂ and (S) have the same meanings as described above. ]
There is provided a method for stereoselectively producing an optically active norstatin derivative represented by the above formula (2), which comprises allowing an enzyme to act on an optically active aldehyde derivative represented by the formula (1) in the presence of a cyanide compound. further

And the following formula (6):

[Chemical 12] [In the formula, R ₁ , R ₂ and (R) have the same meanings as described above. ]
There is provided a stereoselective method for producing an optically active norstatin derivative represented by the above formula (4), wherein an enzyme is allowed to act on the optically active aldehyde derivative represented by the formula (3) in the presence of a cyanide compound. The present invention will be described in detail below.

In the present invention, in the formula, R ₁ represents hydrogen or a protecting group for an amino group, and R ₂ represents a substituted or unsubstituted alkyl group, aryl group or aralkyl group. The carbon atom marked with (S) has the S configuration, and the carbon atom marked with (R) has the R configuration. In this case, as the amino-protecting group, a urethane-type or amide-type protecting group usually used for a primary amino group, particularly preferably, in the production of various protease inhibitors using the compound of the present invention as a starting material Good protecting group, that is, a benzyloxycarbonyl group that can be easily deprotected by hydrogenation,
Alternatively, a tert.-butoxycarbonyl group or the like, which can be easily deprotected by acid decomposition, is desirable.

The alkyl group, aryl group and aralkyl group represented by R ₂ are, for example, an alkyl group having 1 to 5 carbon atoms such as methyl group, ethyl group, isopropyl group and t-butyl group; Further, a cycloalkane-substituted alkyl group having 1 to 5 carbon atoms in an alkyl moiety such as a cyclohexylmethyl group, a cyclopentylmethyl group, or a cyclohexylethyl group, which is substituted with a cycloalkane having 3 to 8 carbon atoms; phenyl group, naphthyl And aryl groups such as group, tolyl group and the like, such as benzyl group, phenethyl group, p-methylbenzyl group and the like.

In this case, the alkyl group, aryl group and aralkyl group may have a functional group. As a functional group,
For example, mono- to tri-substituted, for example, a fluorine group,
Halogen group such as chlorine group, bromine group, methoxy group,
C1-C1 such as ethoxy and isopropoxy groups
5 alkoxy group; carboxyl group, for example, methoxycarbonyl group, ethoxycarbonyl group, isopropoxycarbonyl group and other C1-C5 alkoxycarbonyl group; nitro group, amino group, aminocarbonyl group and the like.

The compounds represented by formula (1), formula (3), formula (5) and formula (6) can be easily produced from the corresponding D or L amino acid by a known method. As the nitrile hydrolase used in the present invention, an enzyme which converts nitrile into a carboxylic acid, that is, a nitrilase, a nitrile hydratase, or an amidase having activity can be used.

For example, Acinetobacter spp, Alcaligenes spp, Pseudomonas spp, Rhodopseudomonas spp, Corynebacterium spp, Bacillus spp, Mycobacterium spp, Rhodococcus spp, Nocardia spp, Arthrobacter spp, Moraxella spp, Klebsiella spp. It is an enzyme of a microorganism selected from microorganisms belonging to the genus, Acremonium or Candida.

Specific microorganisms include Acinetobacter sp. AK226 (FERM BP-245).
1), Alcaligenes faecalis ATCC 875
0, Pseudomonas fluorescens IFO 39
25. Rhodo-Sudomonas Spheroides ATCC
11167, Corynebacterium sp. KO-
2-4 (FERM BP-2353), Bacillus subtilis CN5 (FERM BP-2354), Mycobacterium sp. AC 777 (FERM B)
P-2352), Rhodococcus sp. AK 32
(FERM BP-1046), Nocardia Globerla ATCC 21505, Arthrobacter sp. A7 (Ministry of Microbiology Deposit No. 8927), Moraxella sp. D12 (Ministry of Microbiology Deposit No. 8933),
Kreb Shera SP D5B
932), Acremonium SP D9K (Ministry of Microbiology Deposit No. 8930), Candida tropicalis ATCC 20311 and the like. All of these strains are disclosed in JP-A-2-84198 and JP-A-63-
No. 209,592.

The reaction method in the present invention is carried out in the presence of a nitrile hydrolase, that is, a microorganism or a preparation thereof, and a cyanohydrin derivative represented by the above formula (1) or the above formula (3), or a cyan compound, in the presence of the above formula. (5) Alternatively, it is carried out by contacting the aldehyde derivative represented by the above formula (6). Microorganisms or preparations thereof, specifically, a culture obtained by culturing the above-mentioned microorganism, cells or treated cells collected therefrom (for example, crushed cells or enzymes separated and extracted from cells), Furthermore, it shows a product obtained by immobilizing a bacterium or a treated product of the bacterium on a carrier by an appropriate method.

Cultivation of the microorganism used in the present invention can be carried out according to a known method. As the medium to be used, those known as nutrient sources for general microorganisms can be used, glucose, glycerin, ethanol, sucrose, glutamic acid, acetic acid, carbon sources such as citric acid, ammonium sulfate, ammonium chloride, ammonia, nitrogen sources such as urea. ,
Organic nutrient sources such as yeast extract, malt extract, peptone, and meat extract, and inorganic nutrient sources such as phosphoric acid, magnesium, potassium, iron, cobalt, manganese, and lanthanum can be used in appropriate combination.

Further, as a substance for increasing the reaction activity of the microorganism in the present invention, a cyano compound such as isobutyronitrile or an amide compound such as caprolactam may be added. The pH of the medium may be selected in the range of 5 to 10, and the culture temperature is 18 to 50 ° C, preferably 25 to 40 ° C.
The culture time may be 1 to 10 days until the activity becomes maximum.

The reaction conditions in the present invention will be described below.
The reaction medium is an aqueous medium such as water, a buffer solution or a culture solution, a mixed medium of an aqueous medium and a water-soluble organic solvent such as dimethyl sulfoxide or methanol, and a two-phase system comprising an aqueous medium and a water-insoluble organic solvent. Media can be used. Further, a suitable surfactant may be added to the reaction medium in an amount of 0.01 to 10% by weight. The substrate is added to the reaction medium by adding the compound represented by the above formula (1), (3), (5) or formula (6) in the form of powder or liquid, or by dissolving it in a suitable solvent.

The addition concentration is about 0.01 to 70% by weight, preferably 0.1 to 40% by weight, and it may not be completely dissolved in the reaction medium. When an aldehyde derivative is used as a raw material, a cyanide compound such as sodium cyanide or potassium cyanide is added in an amount of 0.5 to 0.5 with respect to the aldehyde derivative.
The amount is 30 times mol, preferably 1 to 10 times mol.

When bacterial cells are used in the reaction, the bacterial cell concentration is usually 0.01 to 40% by weight, preferably 0.05.
It may be in the range of 20% by weight. The reaction temperature is 5 to 80 ° C, preferably 15 to 60 ° C, and the reaction pH is 4 to 12, preferably 6 to 10. The reaction is usually completed within 1 to 100 hours within a range in which the optical purity of the optically active norstatin derivative of the formula (2) or the formula (4), which is the target product, does not decrease, and the reaction rate is usually 10 -100%, preferably 40-100%. The optical purity of the product at this time is preferably 80% ee or more. The consumed substrate may be added so as to be maintained within the above range.

The reaction mechanism in the present invention is as follows.
Selectively acting on a nitrile having an R configuration at position
That is, it is considered that the reaction rate by the enzyme is very different depending on the asymmetric center. Further, the other cyanohydrin isomer which remains unacted becomes a rachydrin derivative which is naturally racemized by an equilibrium reaction with an aldehyde derivative in the presence of a cyanide compound, and the reaction proceeds. As a result, the reaction rate of the racemate with respect to the raw material can exceed 50%. Therefore, the aldehyde derivative represented by the formula (5) or the formula (6) and the cyan compound can be used as the raw material.

The separation of the target product in the present invention is carried out as follows. After removing insoluble matters such as bacterial cells from the reaction-terminated liquid, the pH is alkaline, preferably 8.5 to
12, and an unreacted material is extracted and removed with an inert solvent immiscible with water, for example, a solvent such as benzene, diethyl ether, chloroform, hexane, ethyl acetate, and the like.
The target substance is separated by adjusting the pH to acidic, preferably 2.0 to 3.0, and extracting with the above solvent. Furthermore, purification of the target product is performed by column chromatography using silica gel, treatment with activated carbon, or the like.

The optical purity of the reaction product and the raw material is, for example, Chiralcel OD-R, Chiralcel OJ, Chiral AG.
P (Daicel Chemical Industry Co., Ltd.), Ceramospher Chira
It can be measured by HPLC analysis using an optical resolution column such as l RU-1 (Shiseido) or SUMICHIRAL OA (Sumitomo Chemical Analysis Center), or Novapack-Rhenyl (Waters).

[0026]

[Reference example]

Reference Example 1 N-tert. -Butoxycarbonyl-L-cyclohexyl
Alaninal (Synthesis of formula (7) below:

[Chemical 13]

N-tert. -Butoxycarbonyl-L-cyclohexyl alaninol 25.73 g (99.98 mm
ol) was added to 120 ml of DMSO and 58 ml of toluene and cooled to 0 ° C. Triethylamine 75.7
After adding ml (541.89 mmol), sulfur trioxide-
86.24 g (541.89 mmol) of pyridine complex was gradually added, and the mixture was stirred at 0 ° C. for 1 hour. After completion of the reaction, add 300 ml of ethyl acetate and 300 ml of ice water to the reaction mixture,
After separating the organic layer, the organic layer was washed three times with 300 ml of saturated saline. The organic layer was dried over anhydrous magnesium sulfate and then concentrated, and the residue was produced by silica gel column chromatography (hexane: ethyl acetate = 10: 1 to 3: 1). -Butoxycarbonyl-L-cyclohexylalaninal 25.03 g (98.02 mmol)
Was obtained with a yield of 98.04%.

IR (KBr): 3300, 2920, 1
730, 1670, 1520, 1370, 1290, 1
175 cm ^-1 NMR (CDCl ₃ ) δ: 0.8-2.0 (m, 13
H), 1.46 (s, 9H), 4.29 (b, 1H),
4.90 (b, 1H), 9.59 (s, 1H). MS m / e: 256 (M + 1) ⁺

Reference Example 2 N-tert. -Butoxycarbonyl-D-cyclohexyl
Alaninal (synthesis of formula (8):

[Chemical 14] N-tert. -Butoxycarbonyl-D-cyclohexylalaninol was treated with N in the same manner as in Reference Example 1.
-Tert. -Butoxycarbonyl-D-cyclohexylalaninal was obtained.

IR (KBr): 3300, 2920, 1
730, 1670, 1520, 1370, 1290, 1
175 cm ^-1 NMR (CDCl ₃ ) δ: 0.8-2.0 (m, 13
H), 1.46 (s, 9H), 4.29 (b, 1H),
4.90 (b, 1H), 9.59 (s, 1H). MS m / e: 256 (M + 1) ⁺

Reference Example 3 3- (S) -tert. -Butoxycarbonylamino-4-
Cyclohexyl-2- (R, S) -hydroxybutyroni
Trill (synthesis of formula (9) below:

[Chemical 15]

N-tert. -Butoxycarbonyl-L-cyclohexylalaninal 13.01 g (50.94 mm
ol) was added to 132 ml of water and 535 ml of chloroform, and the mixture was cooled to 0 ° C. Sodium cyanide 7.4
After adding 9 g (152.82 mmol), 1N-HCl
153 ml was added dropwise, and the mixture was stirred at 0 ° C for 12 hours. After completion of the reaction, the organic layer of the reaction solution was separated, and the organic layer was extracted from the aqueous layer with 300 ml of chloroform. The combined organic layers were washed twice with 300 ml of water, dried over anhydrous magnesium sulfate, and then concentrated to give a colorless transparent oily substance, 3- (S) -ter.
t. -Butoxycarbonylamino-4-cyclohexyl-2- (R, S) -hydroxybutyronitrile 14.3
8 g (50.94 mmol) was obtained with a yield of 100%.

By high performance liquid chromatography analysis, the syn-form (2R, 3S) was 64% and the anti-form (2S, 3S) was
3S) was 36%. The analysis conditions are shown below. (Column: Waters Nova-PaK Pheny 13, 9
× 47 mm _{Eluent: pH = 3.5 0.05M-KH 2} PO 4 -H 3 PO 4 / CH 3 CN =
70/30 Flow rate: 1 ml / min Detection: RI detector Retention time: 26, 1 min (thin), 24.5 min (anti)

IR (NaCl): 3330, 2850,
2250, 1695, 1510, 1455, 1370,
1250, 1170 cm ^-1 NMR (CDCl ₃ ) δ: 0.8: 1.8 (m, 13
H), 1.47 (s, 9H), 3.6-4.1 (m, 1)
H), 4.3-4.7 (m, 1H), 4.78 (d, 1)
H, J = 7.6 Hz), 5.35 (br, 1H) MS m / e: 283 (M + 1) ⁺

Reference Example 4 3- (R) -tert. -Butoxycarbonylamino-4-
Cyclohexyl-2- (R, S) -hydroxybutyroni
Trill (synthesis of formula (10) below:

[Chemical 16]

N-tert. -Butoxycarbonyl-D-cyclohexylalaninal, using the same method as in Reference Example 3, 3- (R) -tert. -Butoxycarbonylamino-4-cyclohexyl-2- (R, S) -hydroxybutyronitrile was obtained. Analysis by high performance liquid chromatography revealed that the syn-form (2S, 3R) was 64% and the anti-form (2R, 3R) was 36%.

IR (NaCl): 3330, 2850,
2250, 1695, 1510, 1455, 1370,
1250, 1170 cm ^-1 NMR (CDCl ₃ ) δ: 0.8: 1.8 (m, 13
H), 1.47 (s, 9H), 3.6-4.1 (m, 1)
H), 4.3-4.7 (m, 1H), 4.78 (d, 1)
H, J = 7.6 Hz), 5.35 (br, 1H) MS m / e: 283 (M + 1) ⁺

[0038]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. (Example 1) 3- (S) -tert. -Butoxycarbonylamino-4-
Cyclohexyl-2- (S) -hydroxybutyric acid (the following formula
Synthesis of (11):

[Chemical 17]

Glucose 1.0%, yeast extract 0.5
%, Polypeptone 0.5%, potassium phosphate 1 0.2
%, Magnesium sulfate 0.02%, sodium chloride 0.
1%, isobutyronitrile 0.1%, pH 7.
1% of a bacterium of the genus Rhodococcus having nitrilase activity, which had been cultivated in the same medium in advance, was inoculated into 100 ml of the sterilized medium defined as No. 5, and cultured at 32 ° C for 48 hours. After completion of the culture, cells were collected by centrifugation, suspended in an Erlenmeyer flask containing 10 ml of tap water, and then dissolved in 10 ml of ethanol to obtain 50 mg of 3- (S) -tert. -Butoxycarbonylamino-4-cyclohexyl-2- (R, S) -hydroxybutyronitrile was added, and the reaction was carried out at 30 ° C for 48 hours.

After completion of the reaction, the bacterial cells were removed by centrifugation and the supernatant was extracted and chromatographed to 16 m.
g of 3- (S) -tert. -Butoxycarbonylamino-
4-Cyclohexyl-2- (S) -hydroxybutyric acid was obtained with a yield of 30%. HPLC analysis of Reference Example 3 (retention time:
The optical purity was 90% ee from 5.9 min.

IR (KBr): 3350,2920,2
850, 1720, 1690, 1510, 1370, 1
255, 1170 cm ^-1 NMR (CDCl ₃ ) δ: 0.8: 2.1 (m, 13
H), 1.44 (s, 9H), 4.1-4.5 (m, 2)
H), 5.1-5.6 (br, 1H), 5.7-6.1.
(Br, 1H) MS m / e: 302 (M + 1) ⁺

(Example 2) 3- (R) -tert. -Butoxycarbonylamino-4-
Cyclohexyl-2- (S) -hydroxybutyric acid (the following formula
Synthesis of (12):

[Chemical 18]

Acinetobacter bacteria having a nitrile hydrolyzing activity, which had been cultured in a medium having the same composition as in Example 1 except that ammonium acetate was used instead of glucose, were cultured and then collected. This was suspended in an Erlenmeyer flask containing 40 ml of water and then dissolved in 10 ml of methanol to prepare 3- (R) -tert. -Butoxycarbonylamino-4-
80 mg of cyclohexyl-2- (R, S) -hydroxybutyronitrile was added, and the reaction was carried out at 30 ° C for 48 hours.

After completion of the reaction, cells were removed by centrifugation, the supernatant was adjusted to pH 10, and 40 ml of chloroform was added to remove unreacted materials. Then, the pH of the water layer was adjusted to 1.
It was made 5 and extracted with 50 ml of chloroform. The organic layer was concentrated to 52 mg of 3- (R) -tert. -Butoxycarbonylamino-4-cyclohexyl-2- (S) -hydroxybutyric acid was obtained with a yield of 60%. The optical purity in HPLC analysis (retention time: 7.0 min) was 91% ee.

IR (KBr): 3350,2920,2
850, 1720, 1690, 1510, 1370, 1
255, 1170 cm ^-1 NMR (CDCl ₃ ) δ: 0.8: 2.1 (m, 13
H), 1.44 (s, 9H), 4.0-4.3 (m, 2)
H), 4.8-5.1 (br, 1H), 6.0-6.3.
(Br, 1H) MS m / e: 302 (M + 1) ⁺

Example 3 Mycobacterium were cultivated in a medium having the same composition as in Example 1 and then collected. This was suspended in an Erlenmeyer flask containing 40 ml of 0.05 M potassium phosphate buffer (pH 7.2) and then dissolved in 10 ml of N-tert. -Butoxycarbonyl-D-cyclohexylalaninal 50
0 mg and 96 mg of sodium cyanide were added, and the temperature was 30 ° C.
The reaction was performed for 40 hours.

After completion of the reaction, the bacterial cells were removed by centrifugation, and the supernatant was extracted and chromatographed to 500
mg 3- (R) -tert. -Butoxycarbonylamino-4-cyclohexyl-2- (S) -hydroxybutyric acid was obtained with a yield of 85%. The optical purity in HPLC analysis is 9
It was 3% ee.

[0048]

According to the present invention, various renin inhibitors, HI
Norstatin derivatives, which are intermediates of V protease inhibitors and carcinostatics, can be produced using nitrile hydrolases under normal temperature and normal pressure reaction conditions. Furthermore, according to the present invention, it became possible to obtain a norstatin derivative having extremely high optical purity with high selectivity and high yield.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:32)

Claims (5)

[Claims] 1. The following formula (1); [In the formula, R ₁ represents hydrogen or an amino group-protecting group, and R ₂ represents a substituted or unsubstituted alkyl group, aryl group, or aralkyl group. Further, the carbon atom marked with (S) has the S configuration. ] A nitrile hydrolase is allowed to act on the cyanohydrin derivative represented by the following formula (2); [In the formula, R ₁ , R ₂ and (S) have the same meanings as described above. ]
A method for stereoselectively producing an optically active norstatin derivative represented by: 2. The following formula (3); [In the formula, R ₁ , R ₂ and (R) have the same meanings as described above,
The carbon atom marked with (R) is in the R configuration. ] A nitrile hydrolase is allowed to act on the cyanohydrin derivative represented by the following formula (4); [In the formula, R ₁ , R ₂ , (R), and (S) have the same meanings as described above. ] The stereoselective manufacturing method of the optically active norstatin derivative shown by these. 3. The following formula (5); [In the formula, R ₁ , R ₂ and (S) have the same meanings as described above. ]
A stereoselective method for producing an optically active norstatin derivative represented by the above formula (2), characterized in that the nitrile hydrolase is allowed to act on the optically active aldehyde derivative represented by the formula (1) in the presence of a cyanide compound. 4. The following formula (6); [In the formula, R ₁ , R ₂ and (R) have the same meanings as described above. ]
A method for stereoselectively producing an optically active norstatin derivative represented by the above formula (4), characterized in that a nitrile hydrolase is allowed to act on the optically active aldehyde derivative represented by. 5. A nitril hydrolase is used in the genera Acinetobacter, Alcaligenes, Pseudomonas, Rhodoschonas, Corynebacterium, Bacillus, Mycobacterium, Rhodococcus, Nocardia, Arthrobacter, The enzyme of a microorganism selected from the microorganisms belonging to the genera Moraxella, Klebsiella, Acremonium, or Candida is used alone or in any combination.
The manufacturing method according to any one of 1.