CN110857276A - Chiral β -hydroxy amide compounds and preparation method and application thereof - Google Patents
Chiral β -hydroxy amide compounds and preparation method and application thereof Download PDFInfo
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- C07C235/40—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of rings other than six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
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- C07C235/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C235/06—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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- C07C69/757—Esters of carboxylic acids having an esterified carboxyl group bound to a carbon atom of a ring other than a six-membered aromatic ring having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
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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
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.
Example 1 preparation of chiral Amidocarboxylic acid Compounds Ia (R) of the formula I-11is-COOH, n is 3)
The chiral amide carboxylic acid compound Ia (R) is prepared according to the reaction equation shown in figure 11is-COOH, n is 3),
the specific implementation method comprises the following steps:
2g of wet Rhodococcus erythropolis AJ270 (viable: 1X 10)7-1×109CFU/g), thawing at 30 ℃ for 30 minutes, washing the thalli into an Erlenmeyer flat-bottomed flask with a threaded opening by using a buffer solution (0.1M, pH7.0,50ml) of dipotassium phosphate and potassium dihydrogen phosphate, dispersing and shaking uniformly, then putting into a shaking table for activating at 30 ℃ for 30 minutes, then adding 1mmol (186mg) of a compound shown by the formula IIa at one time, putting into the shaking table for catalytic hydrolysis at 30 ℃ and 200 rpm. Monitoring the whole reaction by TLC, stopping the reaction after 12h, filtering the obtained reaction solution by a layer of diatomite to remove thalli, washing filter residue for three times by 20mL of water in sequence to obtain the compound (R) shown as the formula I-1 provided by the invention1-COOH, n was 3), 178mg of product Ia in total, 95% yield and 99.5% ee.
The product is a solid;
melting point mp: 145-148 ℃;
1H NMR(500MHz,DMSO-d6)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).
13C NMR(500MHz,DMSO-d6)δ(ppm)177.14,176.68,67.47,47.85,47.55,24.64,22.79,22.26.
HRMS(ESI+)calcd for[M-H]-(C8H12O4N-),186.07718,found 186.07645.
as can be seen from the above, the compound has the correct structure and is represented by Ia.
Wherein the meso-diamide compound represented by the formula IIa as a reactant is prepared by the following method according to the reaction formula shown in FIG. 2:
adding magnesium chips (3.6g,150mmol) into dry ethanol (200mL), using a small amount of iodine simple substance as an initiator, preparing a magnesium alkoxide reagent under a reflux condition until the solution becomes white and turbid, then dropwise adding 1, 3-acetone diethyl dicarboxylate (9.2mL,50mmol), heating and refluxing for 1h, stopping heating, dropwise adding 1, 3-dibromopropane (5mL,50mmol), violently releasing heat during dropwise adding, and heating and refluxing again overnight after dropwise adding. After the heating was stopped and the system was cooled to room temperature, the ethanol solution in the system was distilled off under reduced pressure, the residue was dissolved in 1M hydrochloric acid (150mL), extracted with ethyl acetate (3X 150mL), the organic phases were combined and washed with saturated brine (3X 100mL), and dried over anhydrous magnesium sulfate. After removal of the solvent, the residue was loaded onto 100-200 mesh silica gel and the wash was a mixed solvent of petroleum ether and ethyl acetate (15: 1). The compound cyclohexanone diethyl 2, 6-dicarboxylate (8.8g, 73%) was collected. Sodium borohydride (76mg,2mmol) was added to a solution of the compound diethyl 2, 6-dicarboxylate (484mg,2mmol) in ethanol (12mL) at 0 ℃ for 5min, and after reaction, a saturated ammonium chloride solution (10mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (3X 50mL), and the organic phases were combined, washed with saturated brine (3X 30mL), and dried over anhydrous magnesium sulfate. After removal of the solvent, the residue was loaded on 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 2, 6-cyclohexanol dicarboxylate (180mg, 37%) was collected.
Methanol (10mL) was added to a 10mL reaction vessel, followed by the addition of the cis compound diethyl 2, 6-cyclohexanol dicarboxylate (732mg,3mmol) to completely dissolve the compound, and then 2mL of liquid ammonia was added thereto at low temperature, and the reaction was stirred at room temperature for 5 d. After methanol in the reaction system was removed, ethyl acetate (10mL) was added to the residue, followed by filtration with suction after sonication to give a white solid. Compound IIa (221mg, 40%) was collected.
Example 2 preparation of chiral amide carboxylate Ib (R) of formula I-21is-COOBn, n is 3)
The chiral amide carboxylic acid compound I b (R) is prepared according to the reaction equation shown in FIG. 31is-COOBn, n is 3),
the specific implementation method comprises the following steps:
2g of wet Rhodococcus erythropolis AJ270 strain was thawed at 30 ℃ for 30 minutes, washed into a threaded Erlenmeyer flask with a buffer solution of dipotassium hydrogenphosphate and potassium dihydrogenphosphate (0.1M, pH7.0,50ml), dispersed and shaken, then placed into a shaker for activation at 30 ℃ for 30 minutes, and then 1mmol (186mg) of a compound represented by formula IIa was added at a time, and placed into the shaker at 30 ℃ for catalytic hydrolysis reaction at 200 rpm. Monitoring the whole reaction by TLC, stopping the reaction after 12h, removing thalli from the obtained reaction solution by suction filtration through a layer of diatomite, washing filter residue three times by using 20mL of water in sequence, removing the solvent by using a rotary evaporator, and directly putting the generated monoamide monocarboxylic acid product into the next reaction without separation. To the residue were added DMF (2ml), K2CO3(138mg,1mmol) and benzyl bromide (342mg,2mmol) were stirred at room temperature (25 ℃ C.) for 24 hours to completion. Thereafter adding H2O (25ml), extraction with ethyl acetate (3X 25ml), anhydrous MgSO4Drying, removing the solvent by using a rotary evaporator, carrying out dry-method sample loading, and carrying out flash column chromatography to obtain the compound Ib (R) of the formula I-2 provided by the invention1is-COOBn, R2is-H, n is 3), the yield is 66 percent, and the compound shown in the structure Ib can be subjected to chiral resolution by using a high performance liquid chromatography ODH column, and the ee value of the compound shown in the structure Ib is 99.5 percent.
The product is a solid;
melting point mp: 116 ℃ and 117 ℃;
1H NMR(400MHz,CDCl3,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).
13C NMR(500MHz,CDCl3,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 C15H19NO4:C,64.97；H,6.91；N,5.05.Found:C,64.76H,6.93；N,5.21.
as can be seen from the above, the above-mentioned compound has a correct structure and is a compound represented by Ib.
Example 3 preparation of chiral amide carboxylate Ic (R) of formula I-21is-COOCH2C6H4Br, n is 3)
According to the reaction equation shown in FIG. 4, the chiral amide carboxylic acid compound Ic (R) is prepared1is-COOCH2C6H4Br, n is 3),
the specific implementation method comprises the following steps: 2g of wet Rhodococcus erythropolis AJ270 strain was thawed at 30 ℃ for 30 minutes, washed into a threaded Erlenmeyer flask with a buffer solution of dipotassium hydrogenphosphate and potassium dihydrogenphosphate (0.1M, pH7.0,50ml), dispersed and shaken, then placed into a shaker at 30 ℃ for activation for 30 minutes, and then 1mmol (186mg) of a compound represented by formula IIa was added at a time, and placed into the shaker at 30 ℃ for catalytic hydrolysis reaction at 200 rpm. Monitoring the whole reaction by TLC, stopping the reaction after 12h, removing thalli from the obtained reaction solution by suction filtration through a layer of diatomite, washing filter residue three times by using 20mL of water in sequence, removing the solvent by using a rotary evaporator, and directly putting the generated monoamide monocarboxylic acid product into the next reaction without separation. To the residue were added DMF (5ml), K2CO3(1.38g,10mmol), o-bromobenzyl bromide (1mL,8.6mmol) and stirring at room temperature (25 ℃ C.) were carried out (48 hours). Thereafter adding H2O (25ml), extraction with ethyl acetate (3X 25ml), anhydrous MgSO4Drying, removing the solvent by using a rotary evaporator, carrying out dry-method sample loading, and carrying out flash column chromatography to obtain the compound Ic (R) of the formula I-2 provided by the invention1is-COOCH2C6H4Br, n was 3) in a total of 201mg, yield 57%, ee value 99.5%.
The product is a solid;
melting point mp: 158 ℃ and 159 ℃;
H NMR(400MHz,CDCl3,TMS)δ(ppm)7.59(dd,J＝8.0,1.3Hz,1H),7.41(dd,J＝7.6,1.7Hz,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).
13C NMR(500MHz,CDCl3,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]+(C15H18O4NBrNa+),378.03114,found 378.03052.
Anal.Calcd.for C15H18BrNO4:C,50.58；H,5.09；N,3.93.Found:C,50.47；H,5.26；N,3.86.
as can be seen from the above, the compound has a correct structure and is represented by Ic.
Example 4 preparation of chiral amide carboxylate Id (R) of formula I-21is-COOCH3N is 3)
According to the reaction equation shown in FIG. 5, the chiral amide carboxylic acid compound Id (R) is prepared1is-COOCH3N is 3),
2g of wet Rhodococcus erythropolis AJ270 strain was thawed at 30 ℃ for 30 minutes, washed into a threaded Erlenmeyer flask with a buffer solution of dipotassium hydrogenphosphate and potassium dihydrogenphosphate (0.1M, pH7.0,50ml), dispersed and shaken, then placed into a shaker at 30 ℃ for activation for 30 minutes, then 1mmol (186mg) of a compound represented by formula IIa was added in one portion, and placed into a shaker at 30 DEG CThe catalytic hydrolysis reaction was carried out at 200 rpm. Monitoring the whole reaction by TLC, stopping the reaction after 12h, removing thalli from the obtained reaction solution by suction filtration through a layer of diatomite, washing filter residue three times by using 20mL of water in sequence, removing the solvent by using a rotary evaporator, and directly putting the generated monoamide monocarboxylic acid product into the next reaction without separation. Methanol (2mL) was added to the residue to dissolve it, and CH was slowly added dropwise at 0 deg.C2N2(2M,10mL) ether solution, naturally raising the temperature to room temperature, reacting overnight, removing the solvent by spinning, and performing flash column chromatography to obtain the compound Id (R) of the formula I-2 provided by the invention1is-COOCH3N is 3) in total 46mg, yield 23% and ee value 99.5%.
The product is a solid;
melting point mp: 124-126 ℃;
1H NMR(500MHz,CDCl3,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).
13C NMR(500MHz,CDCl3,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]+(C9H16O4N+),202.10738,found 202.10732.
as can be seen from the above, the compound has a correct structure and is represented by Id.
Example 5 preparation of chiral amide carboxylic ester compound Ie (R) of formula I-21is-COOCH2CH＝CH2N is 3)
According to the reaction equation shown in FIG. 6, preparing the chiral amide carboxylic acid compound Ie (R)1is-COOCH2CH＝CH2N is 3),
the specific implementation method comprises the following steps:
compound Ia (100mg,0.53mmol) was added to DMF (1ml), K2CO3(74mg,0.53mmol), allyl bromide (90uL,1mmol) and stirring at room temperature (25 ℃ C.) for one day (24h) were complete. Thereafter adding H2O(25ml)，Extraction with ethyl acetate (3X 25ml), anhydrous MgSO4Drying, removing the solvent by using a rotary evaporator, carrying out dry-method sample loading, and carrying out flash column chromatography to obtain the compound Ie (R) of the formula I-2 provided by the invention1is-COOCH2CH＝CH2N is 3), yield is 83%, ee value is 99.5%.
The product is a solid;
melting point mp: 119-122 ℃;
1H NMR(500MHz CDCl3,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).
13C NMR(500MHz,CDCl3,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]+(C11H17O4NNa+),250.10498,found 250.10486.
as can be seen from the above, the compound has the correct structure and is a compound represented by Ie.
Example 6 preparation of chiral amidoalcohol Compound If of formula I-3 (R)1is-CH2OH, n is 3)
The chiral amide alcohol compound If (R) is prepared according to the reaction equation shown in FIG. 71is-CH2OH, n is 3),
the specific implementation method comprises the following steps:
substrate Ib (55mg, 0.2mmol) was dissolved in a mixed solvent of EtOH (2ml) and THF (2ml), and LiCl (17mg, 0.4mmol) and NaBH were added4(15.2mg, 0.4mmol), the substrate disappeared after 18h reaction at room temperature, and saturated ammonium chloride solution was added to the reaction system to quench the reaction. Removing the solvent by spinning, loading the sample by a dry method, and performing flash column chromatography to obtain the compound If (R) of the formula I-3 provided by the invention1is-CH2OH, n is 3) in total 33mg, 93% yield. The compound with the structure can be subjected to chiral resolution by using a high performance liquid chromatography ADH column, and the result shows that the structure of If isThe enantioselectivity of the compounds shown is 99.5%.
The product was a colorless oily liquid;
1H NMR(500MHz,DMSO-d6)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).
13C NMR(500MHz,DMSO-d6)δ(ppm)178.20,66.48,63.54,47.57,44.76,25.08,23.63,22.54.
HRMS(ESI+)calcd for[M+Na]+(C8H15O3NNa+),196.09441,found 196.09439.
as can be seen from the above, the compound has the correct structure and is represented by If.
Example 7 preparation of chiral amide carboxylate Ig (R) of formula I-21is-COOBn, n is 3)
The chiral amide carboxylate Ig (R) is prepared according to the reaction equation shown in FIG. 81is-COOBn, n is 3)
The specific implementation method comprises the following steps:
2g of wet Rhodococcus erythropolis AJ270 strain is thawed at 30 ℃ for 30 minutes, washed into an Erlenmeyer flask with a threaded opening by using a buffer solution (0.1M, pH7.0 and 50ml) of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, dispersed and shaken uniformly, then placed into a shaking table for activation at 30 ℃ for 30 minutes, and then 1mmol (186mg) of a compound shown in formula IIb is added at a time, and placed into the shaking table for catalytic hydrolysis reaction at 30 ℃ and 200 rpm. Monitoring the whole reaction by TLC, stopping the reaction after 12h, removing thalli from the obtained reaction solution by suction filtration through a layer of diatomite, washing filter residue three times by using 20mL of water in sequence, removing the solvent by using a rotary evaporator, and directly putting the generated monoamide monocarboxylic acid product into the next reaction without separation. To the residue were added DMF (2ml), K2CO3(138mg,1mmol), benzyl bromide (342mg,2mmol) and stirring at room temperature (25 ℃ C.) was complete (24 h). Thereafter adding H2O (25ml), ethyl acetate (3X 25ml) extractTaking, anhydrous MgSO4Drying, removing the solvent by using a rotary evaporator, carrying out dry-method sample loading, and carrying out flash column chromatography to obtain the compound Ig (R) of the formula I-2 provided by the invention1is-COOBn, n is 3), the yield is 91 percent, and the chiral resolution of the compound shown by the Ig structure can be carried out by utilizing a high performance liquid chromatography ODH column, and the result shows that the enantioselectivity of the compound shown by the Ig structure is 99.5 percent.
The product is a solid;
melting point mp: 128-129 ℃;
1H NMR(400MHz,CDCl3,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).
13C NMR(500MHz,CDCl3,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]+(C15H19O4NNa+),300.12063,found 300.12036.
Anal.Calcd.for C15H19NO4:C,64.97；H,6.91；N,5.05.Found:C,64.91H,6.96；N,5.01.
as can be seen from the above, the above-mentioned compounds have the correct structure and are represented by Ig.
Wherein the meso-diamide compound represented by the formula IIb as a reactant is prepared according to the reaction formula shown in FIG. 9 by the following method:
adding magnesium chips (3.6g,150mmol) into dry ethanol (200mL), preparing a magnesium alkoxide reagent under a reflux condition by taking a small amount of iodine simple substance as an initiator until the solution becomes white and turbid, then dropwise adding 1, 3-acetone diethyl dicarboxylate (9.2mL,50mmol), heating and refluxing for 1h, stopping heating, dropwise adding 1, 3-dibromopropane (5mL,50mmol), violently releasing heat in the dropwise adding process, and heating and refluxing again overnight after the dropwise adding is finished. After the heating was stopped and the system was cooled to room temperature, the ethanol solution in the system was distilled off under reduced pressure, the residue was dissolved in 1M hydrochloric acid (150mL), extracted with ethyl acetate (3X 150mL), the organic phases were combined and washed with saturated brine (3X 100mL), and dried over anhydrous magnesium sulfate. After removal of the solvent, the residue was loaded onto 100-200 mesh silica gel and the wash was a mixed solvent of petroleum ether and ethyl acetate (15: 1). The compound cyclohexanone diethyl 2, 6-dicarboxylate (8.8g, 73%) was collected. Sodium borohydride (76mg,2mmol) was added to a solution of the compound diethyl 2, 6-dicarboxylate (484mg,2mmol) in ethanol (12mL) at 0 ℃ for 5min, and after reaction, a saturated ammonium chloride solution (10mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (3X 50mL), and the organic phases were combined, washed with saturated brine (3X 30mL), and dried over anhydrous magnesium sulfate. After removal of the solvent, the residue was loaded on 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 diethyl 2, 6-dicarboxylate (53mg, 11%) was collected.
After methanol (20mL) was added to a 75mL pressure-resistant bottle and the trans compound diethyl 2, 6-dicarboxylate (2.44g,10mmol) was added to completely dissolve the compound, 4mL of liquid ammonia was added at low temperature and the reaction was stirred at room temperature for 7 days, during which a white solid precipitated. After methanol in the reaction system was removed, ethyl acetate (20mL) was added to the residue, followed by filtration with suction after sonication to give a white solid. Compound IIb (1.41g, 76%) was collected.
Example 8 preparation of chiral amide carboxylate Ih (R) of formula I-21is-COOCH2C6H4Br, n is 3)
The chiral amide carboxylate Ih (R) is prepared according to the reaction equation shown in FIG. 101is-COOCH2C6H4Br, n is 3), the specific implementation method is as follows:
2g of wet Rhodococcus erythropolis AJ270 strain (institute of chemical research, national academy of sciences) was thawed at 30 ℃ for 30 minutes, washed into a threaded Erlenmeyer flask with a buffer solution of dipotassium hydrogenphosphate and potassium dihydrogenphosphate (0.1M, pH7.0,50ml), dispersed and shaken, activated at 30 ℃ for 30 minutes in a shaking table, and then 0.73mmol (136mg) of a compound represented by formula IIb was added at a timeThe resulting mixture was put into a shaker at 30 ℃ and 200rpm to conduct catalytic hydrolysis. Monitoring the whole reaction by TLC, stopping the reaction after 12h, removing thalli from the obtained reaction solution by suction filtration through a layer of diatomite, washing filter residue three times by using 20mL of water in sequence, removing the solvent by using a rotary evaporator, and directly putting the generated monoamide monocarboxylic acid product into the next reaction without separation. To the residue were added DMF (5ml), K2CO3(1.38g,10mmol), o-bromobenzyl bromide (1mL,8.6mmol) and stirring at room temperature (25 ℃ C.) was complete (48 h). Thereafter adding H2O (25ml), extraction with ethyl acetate (3X 25ml), anhydrous MgSO4Drying, removing the solvent by using a rotary evaporator, carrying out dry-method sample loading, and carrying out flash column chromatography to obtain the compound Ih (R) of the formula I-2 provided by the invention1is-COOCH2C6H4Br, n was 3) in a total of 62mg, yield 24%, ee value 99.5%.
The product is a solid;
melting point mp: 181-183 ℃;
1H NMR(500MHz,CDCl3,TMS)δ(ppm)7.57(dd,J＝8.0,1.2Hz,1H),7.40(dd,J＝7.6,1.7Hz,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).
13C NMR(500MHz,CDCl3,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]+(C15H18O4NBrNa+),378.03114,found 378.03050.
Anal.Calcd.for C15H18BrNO4:C,50.58；H,5.09；N,3.93.Found:C,50.42；H,5.06；N,3.83.
as can be seen from the above, the compound has a correct structure and is a compound represented by Ih.
Example 9 preparation of formula I-2 chiral amide carboxylate Ii (R)1is-COOCH3N is 3)
The chiral amide carboxylic acid compound Ia (R) is prepared according to the reaction equation shown in FIG. 111is-COOH, n is 3), the specific implementation method is as follows:
2g of wet Rhodococcus erythropolis AJ270 strain is thawed at 30 ℃ for 30 minutes, washed into an Erlenmeyer flask with a threaded opening by using a buffer solution (0.1M, pH7.0 and 50ml) of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, dispersed and shaken uniformly, then placed into a shaking table for activation at 30 ℃ for 30 minutes, and then 1mmol (186mg) of a compound shown in formula IIb is added at a time, and placed into the shaking table for catalytic hydrolysis reaction at 30 ℃ and 200 rpm. Monitoring the whole reaction by TLC, stopping the reaction after 12h, removing thalli from the obtained reaction solution by suction filtration through a layer of diatomite, washing filter residue three times by using 20mL of water in sequence, removing the solvent by using a rotary evaporator, and directly putting the generated monoamide monocarboxylic acid product into the next reaction without separation. Methanol (2mL) was added to the residue to dissolve it, and CH was slowly added dropwise at 0 deg.C2N2(2M,10mL) ether solution, naturally raising the temperature to room temperature, reacting overnight, removing the solvent, and performing flash column chromatography to obtain the compound Ii (R) of the formula I provided by the invention1is-COOCH3N is 3) in total 42mg, yield 21% and ee value 99.5%.
The product is a solid;
melting point mp: 156 ℃ and 158 ℃;
1H NMR(500MHz,CDCl3,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).
13C NMR(500MHz,CDCl3,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]+(C9H15O4NNa+),224.08933,found 224.08908.
as can be seen from the above, the above compounds have the correct structure and are represented by Ii.
Example 10 preparation of chiral amide carboxylate Ij (R) of formula I-21is-COOBn, n is 2)
According to the reaction equation shown in FIG. 12, chiral amide carboxylic acid compounds Ij (R) are prepared1is-COOBn, n is 2), the specific implementation method comprises the following steps:
2g of wet Rhodococcus erythropolis AJ270 strain is thawed at 30 ℃ for 30 minutes, washed into an Erlenmeyer flask with a threaded opening by using a buffer solution (0.1M, pH7.0 and 50ml) of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, dispersed and shaken uniformly, then placed into a shaking table for activation at 30 ℃ for 30 minutes, and then 1mmol (172mg) of a compound shown in formula IIc is added at a time, and placed into the shaking table for catalytic hydrolysis reaction at 30 ℃ and 200 rpm. Monitoring the whole reaction by TLC, stopping the reaction after 5h, removing thalli from the obtained reaction solution by suction filtration through a layer of diatomite, washing filter residue three times by using 20mL of water in sequence, removing the solvent by using a rotary evaporator, and directly putting the generated monoamide monocarboxylic acid product into the next reaction without separation. To the residue were added DMF (2ml), K2CO3(138mg,1mmol), benzyl bromide (342mg,2mmol) and stirring at room temperature (25 ℃ C.) was complete (24 h). Thereafter adding H2O (25ml), extraction with ethyl acetate (3X 25ml), anhydrous MgSO4Drying, removing the solvent by using a rotary evaporator, carrying out dry-method sample loading, and carrying out flash column chromatography to obtain the compound Ij (R) of the formula I-2 provided by the invention1is-COOBn, n is 2), the yield is 81 percent, and the chiral resolution of the compound shown by the Ij structure can be carried out by utilizing a high performance liquid chromatography ODH column, and the result shows that the enantioselectivity of the compound shown by the Ij structure is 99.5 percent.
The product is a solid;
melting point mp: 84-85 ℃;
1H NMR(400MHz,CDCl3,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).
13C NMR(400MHz,CDCl3,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]+(C14H17O4NNa+),286.10498,found 286.10498.
Anal.Calcd.for C14H17NO4:C,63.87；H,6.51；N,5.32.Found:C,63.80；H,6.49；N,5.36.
as can be seen from the above, the compound has the correct structure and is represented by Ij.
Wherein the meso-diamide compound represented by the formula IIc as a reactant is prepared according to the reaction formula shown in FIG. 13 by the following method:
potassium carbonate (4g,29mmol) was added to acetone (7.5mL), ethyl cyanoacetate (1mL,9.4mmol) was added dropwise under heating and refluxing, and after reaction for half an hour, 1, 2-dibromoethane (1.61mL,18.8mmol) was added dropwise and reacted overnight under refluxing. After the reaction system was returned to room temperature, water was added to quench the reaction (10mL), the mixture was extracted with ethyl acetate (3X 50mL), the organic phases were combined and washed with saturated brine (3X 50mL), and the mixture was dried over anhydrous magnesium sulfate. After removal of the solvent, the residue was loaded onto 100-200 mesh silica gel and the wash was a mixed solvent of petroleum ether and ethyl acetate (15: 1). Cyclopropanecarbonitrile compounds (1.06g, 82%) were collected.
Under anhydrous condition, adding simple substance sodium (288mg,12.5mmol) into dry anhydrous ethanol (4mL), and heating and refluxing at 70 ℃ until the sodium simple substance is completely dissolved. After that, the reaction system was lowered to 50 ℃ and diethyl malonate (2g,12.5mmol) was added, and after reacting for half an hour, cyclopropanecarbonitrile compounds (1.74g,12.5mmol) were added to the system. After 7min of reaction, the reaction was quenched by pouring the system into an ice-water mixture (10mL), extracted with ethyl acetate (3X 50mL), the organic phases were combined and washed with saturated brine (3X 50mL), and dried over anhydrous magnesium sulfate. After removal of the solvent, the residue was loaded onto 100-200 mesh silica gel and the wash was a mixed solvent of petroleum ether and ethyl acetate (15: 1). The imine compound (1.03g, 36%) was collected.
To an imine compound (227mg,1mmol,200uL) was added concentrated HCl (400uL) in twice its volume, and the reaction was stirred at room temperature for 5min, after which it was transferred to boiling water (800uL), followed by immediately cooling in ice water. Water (5mL) was added to the reaction system, followed by extraction with ethyl acetate (3X 20mL), and the organic phases were combined, washed with saturated brine (3X 20mL), and dried over anhydrous magnesium sulfate. After removal of the solvent, the residue was loaded on 100-200 mesh silica gel and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (10: 1). The cyclopentanone dicarboxylic acid diethyl ester compound (190mg, 83%) was collected.
Sodium borohydride (41mg,1.1mmol) was added to a solution of diethyl cyclopentanone dicarboxylate compound (248mg,1.1mmol) in ethanol (10mL) at 0 deg.C, and after 5min of reaction, a saturated ammonium chloride solution (5mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (3X 30mL), and the organic phases were combined, washed with saturated brine (3X 30mL), and dried over anhydrous magnesium sulfate. After removal of the solvent, the residue was loaded on 100-200 mesh silica gel and the washing solution was a mixed solvent of petroleum ether and ethyl acetate (10: 1). The trans-cyclopentanedicarboxylic acid diethyl ester compound (26mg, 10%) was collected.
After methanol (5mL) was added to a 15mL pressure-resistant bottle and then trans-cyclopentanedicarboxylic acid diethyl ester compound (143mg,0.6mmol) was completely dissolved, 3mL of liquid ammonia was added at low temperature and the reaction was stirred at room temperature for 12d, during which a white solid precipitated. After methanol in the reaction system was removed, ethyl acetate (10mL) was added to the residue, followed by filtration with suction after sonication to give a white solid. Compound IIc (91mg, 89%) was collected.
Example 11 preparation of chiral amide carboxylate Ik (R) of formula I-21is-COOBn, n is 0)
The chiral amide carboxylic ester compound Ik (R) is prepared according to the reaction equation shown in figure 141is-COOBn, n is 0), the specific implementation method comprises the following steps:
2g of wet Rhodococcus erythropolis AJ270 strain is thawed at 30 ℃ for 30 minutes, washed into an Erlenmeyer flask with a threaded opening by using a buffer solution (0.1M, pH7.0,50ml) of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, dispersed and shaken uniformly, then put into a shaking table to be activated at 30 ℃ for 30 minutes, then 1mmol (146mg) of a compound shown in formula IId is added at one time, and the mixture is put into the shaking table to be activated at 30 ℃ and 200rpmAnd (4) carrying out catalytic hydrolysis reaction under the condition. Monitoring the whole reaction by TLC, stopping the reaction after 7h, removing thalli from the obtained reaction solution by suction filtration through a layer of diatomite, washing filter residue three times by using 20mL of water in sequence, removing the solvent by using a rotary evaporator, and directly putting the generated monoamide monocarboxylic acid product into the next reaction without separation. To the residue were added DMF (2ml), K2CO3(138mg,1mmol), benzyl bromide (342mg,2mmol) and stirring at room temperature (25 ℃ C.) was complete (24 h). Thereafter adding H2O (25ml), extraction with ethyl acetate (3X 25ml), anhydrous MgSO4Drying, removing the solvent by using a rotary evaporator, carrying out dry-method sample loading, and carrying out flash column chromatography to obtain the compound Ik (R) of the formula I-2 provided by the invention1is-COOBn, n is 0) in a total amount of 213mg, at a yield of 76%, and chiral resolution of the compound represented by the Ik structure was carried out using a high performance liquid chromatography ADH column, and the result showed that the ee value of the compound represented by the Ik structure was 28%.
The product is a solid;
melting point mp: 91-93 ℃;
1H NMR(500MHz,CDCl3,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).
13C NMR(500MHz,CDCl3,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]+(C12H15O4NNa+),260.08933,found 260.08940.
Anal.Calcd.for C12H15NO4:C,60.75；H,6.37；N,5.90.Found:C,60.67；H,6.18；N,5.85.
as can be seen from the above, the compound has the correct structure and is represented by Ik.
Wherein the meso-diamide compound represented by the formula IId as a reactant is prepared according to the reaction formula shown in FIG. 15 by the following method:
sodium borohydride (190mg,5mmol) was added to a solution of compound diethyl 1, 3-acetonedicarboxylate (1.01g,5mmol) in ethanol (30mL) at 0 deg.C, and after 3min of reaction, a saturated solution of ammonium chloride (15mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (3X 50mL), and the organic phases were combined, washed with saturated brine (3X 30mL), and dried over anhydrous magnesium sulfate. After removal of 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-isopropanodicarboxylate compound (970mg, 95%) was collected.
After methanol (10mL) was added to a 25mL pressure-resistant bottle, followed by the addition of a diethyl 1, 3-isopropanodicarboxylate compound (950mg,4.6mmol) to completely dissolve it, 4mL of liquid ammonia was added at low temperature, and the reaction was stirred at room temperature for 6d, during which a white solid precipitated. After methanol in the reaction system was removed, ethyl acetate (20mL) was added to the residue, followed by filtration with suction after sonication to give a white solid. Compound IId (613mg, 90%) was collected.
Example 12 preparation of chiral amide carboxylic acid ester Compound Il (R) of formula I-21is-COOCH2CH＝CH2N is 0)
The chiral amide carboxylate compound Il (R) is prepared according to the reaction equation shown in FIG. 161is-COOCH2CH＝CH2N is 0)
The specific implementation method comprises the following steps:
2g of wet Rhodococcus erythropolis AJ270 strain is thawed at 30 ℃ for 30 minutes, washed into an Erlenmeyer flat-bottomed flask with a threaded opening by using a buffer solution (0.1M, pH7.0 and 50ml) of dipotassium hydrogen phosphate and potassium dihydrogen phosphate, dispersed and shaken uniformly, put into a shaking table for activation at 30 ℃ for 30 minutes, added with 1mmol (146mg) of a compound shown as formula IId at one time, and put into the shaking table for catalytic hydrolysis reaction at 30 ℃ and 200 rpm. Monitoring the whole reaction by TLC, stopping the reaction after 7h, removing thalli from the obtained reaction solution by suction filtration through a layer of diatomite, washing filter residue three times by using 20mL of water in sequence, removing the solvent by using a rotary evaporator, and directly putting the generated monoamide monocarboxylic acid product into the next reaction without separation. To the residue were added DMF (2ml), Cs2CO3(978mg,3mmol), allyl bromide (0.5mL) stirring at room temperature (25 ℃ C.) (24h) and reactionAnd finishing. Thereafter adding H2O (25ml), extraction with ethyl acetate (3X 25ml), anhydrous MgSO4Drying, removing the solvent by using a rotary evaporator, carrying out dry-method sample loading, and carrying out flash column chromatography to obtain the compound Il (R) of the formula I-2 provided by the invention1is-COOCH2CH＝CH2N is 0) in a total of 126mg, yield 67%, ee value 28%.
The product was a colorless oily liquid;
1H NMR(500MHz,CDCl3,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).
13C NMR(500MHz,CDCl3,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]+(C8H13O4NNa+),210.07368,found 210.07363.
as can be seen from the above, the compound has a correct structure and is a compound represented by Il.
Example 13: experiment on antitumor Activity
The chiral β -hydroxyamide compound prepared by the invention is directed to colon cancer cells HCT-116 which come from ATCC, and the anti-tumor experiment is as follows:
the influence of the compound shown in the structural general formula I on the cell activity is determined by using an MTT method. The MTT method is also called MTT colorimetric method, and is a method for detecting cell survival and growth. The detection principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-purple crystalline Formazan (Formazan) and deposit the blue-purple crystalline Formazan in the cells, and dead cells do not have the function. Methanol can dissolve formazan in cells, and the light absorption value of formazan is measured by an enzyme-linked immunosorbent detector at 490nm wavelength, which can indirectly reflect the number of living cells. The operation process is as follows: preparing a single cell suspension by using a culture solution containing 10% fetal calf serum, inoculating 1000-10000 cells per well to a 96-well plate, culturing for 24 hours in a 37-degree cell culture box, adding a drug to be detected, and continuously culturing for 48 hours in the 37-degree cell culture box by using methanol as a control; adding 20ul of MTT solution (5mg/ml prepared by PBS, pH 7.4) into each well (200ul of culture medium), continuing to incubate for 4h, stopping culturing, carefully sucking culture supernatant in each well, adding 150ul of methanol into each well, and shaking by a decoloration shaking table for 10min to fully dissolve crystals; the wavelength of 490nm (570nm) is selected, the light absorption value of each pore is measured on an enzyme-linked immunosorbent instrument, and the result is recorded.
The results of the anti-tumor activity experiment of the chiral β -hydroxyamide compound shown in formula I on colon cancer cell HCT-116, which is prepared by the invention and is examined by MTT method, are shown in the following Table 1, and the experimental results show that Ic, Ie and Ij in the compound shown in the structural general formula of formula I have a relatively obvious cell growth inhibition effect when the concentration is 100 mu mol.
The table shows the antitumor activity of compound Ia-l on colon cancer cell HCT-116: the data in the table refer to the ratio of the light absorption value in the sample solution to the light absorption value of the control group, and can indicate the growth inhibition capacity of the compound on the tumor cells, and the smaller the value, the stronger the inhibition capacity of the compound on the growth of the 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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