JP2005278525A - Method for producing aromatic compound - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new method for production of an aromatic compound expressed by formula (II) (R<SB>1</SB>is amino or nitro; R<SB>2</SB>is hydroxymethyl or carboxyl; and R<SB>3</SB>is hydrogen atom or methyl) usable as a synthetic intermediate for pharmaceuticals by a biological conversion method. <P>SOLUTION: The objective material is produced by incubating an aromatic compound used as a starting raw material in the presence of cultured microbial cells or a processed product of the cultured cells capable of converting the starting compound to the aromatic compound expressed by formula (II) and separating the objective material from the incubated liquid. As an example, bacteria belonging to genus Pseudomonas are mentioned. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to a method for producing aromatic compounds that can be used as synthetic intermediates for pharmaceuticals by biological transformation.

（但し、式中、Ｒ₁はアミノ基またはニトロ基を表し、Ｒ₂はヒドロキシメチル基またはカルボキシル基を表し、Ｒ₃は水素原子またはメチル基を表す） The aromatic compound represented by the formula (II) is mainly used as a synthetic intermediate for various compounds having a quinazoline skeleton. The quinazoline skeleton is useful as a basic skeleton of various drugs. For example, it has been shown that Iressa having a 4-amino-6,7-dialkoxyquinazoline skeleton has a high antitumor effect (see Non-Patent Document 1). ).

(Wherein, R ₁ represents an amino group or a nitro group, R ₂ represents a hydroxymethyl group or a carboxyl group, and R ₃ represents a hydrogen atom or a methyl group)

In particular, 6-amino-m-toluic acid represented by the formula (II-A) is useful as a synthetic intermediate of a 4-quinazolinone derivative having excellent histamine H2 antagonist activity (see Non-Patent Document 2).

As a method for producing such 6-amino-m-toluic acid, there is a method in which a commonly available nitro derivative is converted to an amino group by reduction, and the yield is relatively good, but 6-nitro-m- Toluic acid is very expensive (see Non-Patent Document 3). In addition, to obtain 6-nitro-m-toluic acid, there is a nitration method of 3-methylbenzoic acid with a mixed acid of nitric acid and sulfuric acid, but it is generally necessary to control the reaction such as regioselectivity and excessive nitration. (Refer nonpatent literature 4).

Cancer Res. 62,5749 (2002) Chemical & Pharmaceutical Bulletin 36,2955 (1988) Nishimura, Shigeo, Takagi Gen, “Catalytic Hydrogenation Application to Organic Synthesis”, Tokyo Chemical Doujin Publishing, p. 211 Tamura, "Experimental Chemistry Course (4th edition) Nitro and Nitroso Compounds", page 394

The present invention provides a novel method for producing an aromatic compound represented by the above formula (II) by a biological conversion method, which has few problems with the above-described conventional techniques.

In order to solve the above problems, the present inventors regioselectively oxidize a methyl group adjacent to a substituent R ₁ of an aromatic compound represented by the following formula (I) from a wide range of microorganisms, As a result of searching for a microorganism that can be converted into the aromatic compound represented by II), a microorganism having high conversion ability was found and the present invention was completed.

（但し、式中、Ｒ₁はアミノ基またはニトロ基を表す）で示される芳香族化合物の、
式(II)、

（但し、式中、Ｒ₁はアミノ基またはニトロ基を表し、Ｒ₂はヒドロキシメチル基またはカルボキシル基を表し、Ｒ₃は水素原子またはメチル基を表す）で示される芳香族化合物への生物学的変換方法による、式(II)で示される芳香族化合物の製造方法であって、
(A)前記生物学的変換方法を行うことができるものであって、かつシュードモナス(Pseudomonas)属に属する微生物の培養菌体またはその培養菌体の調製物の存在下で、式(I)で示される芳香族化合物をインキュベーション処理する工程、
(B)インキュベーション処理液から式(II)で示される芳香族化合物を採取する工程、
を含んでなる方法。 That is, the present invention provides the following [1] to [5].
[1] Formula (I),

(Wherein, R ₁ represents an amino group or a nitro group)
Formula (II),

(Wherein R ₁ represents an amino group or a nitro group, R ₂ represents a hydroxymethyl group or a carboxyl group, and R ₃ represents a hydrogen atom or a methyl group) Biology to an aromatic compound A process for producing an aromatic compound represented by formula (II) by a chemical conversion method,
(A) In the presence of a cultured cell of a microorganism belonging to the genus Pseudomonas or a preparation of the cultured cell, which can be subjected to the biological conversion method, and represented by the formula (I) Incubating the indicated aromatic compound;
(B) collecting the aromatic compound represented by the formula (II) from the incubation solution,
Comprising a method.

[2] The method according to [1], wherein the cultured cells of the microorganism used in the biological conversion method are cultured in a medium containing benzoic acid.
[3] Pseudomonas sp. (Pseudomonas sp.) AO11 strain (FERM P-19756) or Pseudomonas sp. (Pseudomonas sp.)
sp.) The method according to [1] or [2], which is AO37 strain (FERM P-19757).
[4] A microorganism belonging to the genus Pseudomonas having an ability to convert the aromatic compound represented by the formula (I) into the aromatic compound represented by the formula (II).
[5] Pseudomonas sp. AO11 strain (FERM P-19756) or Pseudomonas sp.
sp.) The microorganism according to [4], which is AO37 strain (FERM P-19757).

In the biological conversion method of the present invention, culture of microorganisms belonging to the genus Pseudomonas and having the ability to convert the aromatic compound represented by the formula (I) into the aromatic compound represented by the formula (II) If it is a preparation of a microbial cell or its cultivated microbial cell, it can be used without questioning the species and strain type. An enzyme that is isolated from these strains and catalyzes the conversion reaction (hereinafter sometimes simply referred to as oxidase) can also be used.

Preferable examples of such microorganisms include Pseudomonas sp. AO11 strain and Pseudomonas sp.
sp.) AO37 strain. Pseudomonas sp. AO11 strain was established on March 29, 2004 by the National Institute of Advanced Industrial Science and Technology.
It has been deposited as sp. AO11 (accession number FERM P-19756). In addition, Pseudomonas sp. AO37 strain was incorporated into the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center on March 29, 2004.
It has been deposited as sp. AO37 (accession number FERM P-19757).

The sequence of about 500 bases on the 5 ′ end side of the 16srRNA gene of AO11 strain is as shown in SEQ ID NO: 1. As a result of homology search using DNA base sequence database (GenBank / DDBJ / EMBL) using BLAST, the sequence of this strain is Pseudomonas nitroredusense (strain name MS367, Accession
No.AY297786) and 99.8% homology, suggesting the closest relationship. However, since a sequence derived from a known species reference strain that perfectly matched the 16S rDNA of the AO11 strain was not searched, the AO11 strain was judged to belong to the genus Pseudomonas.

The sequence of about 500 bases on the 5 ′ end side of the 16srRNA gene of the AO37 strain is as shown in SEQ ID NO: 2. As a result of homology search using DNA base sequence database (GenBank / DDBJ / EMBL) using BLAST, the sequence of this strain is Pseudomonas nitroredusense (strain name MS367, Accession
No.AY297786) and 99.8% homology, suggesting the closest relationship. However, since a sequence derived from a known species reference strain that perfectly matched the 16S rDNA of the AO37 strain was not searched, the AO37 strain was judged to belong to the genus Pseudomonas.

The sequence was determined as follows. After collecting the culture solution of the test strain, DNA extraction was performed using PrepMan Method (Applied Biosystems). MicroSeq500 for PCR amplification
16SrDNA Kit (Applied Biosystems) was used for PCR product purification and cycle sequencing.
Bacterial Sequencing Kit (Applied Biosystems) was used. GeneAmp PCR for thermal cyclers
System 9600 (Applied Biosystems) with ABI PRISM 310 Genetic Analyzer as DNA sequencer
(Applied Biosystems) was used. In addition, basic operation followed the protocol (P / N4308132 Rev.A) of Applied Biosystems.

According to the present invention, an aromatic compound represented by the formula (I) as a starting material (substrate) is incubated in the presence of a cultured cell of a microorganism having the above-described properties or a preparation of the cultured cell. The This treatment is performed when cultivating the microorganism, or after adding the substrate to the culture solution after culturing, or collecting the cultured cells of the microorganism according to circumstances, for example, as it is or by freeze-drying treatment, spray drying Use after pretreatment such as treatment, organic solvent (eg, acetone) treatment, crushing treatment, etc., or oxidase is roughly purified or purified and isolated, then suspended in buffer, added with substrate, and incubated The reaction can also be carried out.

The substrate may be added to the culture solution at any time before culturing or when a certain period (2 to 4 days) has elapsed after the start of culturing. The cells can be produced by inoculating the above microorganisms in a nutrient source-containing medium and culturing aerobically. The culture of microorganisms for preparing a preparation of such cultured cells and the culture of microorganisms performed in a state where a substrate is added can be performed in principle according to a general microorganism culture method. Usually, it is preferably carried out under aerobic conditions such as shaking culture by liquid culture and aeration and agitation culture. In particular, when a microorganism is cultured using a medium containing benzoic acid or a salt thereof, the conversion ability can be remarkably increased, and the desired aromatic compound represented by the formula (II) can be efficiently obtained. In addition, the addition amount of benzoic acid or a salt thereof to the nutrient source-containing medium is preferably 2 to 4 g / L as a free acid.

As a medium used for the culture, any medium capable of growing these microorganisms may be used, and any of various synthetic media, semi-synthetic media, natural media and the like can be used. As the medium composition, glucose, maltose, xylose, fructose, sucrose, starch, dextrin, glycerol, mannitol, oatmeal and the like as a carbon source can be used alone or in combination, but as described above, benzoic acid or a salt thereof is used. It is preferable.

Nitrogen sources include peptone, meat extract, soy flour, casein, amino acids, malt extract, yeast extract, organic nitrogen sources such as urea, ammonium citrate, ammonium fumarate, sodium nitrate, potassium nitrate, ammonium sulfate, ammonium chloride, phosphoric acid Inorganic nitrogen sources such as ammonium hydrogen and ammonium dihydrogen phosphate can be used alone or in combination. In addition, for example, salts such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, cobalt chloride, and vitamins can be added and used as necessary. In addition, when foaming is remarkable during culture, various known antifoaming agents can be appropriately added to the medium.

Suitable media include, for example, Nutrient Broth media (peptone 5 g / L, meat extract 3 g / L, sodium chloride 5 g / L).

The culture conditions can be appropriately selected within a range in which the microorganism can grow well. Usually, the pH is 5.0 to 10.0, 20 to 30 ° C, preferably pH 6.5 to 8.0, 25 to 28 ° C, and the culture is usually performed for 1 to 3 days, preferably about 3 days. The various culture conditions described above can be appropriately changed according to the type and characteristics of microorganisms used, external conditions, etc., and optimal conditions can be selected.

In addition, the preparation of cultured bacterial cells should be prepared by appropriately collecting bacterial cells separated by centrifugation or filtration after culturing or bacterial cells subjected to pretreatment such as freeze-drying treatment, spray-drying treatment, organic solvent treatment, and crushing treatment. Prepare by suspending in solution. The solution that can be used for suspending the cells is the above-mentioned medium, or a buffer such as Tris-acetic acid, Tris-hydrochloric acid, sodium acetate, sodium citrate, sodium phosphate, potassium phosphate or the like alone or in combination. Is. The pH of the buffer is preferably 5.0 to 9.0, more preferably 7.0 to 8.5.

The aromatic compound represented by the formula (I) as a substrate remains in a liquid state or is diluted with a water-soluble organic solvent such as methanol, ethanol, acetone, dimethylformamide, dimethyl sulfoxide, etc. For example, in the case of a culture solution, the addition amount is 0.1 to 10 g, preferably 1 to 5 g, per liter of the culture solution. The substrate may be added all at once, but may be added several times or continuously when the amount added is relatively large. After the addition of the substrate, the aromatic compound represented by the formula (I) as the substrate is converted to the formula (II) by performing the reaction such as shaking or aeration stirring for 1 to 3 days, preferably 1 day, and allowing the reaction to proceed. ) Can be converted into the desired aromatic compound.

In order to isolate the desired aromatic compound represented by the formula (II) from the reaction mixture, various known purification means can be selected and combined. For example, adsorption and elution on a hydrophobic adsorption resin, solvent extraction using ethyl acetate, n-butanol, etc., column chromatography using silica gel, etc., or thin layer chromatography, high performance liquid chromatography, distillation, etc. Alternatively, combination separation and purification can be performed as appropriate.

Hereinafter, although an example is given and this invention is demonstrated in detail, this invention is not limited to these examples.

Example 1
2 ml of benzoic acid medium (3 g sodium benzoate, 3 g ammonium sulfate, 1.2 g potassium dihydrogen phosphate, 5 g sodium chloride, 0.2 g magnesium sulfate heptahydrate, 0.5 g yeast extract, trace elements Solution (Zinc sulfate heptahydrate 100mg, boric acid 300mg, calcium chloride hexahydrate 200mg, copper chloride dihydrate 10mg, sodium molybdate dihydrate 30mg, nickel chloride hexahydrate 20mg, manganese chloride tetra Hydrate 30 mg, iron sulfate heptahydrate 2 g, distilled water 1 L) 1 ml, distilled water 1 L, pH 7.2, sodium benzoate was dissolved in distilled water and filtered through a disinfecting filter (DISMIC-13CP, ADVANTEC) Thereafter, the tube was inoculated into a centrifuge tube containing a high-pressure steam sterilized medium) and cultured with shaking at 30 ° C. and 220 rpm for 2 days.

For the strains whose growth was confirmed, the preculture was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of the same medium, and cultured with shaking at 30 ° C. and 220 rpm for 2 days. After culturing, the cells were collected using a high-speed centrifuge (8,000 rpm, 20 minutes). The obtained bacterial cells were washed with physiological saline and then suspended in 5 ml of 0.1 M phosphate buffer (pH 7.6) containing 1% (w / v) glucose. 1 ml of the cell suspension was dispensed into a centrifuge tube, and 2,4-dimethylaniline (dissolved in dimethyl sulfoxide) as a substrate was added to 0.1% (w / v). Thereafter, the reaction was carried out at 30 ° C. and 220 rpm for 20 hours.

After the reaction, 500 μl of ethyl acetate was added to the reaction solution for extraction. After extraction, use a high-speed centrifuge (12,000 rpm, 5 minutes), and extract the ethyl acetate layer with thin-layer chromatography (Silica
Gel 60F254, Merck, developing solvent was separated by hexane: ethyl acetate = 1: 1). Substrate reduction and product 2-amino-methylbenzyl alcohol, 6-amino-m-toluic acid and anthranilic acid were detected with 5% phosphomolybdic acid. The analysis conditions by high performance liquid chromatography are as follows.

Column: ZORBAX Rx-SIL (φ4.6 × 250mm, 5μm, Agilent)
Column temperature: 35 ° C
Eluent: Hexane: Isopropyl alcohol: Acetic acid = 50: 50: 0.01
Flow rate: 0.75ml / min
Detection: 240nm
As a result of the analysis, it was confirmed that the products 2-amino-5-methylbenzyl alcohol and 6-amino-m-toluic acid were present in the reaction solution of AO11 strain. It was also confirmed that the product anthranilic acid was present in the reaction solution of the AO37 strain.

Example 2
Pseudomonas sp. Strain AO11 obtained in Example 1 was precultured in a benzoic acid medium in the same manner as in Example 1. Next, the obtained preculture was inoculated into 14 500 ml Erlenmeyer flasks containing 100 ml of the same medium, and cultured with shaking at 30 ° C. and 220 rpm for 2 days to obtain 1.4 L of the culture. This was treated under the same conditions as in Example 1 to prepare 70 ml of a cell suspension. After dispensing this into a centrifuge tube (1 ml / tube), 2,4-dimethylaniline was added to 0.1% (w / v) and reacted at 30 ° C. and 220 rpm for 20 hours. After the reaction, the entire reaction solution was extracted twice with 70 ml of ethyl acetate to obtain an ethyl acetate layer. The ethyl acetate layer was evaporated to dryness, and the dried product was dissolved in a small amount of ethyl acetate, followed by preparative thin layer chromatography (Silica
gel 60F254, Merck, developing solvent is hexane: ethyl acetate = 1: 1), the product 2-amino-5-methylbenzyl alcohol 21.0 mg (yield 30%) and 6-amino-m-toluyl The acid 3.8 mg (yield 5.4%) was isolated, and the structure was confirmed by ¹ H-NMR analysis and ¹³ C-NMR analysis. The structure of 6-amino-m-toluic acid was confirmed by comparing with a commercial product (Tokyo Chemical Industry Co., Ltd.).

¹ H-NMR and ¹³ C-NMR of 2-amino-5-methylbenzyl alcohol (400 MHz, CD ₃ OD, using standard substance TMS):

¹³ C (ppm) ¹ H (ppm)
1 144.34-
2 127.23-
2 -CH ₂ 63.55 4.54 (s)
3 130.56 6.91 (s)
4 128.60-
4 -CH ₃ 20.50 2.19 (s)
5 117.60 6.66 (d), J = 8.15Hz
6 130.20 6.87 (d), J = 8.15Hz

Example 3
Pseudomonas sp. Strain AO37 obtained in Example 1 was pre-cultured in a benzoic acid medium and then main cultured in the same manner as in Example 1 to obtain 400 ml of a culture solution. This was treated under the same conditions as in Example 1 to prepare 20 ml of a cell suspension, dispensed into a centrifuge tube (1 ml / tube), and then 0.05% (w / v) of 2,4-dimethylaniline. ) And reacted at 30 ° C. and 220 rpm for 20 hours. After the reaction, the entire reaction solution was extracted twice with 70 ml of ethyl acetate to obtain an ethyl acetate layer. The ethyl acetate layer was evaporated to dryness, and the dried product was dissolved in a small amount of ethyl acetate.
Gel 60F254, Merck, developing solvent is chloroform: methanol = 10: 1), the product anthranilic acid 0.72mg (yield 7.2%) is isolated, ¹ H-NMR analysis (commercial product (Wako Pure Chemical Industries, Ltd.) The structure was confirmed by comparison with Kogyo Co.).

Example 4
Pseudomonas sp. Strain AO11 was inoculated into 2 ml of a benzoic acid medium containing 0.5% (w / v) glycerol and cultured in the same manner as in Example 1. After culturing, 2,4-dimethylaniline was added to the culture solution at 0.05% ((w / v), approximately 4.1 mM), and the reaction was performed at 30 ° C. and 220 rpm for 20 hours. After the reaction, analysis by high performance liquid chromatography was conducted in the same manner as in Example 1. As a result, it was confirmed that the products 2-amino-5-methylbenzyl alcohol 1.1 mM and 6-amino-m-toluic acid 0.62 mM were formed.

Example 5
Pseudomonas sp. Strain AO11 was cultured in the same manner as in Example 2 except that a benzoic acid medium containing 0.5% (w / v) glycerol was used, and a 1.4 L culture solution was obtained. Subsequently, the cells were collected by a high-speed centrifuge (8,000 rpm, 20 minutes). The obtained cells were washed with physiological saline and suspended in 80 ml of 0.1 M phosphate buffer (pH 7.6). Dispense the cell suspension into a centrifuge tube (1 ml / tube), add 2,4-dimethylaniline to 0.1% (w / v), and react at 30 ° C and 220 rpm for 20 hours. It was. After the reaction, the whole reaction solution was extracted twice with 80 ml of ethyl acetate to obtain an ethyl acetate layer. Thereafter, the product 6-amino-m-toluic acid was isolated by the method shown in Example 2. The structure was confirmed by ¹ H-NMR analysis. As a result, 16.8 mg (yield 21%) of the product 6-amino-m-toluic acid was obtained. By using a medium in which glycerol was added to a benzoic acid medium, the yield of the product 6-amino-m-toluic acid could be increased as compared with Example 2.

Example 6
Pseudomonas sp. Strain AO11 was inoculated into 1.4 L of benzoic acid medium containing 0.5% (w / v) glycerol and cultured according to the method shown in Example 1. After culturing, 80 ml of cell suspension is prepared under the conditions shown in Example 5 and dispensed into a centrifuge tube (1 ml / tube), and then 2,4-dimethylnitrobenzene is adjusted to 0.1% (w / v). And reacted at 30 ° C. and 220 rpm for 20 hours. After the reaction, the whole reaction solution was extracted twice with 80 ml of ethyl acetate to obtain an ethyl acetate layer. The ethyl acetate layer was evaporated to dryness, and the dried product was dissolved in a small amount of ethyl acetate, followed by preparative thin layer chromatography (Silica
gel 60F254, Merck, developing solvent is hexane: ethyl acetate = 1: 1) to isolate 1.9 mg of product 2-nitro-5-methylbenzyl alcohol (yield 2.4%), ¹ H-NMR The structure was confirmed by analysis and ¹³ C-NMR analysis (400 MHz, CDCl ₃ , using standard substance TMS).

¹³ C (ppm) ¹ H (ppm)
1 145.65-
2 136.76-
2 -CH ₂ -OH 62.84 4.95 (d), -OH 2.55 (t), J = 6.66Hz
3 130.72 7.51 (s)
4 145.65-
4 -CH ₃ 21.60 2.47 (s)
5 129.06 7.26 (overlap)
6 125.37 8.04 (d), J = 8.44Hz

Claims (5)

Formula (I),

(Wherein, R ₁ represents an amino group or a nitro group)
Formula (II),

(Wherein R ₁ represents an amino group or a nitro group, R ₂ represents a hydroxymethyl group or a carboxyl group, and R ₃ represents a hydrogen atom or a methyl group) Biology to an aromatic compound A process for producing an aromatic compound represented by formula (II) by a chemical conversion method,
(A) In the presence of a cultured cell of a microorganism belonging to the genus Pseudomonas or a preparation of the cultured cell, which can be subjected to the biological conversion method, and represented by the formula (I) Incubating the indicated aromatic compound;
(B) collecting the aromatic compound represented by the formula (II) from the incubation solution,
Comprising a method. 2. The method according to claim 1, wherein the microbial cell used in the biological conversion method is cultured in a medium containing benzoic acid. Strains capable of performing biological conversion methods are Pseudomonas sp. AO11 strain (FERM P-19756) or Pseudomonas sp.
The method according to claim 1 or 2, which is sp.) AO37 strain (FERM P-19757). A microorganism belonging to the genus Pseudomonas having an ability to convert the aromatic compound represented by the formula (I) into the aromatic compound represented by the formula (II). Pseudomonas sp. AO11 strain (FERM P-19756) or Pseudomonas sp.
sp.) The microorganism according to claim 4, which is AO37 strain (FERM P-19757).