CN110857276A - A class of chiral β-hydroxyamide compounds, preparation method and application thereof - Google Patents

Abstract

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.

Description

A class of chiral β-hydroxyamide compounds, preparation method and application thereof

technical field

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

Background technique

Chiral β-hydroxyamides are widely found in the structures of biologically active drugs and natural products. For example, the anticancer drug Vincaleukoblastine and the antibacterial drug Pneumocandin A0. For the synthesis of chiral β-hydroxyamide compounds, the existing synthetic methods still have the disadvantages of low yield, complicated preparation of raw materials, and low stereoselectivity. Therefore, a new synthesis method for chiral β-hydroxyamide compounds is developed. is necessary.

Biocatalysis is by far the most efficient, selective and environmentally friendly process, and the use of biocatalysis to synthesize some chemicals with high added value, especially chiral chemicals, has important application prospects and significance. Nitriles are a class of important organic synthesis intermediates. The chemical transformation of nitrile requires harsh conditions and poor selectivity, while the biotransformation of nitrile has the advantages of mild conditions and high selectivity. At present, the corresponding carboxylic acids and Amide derivatives, the most famous is the industrialization of microbial synthesis of acrylamide was first realized by Nitto Company of Japan (now renamed Mitsubishi Rayon Company) in 1985. In 1998, the annual output of acrylamide exceeded 40,000 tons, making it one of the largest industrialized bioconversion pathways in the world. Rhodococcus rhodochrous J1 is also used by Lonza AG in Switzerland to industrially produce B vitamins nicotinamide and nicotinic acid, of which the annual output of nicotinamide has exceeded 3,000 tons. However, the biocatalytic reactions of nitriles and amides are relatively rare compared to enzyme-catalyzed ester hydrolysis and ester bond formation reactions.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a chiral beta-hydroxy amide compound and its preparation method and application.

The chiral β-hydroxyamide compound provided by the present invention, its structural formula is shown in formula I:

In the formula I, * represents chirality, which is R or S;

R ¹ is selected from any one of the following groups: -COOH, -COOR ² , -CH ₂ OH; wherein, R ² is selected from any one of the following groups: C ₁ -C ₆ alkyl, Allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl, p-bromobenzyl;

n represents the number of -CH ₂ - and is an integer of 0-4.

The compounds shown in the above formula I can be chiral amide carboxylic acid, chiral amide carboxylic acid ester and chiral amide alcohol compound according to the difference of R ¹ group, which are respectively shown in formula I-1 to formula I-3 compound;

In the formula I-1 to formula I-3, * represents chirality, which is R or S;

R ² is selected from any one of the following groups: C ₁ -C ₆ alkyl, allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl, p-bromobenzyl;

n represents the number of -CH ₂ - and is an integer of 0-4.

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

Under the catalysis of the Rhodococcus catalysis system, the achiral compound represented by the formula II is hydrolyzed to obtain the compound represented by the formula I (that is, the compound represented by the formula I-1) in which the R ¹ is -COOH;

In the formula II, n represents the number of -CH ₂ - and is an integer of 0-4.

In the above method, the Rhodococcus catalytic system is composed of Rhodococcus and a buffer solution with a pH value of 6.0-8.0.

The Rhodococcus can specifically be Rhodococcus erythropolis AJ270.

The catalytic system is prepared by the following method: connect the Rhodococcus in the buffer solution with a pH value of 6.0-8.0 and activate at 30° C. for 30 minutes.

The buffer solution is Na ₂ HPO ₄ -citrate buffer solution, K ₂ HPO ₄ -KH ₂ PO ₄ buffer solution, Tris buffer solution, Hanks' buffer solution or PBS buffer solution, specifically K ₂ HPO ₄ -KH ₂ PO ₄ buffer solution.

In the Rhodococcus catalytic system, the dosage ratio of Rhodococcus to the buffer solution is 2g (wet weight): 50mL-1L; wherein, the bacterial viability of Rhodococcus can be: 1×10 ⁷ -1×10 ⁹ CFU/ g.

The dosage ratio of the Rhodococcus to the compound represented by formula II is 2g:1mmol-1mol, specifically 2g:1mmol.

In the hydrolysis reaction, the temperature may be 20-37°C, specifically 30°C, and the time may be 3-120 hours, specifically 5-20 hours.

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

The present invention also provides a method for preparing the compound represented by formula I (that is, the compound represented by formula I-2) in which R ¹ is -CO ₂ R ² ,

When R ² is methyl, the method is:

a) react the compound of formula I whose R ¹ is -COOH in formula I (that is, the compound of formula I-1) with the ether solution of CH ₂ N ₂ in methanol, and after the reaction is completed, the R ¹ is obtained as The compound represented by formula I of -CO ₂ CH ₃ (that is, the compound represented by formula I-2); or,

b) react the compound shown in formula I whose R ¹ is -COOH in formula I (that is, the compound shown in formula I-1) with a base and methyl iodide in an organic solvent, and after the reaction is completed, obtain that the R ¹ is -CO The compound represented by the formula I of ₂ R ² (that is, the compound represented by the formula I-2)

When R ² is C ₂ -C ₆ alkyl, allyl, propargyl, benzyl, o-bromobenzyl, m-bromobenzyl, p-bromobenzyl, the method is:

a') react the compound of formula I whose R ¹ is -COOH in formula I (that is, the compound of formula I-1) with a base and R ² Br in an organic solvent, and after the reaction is completed, the R ¹ is obtained as The compound represented by formula I of -CO ₂ R ² (that is, the compound represented by formula I-2).

In the method a), the amount ratio of the compound represented by the formula I (that is, the compound represented by the formula I-1) in which R ¹ is -COOH, the diethyl ether solution of CH ₂ N ₂ and the methanol can be 0.1-10 mmol: 0.5- 50ml: 5-50mL, specifically 1mmol: 5mL: 10mL;

The concentration of the ether solution of CH ₂ N ₂ can be 0.1-5mol/L, specifically 2mol/L;

In the reaction, the temperature may be -20°C-30°C, specifically -15°C, and the time may be 1-48 hours, specifically 4 hours;

In described methods b) and a'), the alkali is at least one of potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide or cesium carbonate, specifically potassium carbonate;

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

^R2Br can be: bromoethane-hexane, 3-bromopropene, 3-bromopropyne, benzyl bromide, ortho-bromobenzyl bromide, meta-bromobenzyl bromide, or para-bromobenzyl bromide.

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

In the reaction, the temperature may be -20°C-50°C, specifically 25°C, and the time may be 6-48 hours, specifically 12 hours.

The present invention also provides a method for preparing the compound represented by formula I in which R ¹ is -CH ₂ OH (that is, the compound represented by formula I-3), the method comprising the following steps:

The compound shown in the formula I-2, sodium borohydride and lithium chloride are subjected to a reduction reaction in an organic solvent, and after the reaction is completed, the compound shown in the formula I (that is, the compound shown in the formula I-3) whose R ¹ is -CH ₂ OH is obtained is obtained. compound);

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

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

In the reaction, the temperature may be 0°C-50°C, specifically 25°C, and the time may be 1-48 hours, specifically 16 hours.

The application of the compound represented by the above formula I in the preparation of the following products also belongs to the protection scope of the present invention: 1) an inhibitor of eukaryotic tumor cell proliferation; 2) a drug for preventing and/or treating tumors.

In the application, the eukaryotic organism is a mammal; the tumor cell is a cancer cell; the tumor is a cancer;

The cancer cells are colon cancer cells; the cancer is colon cancer.

The Rhodococcus erythropolis AJ270 sample used in the present invention was originally isolated from an acetonitrile agar medium containing 25 mM in an Anderson bioparticle sampler, and the sample source was originally collected from an abandoned industrial plant near the banks of the Tyne in the United Kingdom. in dry soil. The chemical taxonomic study of mycolic acid and diaminopimelic acid on the cell wall of AJ270 confirmed that it belongs to the species Rhodococcus. Until 2005, it was confirmed that Rhodococcus AJ270 belongs to the Rhodococcus erythropolis strain through the study of its 16S rRNA gene sequence. Specifically refer to the following two documents:

a. Blakey A.J.; Colby J.; Williams E.; O'Reilly C., FEMS Microbiol. 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 shown to be a highly active whole-cell catalyst for nitrile hydratase/amidohydrolase systems. Studies have shown that compared with other strains, Rhodococcus erythropolis AJ270 has a good substrate broad spectrum and can efficiently catalyze the hydrolysis of aliphatic nitrile, aromatic nitrile and aromatic heterocyclic nitrile compounds. (Wang M.-X. Enantioselective biotransformations of nitrile in organic synthesis. Top. Catal. 2005, 35, 117-130).

The raw materials for preparing non-natural amino acid compounds provided by the invention are obtained by catalytically hydrolyzing diamide compounds with Rhodococcus erythropolis AJ270 microorganism system. The amount of Rhodococcus cells used can be adjusted according to the amount of substrate. The reaction solvent is a common buffer solution with pH=6.0-8.0, the temperature is 20-37°C, and the reaction time is 3-120 hours. The Rhodococcus microbial catalytic system has the characteristics of being fermentable, culturing and convenient for storage. The method for preparing chiral amide carboxylic acid, chiral amide carboxylic acid ester or chiral amide alcohol compound by using this biotransformation has the advantages of simple operation, high reaction efficiency, mild reaction conditions, high enantioselectivity, easy product separation and product purity. It has high characteristics and has important application value.

Description of drawings

Fig. 1 is the reaction equation for preparing the chiral amide carboxylic acid compound shown in Ia in Example 1 of the present invention.

Figure 2 is the reaction equation for preparing the meso diamide compound shown in IIa in Example 1 of the present invention.

3 is the reaction equation for preparing the chiral amide carboxylate compound shown in Ib in Example 2 of the present invention.

Figure 4 is the reaction equation for preparing the chiral amide carboxylate compound shown in Ic in Example 3 of the present invention.

Figure 5 is the reaction equation for preparing the chiral amide carboxylate compound shown in Id in Example 4 of the present invention.

Figure 6 is the reaction equation for preparing the chiral amide carboxylate compound shown in Ie in Example 5 of the present invention.

Figure 7 is the reaction equation for preparing the chiral amide alcohol compound shown in If in Example 6 of the present invention.

8 is the reaction equation for preparing the chiral amide carboxylate compound shown in Ig in Example 7 of the present invention.

Figure 9 is the reaction equation for preparing the meso diamide compound represented by formula IIb in Example 7 of the present invention.

Figure 10 is the reaction equation for preparing the chiral amide carboxylate compound represented by formula Ih in Example 8 of the present invention.

Figure 11 is the reaction equation for preparing the chiral amide carboxylate compound represented by formula Ii in Example 9 of the present invention.

Figure 12 is the reaction equation for preparing the chiral amide carboxylate compound represented by formula Ij in Example 10 of the present invention.

Figure 13 is a reaction equation for preparing the meso diamide compound represented by formula IIc in Example 10 of the present invention.

Figure 14 is the reaction equation for preparing the chiral amide carboxylate compound represented by formula Ik in Example 11 of the present invention.

Figure 15 is the reaction equation for preparing the meso diamide compound represented by formula IId in Example 11 of the present invention.

Figure 16 is the reaction equation for preparing the chiral amide carboxylate compound represented by formula II in Example 12 of the present invention.

Detailed ways

The present invention will be described below through specific embodiments, but the present invention is not limited thereto.

The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Preparation of chiral amide carboxylic acid compound Ia represented by formula I-1 (R ¹ is -COOH, n is 3)

According to the reaction equation shown in Figure 1, the chiral amide carboxylic acid compound Ia (R ¹ is -COOH, n is 3) is prepared,

The specific implementation method is:

Take 2 grams of wet weight Rhodococcus erythropolis AJ270 cells (viability: 1 × 10 ⁷ -1 × 10 ⁹ CFU/g), thaw at 30°C for 30 minutes, and use dipotassium hydrogen phosphate and potassium dihydrogen phosphate buffer. The solution (0.1M, pH 7.0, 50ml) was washed into the Erlenmeyer flat-bottomed flask with a threaded mouth, dispersed and shaken, and then placed in a shaker for activation at 30°C for 30 minutes, and then 1 mmol (186 mg) was added at one time. The compound represented by the formula IIa is put into a shaker at 30° C. and under the condition of 200 rpm to carry out the catalytic hydrolysis reaction. The whole reaction was monitored by TLC, the reaction was stopped after 12h, the obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells, and the filter residue was washed three times with 20 mL of water successively to obtain the compound shown in the formula I-1 provided by the present invention (R ¹ is - The product Ia of COOH, n is 3) is 178 mg in total, the yield is 95%, and the ee value is 99.5%.

the product is a solid;

Melting point mp: 145-148℃;

¹ H NMR (500MHz, DMSO-d ₆ )δ(ppm) 7.26(br,s,1H), 6.86(br,s,1H), 4.30(d,J=2.2Hz,1H), 2.11(dddd,J =16.8,12.9,4.2,2.2Hz,2H),1.76–1.39(m,5H),1.18(dt,J=13.1,3.9Hz,1H).

¹³ C NMR (500MHz, DMSO-d ₆ ) δ (ppm) 177.14, 176.68, 67.47, 47.85, 47.55, 24.64, 22.79, 22.26.

HRMS(ESI ⁺ )calcd for[MH] ^- (C ₈ H ₁₂ O ₄ N ^- ),186.07718,found 186.07645.

From the above, it can be seen that the above-mentioned compound has the correct structure and is the compound represented by Ia.

Wherein, the meso diamide compound shown in formula IIa as reactant is prepared by the following method according to the reaction equation shown in Figure 2:

Magnesium chips (3.6g, 150mmol) were added to dry ethanol (200mL), a small amount of iodine was used as an initiator, and a magnesium alkoxide reagent was first prepared under reflux conditions until the solution became white and cloudy, and then 1,3-acetone was added dropwise. Diethyl dicarboxylate (9.2 mL, 50 mmol) was heated to reflux for 1 h, then the heating was stopped, and 1,3-dibromopropane (5 mL, 50 mmol) was added dropwise. During the dropwise addition, there was a violent exotherm, and after the dropwise addition was completed, the heating was reheated. Reflux overnight. After the heating was stopped, the system was cooled to room temperature, the ethanol solution in the system was first distilled off under reduced pressure, the residue was dissolved with 1M hydrochloric acid (150 mL), extracted with ethyl acetate (3×150 mL), and the organic phases were combined with saturated brine ( 3×100 mL), and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was loaded onto 100-200 mesh silica gel, and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (15:1). The compound cyclohexanone diethyl 2,6-dicarboxylate (8.8 g, 73%) was collected. At 0 °C, sodium borohydride (76 mg, 2 mmol) was added to a solution of diethyl cyclohexanone 2,6-dicarboxylate (484 mg, 2 mmol) in ethanol (12 mL), and saturated chlorine was added after the reaction for 5 min. The reaction was quenched with ammonium chloride solution (10 mL). Extracted with ethyl acetate (3×50 mL), the organic phases were combined, washed with saturated brine (3×30 mL), and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was loaded onto 100-200 mesh silica gel, and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (10:1). The cis compound, diethyl cyclohexanol 2,6-dicarboxylate (180 mg, 37%) was collected.

Methanol (10mL) was added to the 10mL reactor, followed by adding cis compound cyclohexanol 2,6-diethyl carboxylate (732mg, 3mmol) to make it completely dissolved, and 2mL of liquid was added under low temperature conditions. Ammonia, the reaction was stirred at room temperature for 5d. After the methanol in the reaction system was removed, ethyl acetate (10 mL) was added to the residue, followed by sonication and suction filtration to obtain a white solid. Compound Ha (221 mg, 40%) was collected.

Example 2. Preparation of chiral amide carboxylate compound Ib represented by formula I-2 (R ¹ is -COOBn, n is 3)

According to the reaction equation shown in Figure 3, the chiral amide carboxylic acid compound I b (R ¹ is -COOBn, n is 3) is prepared,

The specific implementation method is:

Take 2 grams of wet Rhodococcus erythropolis AJ270 cells, thaw at 30°C for 30 minutes, and wash the cells into the threaded port with a buffer solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.1M, pH 7.0, 50ml). Erlenmeyer flat-bottomed flask, dispersed and shaken well, put it into a shaker for activation at 30°C for 30 minutes, then added 1 mmol (186mg) of the compound represented by formula IIa at one time, put it into a shaker at 30°C, and carried out under the condition of 200rpm. Catalytic hydrolysis reaction. The whole reaction was monitored by TLC, and the reaction was stopped after 12 hours of reaction. The obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells. The filter residue was washed with 20 mL of water three times in turn, and the solvent was removed by a rotary evaporator. The generated monoamide monocarboxylic acid product It was directly put into the next reaction without separation. To the residue were added DMF (2 ml), K ₂ CO ₃ (138 mg, 1 mmol) and benzyl bromide (342 mg, 2 mmol) and stirred at room temperature (25° C.) (24 hours) to complete the reaction. Then add H ₂ O (25ml), extract with ethyl acetate (3×25ml), dry with anhydrous MgSO ₄ , remove the solvent with a rotary evaporator, dry sample, and flash column chromatography to obtain the formula I- 2. The compound Ib (R ¹ is -COOBn, R ² is -H, and n is 3) is 182 mg in total, and the yield is 66%. The compound shown in structure 2 can be chiral resolved by using high performance liquid chromatography ODH column , the results show that the ee value of the compound represented by the Ib structure is 99.5%.

the product is a solid;

Melting point mp: 116-117℃;

¹ H NMR (400MHz, CDCl ₃ , TMS)δ(ppm) 7.49-7.28(m,5H), 6.76(br,s,1H), 5.59(br,s,1H), 5.16(s,2H), 4.53 (s, 1H), 2.83 (br, s, 1H), 2.42 (dd, J=12.2, 4.6Hz, 1H), 2.24 (d, J=11.6Hz, 1H), 2.07–1.70 (m, 5H), 1.36(d,J=13.2Hz,1H).

¹³ C NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 177.29, 174.92, 135.51, 128.66, 128.43, 128.13, 66.69, 65.92, 48.29, 46.61, 24.70, 23.46, 22.18.

HRMS(ESI+)calcd for[M+Na]+(C15H19O4NNa+),300.12063,found 300.12018.

Anal.Calcd.for C ₁₅ H ₁₉ NO ₄ : C, 64.97; H, 6.91; N, 5.05. Found: C, 64.76H, 6.93; N, 5.21.

From the above, it can be seen that the structure of the above-mentioned compound is correct, and it is the compound represented by Ib.

Example 3. Preparation of chiral amide carboxylate compound Ic represented by formula I-2 (R ¹ is -COOCH ₂ C ₆ H ₄ Br, n is 3)

The chiral amide carboxylic acid compound Ic was prepared according to the reaction equation shown in Figure 4 (R ¹ is -COOCH ₂ C ₆ H ₄ Br, n is 3),

The specific implementation method is as follows: take 2 grams of wet weight of Rhodococcus erythropolis AJ270 cells, thaw at 30°C for 30 minutes, use a buffer solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.1M, pH7.0, 50ml) to thaw the bacteria The body was washed into an Erlenmeyer flat-bottomed flask with a threaded mouth, dispersed and shaken, and then placed in a shaker for activation at 30°C for 30 minutes, and then 1 mmol (186 mg) of the compound represented by formula IIa was added at one time, and placed in a shaker for 30 minutes. The catalytic hydrolysis was carried out at 200 rpm. The whole reaction was monitored by TLC, and the reaction was stopped after 12 hours of reaction. The obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells. The filter residue was washed with 20 mL of water three times in turn, and the solvent was removed by a rotary evaporator. The generated monoamide monocarboxylic acid product It was directly put into the next reaction without separation. To the residue were added DMF (5 ml), K ₂ CO ₃ (1.38 g, 10 mmol), o-bromobenzyl bromide (1 mL, 8.6 mmol) and stirred at room temperature (25° C.) (48 hours) to complete the reaction. Then add H ₂ O (25ml), extract with ethyl acetate (3×25ml), dry with anhydrous MgSO ₄ , remove the solvent with a rotary evaporator, dry sample, and flash column chromatography to obtain the formula I- 2 The compound Ic (R ¹ is -COOCH ₂ C ₆ H ₄ Br, n is 3) is 201 mg in total, the yield is 57%, and the ee value is 99.5%.

the product is a solid;

Melting point mp: 158-159℃;

H NMR (400MHz, CDCl ₃ , TMS) δ (ppm) 7.59 (dd, J=8.0, 1.3 Hz, 1H), 7.41 (dd, J=7.6, 1.7 Hz, 1H), 7.33 (td, J=7.5, 1.3Hz,1H), 7.21(td,J=7.7,1.8Hz,1H), 6.67(br,s,1H), 5.50(br,s,1H), 5.24(d,J=2.4Hz,2H), 4.56(d,J＝2.0Hz,1H),2.94(s,1H),2.45(ddd,J＝12.2,5.1,1.9Hz,1H),2.32–2.19(m,1H),1.96–1.74(m, 5H),1.38(dd,J=10.7,6.5Hz,1H).

¹³ C NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 177.38, 174.56, 134.80, 132.95, 130.10, 129.99, 127.61, 123.56, 66.32, 65.93, 48.23, 46.65, 24.69, 23.45, 22.17.

HRMS(ESI+)calcd for [M+Na]+(C ₁₅ H ₁₈ O ₄ NBrNa+), 378.03114, found 378.03052.

Anal.Calcd.for C ₁₅ H ₁₈ BrNO ₄ : C, 50.58; H, 5.09; N, 3.93. Found: C, 50.47; H, 5.26; N, 3.86.

From the above, it can be seen that the above-mentioned compound has the correct structure and is the compound represented by Ic.

Example 4. Preparation of chiral amide carboxylate compound Id represented by formula I-2 (R ¹ is -COOCH ₃ , n is 3)

According to the reaction equation shown in Figure 5, the chiral amide carboxylic acid compound Id (R ¹ is -COOCH ₃ , n is 3) is prepared,

Take 2 grams of wet Rhodococcus erythropolis AJ270 cells, thaw at 30°C for 30 minutes, and wash the cells into the threaded port with a buffer solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.1M, pH 7.0, 50ml). Erlenmeyer flat-bottomed flask, dispersed and shaken well, put it into a shaker for activation at 30°C for 30 minutes, then added 1 mmol (186mg) of the compound represented by formula IIa at one time, put it into a shaker at 30°C, and carried out under the condition of 200rpm. Catalytic hydrolysis reaction. The whole reaction was monitored by TLC, and the reaction was stopped after 12 hours of reaction. The obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells. The filter residue was washed with 20 mL of water three times in turn, and the solvent was removed by a rotary evaporator. The generated monoamide monocarboxylic acid product It was directly put into the next reaction without separation. Methanol (2 mL) was added to the residue to dissolve, and CH ₂ N ₂ (2M, 10 mL) ether solution was slowly added dropwise at 0°C, and the solution was allowed to rise to room temperature naturally. A total of 46 mg of the compound Id (R ¹ is -COOCH ₃ , n is 3) of the formula I-2 provided by the present invention was obtained, the yield was 23%, and the ee value was 99.5%.

the product is a solid;

Melting point mp: 124-126℃;

¹ H NMR (500MHz, CDCl ₃ , TMS)δ(ppm) 6.67(br,s,1H), 5.53(br,s,1H), 4.51(s,1H), 3.73(s,3H), 3.24(br , s, 1H), 2.37 (dd, J=12.6, 4.2Hz, 1H), 2.24 (d, J=12.3Hz, 1H), 2.00–1.74 (m, 5H), 1.52–1.20 (m, 1H).

¹³ C NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 177.25, 175.56, 65.96, 52.07, 48.39, 46.54, 24.74, 23.45, 22.16.

HRMS(ESI ⁺ )calcd for[M+H] ⁺ (C ₉ H ₁₆ O ₄ N ⁺ ), 202.10738, found 202.10732.

From the above, it can be seen that the above-mentioned compound has the correct structure and is the compound represented by Id.

Example 5. Preparation of the chiral amide carboxylate compound Ie represented by the general structural formula of formula I-2 (R ¹ is -COOCH ₂ CH=CH ₂ , n is 3)

The chiral amide carboxylic acid compound Ie was prepared according to the reaction equation shown in Figure 6 (R ¹ is -COOCH ₂ CH=CH ₂ , n is 3),

The specific implementation method is:

Compound Ia (100 mg, 0.53 mmol) was added to DMF (1 ml), K ₂ CO ₃ (74 mg, 0.53 mmol), allyl bromide (90 uL, 1 mmol) and stirred at room temperature (25° C.) for one day (24 h) and the reaction was completed. Then add H ₂ O (25ml), extract with ethyl acetate (3×25ml), dry with anhydrous MgSO ₄ , remove the solvent with a rotary evaporator, dry sample, and flash column chromatography to obtain the formula I- 2 The compound Ie (R ¹ is -COOCH ₂ CH=CH ₂ , n is 3) is 100 mg in total, the yield is 83%, and the ee value is 99.5%.

the product is a solid;

Melting point mp: 119-122℃;

¹ H NMR (500MHz CDCl ₃ , TMS)δ(ppm)6.65(br,s,1H),5.91(ddt,J=16.5,11.0,5.7Hz,1H),5.63-5.43(br,m,1H), 5.41–5.16 (m, 2H), 4.63 (d, J=5.7Hz, 2H), 4.53 (s, 1H), 2.98 (br, s, 1H), 2.40 (ddd, J=12.4, 4.4, 1.7Hz, 1H), 2.24 (dt, J=12.8, 2.7Hz, 1H), 2.01–1.74 (m, 5H), 1.38 (ddt, J=13.6, 8.9, 3.5Hz, 1H).

¹³ C NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 177.22, 174.74, 131.71, 118.64, 65.97, 65.49, 48.41, 46.64, 24.73, 23.45, 22.17.

HRMS(ESI ⁺ )calcd for[M+Na] ⁺ (C ₁₁ H ₁₇ O ₄ NNa ⁺ ),250.10498,found 250.10486.

From the above, it can be seen that the structure of the above-mentioned compound is correct, and it is the compound represented by Ie.

Example 6. Preparation of chiral amide alcohol compound If (R ¹ is -CH ₂ OH, n is 3) represented by formula I-3

According to the reaction equation shown in Figure 7, the chiral amide alcohol compound If (R ¹ is -CH ₂ OH, n is 3) is prepared,

The specific implementation method is:

Substrate Ib (55 mg, 0.2 mmol) was dissolved in a mixed solvent of EtOH (2 ml) and THF (2 ml), LiCl (17 mg, 0.4 mmol) and NaBH ₄ (15.2 mg, 0.4 mmol) were added, and the reaction was carried out at room temperature for 18 h. The substrate disappeared, and saturated ammonium chloride solution was added to the reaction system to quench the reaction. Spin off the solvent, dry loading, and flash column chromatography to obtain the compound If (R ¹ is -CH ₂ OH, n is 3) of the formula I-3 provided by the present invention, a total of 33 mg, with a yield of 93%. The compound represented by the structure can be chiral resolved by high performance liquid chromatography ADH column, and the result shows that the enantioselectivity of the compound represented by the If structure is 99.5%.

The product is a colorless oily liquid;

¹ H NMR (500MHz, DMSO-d ₆ )δ(ppm) 7.33(br,s,1H), 6.92(br,s,1H), 4.57(d,J=2.9Hz,1H), 4.31(t,J =5.3Hz,1H),4.04(q,J=2.3Hz,1H),3.40(ddd,J=10.4,7.0,5.5Hz,1H),3.25–3.18(m,1H),2.09(ddd,J= 12.6, 3.9, 2.0Hz, 1H), 1.73–1.60 (m, 2H), 1.49 (dd, J=13.8, 4.0Hz, 1H), 1.42–1.30 (m, 2H), 1.30–1.10 (m, 2H) .

¹³ C NMR (500MHz, DMSO-d ₆ ) δ (ppm) 178.20, 66.48, 63.54, 47.57, 44.76, 25.08, 23.63, 22.54.

HRMS(ESI+)calcd for[M+Na]+(C ₈ H ₁₅ O ₃ NNa+), 196.09441, found 196.09439.

From the above, it can be seen that the above-mentioned compound has the correct structure and is the compound represented by If.

Example 7. Preparation of chiral amide carboxylate compound Ig represented by formula I-2 (R ¹ is -COOBn, n is 3)

The chiral amide carboxylate compound Ig was prepared according to the reaction equation shown in Figure 8 (R ¹ is -COOBn, n is 3)

The specific implementation method is:

Take 2 grams of wet Rhodococcus erythropolis AJ270 cells, thaw at 30°C for 30 minutes, and wash the cells into the threaded port with a buffer solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.1M, pH 7.0, 50ml). Erlenmeyer flat-bottomed flask, dispersed and shaken well, put it into a shaker for activation at 30 °C for 30 minutes, then added 1 mmol (186 mg) of the compound represented by formula IIb at one time, put it into a shaker at 30 °C, and carried out under the condition of 200rpm. Catalytic hydrolysis reaction. The whole reaction was monitored by TLC, and the reaction was stopped after 12 hours of reaction. The obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells. The filter residue was washed with 20 mL of water three times in turn, and the solvent was removed by a rotary evaporator. The generated monoamide monocarboxylic acid product It was directly put into the next reaction without separation. To the residue were added DMF (2 ml), K ₂ CO ₃ (138 mg, 1 mmol), benzyl bromide (342 mg, 2 mmol) and stirred at room temperature (25° C.) (24 h) to complete the reaction. Then add H ₂ O (25ml), extract with ethyl acetate (3×25ml), dry with anhydrous MgSO ₄ , remove the solvent with a rotary evaporator, dry sample, and flash column chromatography to obtain the formula I- 2 The compound Ig (R ¹ is -COOBn, n is 3) is 251 mg in total, and the yield is 91%. The compound shown in the Ig structure can be chiral resolved by using a high performance liquid chromatography ODH column. The enantioselectivity of the compound shown is 99.5%.

the product is a solid;

Melting point mp: 128-129℃;

¹ H NMR (400MHz, CDCl ₃ , TMS)δ(ppm) 7.51-7.30(m,5H), 6.27(br,s,1H), 5.68(br,s,1H), 5.17(d,J=1.8Hz) ,2H),4.03(t,J＝10.2Hz,1H),2.92(br,s,1H),2.44(ddd,J＝12.6,10.1,3.8Hz,1H),2.24(ddd,J＝12.7,10.2 ,3.9Hz,1H),2.04(dd,J=33.9,13.5Hz,2H),1.83(dt,J=13.4,3.3Hz,1H),1.63–1.21(m,3H).

¹³ C NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 176.54, 174.39, 135.71, 128.59, 128.27, 128.03, 71.32, 66.54, 50.58, 50.18, 27.98, 27.92, 24.31.

HRMS(ESI ⁺ )calcd for[M+Na] ⁺ (C ₁₅ H ₁₉ O ₄ NNa ⁺ ),300.12063,found 300.12036.

Anal.Calcd.for C ₁₅ H ₁₉ NO ₄ : C, 64.97; H, 6.91; N, 5.05. Found: C, 64.91H, 6.96; N, 5.01.

From the above, it can be seen that the structure of the above-mentioned compound is correct, and it is a compound represented by Ig.

Wherein, the meso diamide compound shown in formula IIb as reactant is prepared by the following method according to the reaction equation shown in Figure 9:

To dry ethanol (200mL), add magnesium scraps (3.6g, 150mmol) of magnesium scraps, use a small amount of iodine as an initiator, first prepare a magnesium alkoxide reagent under reflux conditions until the solution becomes white and turbid, then dropwise add 1, Diethyl 3-acetone dicarboxylate (9.2 mL, 50 mmol) was heated to reflux for 1 h, then the heating was stopped, and 1,3-dibromopropane (5 mL, 50 mmol) was added dropwise, and the dropwise addition process was vigorously exothermic, and the dropwise addition was completed. Then reheat to reflux overnight. After the heating was stopped, the system was cooled to room temperature, the ethanol solution in the system was first distilled off under reduced pressure, the residue was dissolved with 1M hydrochloric acid (150 mL), extracted with ethyl acetate (3×150 mL), and the organic phases were combined with saturated brine ( 3×100 mL), and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was loaded onto 100-200 mesh silica gel, and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (15:1). The compound cyclohexanone diethyl 2,6-dicarboxylate (8.8 g, 73%) was collected. At 0 °C, sodium borohydride (76 mg, 2 mmol) was added to a solution of diethyl cyclohexanone 2,6-dicarboxylate (484 mg, 2 mmol) in ethanol (12 mL), and saturated chlorine was added after the reaction for 5 min. The reaction was quenched with ammonium chloride solution (10 mL). Extracted with ethyl acetate (3×50 mL), the organic phases were combined, washed with saturated brine (3×30 mL), and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was loaded onto 100-200 mesh silica gel, and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (10:1). The trans compound cyclohexanol 2,6-dicarboxylate diethyl ester (53 mg, 11%) was collected.

Methanol (20 mL) was added to a 75 mL pressure bottle, followed by the trans compound cyclohexanol 2,6-diethyl carboxylate (2.44 g, 10 mmol) to dissolve it completely, and 4 mL of cyclohexanol was added at low temperature. Liquid ammonia was stirred at room temperature for 7 d, and white solid was precipitated during the reaction. After the methanol in the reaction system was removed, ethyl acetate (20 mL) was added to the residue, followed by sonication and suction filtration to obtain a white solid. Compound lib (1.41 g, 76%) was collected.

Example 8. Preparation of chiral amide carboxylate compound Ih represented by formula I-2 (R ¹ is -COOCH ₂ C ₆ H ₄ Br, n is 3)

According to the reaction equation shown in Figure 10, the chiral amide carboxylic acid ester compound Ih (R ¹ is -COOCH ₂ C ₆ H ₄ Br, n is 3) is prepared, and the specific implementation method is:

Take 2 grams of wet weight of Rhodococcus erythropolis AJ270 cells (Institute of Chemistry, Chinese Academy of Sciences), thaw at 30°C for 30 minutes, and use a buffer solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.1M, pH 7.0, 50ml). The bacteria were washed into an Erlenmeyer flat-bottomed flask with a threaded mouth, dispersed and shaken, and then placed in a shaker for activation at 30°C for 30 minutes, and then 0.73 mmol (136 mg) of the compound shown in formula IIb was added at one time, and put into a shaker. The catalytic hydrolysis reaction was carried out under the conditions of 30 °C and 200 rpm. The whole reaction was monitored by TLC, and the reaction was stopped after 12 hours of reaction. The obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells. The filter residue was washed with 20 mL of water three times in turn, and the solvent was removed by a rotary evaporator. The generated monoamide monocarboxylic acid product It was directly put into the next reaction without separation. To the residue were added DMF (5 ml), K ₂ CO ₃ (1.38 g, 10 mmol), o-bromobenzyl bromide (1 mL, 8.6 mmol) and stirred (48 h) at room temperature (25° C.) to complete the reaction. Then add H ₂ O (25ml), extract with ethyl acetate (3×25ml), dry with anhydrous MgSO ₄ , remove the solvent with a rotary evaporator, dry sample, and flash column chromatography to obtain the formula I- 2. The compound Ih (R ¹ is -COOCH ₂ C ₆ H ₄ Br, n is 3) has a total of 62 mg, the yield is 24%, and the ee value is 99.5%.

the product is a solid;

Melting point mp: 181-183℃;

¹ H NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 7.57 (dd, J=8.0, 1.2 Hz, 1H), 7.40 (dd, J=7.6, 1.7 Hz, 1H), 7.31 (td, J=7.5 ,1.2Hz,1H),7.19(td,J=7.7,1.7Hz,1H),6.27(br,s,1H),5.85(br,s,1H),5.33–5.12(m,2H),4.05( t,J＝10.1Hz,1H),3.00(br,s,1H),2.48(ddd,J＝13.4,10.2,3.8Hz,1H),2.23(ddd,J＝13.5,10.2,3.7Hz,1H) ,2.15–2.03(m,1H),1.97(dt,J=14.2,3.1Hz,1H),1.82(dp,J=13.5,3.3Hz,1H),1.62–1.41(m,2H),1.31(dtt ,J=25.1,14.5,5.3Hz,1H).

¹³ C NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 176.65, 174.12, 134.98, 132.86, 129.96, 129.83, 127.57, 123.42, 71.30, 66.21, 50.63, 50.21, 28.08, 27.93, 24.31.

HRMS(ESI ⁺ )calcd for [M+Na] ⁺ (C ₁₅ H ₁₈ O ₄ NBrNa ⁺ ), 378.03114, found 378.03050.

Anal.Calcd.for C ₁₅ H ₁₈ BrNO ₄ : C, 50.58; H, 5.09; N, 3.93. Found: C, 50.42; H, 5.06; N, 3.83.

From the above, it can be seen that the structure of the above compound is correct, and it is the compound represented by Ih.

Example 9. Preparation of chiral amide carboxylate compound Ii represented by formula I-2 (R ¹ is -COOCH ₃ , n is 3)

Prepare chiral amide carboxylic acid compound Ia (R ¹ is -COOH, n is 3) according to the reaction equation shown in Figure 11, and the specific implementation method is:

Take 2 grams of wet Rhodococcus erythropolis AJ270 cells, thaw at 30°C for 30 minutes, and wash the cells into the threaded port with a buffer solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.1M, pH 7.0, 50ml). Erlenmeyer flat-bottomed flask, dispersed and shaken well, put it into a shaker for activation at 30 °C for 30 minutes, then added 1 mmol (186 mg) of the compound represented by formula IIb at one time, put it into a shaker at 30 °C, and carried out under the condition of 200rpm. Catalytic hydrolysis reaction. The whole reaction was monitored by TLC, and the reaction was stopped after 12 hours of reaction. The obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells. The filter residue was washed with 20 mL of water three times in turn, and the solvent was removed by a rotary evaporator. The generated monoamide monocarboxylic acid product It was directly put into the next reaction without separation. Methanol (2 mL) was added to the residue to dissolve, and CH ₂ N ₂ (2M, 10 mL) ether solution was slowly added dropwise at 0°C, and the solution was allowed to rise to room temperature naturally. A total of 42 mg of the compound Ii of formula I provided by the present invention (R ¹ is -COOCH ₃ , and n is 3) was obtained with a yield of 21% and an ee value of 99.5%.

the product is solid;

Melting point mp: 156-158℃;

¹ H NMR (500MHz, CDCl ₃ , TMS)δ(ppm) 6.32(br,s,1H), 5.76(br,s,1H), 4.01(t, J＝10.2Hz,1H), 3.73(s,3H) ),3.31(br,s,1H),2.39(ddd,J=12.5,10.2,3.8Hz,1H),2.24(ddd,J=12.1,10.2,3.8Hz,1H),2.16–1.92(m,2H ),1.84(dq,J=13.3,3.0Hz,1H),1.57–1.14(m,3H).

¹³ C NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 176.60, 175.00, 71.32, 52.03, 50.33, 49.90, 27.90, 27.87, 24.34.

HRMS(ESI ⁺ )calcd for[M+Na] ⁺ (C ₉ H ₁₅ O ₄ NNa ⁺ ),224.08933,found 224.08908.

From the above, it can be seen that the structure of the above compound is correct, and it is the compound represented by Ii.

Example 10. Preparation of chiral amide carboxylate compound Ij represented by formula I-2 (R ¹ is -COOBn, n is 2)

The chiral amide carboxylic acid compound Ij (R ¹ is -COOBn, n is 2) is prepared according to the reaction equation shown in Figure 12, and the specific implementation method is:

Take 2 grams of wet Rhodococcus erythropolis AJ270 cells, thaw at 30°C for 30 minutes, and wash the cells into the threaded port with a buffer solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.1M, pH 7.0, 50ml). Erlenmeyer flat-bottomed flask, dispersed and shaken well, put it into a shaker for activation under 30°C for 30 minutes, then added 1 mmol (172mg) of the compound represented by formula IIc at one time, put it into a shaker at 30°C, and carried out under the condition of 200rpm. Catalytic hydrolysis reaction. The whole reaction was monitored by TLC, and the reaction was stopped after 5 hours of reaction. The obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells. The filter residue was washed with 20 mL of water three times in turn, and the solvent was removed with a rotary evaporator. The generated monoamide monocarboxylic acid product It was directly put into the next reaction without separation. To the residue were added DMF (2 ml), K ₂ CO ₃ (138 mg, 1 mmol), benzyl bromide (342 mg, 2 mmol) and stirred at room temperature (25° C.) (24 h) to complete the reaction. Then add H ₂ O (25ml), extract with ethyl acetate (3×25ml), dry with anhydrous MgSO ₄ , remove the solvent with a rotary evaporator, dry sample, and flash column chromatography to obtain the formula I- 2 The compound Ij (R ¹ is -COOBn, n is 2) is 213 mg in total, and the yield is 81%. The compound shown in the structure of Ij can be chiral resolved by using a high-performance liquid chromatography ODH column. The enantioselectivity of the compound shown is 99.5%.

the product is solid;

Melting point mp: 84-85℃;

¹ H NMR (400MHz, CDCl ₃ , TMS)δ(ppm) 7.45-7.28(m,5H), 6.25(br,s,1H), 5.74(br,s,1H), 5.16(s,2H), 4.38 (t, J=9.3Hz, 1H), 3.27 (br, s, 1H), 2.82 (q, J=9.1Hz, 1H), 2.65 (q, J=9.2Hz, 1H), 2.21–1.71 (m, 4H).

¹³ C NMR (400MHz, CDCl ₃ , TMS) δ (ppm) 175.65, 173.96, 135.66, 128.64, 128.36, 128.08, 77.56, 66.65, 51.41, 50.69, 24.35, 23.59.

HRMS(ESI ⁺ )calcd for[M+Na] ⁺ (C ₁₄ H ₁₇ O ₄ NNa ⁺ ), 286.10498, found 286.10498.

Anal.Calcd.for C ₁₄ H ₁₇ NO ₄ : C, 63.87; H, 6.51; N, 5.32. Found: C, 63.80; H, 6.49; N, 5.36.

From the above, it can be seen that the structure of the above compound is correct, and it is the compound represented by Ij.

Wherein, the meso diamide compound shown in formula IIc as a reactant is prepared by the following method according to the reaction equation shown in Figure 13:

Potassium carbonate (4 g, 29 mmol) was added to acetone (7.5 mL), ethyl cyanoacetate (1 mL, 9.4 mmol) was added dropwise under heating and reflux, and 1,2-dibromoethane ( 1.61 mL, 18.8 mmol), reacted overnight under reflux conditions. After the reaction system returned to room temperature, water was added to quench the reaction (10 mL), extracted with ethyl acetate (3×50 mL), the organic phases were combined, washed with saturated brine (3×50 mL), and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was loaded onto 100-200 mesh silica gel, and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (15:1). The cyclopropanenitrile compound (1.06 g, 82%) was collected.

Under anhydrous conditions, elemental sodium (288 mg, 12.5 mmol) was added to dry anhydrous ethanol (4 mL), and the mixture was heated to reflux at 70° C. until the elemental sodium was completely dissolved. After that, the reaction system was lowered to 50° C., and diethyl malonate (2 g, 12.5 mmol) was added. After half an hour of reaction, a cyclopropanenitrile compound (1.74 g, 12.5 mmol) was added to the system. After 7 min of reaction, the system was poured into ice-water mixture (10 mL) to quench the reaction, extracted with ethyl acetate (3×50 mL), the organic phases were combined, washed with saturated brine (3×50 mL), and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was loaded onto 100-200 mesh silica gel, and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (15:1). The imine compound (1.03 g, 36%) was collected.

To the imine compound (227 mg, 1 mmol, 200 uL), twice the volume of concentrated HCl (400 uL) was added, and the reaction was stirred at room temperature for 5 min, after which it was transferred to boiling water (800 uL), and then immediately cooled in ice water. Water (5 mL) was added to the reaction system, extracted with ethyl acetate (3×20 mL), the organic phases were combined, washed with saturated brine (3×20 mL), and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was loaded onto 100-200 mesh silica gel, and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (10:1). The cyclopentanone dicarboxylate diethyl ester compound (190 mg, 83%) was collected.

At 0°C, sodium borohydride (41 mg, 1.1 mmol) was added to a solution of diethyl cyclopentanone dicarboxylate compound (248 mg, 1.1 mmol) in ethanol (10 mL), and saturated ammonium chloride was added after the reaction for 5 min. The solution (5 mL) was quenched. Extracted with ethyl acetate (3×30 mL), the organic phases were combined, washed with saturated brine (3×30 mL), and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was loaded onto 100-200 mesh silica gel, and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (10:1). The trans-cyclopentanol dicarboxylate diethyl ester compound (26 mg, 10%) was collected.

Methanol (5 mL) was added to a 15 mL pressure bottle, followed by trans-cyclopentanol dicarboxylate diethyl ester compound (143 mg, 0.6 mmol) to dissolve it completely, and 3 mL of liquid ammonia was added at low temperature, The reaction was stirred at room temperature for 12 d, and a white solid was precipitated during the reaction. After the methanol in the reaction system was removed, ethyl acetate (10 mL) was added to the residue, followed by sonication and suction filtration to obtain a white solid. Compound IIc (91 mg, 89%) was collected.

Example 11. Preparation of chiral amide carboxylate compound Ik represented by formula I-2 (R ¹ is -COOBn, n is 0)

According to the reaction equation shown in Figure 14, the chiral amide carboxylate compound Ik (R ¹ is -COOBn, n is 0) is prepared, and the specific implementation method is:

Take 2 grams of wet Rhodococcus erythropolis AJ270 cells, thaw at 30°C for 30 minutes, and wash the cells into the threaded port with a buffer solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.1M, pH 7.0, 50ml). Erlenmeyer flat-bottomed flask, dispersed and shaken well, put it into a shaker for activation at 30°C for 30 minutes, then added 1 mmol (146mg) of the compound represented by formula IId at one time, put it into a shaker at 30°C, and carried out under the condition of 200rpm. Catalytic hydrolysis reaction. The whole reaction was monitored by TLC, and the reaction was stopped after 7 hours of reaction. The obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells. The filter residue was washed with 20 mL of water three times in turn, and the solvent was removed with a rotary evaporator. The generated monoamide monocarboxylic acid product It was directly put into the next reaction without separation. To the residue were added DMF (2 ml), K ₂ CO ₃ (138 mg, 1 mmol), benzyl bromide (342 mg, 2 mmol) and stirred at room temperature (25° C.) (24 h) to complete the reaction. Then add H ₂ O (25ml), extract with ethyl acetate (3×25ml), dry with anhydrous MgSO ₄ , remove the solvent with a rotary evaporator, dry sample, and flash column chromatography to obtain the formula I- 2 The compound Ik (R ¹ is -COOBn, n is 0) is 213 mg in total, and the yield is 76%. The compound shown in the Ik structure can be chiral resolved by using a high-performance liquid chromatography ADH column. The ee value of the compound shown is 28%.

the product is solid;

Melting point mp: 91-93℃;

¹ H NMR (500MHz, CDCl ₃ , TMS)δ(ppm) 7.48-7.30(m,5H), 6.27-6.00(br,m,1H), 5.58(br,s,1H), 5.16(s,2H) ,4.43(tt,J=8.1,4.4Hz,1H),3.57(br,s,1H),2.70–2.53(m,2H),2.51–2.36(m,2H).

¹³ C NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 173.51, 171.91, 135.42, 128.65, 128.46, 128.30, 66.69, 64.99, 41.36, 40.64.

HRMS(ESI ⁺ )calcd for[M+Na] ⁺ (C ₁₂ H ₁₅ O ₄ NNa ⁺ ),260.08933,found 260.08940.

Anal.Calcd.for C ₁₂ H ₁₅ NO ₄ : C, 60.75; H, 6.37; N, 5.90. Found: C, 60.67; H, 6.18; N, 5.85.

From the above, it can be seen that the above-mentioned compound has the correct structure and is the compound represented by Ik.

Wherein, the meso diamide compound shown in formula IId as the reactant is prepared by the following method according to the reaction equation shown in Figure 15:

At 0 °C, sodium borohydride (190 mg, 5 mmol) was added to a solution of diethyl 1,3-acetone dicarboxylate (1.01 g, 5 mmol) in ethanol (30 mL), and saturated chlorinated sodium chloride was added after the reaction for 3 min. Ammonium solution (15 mL) quenched the reaction. Extracted with ethyl acetate (3×50 mL), the organic phases were combined, washed with saturated brine (3×30 mL), and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was loaded onto 100-200 mesh silica gel, and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (5:1). The diethyl 1,3-isopropanol dicarboxylate compound (970 mg, 95%) was collected.

Methanol (10 mL) was added to a 25 mL pressure bottle, followed by adding 1,3-isopropanol dicarboxylate diethyl ester compound (950 mg, 4.6 mmol) to completely dissolve it, and then 4 mL of liquid ammonia was added at low temperature. , the reaction was stirred at room temperature for 6d, and a white solid was precipitated during the reaction. After the methanol in the reaction system was removed, ethyl acetate (20 mL) was added to the residue, followed by sonication and suction filtration to obtain a white solid. Compound Id (613 mg, 90%) was collected.

Example 12. Preparation of the chiral amide carboxylate compound Il represented by the general structural formula of formula I-2 (R ¹ is -COOCH ₂ CH=CH ₂ , n is 0)

The chiral amide carboxylate compound Il was prepared according to the reaction equation shown in Figure 16 (R ¹ is -COOCH ₂ CH=CH ₂ , n is 0)

The specific implementation method is:

Take 2 grams of wet Rhodococcus erythropolis AJ270 cells, thaw at 30°C for 30 minutes, and wash the cells into the threaded port with a buffer solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (0.1M, pH 7.0, 50ml). Erlenmeyer flat-bottomed flask, dispersed and shaken well, put it into a shaker for activation at 30°C for 30 minutes, then added 1 mmol (146mg) of the compound represented by formula IId at one time, put it into a shaker at 30°C, and carried out under the condition of 200rpm. Catalytic hydrolysis reaction. The whole reaction was monitored by TLC, and the reaction was stopped after 7 hours of reaction. The obtained reaction solution was filtered through a layer of diatomaceous earth to remove the bacterial cells. The filter residue was washed with 20 mL of water three times in turn, and the solvent was removed with a rotary evaporator. The generated monoamide monocarboxylic acid product It was directly put into the next reaction without separation. To the residue were added DMF (2 ml), Cs ₂ CO ₃ (978 mg, 3 mmol), allyl bromide (0.5 mL) and stirred at room temperature (25° C.) (24 h) to complete the reaction. Then add H ₂ O (25ml), extract with ethyl acetate (3×25ml), dry with anhydrous MgSO ₄ , remove the solvent with a rotary evaporator, dry sample, and flash column chromatography to obtain the formula I- 2. The compound I1 (R ¹ is -COOCH ₂ CH=CH ₂ , n is 0) has a total of 126 mg, the yield is 67%, and the ee value is 28%.

The product is a colorless oily liquid;

¹ H NMR (500MHz, CDCl ₃ , TMS)δ(ppm) 6.21(br,s,1H),5.92(ddt,J=17.2,10.4,5.8Hz,1H),5.61(br,s,1H),5.41 –5.21(m, 2H), 4.63(dt, J=5.9, 1.4Hz, 2H), 4.43(tt, J=7.8, 4.6Hz, 1H), 2.82–2.68(m, 1H), 2.66–2.53(m ,2H),2.53–2.42(m,2H).

¹³ C NMR (500MHz, CDCl ₃ , TMS) δ (ppm) 173.64, 171.80, 131.65, 118.81, 65.57, 64.95, 41.36, 40.54.

HRMS(ESI ⁺ )calcd for[M+Na] ⁺ (C ₈ H ₁₃ O ₄ NNa ⁺ ),210.07368,found 210.07363.

From the above, it can be seen that the structure of the above compound is correct, and it is the compound represented by I1.

Example 13: Antitumor Activity Experiment

The chiral beta-hydroxyamide compound prepared by the present invention is aimed at colon cancer cell HCT-116, which is from ATCC, and its anti-tumor experiment is as follows:

The MTT method was used to determine the effect of the compound represented by the general structural formula of formula I on cell activity. MTT method, also known as MTT colorimetric method, is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan (Formazan) and deposit in cells, while dead cells have no such function. Methanol can dissolve formazan in cells, and its light absorption value is measured at 490nm wavelength with enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. The operation process is as follows: prepare a single cell suspension with a culture medium containing 10% fetal calf serum, inoculate 1000-10000 cells per well into a 96-well plate, and incubate it in a 37-degree cell incubator for 24 hours. The drug was tested, and methanol was used as a control to continue to culture in a 37-degree cell incubator for 48 hours; add 20 ul (200 ul medium) of MTT solution (5 mg/ml in PBS, pH=7.4) to each well, continue to incubate for 4 h, and terminate the culture , carefully aspirate the culture supernatant in the wells, add 150ul methanol to each well, shake on a decolorizing shaker for 10 minutes to fully dissolve the crystals; select a wavelength of 490nm (570nm), measure the light absorption value of each well on an enzyme-linked immunosorbent monitor, and record result.

The MTT method is used to investigate the results of the anti-tumor activity test of the chiral β-hydroxyamide compounds of formula I prepared and provided in the present invention against colon cancer cells HCT-116, as shown in Table 1 below. When , Ic, Ie, and Ij in the compounds represented by the general structural formula of formula I have obvious inhibitory effects on cell growth.

The table shows the antitumor activity experiment of compound Ia-1 on colon cancer cells HCT-116: The data in the table refers to the ratio of the light absorption value in the sample solution to the light absorption value of the control group, which can indicate the growth inhibition ability of the compound on tumor cells , the smaller the value, the stronger the ability to inhibit the growth of tumor cells.

Claims (10)

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.

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