CN103288674B - Functionalized unnatural amino acids with quaternary carbon centers and biocatalytic desymmetrization preparation method thereof - Google Patents

Functionalized unnatural amino acids with quaternary carbon centers and biocatalytic desymmetrization preparation method thereof Download PDF

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CN103288674B
CN103288674B CN201310053582.5A CN201310053582A CN103288674B CN 103288674 B CN103288674 B CN 103288674B CN 201310053582 A CN201310053582 A CN 201310053582A CN 103288674 B CN103288674 B CN 103288674B
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CN103288674A (en
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王梅祥
王德先
张立彬
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Institute of Chemistry CAS
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Abstract

The invention discloses a functionalized unnatural chiral amino acid compounds with quaternary carbon centers and a preparation method thereof. In the invention, a Rhodococcus erythropolis AJ270 microbial system is taken for catalytic hydrolysis of prochiral malonamide compounds with different substituent groups so as to obtain corresponding unnatural amino acids with an amide group. The dosage of the Rhodococcus erythropolis can be adjusted according to the dosage of a substrate. The reaction solvent is a common buffer solution with a pH value of 6.0-8.0, and the reaction is performed at 20-37DEG C for 0.1-120h. The Rhodococcus erythropolis microbial catalytic system has the characteristics of fermentable cultivation and convenient preservation. The method for preparing monoamide carboxylic acid and dicarboxylic acid through bioconversion has the characteristics of simple operation, high efficiency reaction, mild reaction conditions, high enantioselectivity, easy separation of products, and high product purity, and can be used for synthesis of other unnatural chiral amino acids with a quaternary carbon center.

Description

The functionalized alpha-non-natural amino acid with quaternary carbon center and biocatalysis desymmetrization preparation method thereof
Technical field
The present invention relates to the functionalized alpha-non-natural amino acid with quaternary carbon center and biocatalysis desymmetrization preparation method thereof.
Background technology
Amino acids is the compounds that life entity the most extensively exists, and be the important composition material of life entity, it plays very important effect in vital process.And alpha-non-natural amino acid due to its structure and natural amino acid similar, often can as the surrogate of natural amino acid, the effect of research different aminoacids in vital process, alpha-non-natural amino acid has demonstrated certain pharmaceutical activity as the synthesis material of some growth factor receptor protein.Commercial alpha-non-natural amino acid has 2-Amino-2-methyl-3-phenylpropanoic acid (U.S. Pure Chemistry Scientific Inc sold) and 2-Amino-2-methyl-4-pentenoic acid (U.S. hexagon labs sold) at present.
Biocatalysis is the most efficient, highly selective and eco-friendly process up to now, utilize the method for biocatalysis to synthesize chemical that some have high added value, and particularly chiral chemistry product have important application prospect and meaning.Acid amides is the important organic synthesis intermediate of a class, the chemical conversion requirement condition of acid amides is harsh and selectivity is very poor, and the bioconversion reaction of acid amides has the advantage such as mild condition, highly selective, industrial can realization prepares corresponding carboxylic acid and amide derivatives from nitrile at present, and prepare carboxylic acid from acid amides, especially adopt the preparation of bio-transformation desymmetrization method with the chiral amides carboxylic acid compound of quaternary carbon center and the report of amino acid derivatives also little
Summary of the invention
The object of this invention is to provide a kind of functionalized Non-natural chiral amino acid with quaternary carbon center and biocatalysis desymmetrization preparation method thereof.
Compound shown in formula I provided by the invention,
(formula I)
In described formula I, * represents chirality, is R or S;
R 1be selected from-CH 3,-CH 2cH 3,-CH 2cH 2cH 3,-CH 2cH 2cH 2cH 3,-CH 2cH=CH 2,-CH 2cH=CHCH 3,-CH 2cH 2cH=CH 2,-CH 2cH 2cH 2cH=CH 2,-CH 2c ≡ CH ,-CH 2cH 2c ≡ CH ,-CH 2cH 2cH 2c ≡ CH ,-CH 2c ≡ CCH 3,-CH 2c ≡ CCH 2cH 3,-CH 2c ≡ CCH 2cH 2cH 3,-CH 2c 6h 5,-CH 2cH 2c 6h 5,-CH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2cH 2c 6h 5,-(CH 2) ncH=CHCH 3,-(CH 2) nc (CH 3)=CH 2with-(CH 2) ncH=CHC 6h 5in any one,
R 2be selected from-NH 2,-NHCH 3,-N (CH 3) 2,-NHCH 2cH 3,-N (CH 2cH 3) 2,-NHCH 2cH 2cH 3,-N (CH 2cH 2cH 3) 2,-NHCH 2cH 2cH 2cH 3,-N (CH 2cH 2cH 2cH 3) 2,-NHCH 2cH=CH 2,-N (CH 2cH=CH 2) 2, NH-CH 2cH=CHCH 3, N (-CH 2cH=CHCH 3) 2,-NHCH 2cH 2cH=CH 2,-N (CH 2cH 2cH=CH 2) 2,-NHCH 2cH 2cH 2cH=CH 2,-N (CH 2cH 2cH 2cH=CH 2) 2,-NHCH 2c ≡ CH ,-N (CH 2c ≡ CH) 2,-NHCH 2cH 2c ≡ CH ,-N (CH 2cH 2c ≡ CH) 2,-NHCH 2cH 2cH 2c ≡ CH ,-N (CH 2cH 2cH 2c ≡ CH) 2,-NHCH 2c ≡ CCH 3,-N (CH 2c ≡ CCH 3) 2,-NHCH 2c ≡ CCH 2cH 3,-N (CH 2c ≡ CCH 2cH 3) 2,-NHCH 2c ≡ CCH 2cH 2cH 3,-N (CH 2c ≡ CCH 2cH 2cH 3) 2,-NHCH 2c 6h 5,-N (CH 2c 6h 5) 2,-NHCH 2cH 2c 6h 5,-N (CH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2cH 2c 6h 5) 2,-NH (CH 2) ncH=CHCH 3,-N ((CH 2) ncH=CHCH 3) 2,-NH (CH 2) nc (CH 3)=C and-N ((CH 2) nc (CH 3)=C) 2in any one, n is the integer of 1-3;
X 1and X 2be selected from-CONH 2,-CH 2nH 2,-CO 2h, CH 2oH or-CO 2r 3in any one.R 3for-CH 3or-CH 2c 6h 5(being also-Bn).
Compound shown in above-mentioned formula I, according to its X 1and X 2group is different, can be chiral amides carboxylic acid, chiral amides carboxylicesters, chiral amides alcohol, chiral carboxylic acids alcohol or chiral amino alcohol compounds, is respectively compound shown in formula I-1 to formula I-5.
X in the above-mentioned formula I of preparation provided by the invention 1for-CONH 2, X 2for-CO 2the method of compound shown in the formula I of H (being also compound shown in formula I-1), comprises the steps:
Carry out catalytic hydrolysis reaction with compound shown in rhodococcus (Rhodococcus erythropolis AJ270) catalyst system and formula VI, react complete and obtain described X 1for-CONH 2, X 2for-CO 2compound shown in the formula I of H (being also compound shown in formula I-1);
(formula VI)
R 1be selected from-CH 3,-CH 2cH 3,-CH 2cH 2cH 3,-CH 2cH 2cH 2cH 3,-CH 2cH=CH 2,-CH 2cH=CHCH 3,-CH 2cH 2cH=CH 2,-CH 2cH 2cH 2cH=CH 2,-CH 2c ≡ CH ,-CH 2cH 2c ≡ CH ,-CH 2cH 2cH 2c ≡ CH ,-CH 2c ≡ CCH 3,-CH 2c ≡ CCH 2cH 3,-CH 2c ≡ CCH 2cH 2cH 3,-CH 2c 6h 5,-CH 2cH 2c 6h 5,-CH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2cH 2c 6h 5,-(CH 2) ncH=CHCH 3,-(CH 2) nc (CH 3)=CH 2with-(CH 2) ncH=CHC 6h 5in any one,
R 2be selected from-NH 2,-NHCH 3,-N (CH 3) 2,-NHCH 2cH 3,-N (CH 2cH 3) 2,-NHCH 2cH 2cH 3,-N (CH 2cH 2cH 3) 2,-NHCH 2cH 2cH 2cH 3,-N (CH 2cH 2cH 2cH 3) 2,-NHCH 2cH=CH 2,-N (CH 2cH=CH 2) 2, NH-CH 2cH=CHCH 3, N (-CH 2cH=CHCH 3) 2,-NHCH 2cH 2cH=CH 2,-N (CH 2cH 2cH=CH 2) 2,-NHCH 2cH 2cH 2cH=CH 2,-N (CH 2cH 2cH 2cH=CH 2) 2,-NHCH 2c ≡ CH ,-N (CH 2c ≡ CH) 2,-NHCH 2cH 2c ≡ CH ,-N (CH 2cH 2c ≡ CH) 2,-NHCH 2cH 2cH 2c ≡ CH ,-N (CH 2cH 2cH 2c ≡ CH) 2,-NHCH 2c ≡ CCH 3,-N (CH 2c ≡ CCH 3) 2,-NHCH 2c ≡ CCH 2cH 3,-N (CH 2c ≡ CCH 2cH 3) 2,-NHCH 2c ≡ CCH 2cH 2cH 3,-N (CH 2c ≡ CCH 2cH 2cH 3) 2,-NHCH 2c 6h 5,-N (CH 2c 6h 5) 2,-NHCH 2cH 2c 6h 5,-N (CH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2cH 2c 6h 5) 2,-NH (CH 2) ncH=CHCH 3,-N ((CH 2) ncH=CHCH 3) 2,-NH (CH 2) nc (CH 3)=C and-N ((CH 2) nc (CH 3)=C) 2in any one, n is the integer of 1-3;
In aforesaid method, the buffered soln that described rhodococcus (Rhodococcus erythropolis AJ270) catalyst system is 6.0-8.0 by rhodococcus and pH value forms, and this catalyst system is specially and described rhodococcus is connected to described pH value is that in the buffered soln of 6.0-8.0,30 DEG C of activation obtain for 30 minutes; Described buffered soln is Na 2hPO 4-citric acid solution, K 2hPO 4-KH 2pO 4buffered soln, Tris buffered soln, Hanks ' buffered soln or PBS buffered soln, preferred K 2hPO 4-KH 2pO 4buffered soln; The amount ratio of compound shown in described rhodococcus and formula VI is 2g: 1mmol-10mol, preferred 2g: 2mmol; In described rhodococcus catalyst system, the amount ratio of rhodococcus and described buffered soln is 2g: 50mL-1L, preferred 2g: 50mL; In described catalytic hydrolysis reaction step, temperature is 20-37 DEG C, preferably 30 DEG C, and the time is 0.1-240 hour, specifically can be 0.5-25 hour, 0.7-8 hour, 1-8 or 1.3-5 hour, preferred 0.5-10 hour.Different substrate and the preferred different time of consumption, make reaction product enantioselectivity between 90-99.5%;
Preparation X provided by the invention 1for-CONH 2, X 2for-CO 2r 3formula I shown in namely the method for compound (also formula I-2 shown in compound), be following method a and b
Wherein, method a comprises the steps:
By X in formula I 1for-CONH 2, X 2for-CO 2compound shown in the formula I of H (being also compound shown in formula I-1) and CH 2n 2diethyl ether solution react in methyl alcohol, react complete and obtain described X 1for-CONH 2, X 2for-CO 2cH 3formula I shown in namely compound (also formula I-2 shown in compound);
Described method b comprises the steps:
By X in formula I 1for-CONH 2, X 2for-CO 2compound shown in the formula I of H (being also compound shown in formula I-1) reacts in organic solvent with methyl iodide or benzyl bromine, reacts complete and obtains described X 1for-CONH 2, X 2for-CO 2cH 2c 6h 5formula I shown in namely compound (also formula I-2 shown in compound).
R 1be selected from-CH 3,-CH 2cH 3,-CH 2cH 2cH 3,-CH 2cH 2cH 2cH 3,-CH 2cH=CH 2,-CH 2cH=CHCH 3,-CH 2cH 2cH=CH 2,-CH 2cH 2cH 2cH=CH 2,-CH 2c ≡ CH ,-CH 2cH 2c ≡ CH ,-CH 2cH 2cH 2c ≡ CH ,-CH 2c ≡ CCH 3,-CH 2c ≡ CCH 2cH 3,-CH 2c ≡ CCH 2cH 2cH 3,-CH 2c 6h 5,-CH 2cH 2c 6h 5,-CH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2cH 2c 6h 5,-(CH 2) ncH=CHCH 3,-(CH 2) nc (CH 3)=CH 2with-(CH 2) ncH=CHC 6h 5in any one,
R 2be selected from-NH 2,-NHCH 3,-N (CH 3) 2,-NHCH 2cH 3,-N (CH 2cH 3) 2,-NHCH 2cH 2cH 3,-N (CH 2cH 2cH 3) 2,-NHCH 2cH 2cH 2cH 3,-N (CH 2cH 2cH 2cH 3) 2,-NHCH 2cH=CH 2,-N (CH 2cH=CH 2) 2, NH-CH 2cH=CHCH 3, N (-CH 2cH=CHCH 3) 2,-NHCH 2cH 2cH=CH 2,-N (CH 2cH 2cH=CH 2) 2,-NHCH 2cH 2cH 2cH=CH 2,-N (CH 2cH 2cH 2cH=CH 2) 2,-NHCH 2c ≡ CH ,-N (CH 2c ≡ CH) 2,-NHCH 2cH 2c ≡ CH ,-N (CH 2cH 2c ≡ CH) 2,-NHCH 2cH 2cH 2c ≡ CH ,-N (CH 2cH 2cH 2c ≡ CH) 2,-NHCH 2c ≡ CCH 3,-N (CH 2c ≡ CCH 3) 2,-NHCH 2c ≡ CCH 2cH 3,-N (CH 2c ≡ CCH 2cH 3) 2,-NHCH 2c ≡ CCH 2cH 2cH 3,-N (CH 2c ≡ CCH 2cH 2cH 3) 2,-NHCH 2c 6h 5,-N (CH 2c 6h 5) 2,-NHCH 2cH 2c 6h 5,-N (CH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2cH 2c 6h 5) 2,-NH (CH 2) ncH=CHCH 3,-N ((CH 2) ncH=CHCH 3) 2,-NH (CH 2) nc (CH 3)=C and-N ((CH 2) nc (CH 3)=C) 2in any one, n is the integer of 1-3;
In described method a, X 1for-CONH 2, X 2for-CO 2compound shown in the formula I of H (being also compound shown in formula I-1), CH 2n 2diethyl ether solution and the amount ratio of methyl alcohol be 0.1-10mmol: 0.5-50ml: 5-50mL, preferred 1mmol: 5mL: 10mL; Described CH 2n 2the concentration of diethyl ether solution be 0.1-5mol/L, preferred 2mol/L; In described reactions steps, temperature is-20 DEG C-30 DEG C, preferably 0 DEG C, and the time is 4-48 hour, preferably 12 hours;
In described method b, described alkali is salt of wormwood, sodium carbonate, potassium hydroxide, sodium hydroxide or cesium carbonate, preferred salt of wormwood; Described organic solvent is selected from least one in ether, tetrahydrofuran (THF), dioxane, DMF, preferred DMF; Described X 1for-CONH 2, X 2for-CO 2the amount ratio of compound shown in the formula I of H (being also compound shown in formula I-1), methyl iodide or benzyl bromine, alkali, organic solvent is 0.1-10mmol: 0.13-15mL: 0.14-14 g: 1-100mL, preferred 1mmol: 0.13-1mL: 0.27-1.38g: 2-5mL; In described reactions steps, temperature is-20 DEG C-50 DEG C, preferably 25 DEG C, and the time is 6-48 hour, preferably 12 hours.
Preparation X provided by the invention 1for-CONH 2, X 2for-CH 2the method of compound shown in the formula I of OH (being also compound shown in formula I-3) is following method:
Make X in formula I 1for-CONH 2, X 2for-CO 2r 3formula I shown in namely compound (also formula I-2 shown in compound) be reduced agent and reduce in a certain amount of organic solvent, react complete and obtain described X 1for-CONH 2, X 2for-CH 2compound shown in the formula I of OH (being also compound shown in formula I-3);
R 1be selected from-CH 3,-CH 2cH 3,-CH 2cH 2cH 3,-CH 2cH 2cH 2cH 3,-CH 2cH=CH 2,-CH 2cH=CHCH 3,-CH 2cH 2cH=CH 2,-CH 2cH 2cH 2cH=CH 2,-CH 2c ≡ CH ,-CH 2cH 2c ≡ CH ,-CH 2cH 2cH 2c ≡ CH ,-CH 2c ≡ CCH 3,-CH 2c ≡ CCH 2cH 3,-CH 2c ≡ CCH 2cH 2cH 3,-CH 2c 6h 5,-CH 2cH 2c 6h 5,-CH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2cH 2c 6h 5,-(CH 2) ncH=CHCH 3,-(CH 2) nc (CH 3)=CH 2with-(CH 2) ncH=CHC 6h 5in any one,
R 2be selected from-NH 2,-NHCH 3,-N (CH 3) 2,-NHCH 2cH 3,-N (CH 2cH 3) 2,-NHCH 2cH 2cH 3,-N (CH 2cH 2cH 3) 2,-NHCH 2cH 2cH 2cH 3,-N (CH 2cH 2cH 2cH 3) 2,-NHCH 2cH=CH 2,-N (CH 2cH=CH 2) 2, NH-CH 2cH=CHCH 3, N (-CH 2cH=CHCH 3) 2,-NHCH 2cH 2cH=CH 2,-N (CH 2cH 2cH=CH 2) 2,-NHCH 2cH 2cH 2cH=CH 2,-N (CH 2cH 2cH 2cH=CH 2) 2,-NHCH 2c ≡ CH ,-N (CH 2c ≡ CH) 2,-NHCH 2cH 2c ≡ CH ,-N (CH 2cH 2c ≡ CH) 2,-NHCH 2cH 2cH 2c ≡ CH ,-N (CH 2cH 2cH 2c ≡ CH) 2,-NHCH 2c ≡ CCH 3,-N (CH 2c ≡ CCH 3) 2,-NHCH 2c ≡ CCH 2cH 3,-N (CH 2c ≡ CCH 2cH 3) 2,-NHCH 2c ≡ CCH 2cH 2cH 3,-N (CH 2c ≡ CCH 2cH 2cH 3) 2,-NHCH 2c 6h 5,-N (CH 2c 6h 5) 2,-NHCH 2cH 2c 6h 5,-N (CH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2cH 2c 6h 5) 2,-NH (CH 2) ncH=CHCH 3,-N ((CH 2) ncH=CHCH 3) 2,-NH (CH 2) nc (CH 3)=C and-N ((CH 2) nc (CH 3)=C) 2in any one, n is the integer of 1-3;
In described method, reductive agent is lithium aluminum hydride, sodium borohydride, itrile group sodium borohydride, acetic acid sodium borohydride or borine, preferred sodium borohydride; Described organic solvent is selected from least one in ether, tetrahydrofuran (THF), dioxane, DMF, preferred tetrahydrofuran (THF); Described X 1for-CONH 2, X 2for-CO 2r 3formula I shown in namely compound (also formula I-2 shown in compound): reductive agent: the amount ratio of organic solvent is 0.1-10mmol: 0.04-14g: 1-100mL, preferred 1mmol: 0.08-1.38g: 2-50mL; In described reactions steps, temperature is-20 DEG C-50 DEG C, preferably 25 DEG C, and the time is 6-48 hour, preferably 12 hours.
Preparation X provided by the invention 1for-COOH, X 2for-CH 2the method of compound shown in the formula I of OH (being also compound shown in formula I-4) is following method a, b or c.
Wherein, method a comprises the steps:
By X in formula I 1for-CONH 2, X 2for-CH 2compound shown in the formula I of OH (being also compound shown in formula I-3) reacts under strong acid or basic conditions, reacts complete and obtains described X 1for-COOH, X 2for-CH 2compound shown in the formula I of OH (being also compound shown in formula I-4).
R 1be selected from-CH 3,-CH 2cH 3,-CH 2cH 2cH 3,-CH 2cH 2cH 2cH 3,-CH 2cH=CH 2,-CH 2cH=CHCH 3,-CH 2cH 2cH=CH 2,-CH 2cH 2cH 2cH=CH 2,-CH 2c ≡ CH ,-CH 2cH 2c ≡ CH ,-CH 2cH 2cH 2c ≡ CH ,-CH 2c ≡ CCH 3,-CH 2c ≡ CCH 2cH 3,-CH 2c ≡ CCH 2cH 2cH 3,-CH 2c 6h 5,-CH 2cH 2c 6h 5,-CH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2cH 2c 6h 5,-(CH 2) ncH=CHCH 3,-(CH 2) nc (CH 3)=CH 2with-(CH 2) ncH=CHC 6h 5in any one,
R 2be selected from-NH 2,-NHCH 3,-N (CH 3) 2,-NHCH 2cH 3,-N (CH 2cH 3) 2,-NHCH 2cH 2cH 3,-N (CH 2cH 2cH 3) 2,-NHCH 2cH 2cH 2cH 3,-N (CH 2cH 2cH 2cH 3) 2,-NHCH 2cH=CH 2,-N (CH 2cH=CH 2) 2, NH-CH 2cH=CHCH 3, N (-CH 2cH=CHCH 3) 2,-NHCH 2cH 2cH=CH 2,-N (CH 2cH 2cH=CH 2) 2,-NHCH 2cH 2cH 2cH=CH 2,-N (CH 2cH 2cH 2cH=CH 2) 2,-NHCH 2c ≡ CH ,-N (CH 2c ≡ CH) 2,-NHCH 2cH 2c ≡ CH ,-N (CH 2cH 2c ≡ CH) 2,-NHCH 2cH 2cH 2c ≡ CH ,-N (CH 2cH 2cH 2c ≡ CH) 2,-NHCH 2c ≡ CCH 3,-N (CH 2c ≡ CCH 3) 2,-NHCH 2c ≡ CCH 2cH 3,-N (CH 2c ≡ CCH 2cH 3) 2,-NHCH 2c ≡ CCH 2cH 2cH 3,-N (CH 2c ≡ CCH 2cH 2cH 3) 2,-NHCH 2c 6h 5,-N (CH 2c 6h 5) 2,-NHCH 2cH 2c 6h 5,-N (CH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2cH 2c 6h 5) 2,-NH (CH 2) ncH=CHCH 3,-N ((CH 2) ncH=CHCH 3) 2,-NH (CH 2) nc (CH 3)=C and-N ((CH 2) nc (CH 3)=C) 2in any one, n is the integer of 1-3;
Described method b comprises the steps:
Under the effect of Pd-C catalyzer, by described X 1for-CONH 2, X 2for-CH 2oH, R 1for-CH 2cH=CH 2,-CH 2cH 2cH=CH 2,-CH 2cH 2cH 2cH=CH 2,-CH 2c ≡ CH ,-CH 2cH 2c ≡ CH ,-CH 2cH 2cH 2c ≡ CH or-CH 2c ≡ CCH 3, R 2for-NH 2,-NHCH 3,-N (CH 3) 2,-NHCH 2c 6h 5or-N (CH 2c 6h 5) 2formula I shown in compound in H 2react in organic solvent in atmosphere, react with strong acid or highly basic more after completion of the reaction, react complete and obtain described X 1for-COOH, X 2for-CH 2oH, R 1for-CH 2cH 2cH 2,-CH 2cH 2cH 2cH 2,-CH 2cH 2cH 2cH 2cH 2,-CH 2cH 2cH ,-CH 2cH 2cH 2cH ,-CH 2cH 2cH 2cH 2cH or-CH 2cH 2cCH 3, R 2for-NH 2,-NHCH 3,-N (CH 3) 2or-NH 2formula I shown in compound;
Described method c comprises the steps:
By X in described formula I 1for-CONH 2, X 2for-CO 2compound shown in the formula I of R3 (being also compound shown in formula I-2) mixes with reductive agent and carries out reduction reaction in organic solvent, reacts after completion of the reaction again, react complete and obtain described X with the aqueous solution of strong acid or highly basic 1for-CONH 2, X 2for-CH 2compound shown in the formula I of OH (being also compound shown in formula I-3)
R 1be selected from-CH 3,-CH 2cH 3,-CH 2cH 2cH 3,-CH 2cH 2cH 2cH 3,-CH 2cH=CH 2,-CH 2cH=CHCH 3,-CH 2cH 2cH=CH 2,-CH 2cH 2cH 2cH=CH 2,-CH 2c ≡ CH ,-CH 2cH 2c ≡ CH ,-CH 2cH 2cH 2c ≡ CH ,-CH 2c ≡ CCH 3,-CH 2c ≡ CCH 2cH 3,-CH 2c ≡ CCH 2cH 2cH 3,-CH 2c 6h 5,-CH 2cH 2c 6h 5,-CH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2cH 2c 6h 5,-(CH 2) ncH=CHCH 3,-(CH 2) nc (CH 3)=CH 2with-(CH 2) ncH=CHC 6h 5in any one,
R 2be selected from-NH 2,-NHCH 3,-N (CH 3) 2,-NHCH 2cH 3,-N (CH 2cH 3) 2,-NHCH 2cH 2cH 3,-N (CH 2cH 2cH 3) 2,-NHCH 2cH 2cH 2cH 3,-N (CH 2cH 2cH 2cH 3) 2,-NHCH 2cH=CH 2,-N (CH 2cH=CH 2) 2, NH-CH 2cH=CHCH 3, N (-CH 2cH=CHCH 3) 2,-NHCH 2cH 2cH=CH 2,-N (CH 2cH 2cH=CH 2) 2,-NHCH 2cH 2cH 2cH=CH 2,-N (CH 2cH 2cH 2cH=CH 2) 2,-NHCH 2c ≡ CH ,-N (CH 2c ≡ CH) 2,-NHCH 2cH 2c ≡ CH ,-N (CH 2cH 2c ≡ CH) 2,-NHCH 2cH 2cH 2c ≡ CH ,-N (CH 2cH 2cH 2c ≡ CH) 2,-NHCH 2c ≡ CCH 3,-N (CH 2c ≡ CCH 3) 2,-NHCH 2c ≡ CCH 2cH 3,-N (CH 2c ≡ CCH 2cH 3) 2,-NHCH 2c ≡ CCH 2cH 2cH 3,-N (CH 2c ≡ CCH 2cH 2cH 3) 2,-NHCH 2c 6h 5,-N (CH 2c 6h 5) 2,-NHCH 2cH 2c 6h 5,-N (CH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2cH 2c 6h 5) 2,-NH (CH 2) ncH=CHCH 3,-N ((CH 2) ncH=CHCH 3) 2,-NH (CH 2) nc (CH 3)=C and-N ((CH 2) nc (CH 3)=C) 2in any one, n is the integer of 1-3;
In described method a, H in strong acid or strong alkali aqueous solution +or OH -concentration be 1-5M, preferred 2M; The amount ratio of compound and strong acid or strong alkali aqueous solution shown in described formula I-3 is 0.1-10m mol: 3-30mL, preferred 1mmol: 2mL; In described reactions steps, temperature is-20 DEG C-100 DEG C, preferably 40 DEG C-90 DEG C; Time is 1-24 hour, preferred 5-10 hour.
In described method b, in Pd-C catalyzer, the mass percent of Pd is 10%; Described organic solvent is all selected from least one in ethanol, methyl alcohol, DMF, tetrahydrofuran (THF), trichloromethane, tetracol phenixin, Benzene and Toluene, particular methanol or DMF; H in strong acid or strong alkali aqueous solution +or OH -concentration be 1-5M, preferred 2M; The amount ratio of compound and strong acid or strong alkali aqueous solution shown in described formula I-3 is 0.1-10m mol: 3-30mL, preferred 1mmol: 2mL; Described with strong acid or the aqueous solution of highly basic mix and carry out in reactions steps, temperature is-20 DEG C-100 DEG C, preferably 40 DEG C-90 DEG C; Time is 1-24 hour, preferred 5-10 hour.
In described method c, described reductive agent is lithium aluminum hydride, sodium borohydride, itrile group sodium borohydride, acetic acid sodium borohydride or borine, preferred sodium borohydride; Described organic solvent is selected from least one in ether, tetrahydrofuran (THF), dioxane, DMF, preferred tetrahydrofuran (THF); Described X 1for-CONH 2, X 2for-CO 2r 3formula I shown in namely compound (also formula I-2 shown in compound): reductive agent: the amount ratio of organic solvent is 0.1-10mmol: 0.04-14g: 1-100mL, preferred 1mmol: 0.08-1.38g: 2-5mL; In described reactions steps, temperature is-20 DEG C-50 DEG C, preferably 25 DEG C, and the time is 6-48 hour, preferably 12 hours; H in strong acid or strong alkali aqueous solution +or OH -concentration be 1-5M, preferred 2M; The amount ratio of compound and strong acid or strong alkali aqueous solution shown in described formula I-2 is 0.1-10mmol: 3-30mL, preferred 1mmol: 2mL; Described with strong acid or the aqueous solution of highly basic mix and carry out in reactions steps, temperature is-20 DEG C-100 DEG C, preferably 20 DEG C-90 DEG C; Time is 1-24 hour, preferred 5-10 hour.
Preparation X provided by the invention 1for-CH 2nH 2, X 2for-CH 2the method of compound shown in the formula I of OH (being also compound shown in formula I-5), comprises the steps:
By X in formula I 1for-CONH 2, X 2for-CH 2compound shown in the formula I of OH (being also compound shown in formula I-3) carries out reduction reaction with reductive agent in organic solvent, reacts complete and obtains described X 1for-CH 2nH 2, X 2for-CH 2compound shown in the formula I of OH (being also compound shown in formula I-5).
R 1be selected from-CH 3,-CH 2cH 3,-CH 2cH 2cH 3,-CH 2cH 2cH 2cH 3,-CH 2cH=CH 2,-CH 2cH=CHCH 3,-CH 2cH 2cH=CH 2,-CH 2cH 2cH 2cH=CH 2,-CH 2c ≡ CH ,-CH 2cH 2c ≡ CH ,-CH 2cH 2cH 2c ≡ CH ,-CH 2c ≡ CCH 3,-CH 2c ≡ CCH 2cH 3,-CH 2c ≡ CCH 2cH 2cH 3,-CH 2c 6h 5,-CH 2cH 2c 6h 5,-CH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2cH 2c 6h 5,-(CH 2) ncH=CHCH 3,-(CH 2) nc (CH 3)=CH 2with-(CH 2) ncH=CHC 6h 5in any one,
R 2be selected from-NH 2,-NHCH 3,-N (CH 3) 2,-NHCH 2cH 3,-N (CH 2cH 3) 2,-NHCH 2cH 2cH 3,-N (CH 2cH 2cH 3) 2,-NHCH 2cH 2cH 2cH 3,-N (CH 2cH 2cH 2cH 3) 2,-NHCH 2cH=CH 2,-N (CH 2cH=CH 2) 2, NH-CH 2cH=CHCH 3, N (-CH 2cH=CHCH 3) 2,-NHCH 2cH 2cH=CH 2,-N (CH 2cH 2cH=CH 2) 2,-NHCH 2cH 2cH 2cH=CH 2,-N (CH 2cH 2cH 2cH=CH 2) 2,-NHCH 2c ≡ CH ,-N (CH 2c ≡ CH) 2,-NHCH 2cH 2c ≡ CH ,-N (CH 2cH 2c ≡ CH) 2,-NHCH 2cH 2cH 2c ≡ CH ,-N (CH 2cH 2cH 2c ≡ CH) 2,-NHCH 2c ≡ CCH 3,-N (CH 2c ≡ CCH 3) 2,-NHCH 2c ≡ CCH 2cH 3,-N (CH 2c ≡ CCH 2cH 3) 2,-NHCH 2c ≡ CCH 2cH 2cH 3,-N (CH 2c ≡ CCH 2cH 2cH 3) 2,-NHCH 2c 6h 5,-N (CH 2c 6h 5) 2,-NHCH 2cH 2c 6h 5,-N (CH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2cH 2c 6h 5) 2,-NH (CH 2) ncH=CHCH 3,-N ((CH 2) ncH=CHCH 3) 2,-NH (CH 2) nc (CH 3)=C and-N ((CH 2) nc (CH 3)=C) 2in any one, n is the integer of 1-3;
In described method, described reductive agent is lithium aluminum hydride, sodium borohydride, itrile group sodium borohydride, acetic acid sodium borohydride or borine, preferred lithium aluminum hydride; Described organic solvent is selected from least one in ether, tetrahydrofuran (THF), dioxane, DMF, preferred tetrahydrofuran (THF); Described X 1for-CONH 2, X 2for-CH 2compound shown in the formula I of OH (being also compound shown in formula I-3): reductive agent: the amount ratio of organic solvent is 0.1-10mmol: 0.04-14g: 1-100mL, preferred 1mmol: 0.08-1.38g: 2-5mL; In described reactions steps, temperature is-20 DEG C-50 DEG C, preferably 25 DEG C, and the time is 6-96 hour, preferably 48 hours.
In addition, shown in the formula I that the invention described above provides, compound is preparing the application in inhibition tumor cell product, also belongs to protection scope of the present invention.Wherein, described tumour cell is specially colon cancer cell, is more specifically colon cancer cell HCT-116.
Rhodococcus Rhodococcus erythropolisAJ270 sample is use in Anderson biomone sampling thief to obtain from the acetonitrile nutrient agar separation containing 25mM at first, and sample source is then collected in the soil of the drying near the industrial working discarded at Britain's Tyne side at first.The mycolic acids of its cell walls of AJ270 bacterium and the research of diaminopimelic acid chemotaxonomy are confirmed it and belongs to Rhodococcus bacterial classification.Until 2005, by the research to its 16SrRNA gene order, confirm Rhodococcus AJ270 and belong to Rhodococcus erythropolis fungus strain.Concrete with reference to following two sections of 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 vanLeeuwenhoek2005，87，221-232.
Rhodococcus AJ270 is a kind of microorganism being derived from soil, and is proved to be a kind of highly active intact cell catalysis agent containing Nitrile hydratase/amide hydrolysis enzyme system.There are some researches show, compared with other bacterial strains, rhodococcus Rhodococcuserythropolis AJ270 has good substrate broad spectrum, can the hydrolysis of catalyze fatty nitrile, aromatic nitriles and fragrant heterocyclic nitrile compounds efficiently.(Wang M.-X.Enantioselective biotransformations of nitrile in organic synthesis.Top.Catal.2005，35，117-130)。
The present invention uses rhodococcus Rhodococcus erythropolis AJ270 microorganism system catalytic hydrolysis to obtain accordingly with the alpha-non-natural amino acid of amide group with the prochirality propane diamide compound of different substituents.Rhodococcus thalline consumption used can regulate according to the consumption of substrate.Reaction solvent is the conventional buffered soln of pH value 6.0-8.0, and temperature is 20-37 DEG C, and the reaction times is 0.1-120 hour.This rhodococcus microorganism catalysis system has can fermentation culture and preserve feature easily.Use this bio-transformation to prepare the method for chirality monoamide carboxylic acid, dicarboxylic acid, have easy and simple to handle, reaction is efficient, and reaction conditions is gentle, and enantioselectivity is high, and product is easily separated, the feature that product purity is high, has important using value.
Accompanying drawing explanation
Fig. 1 is the reaction process of method a;
Fig. 2 is the reaction process of method b;
Fig. 3 is the reaction process of method c;
Embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated, but the present invention is not limited to following examples.Described method is ordinary method if no special instructions.Described reactant all can obtain from open commercial sources if no special instructions.
The preparation method of biocatalysis substrate shown in formula VI described in mentioned microorganism catalytic process is following method a, b or c.
Wherein, method a comprises reaction process as shown in Figure 1:
The concrete steps of method a are as follows:
EtONa (10mmol) is joined in the EtOH (30ml) being positioned over ice-water bath, afterwards by raw material A (1.05g, 5mmol) join in system, slow dropping RBr (6mmol) ethanol (3ml) solution, after reacting about 3h, reaction system slowly adds water (100ml), and EA extracts (3 × 50ml), MgSO 4drying, revolves desolventizing, rapid column chromatography (filler silica gel: 100-200 order; Developping agent: PE/EA=8/1 ~ 4/1), obtain product B.Substrate B (2mmol) is joined MeOH and NH 3/ H 2in the mixed system of O (4ml/4ml), stir under ice-water bath, treat disappearance of substrate, add 1N HCl and adjust system ph=7, revolve most of solvent, rapid column chromatography, (filler silica gel: 100-200 order; Developping agent: EA), obtain white solid product VI.
Aforesaid method can R group be compound shown in allyl group, E-but-2-ene base, 2-methacrylic, propargyl equation VI for the preparation.
Wherein, method b comprises reaction process as shown in Figure 2:
The concrete steps of method b are as follows:
Substrate A (5mmol), benzophenone (5mmol) and tosic acid (0.5mmol) are put into there-necked flask, add the backflow of toluene post-heating, with fraction water device water-dividing, after reaction is spent the night, revolve desolventizing, rapid column chromatography, (filler silica gel: 100-200 order; Developping agent: PE: EA=15: 1 ~ 10: 1), obtains white solid product C (53%).Substrate C (25mmol) is dissolved in THF (125ml) and DMF (15ml) mixed solvent, add NaH (26mmol), slow dropping RBr (30mmol), after under room temperature, reaction is spent the night, add water (300ml), EA extracts (3 × 150ml), MgSO 4drying, revolves desolventizing, rapid column chromatography (filler silica gel: 100-200 order; Developping agent: PE/EA=30/1 ~ 20/1), obtain white solid D.Substrate D (1mmol) is dissolved in ether (5ml), adds 1N HCl (1ml) room temperature for overnight, be adjusted to ph=8 with the NaOH aqueous solution, add water (10ml), and EA extracts (3 × 20ml), MgSO 4drying, revolves desolventizing, rapid column chromatography (filler silica gel: 100-200 order; Developping agent: PE/EA=30/1 ~ 20/1), obtain white solid B.Substrate B (2mmol) is joined MeOH and NH 3/ H 2in the mixed system of O (4ml/4ml), stir under ice-water bath, treat disappearance of substrate, add 1N HCl and adjust system ph=7, revolve most of solvent, rapid column chromatography, (filler silica gel: 100-200 order; Developping agent: EA), obtain white solid product VI.
Aforesaid method can R group be compound shown in benzyl, 2-cinnamyl group, high allyl, 2-alkynes butyl equation VI for the preparation.
Wherein, method c comprises reaction process as shown in Figure 3:
The concrete steps of method c are as follows:
Claim B-1 (2.15g, 10mmol) add in single port bottle after methyl alcohol (50ml) stirred at ambient temperature dissolves and add palladium carbon (10%, 200mg), suspend and stir, air hydrogen exchange (with hydrogen balloon) is stirred, after disappearance of substrate afterwards, suction filtration, revolve desolventizing, obtain product B-2 (2.15g, 99%).Substrate B-2 (217mg, 1mmol) is placed in MeOH (1ml) and NH 3/ H 2in the mixed system of O (1ml), stir under ice-water bath, treat disappearance of substrate, add 1N HCl and adjust system ph=7, revolve most of solvent, rapid column chromatography, (filler silica gel: 100-200 order; Developping agent: EA), obtain white solid product VI-9 (74mg, 34%).
Embodiment 1: prepare compound 1
Specific implementation method:
Get Rhodococcus erythropolis AJ270 thalline (Institute of Chemistry, Academia Sinica) of 2 grams of weight in wet bases, thaw under 30 DEG C of conditions 30 minutes, with the buffered soln (0.1M of dipotassium hydrogen phosphate and potassium primary phosphate, pH7.0,50ml) thalline is washed in the Erlenmeyer Florence flask of threaded mouth, 30 minutes are activated under putting into shaking table 30 DEG C of conditions after dispersion shakes up, then compound shown in the disposable formula VI-1 adding 2mmol (314mg), put into shaking table 30 DEG C, under 200rpm condition, carry out catalytic hydrolysis reaction.Whole reaction TLC monitors, and reacts stopped reaction after 40 minutes, and gained reaction solution, by one deck suction filtered through kieselguhr removing thalline, washs filter residue three times with 20 milliliters, water successively, obtains compound (R shown in formula 1 provided by the invention 1for-CH 2cH=CH 2, R 2for-NH 2), product 275mg, productive rate is 87%.
Compound 1solid, mp145-146 DEG C; [α] 25d:-22.4 ° of (c1.70, H 2o); IR (KBr) ν 3397cm -1, 1668cm -1, 1516cm- 1; 1h NMR (300MHz, D 2o) 5.67-5.53 (m, 1H), 5.25-5.19 (m, 2H), 2.84-2.69 (m, 2H); (i)cNMR (75MHz, D 2o) 170.9,170.5,129.2,122.1,67.0,40.3; MS (ESI) m/z (%) 157 [M-1] +(100) .Anal.Calcd.for C 6h 10n 2o 3: 159.0765 [M+1] +.Found:157.0764 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 1.
Embodiment 2: prepare compound 2
Specific implementation method:
Compound 1 (100mg) is added DMF (5ml), K 2cO 3(1.5mmol), stir spend the night (12 hours) under benzyl bromine (2mmol) room temperature (25 DEG C) and react complete.Thereafter H is added 2o (25ml), ethyl acetate (3 × 25ml) extracts, anhydrous MgSO 4drying, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (R described in formula I-2 provided by the invention 1for-CH 2cH=CH 2, R 2for-NH 2, R 3for-Bn) 196mg, productive rate: 91%, ee97.0%.
Compound 2solid, mp145-146 DEG C; [α] 25 d:+17.9 ° of (c1.90, CHCl 3); Ee=97.0% (chiral HPLCanalysis); IR (KBr) ν 3437cm -1, 1742cm -1, 1689cm -1, 1202cm -1; 1h NMR (300MHz, CDCl 3) 7.32-7.19 (m, 10H), 7.08 (s, 1H), 5.80-5.66 (m, 2H), 5.29-5.13 (m, 4H), 3.61 (dd, J=26.7,12.0Hz, 2H), 2.97-2.84 (m, 2H) 2.31 (s, 1H); 13c NMR (75MHz, CDCl 3) 172.2,170.7,135.4,131.5,128.63,128.58,128.51,128.47,128.2,128.1,127.5,119.7,70.5,67.5,47.6,36.7; MS (ESI) m/z (%) 339 [M+H] +(100) .Anal.Calcd.for C 20h 22n 2o 3: C, 70.99; H, 6.55; N, 8.28.Found:C, 70.81; H, 6.57; N, 8.25.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 2.
Embodiment 3: prepare compound 3
1) Rhodococcus erythropolis AJ270 thalline (Institute of Chemistry, Academia Sinica) of two grams of weight in wet bases is got, thaw under 30 DEG C of conditions 30 minutes, with the buffered soln (0.1M of dipotassium hydrogen phosphate and potassium primary phosphate, pH7.0,50ml) thalline is washed in the Erlenmeyer Florence flask of 150 milliliters of threaded mouths, 30 minutes are activated under putting into shaking table 30 DEG C of conditions after dispersion shakes up, then compound shown in the disposable formula VI-2 adding 2mmol (342mg), put into shaking table 30 DEG C, under 200rpm condition, carry out catalytic hydrolysis reaction.Whole reaction TLC monitors, stopped reaction after reaction 50min, and gained reaction solution, by one deck suction filtered through kieselguhr removing thalline, washs filter residue three times with 20 milliliters, water successively, obtains compound (R shown in formula 3 provided by the invention 1for-CH 2cH=CHCH 3, R 2for-NH 2) 320mg, productive rate is 93%.
Compound 3solid, mp148-149 DEG C; [α] 25 d:-18.7 ° of (c1.50, H 2o); IR (KBr) ν 3326cm -1, 1685cm -1, 1508cm -1; 1h NMR (300MHz, D 2o) 5.78-5.66 (m, 1H), 5.32-5.22 (m, 1H), 2.87-2.66 (m, 2H), 1.61 (d, J=6.3Hz, 3H); 13c NMR (75MHz, D 2o) 170.9,170.6,134.1,121.2,67.3,39.4,17.3; MS (ESI) m/z (%) 195 [M+Na] +(33), 173 [M+H] +(100) .Anal.Calcd.for C 7h 12n 2o 3: 173.0921 [M+1] +.Found:173.0922 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 3.
Embodiment 4: prepare compound 4
Specific implementation method:
Compound 3 (100mg) is added DMF (5ml), K 2cO 3(1.5mmol), stir spend the night (12 hours) under benzyl bromine (2mmol) room temperature (25 DEG C) and react complete.Thereafter H is added 2o (25ml), ethyl acetate (3 × 25ml) extracts, anhydrous MgSO 4drying, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (R described in formula 4 provided by the invention 1for-CH 2cH=CH 2cH 3, R 2for-NHBn, R 3for-Bn) 127mg, productive rate: 62%, ee97.0%.
Compound 4solid, mp88-89 DEG C; [α] 25 d:+17.5 ° of (c1.60CHCl 3); Ee=97.4% (chiral HPLC analysis); IR (KBr) ν 3433cm -1, 1745cm -1, 1683cm -1, 1202cm -1; 1h NMR (300MHz, CDCl 3) 7.36-7.20 (m, 10H), 7.11 (s, 1H), (5.75 s, 1H), 5.60-5.52 (m, 1H), 5.34-5.15 (m, 3H), 3.60 (dd, J=20.7,12.3Hz, 2H), 2.86-2.80 (m, 2H), 2.31 (s, 1H), 1.63 (d, J=6, Hz, 3H); 13c NMR (75MHz, CDCl 3) 172.3,170.8,139.0,135.4,130.5,128.6,128.55,128.47,128.2,127.4,123.7,70.6,67.4,47.6,35.6,18.1; MS (ESI) m/z (%) 375 [M+Na] +(17), 353 [M+H] +(100) .Anal.Calcd.for C 21h 24n 2o 3: C, 71.57; H, 6.86; N, 7.95.Found:C, 71.50; H, 6.87; N, 8.02.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 4.
Embodiment 5: prepare compound 5
Get the Rhodococcus erythropolisAJ270 thalline (Institute of Chemistry, Academia Sinica) of two grams of weight in wet bases, thaw under 30 DEG C of conditions 30 minutes, with the buffered soln (0.1M of dipotassium hydrogen phosphate and potassium primary phosphate, pH7.0,50ml) thalline is washed in the Erlenmeyer Florence flask of 150 milliliters of threaded mouths, 30 minutes are activated under putting into shaking table 30 DEG C of conditions after dispersion shakes up, then compound shown in the disposable formula VI-3 adding 2mmol (342mg), put into shaking table 30 DEG C, under 200rpm condition, carry out catalytic hydrolysis reaction.Whole reaction TLC monitors, stopped reaction after reaction 40min, and gained reaction solution, by one deck suction filtered through kieselguhr removing thalline, washs filter residue three times with 20 milliliters, water successively, obtains compound (R shown in formula 5 provided by the invention 1for-CH 2c (CH 3)=CH 2, R 2for-NH 2) 337mg, productive rate is 98%.
Compound 5solid, mp148-149 DEG C; [α] 25 d:-18.7 ° of (c1.50, H 2o); IR (KBr) ν 3326cm -1, 1685cm -1, 1508cm -1; 1h NMR (300MHz, D 2o) 5.78-5.66 (m, 1H), 5.32-5.22 (m, 1H), 2.87-2.66 (m, 2H), 1.61 (d, J=6.3Hz, 3H); 13c NMR (75MHz, D 2o) 170.9,170.6,134.1,121.2,67.3,39.4,17.3; MS (ESI) m/z (%) 195 [M+Na] +(33), 173 [M+H] +(100) .Anal.Calcd.for C 7h 12n 2o 3: 173.0921 [M+1] +.Found:173.0922 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 5.
Embodiment 6: prepare compound 6
Specific implementation method:
Compound 5 (100mg) is added DMF (5ml), K 2cO 3(1.5mmol), stir spend the night (12 hours) under benzyl bromine (2mmol) room temperature (25 DEG C) and react complete.Thereafter H is added 2o (25ml), ethyl acetate (3 × 25ml) extracts, anhydrous MgSO 4drying, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (R described in formula 6 provided by the invention 1for-CH 2c (CH 3)=CH 2, R 2for-NHBn, R 3for-Bn) 137mg, productive rate: 67%, ee98.6%.
Compound 5solid, mp139-140 DEG C; [α] 25 d:+7.62 ° of (c1.45, CHCl 3); Ee=98.6% (chiral HPLCanalysis); IR (KBr) ν 3382cm -1, 1740cm -1, 1680cm -1, 1213cm -1; 1h NMR (300MHz, CDCl 3) 7.35-7.21 (m, 11H), 5.67 (s, 1H), 5.21 (dd, J=31.2,12.3Hz, 2H), 4.88 (d, J=17.4, Hz, 2H), 3.64 (dd, J=19.1,12Hz, 2H), 2.93 (dd, J=18.6,15.0Hz, 2H), (2.34 s, 1H), 1.76 (s, 3H); 13c NMR (75MHz, CDCl 3) 172.3,170.9,140.3,139.0,135.2,128.60,128.55,128.5,128.4,128.2,127.4,115.6,70.6,67.5,47.9,39.7,23.7; MS (ESI) m/z (%) 375 [M+Na] +(17), 353 [M+H] +(100) .Anal.Calcd.for C 21h 24n 2o 3: C, 71.57; H, 6.86; N, 7.95.Found:C, 71.59; H, 6.78; N, 7.81.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 6.
Embodiment 7: prepare compound 7
Get the Rhodococcus erythropolisAJ270 thalline (Institute of Chemistry, Academia Sinica) of two grams of weight in wet bases, thaw under 30 DEG C of conditions 30 minutes, with the buffered soln (0.1M of dipotassium hydrogen phosphate and potassium primary phosphate, pH7.0,50ml) thalline is washed in the Erlenmeyer Florence flask of 150 milliliters of threaded mouths, 30 minutes are activated under putting into shaking table 30 DEG C of conditions after dispersion shakes up, then compound shown in the disposable formula VI-4 adding 2mmol (466mg), put into shaking table 30 DEG C, under 200rpm condition, carry out catalytic hydrolysis reaction.Whole reaction TLC monitors, stopped reaction after reaction 330min, and gained reaction solution, by one deck suction filtered through kieselguhr removing thalline, washs filter residue three times with 20 milliliters, water successively, obtains compound (R shown in formula 7 provided by the invention 1for-CH 2cH=CHC 6h 5, R 2for-NH 2) 346mg, productive rate is 74%.
Compound 7solid, mp162-163 DEG C; [α] 25 d:+11.8 ° (c0.85, DMF); IR (KBr) ν 3140cm -1, 1682cm -1, 1507cm -1; 1h NMR (300MHz, d 6-DMSO) 7.36-7.19 (m, 5H), 6.50 (d, J=15.6Hz, 1H), 6.20-6.10 (m, 1H), 2.88 (d, J=6Hz, 2H); 13c NMR (75MHz, MeOD/d 6-DMSO) 170.7,168.8,137.3,134.2,128.7,127.6,126.5,123.4,66.8,40.6; MS (ESI) m/z (%) 232 [M-1] +(100) .Anal.Calcd.for C 12h 14n 2o 3: 235.1077 [M+1] +.Found:235.1079 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 7.
Embodiment 8: prepare compound 8
Specific implementation method:
Compound 7 (100mg) is added DMF (5ml), K 2cO 3(1.5mmol), stir spend the night (12 hours) under benzyl bromine (2mmol) room temperature (25 DEG C) and react complete.Thereafter H is added 2o (25ml), ethyl acetate (3 × 25ml) extracts, anhydrous MgSO 4drying, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (R described in formula 8 provided by the invention 1for-CH 2cH=CHC 6h 5, R 2for-NHBn, R 3for-Bn) 131mg, productive rate: 74%, ee94.0%.
Compound 8solid, mp125-126 DEG C; [α] 25 d:+16.9 ° of (c1.30, CHCl 3); Ee=94.0% (chiral HPLC analysis); IR (KBr) ν 3423cm -1, 1727cm -1, 1696cm -1, 1190cm -1; 1h NMR (300MHz, CDCl 3) 7.37-7.22 (m, 15H), 7.10 (s, 1H), 6.48 (d, J=15.9, Hz, 1H), (6.11-6.00 m, 1H), 5.76 (s, 1H), 5.23 (dd, J=23.1,12Hz, 2H), 3.67 (dd, J=25.8,12.3Hz, 2H), (3.13-2.97 m, 2H), 2.36 (s, 1H); 13c NMR (75MHz, CDCl 3) 172.1,170.7,138.9,137.0,135.4,134.5,128.7,128.60,128.58,128.5,128.2,127.52,127.49,126.3,122.9,70.9,67.6,47.8,36.0; MS (ESI) m/z (%) 415 [M+H] +(100) .Anal.Calcd.forC 26h 26n 2o 3: C, 75.34; H, 6.32; N, 6.76.Found:C, 75.14; H, 6.29; N, 6.83.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 8.
Embodiment 9: prepare compound 9
Get Rhodococcus erythropolis AJ270 thalline (Institute of Chemistry, Academia Sinica) of two grams of weight in wet bases, thaw under 30 DEG C of conditions 30 minutes, with the buffered soln (0.1M of dipotassium hydrogen phosphate and potassium primary phosphate, pH7.0,50ml) thalline is washed in the Erlenmeyer Florence flask of 150 milliliters of threaded mouths, 30 minutes are activated under putting into shaking table 30 DEG C of conditions after dispersion shakes up, then compound shown in the disposable formula VI-5 adding 2mmol (342mg), put into shaking table 30 DEG C, under 200rpm condition, carry out catalytic hydrolysis reaction.Whole reaction TLC monitors, stopped reaction after reaction 25min, and gained reaction solution, by one deck suction filtered through kieselguhr removing thalline, washs filter residue three times with 20 milliliters, water successively, obtains compound (R shown in formula 9 provided by the invention 1for-CH 2cH 2cH=CH 2, R 2for-NH 2) 330mg, productive rate is 96%.
Compound 8solid, mp158-159 DEG C; [α] 25 d:-28.1 ° of (c1.85, H 2o); IR (KBr) ν 3332cm -1, 1682cm -1, 1508cm -1; 1h NMR (300MHz, D 2o) 5.86-5.72 (m, 1H), 5.09-4.98 (m, 2H), 2.15 (d, J=8.4Hz, 2H), 2.07 (t, J=5.4Hz, 2H); 13c NMR (75MHz, D 2o) 171.2,170.7,136.3,116.1,67.6,35.2,27.6; MS (ESI) m/z (%) 171 [M-1] +(100) .Anal.Calcd.for C 7h 12n 2o 3: 173.0921 [M+1] +.Found:173.0922 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 9.
Embodiment 10: prepare compound 10
Specific implementation method:
Compound 9 (100mg) is added DMF (5ml), K 2cO 3(1.5mmol), stir spend the night (12 hours) under benzyl bromine (2mmol) room temperature (25 DEG C) and react complete.Thereafter H is added 2o (25ml), ethyl acetate (3 × 25ml) extracts, anhydrous MgSO 4drying, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (R described in formula 10 provided by the invention 1for-CH 2cH 2cH=CH 2, R 2for-NHBn, R 3for-Bn) 126mg, productive rate: 61%, ee > 99.5%.
Compound 10solid, mp78-79 DEG C; [α] 25 d:+12.4 ° of (c1.45, CHCl 3); Ee > 99.5% (chiral HPLCanalysis); IR (KBr) ν 3439cm -1, 1729cm -1, 1644cm -1, 1221cm -1; 1h NMR (300MHz, CDCl 3) 7.64-7.19 (m, 10H), 7.09 (s, 1H), 5.83-5.72 (m, 2H), 5.24 (dd, J=20.6,12Hz, 2H), 5.15-4.95 (m, 2H), 3.54 (dd, J=34.2,12Hz, 2H), 2.23-1.64 (m, 5H); 13c NMR (75MHz, CDCl 3) 172.4,171.1,138.8,137.2,135.4,128.63,128.59,128.50,128.46,128.2,127.5,115.3,70.5,67.4,47.8,31.2,27.7; MS (ESI) m/z (%) 375 [M+Na] +(12), 353 [M+H] +(100) .Anal.Calcd.for C 21h 24n 2o 3: C, 71.57; H, 6.86; N, 7.95.Found:C, 71.56; H, 6.77; N, 8.02.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 10.
Embodiment 11: prepare compound 11
Get Rhodococcus erythropolis AJ270 thalline (Institute of Chemistry, Academia Sinica) of two grams of weight in wet bases, thaw under 30 DEG C of conditions 30 minutes, with the buffered soln (0.1M of dipotassium hydrogen phosphate and potassium primary phosphate, pH7.0,50m1) thalline is washed in the Erlenmeyer Florence flask of 150 milliliters of threaded mouths, 30 minutes are activated under putting into shaking table 30 DEG C of conditions after dispersion shakes up, then compound shown in the disposable formula VI-6 adding 2mmol (310mg), put into shaking table 30 DEG C, under 200rpm condition, carry out catalytic hydrolysis reaction.Whole reaction TLC monitors, stopped reaction after reaction 25min, and gained reaction solution, by one deck suction filtered through kieselguhr removing thalline, washs filter residue three times with 20 milliliters, water successively, obtains compound (R shown in formula 11 provided by the invention 1for-CH 2c ≡ CH, R 2for-NH 2) 248mg, productive rate is 80%.
Compound 11solid, mp125-126 DEG C; [α] 25 d:-5.7 ° of (c1.75, H 2o); IR (KBr) ν 3395cm -1, 1671cm -1, 1513cm -1; 1h NMR (300MHz, D 2o) 2.98 (s, 2H), 2.49 (s, 1H); 13c NMR (75MHz, D 2o) 171.4,170.9,77.1,74.0,66.0,26.5; MS (ESI) m/z (%) 155 [M-1] +(100) .Anal.Calcd.for C 6h 8n 2o 3: 157.0608 [M+1] +.Found:157.0609 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 11.
Embodiment 12: prepare compound 12
Compound 11 (100mg) is added DMF (5ml), K 2cO 3(1.5mmol), stir spend the night (12 hours) under benzyl bromine (2mmol) room temperature (25 DEG C) and react complete.Thereafter H is added 2o (25ml), ethyl acetate (3 × 25ml) extracts, anhydrous MgSO 4drying, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (R described in formula 12 provided by the invention 1for-CH 2c ≡ CH, R 2for-NHBn, R 3for-Bn) 126mg, productive rate: 61%, ee96.8%.
Compound 12solid, mp113-114 DEG C; [α] 25 d:+24.6 ° of (c1.30, CHCl 3); Ee=96.8% (chiral HPLCanalysis); IR (KBr) ν 3430cm -1, 1729cm -1, 1701cm -1, 1026cm -1; 1h NMR (300MHz, CDCl 3) 7.35-7.26 (m, 10H), 7.21 (s, 1H), (5.76 s, 1H), 5.23 (dd, J=17.1,12.3Hz, 2H), 3.64 (dd, J=19.8,11.8Hz, 2H), 3.14 (ddd, J=49.2,17.7,2.6Hz, 2H) and .2.65 (s, 1H), 2.07 (t, J=2.7Hz, 1H); 13cNMR (75MHz, CDCl 3) 170.9,169.3,138.7,135.1,128.6,128.5,128.3,128.2,127.5,78.3,72.4,69.6,67.9,47.3,22.5; MS (ESI) m/z (%) 359 [M+Na] +(40), 337 [M+H] +(100) .Anal.Calcd.forC 20h 20n 2o 3: C, 71.41; H, 5.99; N, 8.33.Found:C, 71.62; H, 6.07; N, 8.25.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 12.
Embodiment 13: prepare compound 13
Get the Rhodococcus erythropolisAJ270 thalline (Institute of Chemistry, Academia Sinica) of two grams of weight in wet bases, thaw under 30 DEG C of conditions 30 minutes, with the buffered soln (0.1M of dipotassium hydrogen phosphate and potassium primary phosphate, pH7.0,50ml) thalline is washed in the Erlenmeyer Florence flask of 150 milliliters of threaded mouths, 30 minutes are activated under putting into shaking table 30 DEG C of conditions after dispersion shakes up, then compound shown in the disposable formula VI-7 adding 2mmol (338mg), put into shaking table 30 DEG C, under 200rpm condition, carry out catalytic hydrolysis reaction.Whole reaction TLC monitors, stopped reaction after reaction 34min, and gained reaction solution, by one deck suction filtered through kieselguhr removing thalline, washs filter residue three times with 20 milliliters, water successively, obtains compound (R shown in formula 13 provided by the invention 1for-CH 2c ≡ CCH 3, R 2for-NH 2) 320mg, productive rate is 94%.
Compound 12solid, mp129-130 DEG C; [α] 25 d:-27.7 ° of (c1.56, H 2o); IR (KBr) ν 3382cm -1, 1697cm -1, 1335cm -1; 1h NMR (300MHz, D 2o) 2.91 (s, 2H), 1.69 (s, 3H); 13c NMR (75MHz, D 2o) 171.0,170.6,82.4,71.1,66.5,26.9,2.5; MS (ESI) m/z (%) 169 [M-1] +(100) .Anal.Calcd.for C 7h 10n 2o 3: 171.0764 [M+1] +.Found:171.0762 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 13.
Embodiment 14: prepare compound 14
Compound 13 (100mg) is added DMF (5ml), K 2cO 3(1.5mmol), stir spend the night (12 hours) under benzyl bromine (2mmol) room temperature (25 DEG C) and react complete.Thereafter H is added 2o (25ml), ethyl acetate (3 × 25ml) extracts, anhydrous MgSO 4drying, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (R described in formula 14 provided by the invention 1for-CH 2c ≡ CCH 3, R 2for-NHBn, R 3for-Bn) 122mg, productive rate: 59%, ee96.8%.
Compound 14solid, mp95-96 DEG C; [α] 25 d:+31.1 ° of (c1.80, CHCl 3); Ee=96.8% (chiral HPLCanalysis); IR (KBr) ν 3430cm -1, 3252cm -1, 1702cm -1, 1185cm -1; 1h NMR (300MHz, CDCl 3) 3430cm -1, 3252cm -1, 1702cm -1, 1185cm -1; 1h NMR (300MHz, CDCl 3) δ 7.33-7.21 (m, 11H), 6.11 (s, 1H), 5.21 (s, 2H), 3.64 (dd, J=19.8,11.8Hz, 2H), 3.06 (ddd, J=19.2,17.3,1.9Hz, 1H), 2.59 (s, 1H), 1.73 (s, 1H); 13c NMR (75MHz, CDCl 3) 171.5,169.7,139.1,135.3,128.5,128.3,128.2,128.1,127.4,79.8,72.8,69.9,67.6,47.2,23.0,3.6; MS (ESI) m/z (%) 373 [M+Na] +(22), 351 [M+H] +(100) .Anal.Calcd.for C 21h 22n 2o 3: C, 71.98; H, 6.33; N, 7.99.Found:C, 71.98; H, 6.18; N, 8.15.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 14.
Embodiment 15: prepare compound 15
Get the Rhodococcus erythropolisAJ270 thalline (Institute of Chemistry, Academia Sinica) of two grams of weight in wet bases, thaw under 30 DEG C of conditions 30 minutes, with the buffered soln (0.1M of dipotassium hydrogen phosphate and potassium primary phosphate, pH7.0,50ml) thalline is washed in the Erlenmeyer Florence flask of 150 milliliters of threaded mouths, 30 minutes are activated under putting into shaking table 30 DEG C of conditions after dispersion shakes up, then compound shown in the disposable formula VI-8 adding 2mmol (414mg), put into shaking table 30 DEG C, under 200rpm condition, carry out catalytic hydrolysis reaction.Whole reaction TLC monitors, stopped reaction after reaction 60min, and gained reaction solution, by one deck suction filtered through kieselguhr removing thalline, washs filter residue three times with 20 milliliters, water successively, obtains compound (R shown in formula 15 provided by the invention 1for-CH 2c 6h 5, R 2for-NH 2) 345mg, productive rate is 83%.
Compound 15solid, mp148-149 DEG C; [α] 25 d:-23.3 ° (c0.60, DMF); IR (KBr) ν 3377cm -1, 1682cm -1, 1503cm -1; 1h NMR (300MHz, d 6-DMSO) 7.34-7.22 (m, 5H), 3.39 (dd, J=14.4,14.4Hz, 2H); 13cNMR (75MHz, d 6-DMSO) 171.0,170.6,132.9,129.9,129.1,128.3,68.3,41.6; MS (ESI) m/z (%) 207 [M-1] +(100) .Anal.Calcd.for C 10h 12n 2o 3: 209.0921 [M+1] +.Found:209.0921 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 15.
Embodiment 16: prepare compound 16
Compound 15 (100mg) is added DMF (5ml), K 2cO 3(1.5mmol), stir spend the night (12 hours) under benzyl bromine (2mmol) room temperature (25 DEG C) and react complete.Thereafter H is added 2o (25ml), ethyl acetate (3 × 25ml) extracts, anhydrous MgSO 4drying, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (R described in formula 16 provided by the invention 1for-CH 2c 6h 5, R 2for-NHBn, R 3for-Bn) 122mg, productive rate: 59%, ee > 99.5%.
Compound 16solid, mp81-82 DEG C; [α] 25 d:+16.0 ° of (c2.00, CHCl 3); Ee > 99.5% (chiral HPLCanalysis); IR (KBr) ν 3440cm -1, 1714cm -1, 1680cm -1, 1186cm -1; 1h NMR (300MHz, CDCl 3) 7.35-7.14 (m, 15H), 6.89 (s, 1H), 5.45 (s, 1H), 5.21 (dd, J=12.5,12.1Hz, 2H), 3.74 (dd, J=12.4,12.2Hz, 2H), 3.47 (s, 2H), 2.19 (s, 1H); 13c NMR (75MHz, CDCl 3) 171.9,170.6,135.2,130.1,128.64,128.63,128.56,128.4,128.1,127.5,127.1,72.0,67.6,48.1,38.0; MS (ESI) m/z (%) 389 [M+H] +(100) .Anal.Calcd.for C 24h 24N 2o 3: C, 74.21; H, 6.23; N, 7.21.Found:C, 74.17; H, 6.26; N, 7.10.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 16.
Embodiment 17: prepare compound 17
Get the Rhodococcus erythropolisAJ270 thalline (Institute of Chemistry, Academia Sinica) of two grams of weight in wet bases, thaw under 30 DEG C of conditions 30 minutes, with the buffered soln (0.1M of dipotassium hydrogen phosphate and potassium primary phosphate, pH7.0,50ml) thalline is washed in the Erlenmeyer Florence flask of 150 milliliters of threaded mouths, 30 minutes are activated under putting into shaking table 30 DEG C of conditions after dispersion shakes up, then compound shown in the disposable formula VI-9 adding 2mmol (318mg), put into shaking table 30 DEG C, under 200rpm condition, carry out catalytic hydrolysis reaction.Whole reaction TLC monitors, stopped reaction after reaction 35min, and gained reaction solution, by one deck suction filtered through kieselguhr removing thalline, washs filter residue three times with 20 milliliters, water successively, obtains compound (R shown in formula 17 provided by the invention 1for-CH 2cH 2cH 3, R 2for-NH 2) 269mg, productive rate is 84%.
Compound 16solid, mp163-164 DEG C; [α] 25 d:-22.1 ° of (c1.90, H 2o); IR (KBr) ν 3441cm -1, 1681cm -1, 1508cm -1; 1h NMR (300MHz, D 2o) 2.05-1.88 (m, 2H), 1.32-1.08 (m, 2H), 0.81 (t, J=7.5Hz, 3H); 13c NMR (75MHz, D 2o) 171.5,171.1,67.8,38.1,16.9,12.9; MS (ESI) m/z (%) 159 [M-1] +(100) .Anal.Calcd.for C 6h 12n 2o 3: 161.0921 [M+1] +.Found:161.0921 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 17.
Embodiment 18: prepare compound 18
Compound 17 (100mg) is added DMF (5ml), K 2cO 3(1.5mmol), stir spend the night (12 hours) under benzyl bromine (2mmol) room temperature (25 DEG C) and react complete.Thereafter H is added 2o (25ml), ethyl acetate (3 × 25ml) extracts, anhydrous MgSO 4drying, with Rotary Evaporators except desolventizing, dry method loading, rapid column chromatography obtains compound (R described in formula 18 provided by the invention 1for-CH 2cH 2cH 3, R 2for-NHBn, R 3for-Bn) 123mg, productive rate: 58%, ee97.8%.
Compound 16, mp163-164 DEG C; [α] 25 d:+16.5 ° of (c1.70, CHCl 3); Ee=97.8% (chiral HPLCanalysis); IR (KBr) ν 3440cm -1, 1728cm -1, 1642cm -1, 1218cm -1; 1h NMR (300MHz, CDCl 3) 7.33-7.16 (m, 10H), 7.12 (s, 1H), (5.93 s, 1H), 5.22 (dd, J=33.9,12.3Hz, 2H), 3.52 (dd, J=33.9,12.0Hz, 2H), 2.28 (s, 1H), (2.10-2.05 m, 2H), 1.34-1.25 (m, 2H), 0.93 (t, J=7.2Hz, 3H); 13cNMR (75MHz, CDCl 3) 172.8,171.4,135.5,128.61,128.56,128.5,128.4,128.2,127.5,70.8,67.3,47.8,34.2,16.8,14.1; MS (ESI) m/z (%) 341 [M+H] +(100) .Anal.Calcd.for C 20h 24n 2o 3:: 341.1860 [M+1] +.Found:341.1857 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 18.
Embodiment 19: prepare compound 19
Compound 1 (316mg, 2mmol) is dissolved in methyl alcohol (20ml), at 0 DEG C, slowly drips CH 2n 2(2M, 10ml) diethyl ether solution, rises to room temperature after keeping temperature 0.5h, and solvent revolves after spending the night by reaction, rapid column chromatography (filler silica gel: 100-200 order; Developping agent: PE/EA=1/1 ~ 1/2), obtain white solid 19 (269mg, 78%)
Compound 19, mp66-67 DEG C; [α] 25D:+5.0 ° (c2.00, CHCl3); IR (KBr) ν 3407cm-1,1730cm-1,1633cm-1; 1H NMR (300MHz, CDCl3) 7.42 (s, 1H), 6.27 (s, 1H), 5.79-5.71 (m, 2H), 5.23-5.18 (m, 2H), 3.81 (s, 3H), 2.85-2.62 (m, 2H), 2.10 (s, 2H); 13C NMR (75MHz, CDCl3) 173.7,172.4,131.9,120.8,65.0,53.2,41.7; MS (ESI) m/z (%) 173 [M+H] +(100) .Anal.Calcd.for C 7h 12n 2o 3: 173.0921 [M+1] +.Found:173.0915 [M+1] +.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 19.
Embodiment 20: the synthesis of compound 20
Substrate 19 (50mg, 0.3mmol) is dissolved in the mixed solvent of EtOH (4ml) and THF (4ml), adds LiClH 2o (30mg, 0.5mmol) and NaBH 4(38mg, 1mmol), reacts under room temperature, revolves desolventizing after disappearance of substrate, is incorporated in methyl alcohol (1ml) and 2N NaOH (1ml), is adjusted to neutrality (HCl/H at 60 DEG C after reacting 5h in system 2o), revolve desolventizing, make spent ion exchange resin carry out being separated (CAS:11119-67-8; Developping agent 10% ammoniacal liquor), obtain white solid 20 (46mg, 99%).
Compound 20, mp178-179 DEG C; [α] 25 d:-34.7 ° of (c0.75, H 2o); IR (KBr) ν 3080cm -1, 1620cm -1; 1hNMR (300MHz, D 2o) 5.72-5.58 (m, 1H), 5.28-5.18 (m, 2H), 3.75 (dd, J=35.1,12.3Hz, 2H), 2.58-2.32 (m, 2H); MS (ESI) m/z (%) 168 [M+Na] +(23), 146 [M+H] +(100).
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 20.
Embodiment 21: the synthesis of compound 21
By substrate 20 (30mg, 0.2mmol) be dissolved in methyl alcohol (50ml), stirred at ambient temperature adds palladium carbon (10%, 10mg) after dissolving, and suspends and stirs, air hydrogen exchange (with hydrogen balloon) is stirred afterwards, after disappearance of substrate, suction filtration, revolves desolventizing, 21 (30mg, 99%) must be produced.
Compound 21, mp188-189 DEG C; { lit 5.mp279 DEG C }; [α] 25 d:+9.5 ° (c1.05,1N HCl); IR (KBr) ν 3140cm -1, 3044cm -1, 1405cm -1; 1h NMR (300MHz, D 2o) 3.65 (dd, J=73.5,12.0Hz, 2H), 1.67-1.46 (m, 2H), 1.30-1.00 (m, 2H), 0.78 (t, J=7.5Hz, 3H); 13c NMR (75MHz, D 2o) 174.5,66.4,64.3,34.3,16.5,13.4; MS (ESI) m/z (%) 148 [M+H] +(100).
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 21.
Embodiment 22: the synthesis of compound 22
Substrate 2 (170mg, 0.5mmol) is dissolved in the mixed solvent of EtOH (5ml) and THF (5ml), adds LiClH 2o (30mg, 0.5mmol) and NaBH 4(38mg, 1mmol), reacts under room temperature, and add water after disappearance of substrate (20ml), and EA extracts (3 × 20ml), MgSO 4drying, revolves desolventizing, rapid column chromatography (filler silica gel: 100-200 order; Developping agent: PE/EA=1/1 ~ 1/2), obtain white solid 22 (116mg, 99%).
Compound 22, mp95-96 DEG C; [α] 25 d:+2.05 ° of (c1.95, CHCl 3); IR (KBr) ν 3443cm -1, 3285cm -1, 1659cm -1; 1h NMR (300MHz, CDCl 3) 77.36-7.24 (m, 6H), 6.00 (s, 1H), 5.92-5.78 (m, 1H), 5.20-5.15 (m, 2H), 3.84 (s, 2H), 3.75 (dd, J=24.6,12.3Hz, 2H), 2.58-2.41 (m, 2H); 13c NMR (75MHz, CDCl 3) 178.1,139.9,132.4,128.6,128.0,127.3,119.4,65.1,64.1,46.9,38.2; MS (ESI) m/z (%) 257 [M+Na] +(28), 235 [M+H] +(100) .Anal.Calcd.for C 13h 18n 2o 2: C, 66.64; H, 7.74; N, 11.96.Found:C, 66.72; H, 7.67; N, 11.92.
As from the foregoing, above-claimed cpd structure is correct, is compound shown in 22.
Embodiment 23: the synthesis of compound 23
Substrate 22 (117mg, 0.5mmol) is weighed into two-mouth bottle fast, uses argon shield with after high-purity argon gas displaced air, add dry THF (10ml), by LiAlH 4(1N THF solution, 5ml) join in above-mentioned two-mouth bottle with syringe, after reflux 48h, add EtOH (2ml) cancellation reaction, add 2N NaOH (3ml), by system suction filtration, EtOH washs (3 × 10ml), revolve desolventizing, rapid column chromatography (filler silica gel: 100-200 order; Developping agent: EA/EtOH/ ammoniacal liquor=100/5/1), obtain product 23 (72mg, 65%).
Compound 23, mp49-50 DEG C; [α] 25 d:+10.0 ° (c1.60, MeOH); IR (KBr) ν 3359cm -1, 2864cm -1, 1639cm -1; 1h NMR (300MHz, CDCl 3) 7.38-7.21 (m, 5H), 5.97-5.86 (m, 1H), (5.21-5.13 m, 2H), 3.70 (dd, J=12.0,3.6Hz, 2H), 3.53 (dd, J=11.2,8.4Hz, 2H), 2.70 (dd, J=13.6,6.0Hz, 2H), (2.28 d, J=7.2Hz, 2H); 13c NMR (75MHz, CDCl 3) 140.4,133.3,128.11,128.06,126.7,117.5,63.0,58.5,45.1,43.6,36.1; MS (ESI) m/z (%) 221 [M+H] +(100) .Anal.Calcd.for C 18h 20n 2o 1: 221.1648 [M+1] +.Found:221.1656 [M+1] +.
As from the foregoing, this compound structure is correct, is compound shown in 23.
Embodiment 24, anti-tumor activity are tested
The functionalized alpha-non-natural amino acid with quaternary carbon center prepared by the present invention is for colon cancer cell HCT-116, and this cell is from ATCC, and the experiment of its anti-tumor activity is as follows:
Mtt assay is utilized to measure the impact of compound on intracellular activity shown in formula I general structure.Mtt assay, also known as MTT colorimetry, is a kind of method detecting cell survival and growth.Its Cleaning Principle is that the succinodehydrogenase in viable cell plastosome can make exogenous MTT be reduced to water-insoluble bluish voilet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect viable cell quantity.Operating process is as follows: be made into individual cells suspension with the nutrient solution containing 10% tire calf serum, 96 orifice plates are inoculated into every hole 1000-10000 cell, after cultivating 24 hours in 37 degree of cell culture incubators, add medicine to be measured, with dimethyl sulfoxide (DMSO) (DMSO) in contrast, continue to cultivate 48 hours in 37 degree of cell culture incubators; Every hole adds MTT solution (5mg/ml PBS prepares, pH=7.4) 20ul (200ul substratum), continues to hatch 4h, stop cultivating, careful suction abandons culture supernatant in hole, and every hole adds 150ul DMSO, decolorization swinging table vibration 10min, makes crystallisate fully dissolve; Select 490nm (570nm) wavelength, enzyme linked immunological monitor measures each hole absorbance value, record result; Further experimental result is processed: the ratio calculating each experimental group and con, with this ratio for ordinate zou, with different pharmaceutical or drug level for X-coordinate does bar graph or broken line graph.
Investigate the functionalized alpha-non-natural amino acid with quaternary carbon center prepared by the present invention by mtt assay to test for the anti-tumor activity of colon cancer cell HCT-116, experimental result shows, when concentration is 100 μ g/ml, shown in formula I general structure, compound all shows the lethality to colon cancer cell HCT-116.Wherein, embodiment 7 prepares the medicine median lethal concentration IC of compound 3 couples of colon cancer cell HCT-116 shown in compound 7 shown in gained formula I general structure and embodiment 3 gained formula I 50between 50-75 μ g/ml, other embodiments prepare the activity that gained formula I structure general formula also shows certain inhibition tumor cell growth.

Claims (2)

1. compound shown in formula I is preparing the application in inhibition tumor cell product, and described tumour cell is colon cancer cell;
In described formula I, * represents chirality, is R or S;
R 1be selected from-CH 3,-CH 2cH 3,-CH 2cH 2cH 3,-CH 2cH 2cH 2cH 3,-CH 2cH=CH 2,-CH 2cH 2cH=CH 2,-CH 2cH 2cH 2cH=CH 2,-CH 2c ≡ CH ,-CH 2cH 2c ≡ CH ,-CH 2cH 2cH 2c ≡ CH ,-CH 2c ≡ CCH 3,-CH 2c ≡ CCH 2cH 3,-CH 2c ≡ CCH 2cH 2cH 3,-CH 2c 6h 5,-CH 2cH 2c 6h 5,-CH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2c 6h 5,-CH 2cH 2cH 2cH 2cH 2c 6h 5,-(CH 2) ncH=CHCH 3,-(CH 2) nc (CH 3)=CH 2with-(CH 2) ncH=CHC 6h 5in at least one;
R 2be selected from-NH 2,-NHCH 3,-N (CH 3) 2,-NHCH 2cH 3,-N (CH 2cH 3) 2,-NHCH 2cH 2cH 3,-N (CH 2cH 2cH 3) 2,-NHCH 2cH 2cH 2cH 3,-N (CH 2cH 2cH 2cH 3) 2,-NHCH 2cH=CH 2,-N (CH 2cH=CH 2) 2,-NHCH 2cH 2cH=CH 2,-N (CH 2cH 2cH=CH 2) 2,-NHCH 2cH 2cH 2cH=CH 2,-N (CH 2cH 2cH 2cH=CH 2) 2,-NHCH 2c ≡ CH ,-N (CH 2c ≡ CH) 2,-NHCH 2cH 2c ≡ CH ,-N (CH 2cH 2c ≡ CH) 2,-NHCH 2cH 2cH 2c ≡ CH ,-N (CH 2cH 2cH 2c ≡ CH) 2,-NHCH 2c ≡ CCH 3,-N (CH 2c ≡ CCH 3) 2,-NHCH 2c ≡ CCH 2cH 3,-N (CH 2c ≡ CCH 2cH 3) 2,-NHCH 2c ≡ CCH 2cH 2cH 3,-N (CH 2c ≡ CCH 2cH 2cH 3) 2,-NHCH 2c 6h 5,-N (CH 2c 6h 5) 2,-NHCH 2cH 2c 6h 5,-N (CH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2c 6h 5) 2,-NHCH 2cH 2cH 2cH 2cH 2c 6h 5,-N (CH 2cH 2cH 2cH 2cH 2c 6h 5) 2,-NH (CH 2) ncH=CHCH 3,-N ((CH 2) ncH=CHCH 3) 2,-NH (CH 2) nc (CH 3)=CH 2with-N ((CH 2) nc (CH 3)=CH 2) 2in at least one;
N is the integer of 1-3;
X 1be selected from-CONH 2,-CH 2nH 2with-CO 2at least one in H;
X 2be selected from-CO 2h ,-CH 2oH and-CO 2r 3in at least one; R 3for-CH 3or-CH 2c 6h 5.
2. application according to claim 1, is characterized in that: described colon cancer cell is colon cancer cell HCT-116.
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