JP2000279190A - Production of optical active 3-substituted-(2- hydroxyalkyl)indole - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject optically active 3-substituted-(2-hydroxyalkyl) indole of high optical purity that is useful as a raw material for medicines or the like by bringing a specific 3-substituted indole bearing a carbonyl group into contact with a culture mixture of a microorganism in Nocardia or the like capable of converting a compound bioreductively. SOLUTION: A substrate represented by formula I (R1 is a lower alkyl; R2 is H, a lower alkyl; R3 and R4 are each H, a substituent that has no adverse effect on the bioreductive conversion) is brought into contact with a culture mixture of at least one selected from the group consisting of microorganisms that can bioreductively convert this substrate into 3-substituted indole represented by formula II, selected from the group of Nocardia, Aspergillus and Sclerotinia to obtain the objective 3-substituted indole of high purity that is represented by formula II and is useful as a precursor of a compound having the action to stimulate β-adrenaline receptor.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a bioreductive conversion method of an organic compound using a microorganism, and more particularly to a method for producing 3- (2-hydroxyalkyl) indole having high optical purity using a microorganism.

[0002]

2. Description of the Related Art Among indole derivatives, there are many compounds that have extremely interesting biological activities and are currently used as pharmaceuticals or are being developed as pharmaceuticals. Such derivatives include those of the general formula

[0003]

Embedded image (Where A and B are one or more substituents containing a hydrogen atom), including drugs acting on each subclass of β-adrenergic receptor (WO 96/16938,
J. Med. Chem., 25 , 670-679 (1982);
UK Patent No. 861,428). The carbon at the 2-position of the 2-aminopropane moiety bonded to the 3-position of the indole ring of these compounds is an asymmetric center, which may result in a plurality of stereoisomers. As is common with materials, certain isomers are often desirable for biological activity. In fact, WO96 / 1693
The compound having a β ₃ adrenergic receptor stimulating action described in No. 8 has a configuration at the 2-position of the above moiety which is R
-Or S- is preferable. Therefore, the 2-aminopropane moiety is 2 (R) or 2 (R).
To provide the indole derivative having the configuration of (S), it is necessary to subject the final compound or the synthetic intermediate having the 2-aminopropane moiety to optical resolution.

[0004] By the way, WO 96/16938 includes:

[0005]

Embedded image Forming a moiety from the corresponding amine compound;

[0006]

Embedded image Methods have been proposed for forming moieties from the corresponding oxirane compound or carboxylic acid compound. According to such a method, 2R may be used as the amine compound depending on the purpose.
If the-or 2S- isomer is used, the desired optical isomer can be obtained as the final compound. But,
A method for selectively obtaining such an amine compound rich in either the 2R- or 2S-form is not known.

In general, microorganisms or enzymes may be used successfully in providing certain optical isomers.

[0008]

An object of the present invention is to provide, for example, a compound enriched in any of the optical isomers which can be a precursor of an amine compound for synthesizing the indole derivative.
-To provide a method for producing a substituted (2-hydroxyalkyl) indole.

[0009]

Means for Solving the Problems The present inventors have found that 3- (2
Using (-oxoalkyl) -substituted indole as a substrate and searching for microorganisms capable of stereospecifically converting the oxo group to a hydroxyl group, specific actinomycetes and molds found that the substrate could be converted to the corresponding hydroxy compound with high optical purity. I found something that could be converted.

Thus, according to the present invention, the compound of formula (I)

[0011]

Embedded image (Wherein R ₁ is a lower alkyl group, R ₂ is a hydrogen atom or a lower alkyl group, and R ₃ and R ₄ are independently
A hydrogen atom or a substituent that does not adversely affect bioreductive conversion), wherein the reduction reaction is represented by the formula (II):

[0012]

Embedded image (Wherein R ₁ , R ₂ , R ₃ and R ₄ have the same meanings as defined for formula (I)) from a 3-substituted compound represented by formula (I) Nocardia, Aspergillus, and Sclerotinia (Scler) that have the ability to bioreductively convert to indole
otinia), wherein the method is carried out by contacting with a culture of at least one microorganism selected from the group consisting of microorganisms belonging to biotin-reducing conversion conditions.

According to the method of the present invention, the compound represented by the formula (I) resulting from the 3-position substituent of the 3-substituted indole represented by the formula (I)
Wherein the optical purity of the compound is at least 50% ee at either the S-form or the R-position. The compounds thus obtained can be converted into the corresponding amino compounds with their configuration substantially retained or inverted by chemical reduction methods known per se for hydroxyl groups. Therefore, the compounds obtained by the method of the present invention are useful as raw materials for various drugs including drugs described in WO 96/16938 and other known publications, or precursors thereof.

As used herein, the term "bioreductive conversion" is a concept opposed to chemical reduction,
It means reducing a substrate using an organism, particularly a microorganism. More specifically, a certain enzyme activity of a microorganism is used for the reduction. Therefore, the culture referred to in the present invention also includes living or dead microorganisms or processed cells prepared therefrom, as long as they have the above-mentioned activity. Further, such a microorganism may be derived from any of those in the logarithmic growth phase and those in the stationary phase when cultured in a nutrient medium. As the nutrient medium, a known liquid medium appropriately containing a carbon source, a nitrogen source, a trace metal, and other nutrients depending on the microorganism to be used is used.

The culture used in the present invention belongs to the genus Nocardia, the genus Aspergillus and the genus Sclerotinia and has the formula (I)
It may be derived from any of the strains capable of converting the substrate represented by I) into the 3-substituted indole represented by the formula (I), or from two or more strains thereof.

Representative of these strains is Nocardia orientalis A-6.
40 (FERM P-17341), Aspergillus
Glaucus (Aspergillus gloucus) F-78 (FER
MP-17340) and Sclerotinia araehinidis F-786 (FER
MP-17342), and mutant strains having the bioreductive conversion ability thereof. Mutant strains include those that occur naturally and those that are artificially obtained, for example, using mutagen or genetic engineering. The strains to which the above FERMP P-numbers are assigned are deposited on March 26, 1999 with the Institute of Biotechnology and Industrial Science and Technology, and given the accession numbers described respectively. Each of the above strains has been selected from a stock culture of one of the applicants or isolated from soil. In addition, the genera and species names of the described strains were identified from the respective mycological properties by referring to the literature described later. Specifically, for the actinomycetes, references (1) to (8) were referred.

According to the invention, a substrate of the formula (II) is brought into contact with a culture of said strain under bioreductive conversion conditions. The contact under such conditions may be carried out in such a manner that the culture of the strain having bioreductive conversion ability may contain an organic solvent miscible with water adjusted to pH 6.5 to 7.5. In a temperature of 25 to 35 ° C. with the substrate of formula (II). As mentioned above, the culture can be the culture itself, including the grown strain, or the cells isolated from the culture or, for example, a homogenate of the cells. When the culture itself (culture solution) is used, an aqueous medium may be prepared including an organic solvent miscible with water to enhance the solubility of the substrate. When the isolated cells are used, an aqueous medium may be prepared containing the nutrient medium of the strain to be used and the organic solvent as described above. The “water-miscible organic solvent” includes, but is not limited to, aprotic polar solvents such as dimethylformamide (DMF) and dimethylsulfoxide (DMSO).

The contact between the culture and the substrate in the medium as described above can be carried out by shaking or stirring the medium containing both if necessary. The optimal time for the contact is not limited because it depends on the culture and the substrate used, but generally the growth temperature of the bacterium to be used is 25-3.
12-72 hours at 5 ° C. In any case, those skilled in the art can select optimal conditions by conducting small experiments with reference to examples described later.

In specifying the substrate represented by the formula (II) and the compound represented by the formula (I) and "substituents which do not adversely affect the bioreductive conversion" in the present specification,
A group with the term "lower" means that the group has 1-4 carbon atoms. Specific examples of “lower alkyl group” include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-
Although butyl and the like can be mentioned, methyl, ethyl, propyl and isopropyl are preferable. Specific examples of the “lower alkyl group substituted with a hydroxyl group” include hydroxymethyl, 2-hydroxyethyl and the like. Specific examples of the “lower alkylsulfonylamino group” include methylsulfonylamino, ethylsulfonylamino and the like. Specific examples of the "mono- or di-lower alkylaminosulfonyl group" include monomethylaminosulfonyl, dimethylaminosulfonyl, monoethylaminosulfonyl, diethylaminosulfonyl and the like. Specific examples of the “lower alkoxycarbonyl group” include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl and the like. Specific examples of the “lower alkanoyl group” include acetyl, propionyl and the like. Specific examples of "mono- or di-lower alkylaminocarbonyl group" include:
Methylaminocarbonyl, dimethylaminocarbonyl,
Methylethylaminocarbonyl and the like. "Halogen atom" means fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine, and particularly preferably fluorine or chlorine. Specific examples of the “lower alkoxy group” include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, t
ert-butoxy and the like. Specific examples of the “lower alkoxycarbonyl lower alkoxy group” include methoxycarbonylmethoxy, 1-methoxycarbonylethoxy, 2-methoxycarbonylethoxy, 1-methoxycarbonylpropoxy, 3-methoxycarbonylpropoxy and the like. Specific examples of the “carboxy lower alkoxy group” include carboxymethoxy, 1-carboxyethoxy, 2-carboxyethoxy, 1-carboxypropoxy, 3-carboxypropoxy and the like. Specific examples of the “phenyl lower alkoxy group” include benzyloxy, phenethyloxy and the like.

In formulas (I) and (II), one of R ₃ and R ₄ is a hydrogen atom, and the other is a compound of the formula —O (CH ₂ ) _p — bonded to the 7-position of the indole ring.
A compound having R _c (where R _c and p are as defined above) can be efficiently produced in the method of the present invention, and can be a substrate therefor. Further, a compound to which the method of the present invention is more efficiently applied is a compound in which R _c is a phenyl group or a di-lower alkylaminocarbonyl group (eg, diethylaminocarbonyl group).

The compound represented by the formula (II) can be synthesized by a method known per se from J. Org. Chem. , 25 1548-1
558 (1961), etc., can be produced by hydrolyzing an enamine formed after reducing the amount of nitro with an iron-hydrochloric acid system.

The hydroxyl group in the substituent at the 3-position in the thus-obtained compound represented by the formula (I) is converted to the corresponding amine compound by a known chemical conversion method from a hydroxyl group to an amino group. it can. For example,
The compound represented by the formula (I) is treated with diethyl azodicarboxylate, triphenylphosphine and a phthalimide obtained by a reaction with phthalimide with hydrazine, or after hydrogenation via p-toluenesulfonylation and azidation. What is necessary is just to add.

[0023]

Now, the present invention will be described more specifically below with reference to representative examples. Examples 1-4:

[0024]

Embedded image [A:

[0025]

Embedded image (Respectively referred to as A-1 substrate and A-1 compound) or A: (CH ₃ CH ₂ ) NC (O) CH ₂ — (respectively A
(Referred to as "-2 substrate and A-2 compound")] (1) Preparation of culture solution <Actinobacteria> Nocardia orientalis A-640
(FERM P-17341) (hereinafter referred to as A-640) was prepared by sterilizing 30 ml of C medium in a 250 ml Meyer, inoculating one loopful of slant medium, and culturing with shaking at 28 ° C. for 3 days. This was adjusted to pH 7 and used for the conversion reaction. <Mold> Aspergillus glaucus F-78 (F
ERM P-17340) (hereinafter referred to as F-78) and Sclerotinia alehinidis F-786 (FE
RM P-17342) (hereinafter referred to as F-786)
Was inoculated from a slant medium into 2 ml of an FI medium in a centrifuge tube, cultured for 1 day, and the whole amount was transferred to 30 ml of a medium in a 250 ml Meyer, and cultured for 3 days. This was adjusted to pH 7 and used for the conversion reaction.

[0026] The culture destination pairs formed YM medium: (%) glucose 1.0 polypeptone 0.5 yeast extract 0.3 Malt extract 0.3 (pH = 5.5) BHI medium: (%) Brain Heart Infusion 3. 7 MRS medium: (%) NZ CASE (casein peptone) 1.0 malt extract 1.0 yeast extract 0.5 glucose 2.0 Tween 80 (TM) 0.1 K ₂ HPO ₄ 0.2 sodium acetate 0.5 Diammonium citrate 0.2 MgSO ₄ .7H ₂ O 0.2 MnSO ₄ .H ₂ O 0.005 (pH = 6.0-6.5) C medium: (%) Potato starch 2.0 Glucose 2. 0 soybean meal 2.0 yeast extract _{0.5 NaCl 0.25 CaCO 3 0.32 FeSO 4} · 7H 2 O 0.0005 MnSO 4 · 4H 2 O 0.0005 ZnSO 4 0.0005 (pH = 7.4) FI medium: (%) Potato starch 2.0 Soybean meal 2.0 glucose _{1.0 KH 2 PO 4 0.1 MgSO 4} · 7H 2 O 0.05 ADEKANOL LG109 (TM) 0.05 (2) conversion reaction and Assay Conversion Reaction and Assay Broth 30 in each 250 ml Meyer from (1) above
Transfer 2 to 2 ml to a test tube, add 0.05 ml of dimethylformamide (DMF) for the A-1 substrate and dimethylsulfoxide (DMS) for the A-2 substrate.
O) 2 mg of each substrate dissolved in 0.05 ml was added, and the mixture was shaken at 28 ° C. for one day. Assays were performed by extracting the culture with 0.5 ml of butyl acetate, TLC analysis and HPLC.
Analysis was performed to examine the absolute configuration and optical purity of the target compound. Depending on the microorganism used, the conversion from substrate to product under the above conditions is generally about 30%. TLC conditions Thin layer: Merck thin layer chromatography 5715 Developing system: hexane / ethyl acetate / acetic acid = 20/10/1 Detection: UV HPLC conditions Column: DAISEL CHIRALCEL OD Eluent: substrate A hexane / isopropanol = 10/90 Substrate D Hexane / isopropanol = 50/50 Flow rate: 0.4 ml / min Temperature: room temperature (3) Result 1 A-640 A-1 compound S-64% ee 2 F-78 A-1 compound S-54% ee 3 F-786 A-1 compound S-59% ee 4 F-786 A-2 compound R-100 % Ee Reference Example 1: Conversion of hydroxy form to amino form A-2 compound obtained in Example 4 (R-100% ee)
13.4 mg was inverted in configuration by tosylation and azidation, and reduced with lithium aluminum hydride.
-2 corresponding amino compound of compound [(S) -58% ee]
was gotten. Reference Example 2: Example of substrate synthesis

[0027]

Embedded image (1) DMF (500 ml) was cooled in an ice-salt bath, and phosphorus oxychloride (168 ml, 1.8 mol) was added to 1 ml.
It was dropped over time. 7-benzyloxyindole (3
35 g (1.5 mol) of DMF (600 ml) was added dropwise over 2 hours, followed by stirring at room temperature for 3 hours. Ice-cooled 3N
The reaction solution was poured into an aqueous NaOH solution (2.25 l, 6.75 mol), heated at 100 ° C. for 0.5 hour, then poured into ice water and stirred. The precipitated solid was collected by filtration, washed with water, and dried to obtain 7-benzyloxy-3-formylindole (368 g, 98%) as pale yellow-white crystals. (2) The product of the above (1) (75.3 g, 0.3 mol)
l), diammonium hydrogen phosphate (43.6 g, 0.33
mol), nitroethane (150 ml), acetic acid (180
ml) was heated at reflux for 3 hours. After adding 70% ethanol (200 ml) to the reaction solution, the mixture was stirred under ice cooling for 0.5 hour. The precipitated solid was collected by filtration and dried to give 7-benzyloxy-3- (nitropropenyl) indole (46.3 g, 50%) as yellow crystals. (3) The product of the above (2) (3.08 g, 10 mmol)
l), a mixture of reduced iron (2 g), acetic acid (10 ml), and methanol (20 ml) was heated under reflux for 2 hours. After allowing to cool to room temperature, insolubles were removed by filtration, and the filtrate was distilled off under reduced pressure. Toluene was added to the residue, washed with water, an aqueous NaOH solution and saturated saline in this order, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and 7-benzyloxy-3-
(2-Propanone) indole (2.1 g, 75%) was obtained as a brown solid. Melting point: 105-106 ° C (crystallized from toluene) ¹ H-NMR spectrum (200 MHz, CDCl ₃ , δ)
ppm): 2.15 (3H, s), 3.78 (2H,
s), 5.19 (2H, s), 6.73 (1H, d, J =
8Hz), 7.03 (1H, t, J = 8Hz), 7.08
(1H, d, J = 3 Hz), 7.16 (1H, d, J =
8Hz), 7.32-7.51 (5H, m), 8.37
(1H, s) Elemental analysis: Calculated for C ₁₈ H ₁₇ NO ₂ : C, 77.4
0; H, 6.13; N, 5.01. Found: C, 77.2
3; H, 5.95; N, 4.90. Reference Example 3: Example of substrate synthesis

[0028]

Embedded image (1) 7-benzyloxy-3- (2-propanone) indole (3.4 g, 12 mmol) was added to methanol (2
0%) and 10% palladium on carbon (0.3 g)
And reduced at normal pressure at room temperature. After the theoretical amount of hydrogen had been absorbed, the catalyst was filtered off and the filtrate was evaporated under reduced pressure to give crude 7-hydroxy-3- (2-propanone) indole. (2) After dissolving the above product (1) in acetone (30 ml), potassium carbonate (2.1 g, 15 mmol), chloroacetic acid diethylamide (2.18 g, 15 mmol) and potassium iodide (0.25 g) were added. The mixture was heated under reflux for 3 hours. After cooling the reaction solution with ice, insolubles were removed by filtration, and the solvent was distilled off under reduced pressure. Chloroform (100 ml) and water (30 ml) were added to the residue, and after stirring, the chloroform layer was separated, dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. Diisopropyl ether (100 ml) was added to the residue, and the precipitated solid was collected by filtration, and 7- [3- (2-propanone) indolyl] oxyacetic acid diethylamide (2.4 g, 65
%) As a light brown solid. Melting point: 129 ° C. (crystallized from ethyl acetate-diisopropyl ether) Elemental analysis: Calculated for C ₁₇ H ₂₂ N ₂ O ₃ .0.5H ₂ O: C, 65.57; H, 7.45; N, 9.00. Found: C, 65.75; H, 7.31; N, 9.27. Description of cited references: The references cited herein are listed below.

(1) Bergey's Manual of Systematic
Bacteriology, vol.4, Williams and Wilkins Co., Bal
timore, 1989, (2) Shirling, EB & D. Gottlieb; Cooperative
description of typecultures of Streptomyces. II S
pecies descriptions from first study. Int. J. Sys
t. Bacteriol. Vol.18, No.2, 69-189, 1968, (3) Shirling, EB & D. Gottlieb; Cooperative
description of typecultures of Streptomyces. III
Additional species descriptions fromfirst study an
d second studies, (4) Int. J. Syst. Bacteriol. Vol. 18, No. 4, 279
392, 1968 (5) Shirling, EB & D. Gottlieb; Cooperativ
e description of typecultures of Streptomyces. IV
Species descriptions from the second and third and
fourth studies, (6) Int. J. Syst. Bacteriol. Vol.19, No.4, 391
~ 512, 1969, (7) Shirling, EB & D. Gottlieb; Cooperative
description of typecultures of Streptomyces. V Ad
ditional descriptions, (8) Int. J. Syst. Bacteriol. Vol. 22, No. 4, 265
~ 394, 1972

[0030]

According to the method of the present invention, corresponding precursors which can be used as intermediates for synthesizing various pharmaceuticals can be used as optical isomers of 1- (mono- or di-substituted indol-3-yl) -2-aminoalkanes. An optically active 2-hydroxyalkane compound can be efficiently produced.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12P 17/10 C12R 1: 645) (72) Inventor Akihito Fujii 3-905 Hozumidai, Ibaraki-shi, Osaka (72) Inventor Sozaburo Ohashi 2-23-47- 203, Nagatahigashi, Minami-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Kunio Isshiki 2-2-17, Minamikurihara, Zama-shi, Kanagawa Prefecture (72) Inventor Takeo Yoshioka, Kanagawa Prefecture 1782-10 Yoshioka, Ayase-shi F term (reference) 4B064 AE48 CA03 CA05 CB18 CC03 CC06 CC07 CD12 DA01

Claims (7)

[Claims] 1. Formula (I) via a reduction reaction (Wherein R ₁ is a lower alkyl group, R ₂ is a hydrogen atom or a lower alkyl group, and R ₃ and R ₄ are independently
A hydrogen atom or a substituent that does not adversely affect bioreductive conversion), wherein the reduction reaction is carried out according to the formula (II) (Wherein R ₁ , R ₂ , R ₃ and R ₄ have the same meanings as defined for formula (I)) from a 3-substituted compound represented by formula (I) Nocardia, Aspergillus, and Sclerotinia (Scler) that have the ability to bioreductively convert to indole
otinia), which is carried out by contacting with a culture of at least one microorganism selected from the group consisting of microorganisms belonging to the group consisting of microorganisms belonging to the biotin-reducing conversion condition. 2. A lower alkyl group, a phenylsulfonylamino group or a lower alkylsulfonylamino group, wherein one of the substituents R ₃ and R ₄ which do not adversely affect bioreductive conversion may be substituted with a hydroxyl group. , the group represented by mono- or whether it is a di-lower alkylaminosulfonyl group, or (a) formula -X-R _a (wherein, X
Is O, S or NH, and _Ra is a hydrogen atom or a lower alkyl group, provided that when X is S, _Ra is a lower alkyl group), (b) a compound represented by the following formula: Wherein R _b is a hydrogen atom, a lower alkyl group, a lower alkoxycarbonyl group or a carboxyl group, R _bb is a lower alkoxycarbonyl group or a carboxyl group, m is an integer of 0 to 3, n is 0 or (C) a group represented by the formula —O (CH ₂ ) _p —R _c , wherein R _c is a lower alkanoyl group, a hydroxyl group,
A cyano group, a phenyl group, a mono- or di-lower alkylaminocarbonyl group, or a compound represented by the following formula: (Wherein, R _A is a hydrogen atom or a lower alkyl group) is a group represented by, p is an integer of 1 to 4] and equation (d) -Y- (CH _{2) q} -R _d Wherein Y is NH or S, R _d is a carboxyl group or a lower alkoxycarbonyl group, and q is an integer of 1 to 4, provided that One of them represents a hydrogen atom, a halogen atom, a lower alkyl group which may be substituted with a hydroxyl group, a hydroxyl group, a lower alkoxy group, or the same group as the above (b) or (c), or The method according to claim 1, wherein the methylenedioxy group is substituted with a carboxyl group or a lower alkoxycarbonyl group. 3. One of R ₃ and R ₄ is a hydrogen atom,
(Wherein, R _c and p are defined above and is synonymous) formula -O (CH _{2) p} -R _c of the other that one is attached to the 7-position of the indole ring (c) according to claim 1, which is a the method of. 4. The method according to claim 3, wherein in the formula —O (CH ₂ ) _p —R _c , R _c is a phenyl group or a di-lower alkylaminocarbonyl group, and p is an integer of 1. 5. Cultures comprising Nocardia orientalis A-640, Aspergillus gloucus F-78 and Sclerotinia araehin.
idis) a culture obtained by culturing at least one strain selected from the group consisting of F-786 and a mutant strain having the bioreductive conversion ability in a nutrient medium or a cell preparation derived from the culture; A method according to any of the preceding claims. 6. An aqueous medium which may contain an organic solvent miscible with water, wherein the bioreductive conversion condition is pH 6.
The method according to any one of claims 1 to 5, which is a medium adjusted to 5 to 7.5 and a temperature of 25 to 35 ° C. 7. The optical purity of the compound of the formula (I) resulting from the 3-position substituent of the 3-substituted indole represented by the formula (I) is at least 50 in either the S form or the R form.
% Ee.