阅读说明：本技术 一类手性β-羟基酰胺类化合物及其制备方法与应用 (Chiral β -hydroxy amide compounds and preparation method and application thereof ) 是由 胡慧娟 敖宇飞 王德先 王其强 于 2018-08-22 设计创作，主要内容包括：本发明公开了一种手性β-羟基酰胺类化合物及其制备方法。该化合物如式I所示。本发明提供的制备非天然手性氨基酸类合物的原料是用红球菌Rhodococcus erythropolis AJ270微生物体系催化水解带有不同取代基的前手性二酰胺类化合物II得到。所用红球菌菌体用量可根据底物的用量来进行调节。反应溶剂为pH值6.0-8.0的常用缓冲溶液,温度为20-37℃,反应时间为3-120小时。该红球菌微生物催化体系具有可发酵培养和保存方便的特点。运用此生物转化制备手性单酰胺羧酸、二羧酸的方法,具有操作简便,反应高效,反应条件温和,对映选择性高,产物易分离,产物纯度高的特点,具有很好的应用前景。<Image he="168" wi="700" file="DDA0001773522500000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention discloses a chiral β -hydroxy amide compound and a preparation method thereof, wherein the compound is shown as a formula I, and the raw material for preparing the non-natural chiral amino acid compound provided by the invention is Rhodococcus erythropolis AJ270The microbial system catalyzes and hydrolyzes prochiral diamide compounds II with different substituents to obtain the compound. The dosage of the rhodococcus thallus can be adjusted according to the dosage of the substrate. The reaction solvent is common buffer solution with pH value of 6.0-8.0, the temperature is 20-37 ℃, and the reaction time is 3-120 hours. The Rhodococcus microorganism catalysis system has the characteristics of fermentation culture and convenient preservation. The method for preparing the chiral monoamide carboxylic acid and the chiral dicarboxylic acid by biotransformation has the characteristics of simple and convenient operation, high reaction efficiency, mild reaction conditions, high enantioselectivity, easy separation of products and high product purity, and has good application prospect.)

1. A chiral β -hydroxyamide compound of formula I:

in the formula I, the represents chirality and is R or S;

R¹any one selected from the following groups: -COOH, -COOR²、-CH₂OH; wherein R is²Any one selected from the following groups: c₁-C₆Alkyl, allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl, p-bromobenzyl;

n represents-CH₂-a number of 0-4;

according to R¹Of radicalsThe compound shown in the formula I is: chiral amide carboxylic acid, chiral amide carboxylic ester and chiral amide alcohol compounds which are respectively compounds shown in formulas I-1 to I-3;

in the formulae I-1, I-2 and I-3, n n represents-CH₂-a number of 0-4;

in the formula I-2, R²Any one selected from the following groups: c₁-C₆Alkyl, allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl, p-bromobenzyl.

2. A process for preparing a compound of formula I-1 according to claim 1, comprising:

under the catalysis of a rhodococcus catalytic system, the achiral compound shown in the formula II is subjected to hydrolysis reaction to obtain a compound shown in the formula I-1;

in the formula II, n represents-CH₂-the number of (a) is an integer from 0 to 4.

3. The method of claim 2, wherein: the rhodococcus catalytic system consists of rhodococcus and buffer solution with the pH value of 6.0-8.0;

the Rhodococcus is Rhodococcus erythropolis AJ 270;

the catalytic system is prepared by the following method: inoculating the rhodococcus to the buffer solution with the pH value of 6.0-8.0, and activating for 30 minutes at 30 ℃;

the buffer solution is Na₂HPO₄Citric acid buffer solution, K₂HPO₄-KH₂PO₄Buffer solution, Tris buffer solution, Hanks' buffer solution or PBS buffer solution;

in the Rhodococcus catalytic system, RhodococcusThe dosage ratio of the cocci to the buffer solution is 2 g: 50 mL-1L; wherein, the bacteria activity of the rhodococcus is as follows: 1X 10⁷-1×10⁹CFU/g。

4. A method according to claim 2 or 3, characterized in that:

the dosage ratio of the rhodococcus to the compound shown in the formula II is 2 g: 1mmol-1 mol;

in the hydrolysis reaction, the temperature is 20-37 ℃ and the time is 3-120 hours.

5. A process for the preparation of a compound of formula I-2 according to claim 1,

when R is²When the methyl is adopted, the method comprises the following steps:

a) reacting a compound of formula I-1 according to claim 1 or a compound of formula I-1 prepared according to any one of claims 2 to 4 with CH₂N₂The ether solution is reacted in methanol to obtain the product; or the like, or, alternatively,

b) reacting a compound represented by a formula I-1 in claim 1 or a compound represented by a formula I-1 prepared by the method in any one of claims 2 to 4 with a base and methyl iodide in an organic solvent to obtain the compound;

when R is²Is C₂-C₆Alkyl, allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl and p-bromobenzyl, the method is as follows:

a') reacting a compound of formula I-1 according to claim 1 or a compound of formula I-1 prepared according to any one of claims 2 to 4 with a base and R²Br is reacted in an organic solvent to obtain a compound shown as a formula I-2 after the reaction is finished, wherein R is²Is C₂-C₆Alkyl, allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl, p-bromobenzyl.

6. The method of claim 5, wherein: in the process a), a compound of formula I-1 or a compound of formula I-1 prepared by the process according to any one of claims 2 to 4, CH₂N₂The dosage ratio of the ether solution to the methanol is 0.1-10 mmol: 0.5-50 ml: 5-50 mL;

the CH₂N₂The concentration of the ether solution is 0.1-5 mol/L;

in the reaction, the temperature is-20-30 ℃, and the time is 1-48 hours;

in the methods b) and a'), the base is at least one of potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide or cesium carbonate;

the organic solvent is at least one selected from acetone, N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran;

a compound of formula I-1 or a compound of formula I-1 prepared according to the process of any one of claims 2 to 4 with methyl iodide or R²The dosage ratio of Br, alkali and organic solvent is 0.1-10 mmol: 0.13-15 mL: 0.14-14 g: 1-100 mL;

in the reaction, the temperature is-20-50 ℃ and the time is 6-48 hours.

7. A process for the preparation of a compound of formula I-3 according to claim 1, comprising: carrying out reduction reaction on a compound shown as a formula I-2 in claim 1 or a compound shown as a formula I-2 prepared by the method in claim 5 or 6, sodium borohydride and lithium chloride in an organic solvent, and obtaining a compound shown as a formula I-3 after the reaction is finished.

8. The method of claim 7, wherein: the compound shown as the formula I-2 or the compound shown as the formula I-2 prepared by the method according to claim 5 or 6, sodium borohydride, lithium chloride and an organic solvent are used in a ratio of: 0.3-10.0 mmol: 73mg-730 mg: 0.13-1.4 g: 4-40 mL;

the organic solvent is at least one selected from ethanol, methanol, N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran;

in the reaction, the temperature is 0-50 ℃ and the time is 1-48 hours.

9. Use of a compound of formula I according to claim 1 for the preparation of: 1) an inhibitor of proliferation of eukaryotic tumor cells; 2) a preventive and/or therapeutic agent for tumor; the eukaryote is a mammal; the tumor cell is a cancer cell; the tumor is a carcinoma.

10. Use according to claim 9, characterized in that: the cancer cell is a colon cancer cell; the cancer is colon cancer.

Technical Field

The invention belongs to the field of organic chemistry, and particularly relates to a chiral β -hydroxyamide compound and a preparation method and application thereof.

Background

For the synthesis of chiral β -hydroxyamide compounds, the existing synthetic methods still have the disadvantages of low yield, complex preparation of raw materials and low stereoselectivity, so that the development of new synthetic methods of chiral β -hydroxyamide compounds is necessary.

Biocatalysis is the most efficient, high-selectivity and environment-friendly process so far, and the biocatalysis method is utilized to synthesize some chemicals with high added values, particularly chiral chemicals, so that the method has important application prospects and significance. Nitrile is an important organic synthesis intermediate, the chemical conversion of nitrile requires harsh conditions and has poor selectivity, the biotransformation reaction of nitrile has the advantages of mild conditions, high selectivity and the like, and the nitrile is applied to the industrialized preparation of corresponding carboxylic acid and amide derivatives at present, and the most notable is that the industrialization of synthesizing acrylamide by a microbiological method is realized by Nitto company (now more commonly named Mitsubishi Rayon company) in Japan in 1985. Annual acrylamide production in 1998 exceeds 4 million tons, and is one of the largest industrial biotransformation routes in the world at present. Rhodococcus rhodochrous J1 is also used by Lonza AG of Switzerland for the industrial production of the B vitamins nicotinamide and nicotinic acid, with annual production of nicotinamide already exceeding 3000 tons. But the biocatalytic reactions of nitriles with amides are also relatively rare compared to enzymatic ester hydrolysis and ester bond formation reactions.

Disclosure of Invention

The invention aims to provide a chiral β -hydroxyamide compound and a preparation method and application thereof.

The structural formula of the chiral β -hydroxyamide compound provided by the invention is shown as the formula I:

in the formula I, the represents chirality and is R or S;

R¹any one selected from the following groups: -COOH, -COOR²、-CH₂OH; wherein R is²Any one selected from the following groups: c₁-C₆Alkyl, allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl, p-bromobenzyl;

n represents-CH₂-the number of (a) is an integer from 0 to 4.

A compound of the formula I as described above, according to R¹The groups are different and can be chiral amide carboxylic acid, chiral amide carboxylic ester and chiral amide alcohol compounds which are respectively compounds shown in formulas I-1 to I-3;

in the formulas I-1 to I-3, the chiral is R or S;

R²any one selected from the following groups: c₁-C₆Alkyl, allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl, p-bromobenzyl;

n represents-CH₂-the number of (a) is an integer from 0 to 4.

The compound shown in the formula I-1 is prepared by a method comprising the following steps:

under the catalysis of a rhodococcus catalytic system, the achiral compound shown in the formula II undergoes hydrolysis reaction to obtain the R¹A compound of formula I which is-COOH (i.e., a compound of formula I-1);

in the formula II, n represents-CH₂-the number of (a) is an integer from 0 to 4.

In the method, the rhodococcus catalytic system consists of rhodococcus and buffer solution with the pH value of 6.0-8.0.

The Rhodococcus erythropolis AJ270 can be specifically Rhodococcus erythropolis.

The catalytic system is prepared by the following method: inoculating the rhodococcus to the buffer solution with the pH value of 6.0-8.0, and activating for 30 minutes at 30 ℃.

The buffer solution is Na₂HPO₄Citric acid buffer solution, K₂HPO₄-KH₂PO₄Buffer solution, Tris buffer solution, Hanks' buffer solution or PBS buffer solution, specifically K₂HPO₄-KH₂PO₄And (4) buffer solution.

In the rhodococcus catalytic system, the dosage ratio of the rhodococcus to the buffer solution is 2g (wet weight): 50 mL-1L; wherein, the bacteria activity of the rhodococcus can be: 1X 10⁷-1×10⁹CFU/g。

The dosage ratio of the rhodococcus to the compound shown in the formula II is 2 g: 1mmol-1mol, specifically 2 g: 1 mmol.

In the hydrolysis reaction, the temperature can be 20-37 ℃, particularly 30 ℃, and the time can be 3-120 hours, particularly 5-20 hours.

Different substrates and amounts are preferably used for different times so that the enantioselectivity of the reaction product is between 28 and 99.5%.

The invention also provides a method for preparing R¹is-CO₂R²A compound of formula I (i.e., a compound of formula I-2),

when R is²When the methyl is adopted, the method comprises the following steps:

a) r in the formula I¹A compound of formula I which is-COOH (i.e., a compound of formula I-1) with CH₂N₂The ether solution is reacted in methanol to obtain the R¹is-CO₂CH₃A compound of formula I (i.e., a compound of formula I-2); or the like, or, alternatively,

b) r in the formula I¹Reacting a compound shown as a formula I (namely a compound shown as a formula I-1) which is-COOH with alkali and methyl iodide in an organic solvent to obtain the R after the reaction is finished¹is-CO₂R²Is a compound of the formula I (i.e., a compound of the formula I-2)

When R is²Is C₂-C₆Alkyl, allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl and p-bromobenzyl, the method is as follows:

a') A) is a reaction of R in the formula I¹A compound of formula I which is-COOH (i.e. a compound of formula I-1) with a base and R²Br is reacted in an organic solvent to obtain the R¹is-CO₂R²Is a compound of formula I (i.e., a compound of formula I-2).

In the process a), R¹A compound of formula I which is-COOH (i.e., a compound of formula I-1), CH₂N₂The ratio of the diethyl ether solution to the methanol can be 0.1-10 mmol: 0.5-50 ml: 5-50mL, specifically 1 mmol: 5mL of: 10 mL;

the CH₂N₂The concentration of the ether solution can be 0.1-5mol/L, and specifically can be 2 mol/L;

in the reaction, the temperature can be-20-30 ℃, particularly-15 ℃, and the time can be 1-48 hours, particularly 4 hours;

in the methods b) and a'), the base is at least one of potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide or cesium carbonate, and specifically can be potassium carbonate;

the organic solvent is at least one selected from acetone, N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran, and specifically can be N, N-dimethylformamide;

R²br can be: bromoethane-hexane, 3-bromopropene, 3-bromopropyne, benzyl bromide, o-bromobenzyl bromide, m-bromobenzyl bromide or p-bromobenzyl bromide.

The compound shown as the formula I-1, methyl iodide or R²The dosage ratio of Br, alkali and organic solvent can be 0.1-10 mmol: 0.13-15 mL: 0.14-14 g: 1-100mL, specifically 1 mmol: 0.13-1 mL: 0.27-1.38 g: 2-5 mL;

in the reaction, the temperature can be-20-50 ℃, specifically 25 ℃, and the time can be 6-48 hours, specifically 12 hours.

The invention also provides a method for preparing R¹is-CH₂A method of OH of a compound of formula I (i.e., a compound of formula I-3), the method comprising the steps of:

carrying out reduction reaction on a compound shown as a formula I-2, sodium borohydride and lithium chloride in an organic solvent to obtain the R after the reaction is finished¹is-CH₂OH (i.e., a compound represented by formula I-3);

in the above method, the dosage ratio of the compound shown in formula I-2, sodium borohydride, lithium chloride, and organic solvent may be: 0.3-10.0 mmol: 73mg-730 mg: 0.13-1.4 g: 4-40mL, which can be specifically: 0.35 mmol: 73 mg: 0.134 g: 5 mL;

the organic solvent can be at least one selected from ethanol, methanol, N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran, and specifically can be N, N-dimethylformamide;

in the reaction, the temperature can be 0-50 ℃, specifically 25 ℃, and the time can be 1-48 hours, specifically 16 hours.

The use of the compounds of formula I above for the preparation of the following products also falls within the scope of the present invention: 1) an inhibitor of proliferation of eukaryotic tumor cells; 2) a medicine for preventing and/or treating tumor.

In the application, the eukaryote is a mammal; the tumor cell is a cancer cell; the tumor is a carcinoma;

the cancer cell is a colon cancer cell; the cancer is colon cancer.

Rhodococcus erythropolis AJ270 samples used in the present invention were initially isolated from acetonitrile agar medium containing 25mM using an Anderson bioparticle sampler, and the sample source was initially collected in dry soil near the industrial plant area abandoned in Taien riverside, England. Chemical taxonomic studies of mycolic acid and diaminopimelic acid of the cell wall of AJ270 strain confirmed that it belongs to the Rhodococcus species. Until 2005, it was confirmed that Rhodococcus AJ270 belongs to the Rhodococcus erythropolis strain line by studying its 16SrRNA gene sequence. Specific reference is made to the following two documents:

a.Blakey A.J.；Colby J.；Williams E.；O’Reilly C.,FEMSMicrobiol.Lett.1995,129,57-61.

b.O’Mahony R.；Doran J.；Coffey L.；Cahill O.J.；Black G.W.；O’Reilly C.；Antonie van Leeuwenhoek 2005,87,221-232.

rhodococcus erythropolis AJ270 is a soil-derived microorganism and has been demonstrated to be a high activity whole cell catalyst containing a nitrile hydratase/amidohydrolase system. Research has shown that, compared with other strains, Rhodococcus erythropolis AJ270 has very good substrate broad spectrum, and can efficiently catalyze the hydrolysis of aliphatic nitrile, aromatic nitrile and aromatic heterocyclic nitrile compounds. (Wang M. -X. Enantioselective biological transformations of nitrile in organic synthesis. Top. Cat. 2005,35, 117. Surge. 130).

The raw material for preparing the non-natural amino acid compounds is obtained by catalyzing and hydrolyzing diamide compounds by using a Rhodococcus erythropolis AJ270 microbial system. The dosage of the rhodococcus thallus can be adjusted according to the dosage of the substrate. The reaction solvent is common buffer solution with pH value of 6.0-8.0, temperature of 20-37 deg.C, and reaction time of 3-120 hr. The Rhodococcus microorganism catalysis system has the characteristics of fermentation culture and convenient preservation. The method for preparing the chiral amide carboxylic acid, the chiral amide carboxylic ester or the chiral amide alcohol compound by biotransformation has the characteristics of simple and convenient operation, high reaction efficiency, mild reaction conditions, high enantioselectivity, easy separation of products and high product purity, and has important application value.

Drawings

FIG. 1 is a reaction equation for producing chiral amide carboxylic acid compounds represented by Ia in example 1 of the present invention.

FIG. 2 shows the reaction scheme for the preparation of the meso-diamide compound represented by IIa in example 1 of the present invention.

FIG. 3 is a reaction equation for preparing chiral amide carboxylate compounds represented by Ib in example 2 of the present invention.

FIG. 4 is a reaction equation for preparing the chiral amide carboxylate compound Ic in example 3 of the present invention.

FIG. 5 shows the reaction scheme for preparing chiral amide carboxylate compounds represented by Id in example 4 of the present invention.

FIG. 6 shows the reaction equation for preparing the chiral amide carboxylate compound of Ie in example 5 of the present invention.

FIG. 7 is a reaction equation for preparing chiral amide alcohol compound represented by If in example 6 of the present invention.

FIG. 8 is a reaction equation for preparing chiral amide carboxylate compounds represented by Ig in example 7 of the present invention.

FIG. 9 shows the reaction scheme for preparing the meso-diamide compound of formula IIb in example 7 of the present invention.

FIG. 10 shows the reaction scheme for preparing chiral amide carboxylate compounds of formula Ih in example 8 according to the present invention.

FIG. 11 shows the reaction scheme for preparing chiral amide carboxylate compounds represented by formula Ii in example 9 of the present invention.

FIG. 12 shows the reaction scheme for preparing chiral amide carboxylate compounds represented by formula Ij in example 10.

FIG. 13 shows the reaction scheme for preparing the meso-diamide compound of formula IIc in example 10 of the present invention.

FIG. 14 shows the reaction scheme for preparing chiral amide carboxylate compounds of formula Ik in example 11.

FIG. 15 shows the reaction scheme for preparing the meso-diamide compound of formula IId in example 11 of the present invention.

FIG. 16 shows the reaction scheme for preparing chiral amide carboxylate compounds of formula Il in example 12.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.