CN110229168A - 11,20- dicarbapentaborane Jiyuan Oridonin and its l-amino acid -14- ester trifluoroacetate - Google Patents

Abstract

The invention relates to 11,20-dicarbonyl Jiyuan oridonin and its L-amino acid-14-ester trifluoroacetate and its preparation method and use, belonging to the field of pharmaceutical compounds. Its preparation method: take Jiyuan Rubescensine A as raw material, and oxidize it with Jones reagent to obtain 11,20-dicarbonyl Jiyuan Rubescensin A, whose structure is as follows: Then it reacts with N-BOC-L-amino acid at 14-position esterification to obtain N-BOC-L-amino acid-11,20-dicarbonyl Jiyuan oridonin (III), and then in trifluoroacetic acid Remove the BOC protecting group in the medium, and form a salt to obtain L-amino acid-14-(11,20-dicarbonyl Jiyuan Rubescensin A) ester trifluoroacetate. It has the following general formula: This type of compound has good stability and anti-tumor activity, which provides a basis for screening anti-tumor drugs such as esophageal cancer, gastric cancer, primary liver cancer, pancreatic cancer, cardia cancer, colorectal cancer, bladder cancer, breast cancer, and acute myeloid leukemia .

Description

11,20-dicarbonyl oridonin and its L-amino acid-14-ester trifluoroethyl salt

technical field

The present invention relates to the field of pharmaceutical compounds, in particular to novel Jiyuan Rubescensin A compounds: 11,20-dicarbonyl Jiyuan Rubescensin A and its L-amino acid-14-ester trifluoroacetate compounds, preparation methods and its application.

Background technique

As early as the 1970s, the good anti-cancer activity of Lamiaceae Lamiaceae plant Oryx sativa has attracted people's attention. It is a commonly used Chinese herbal medicine in Henan Province. After long-term research by pharmacists, it has been confirmed that It has a certain anti-tumor effect, and the main anti-tumor effect is the diterpenoids. Jiyuan Rubescensin A is a natural organic compound of kaurene diterpenoids extracted from Rubescens sativa, and it is one of the main anti-tumor active ingredients. Jiyuan Rubescensin A is the C-20 oxidized ent-kaurene diterpenoid extracted and purified from Jiyuan Rubescens for the first time by our research group. In the in vitro activity test, it was found that it showed good cytotoxic activity against various tumor cells such as EC-109 cells. The follow-up study found that the content of Jiyuan oridonin in the leaves of Rubescensus japonicus was higher than that of oridonin in a specific place of origin and growth period, which could reach more than 5‰.

The tautomeric formula of aldehyde and hemiacetal of Oridonium japonicus from Jiyuan

However, Jiyuan Rubescensin A is an equilibrium mixture of aldehyde and hemiacetal, mainly exists in hemiacetal, which is very unstable. Oral bioavailability of Jiyuan Rubescensin A is less than 5%, and effective blood concentration cannot be achieved; at the same time, it is almost insoluble in water, so it is difficult to administer intravenously. To modify its structure, improve its water solubility and activity, and develop it into a new antitumor drug is an important research direction of Jiyuan Rubescensin A at present.

Contents of the invention

The purpose of the present invention is to provide a new ent-kaurene-type diterpene compound and a series of derivatives of the diterpene compound; another purpose is to provide its preparation method and application, so as to provide the possibility for current clinical anticancer drug screening.

In the present invention, Jiyuan Rubescensin A is used as raw material, and its 11,20-position oxidation product 11,20-dicarbonyl Jiyuan Rubescensin A is obtained through Jones reagent oxidation, which is a new enantio-kaurene di Terpenes, whose structure is shown below:

In order to achieve the purpose of the present invention, its structure was modified on the basis of the obtained 11,20-dicarbonyl Jiyuan Rubescensine A, and its L-amino acid-14-ester trifluoroacetate compound was obtained. Its structure is generally The formula is as follows:

R is a 5-10 membered heterocycloalkyl group containing 1-2 N, O or S heteroatoms; or R is Wherein R is hydrogen, straight or branched _C1 -C10 alkyl or C1-C10 straight or branched alkyl substituted thiol, amino substituted C1-C10 alkyl, phenyl substituted C1-C5 alkane Base; R' is hydrogen or C1-C3 alkyl.

Preferably: R is a 5-6 membered heterocycloalkyl group containing 1 N heteroatom; or R is

R' is hydrogen or methyl, R1 is hydrogen, methyl or _one of the following substituents:

For preparation needs, the present invention also needs to further protect the pharmaceutically acceptable salts of the above-mentioned compounds, acceptable salts are organic acid salts, inorganic acid salts, organic alkali salts or inorganic alkali salts, wherein organic acids include acetic acid, methanesulfonic acid, lemon acid, fumaric acid, maleic acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, methanesulfonic acid, malonic acid, lipoic acid; inorganic acids include hydrochloric acid, hydrobromic acid, Nitric acid, sulfuric acid, phosphoric acid; organic bases include meglumine, glucosamine; inorganic bases include alkaline compounds of alkali metals sodium, potassium, barium, calcium, magnesium, and zinc.

The present invention provides the preparation method of above-mentioned compound, synthesize through following reaction route:

(1) Using Jiyuan Rubescensine A (compound 1, abbreviated as JDA) as raw material, acetone as solvent, dissolve it completely under stirring conditions, and then add Jones reagent diluted with acetone in ice bath conditions to generate 11, 20-dicarbonyl Jiyuan oridonin (compound 2, abbreviated as JDAO).

(2) 11,20-dicarbonyl oridonin in dichloromethane, with EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), DMAP (4-Dimethylaminopyridine) and DIPEA (N,N-diisopropylethylamine) as a catalyst, adding N-Boc-amino acid reaction, after purification by column chromatography, N-Boc-L-amino acid-14- (11,20-Dicarbonyl Jiyuan Rubescensin A) ester compound (compound 3);

(3) N-Boc-L-amino acid-14-(11,20-dicarbonyl Jiyuan Rubescensin A) ester compound is ice bathed in dichloromethane, and dichloromethane and trifluoroacetic acid are added to the system The solutions were mixed, and the reaction was stirred in an ice bath. After the completion of the reaction was determined by thin-layer chromatography, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and then diisopropyl ether was added with stirring, and a large amount of white solid was precipitated, which was the product (compound 4).

Advantages and innovations of the present invention are:

1. Jiyuan Rubescensin A itself has poor stability. Partial groups of Jiyuan Rubescensin A are modified, and the 11th and 20th hydroxyl groups are oxidized into carbonyl groups, which greatly improves its stability and enhances its antitumor activity , and then esterified with N-Boc-L-amino acid to form a salt to prepare Jiyuan Rubescensin A compound. The product not only has good water solubility, but the conversion rate in rats and dogs can reach 94.1% and 128%, respectively. Moreover, the compounds have anti-tumor activity and provide a basis for screening anti-tumor drugs.

2. The preparation method of the route is simple and easy. The initial raw material is prepared from Jiyuan Rubescensin A, and then the Jiyuan Rubescensin A derivative that is esterified with the 11th and 20th hydroxyl oxidant and the 14th hydroxyl amino acid is obtained through a modification reaction , the yield is more than 75%.

3. As verified by pharmacology and toxicology experiments, this compound not only has similar activity to 11,20-dicarbonyl Jiyuandonglingin, but also can be rapidly transformed into 11,20-dicarbonyljiyuandonglingin in vitro plasma and in vivo Rubescensine A exerts the pharmacological activity of 11,20-dicarbonyl Jiyuan Rubescensin A; toxicity studies show that this product is less toxic and has no concerns about genotoxicity; Sexual solvents cause phlebitis and side effects of allergies caused by special preparations. Rabbit blood vessel irritation test shows that this product has no irritation to blood vessels, and guinea pig systemic allergy test has no allergic phenomenon, which effectively solves the above-mentioned problems of Jiyuan Rubescensin A patent medicine. question.

The chemical structure and NMR data of the preferred compounds synthesized by the present invention are as follows in Table 1:

Detailed ways

The present invention will be further described below in conjunction with the examples.

Example 1:

Preparation of 11,20-Dicarbonyl Jiyuan Oridonin (Compound 2, JDAO)

Weigh 1 g of Jiyuan Rubescensine A (JDA), add 3.5 mL of acetone to dissolve it, and add an appropriate amount of Jones reagent dropwise to the system under stirring in an ice bath, and react for about 10 minutes. After the completion of the reaction was determined by thin-layer chromatography, the system was diluted with isopropanol, and the solvent was evaporated to dryness by rotary evaporation. After diluting with 50 mL of ethyl acetate, it was washed 5 times with water and stripped once. The ester layers were combined, dried with anhydrous sodium sulfate for 1 hour, and rotary evaporated to obtain 750 mg of JDAO (11,20-dicarbonyl oridonin, compound 2) as a white solid, with a yield of 95%.

Example 2:

Preparation of L-alanine-14-(11,20-dicarbonyl Jiyuan oridonin) ester trifluoroacetate (compound Ⅰ-1)

Weigh 150mg JDAO, dissolve it with 2mL dichloromethane, add Boc-L-alanine, EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride successively under stirring at room temperature ), DMAP (4-dimethylaminopyridine) and DIPEA (N,N-diisopropylethylamine). The reaction can be completed after 1-2 hours. After the completion of the reaction was determined by thin-layer chromatography, 30 mL of dichloromethane was added to dilute the reaction system, and the reaction system was washed three times with saturated ammonium chloride solution, and the aqueous layers were combined and back-extracted once with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate for 1 hour, and purified by column chromatography.

The resulting white solid was dissolved in dichloromethane and ice bathed. When the temperature of the system dropped to 5°C, a mixed solution of dichloromethane:trifluoroacetic acid 1:1 was added to the system, and stirred in an ice bath for 20-30 minutes. After the completion of the reaction was determined by thin-layer chromatography, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and the system was concentrated to dark yellow. Then add isopropyl ether under stirring, at this time, a large amount of white solid can be precipitated, which is the product. After stirring for 1 hour, the product was collected by suction filtration, washed with isopropyl ether and dried in a vacuum oven for 24 hours.

Example 3:

Preparation of L-valine-14-(11,20-dicarbonyl Jiyuan oridonin A) ester trifluoroacetate (compound I-2)

Weigh 150mg of JDAO, dissolve it in 2mL of dichloromethane, add Boc-L-valine, EDCI, DMAP and DIPEA successively under stirring at room temperature. The reaction can be completed after 1-2 hours. After the completion of the reaction was determined by thin-layer chromatography, 30 mL of dichloromethane was added to dilute the reaction system, and the reaction system was washed three times with saturated ammonium chloride solution, and the aqueous layers were combined and back-extracted once with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate for 1 hour, and purified by column chromatography.

The resulting white solid was dissolved in dichloromethane and ice bathed. When the temperature of the system dropped to 5°C, a mixed solution of dichloromethane:trifluoroacetic acid 1:1 was added to the system, and stirred in an ice bath for 20-30 minutes. After the completion of the reaction was determined by thin-layer chromatography, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and the system was concentrated to dark yellow. Then add isopropyl ether under stirring, at this time, a large amount of white solid can be precipitated, which is the product. After stirring for 1 hour, the product was collected by suction filtration, washed with isopropyl ether and dried in a vacuum oven for 24 hours.

Example 4:

Preparation of L-phenylalanine-14-(11,20-dicarbonyl oridonin) ester trifluoroacetate (compound I-3)

Weigh 150mg of JDAO, dissolve it in 2mL of dichloromethane, add Boc-L-phenylalanine, EDCI, DMAP and DIPEA successively under stirring at room temperature. The reaction can be completed after 1-2 hours. After the completion of the reaction was determined by thin-layer chromatography, 30 mL of dichloromethane was added to dilute the reaction system, and the reaction system was washed three times with saturated ammonium chloride solution, and the aqueous layers were combined and back-extracted once with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate for 1 hour, and purified by column chromatography.

The resulting white solid was dissolved in dichloromethane and ice bathed. When the temperature of the system dropped to 5°C, a mixed solution of dichloromethane:trifluoroacetic acid 1:1 was added to the system, and stirred in an ice bath for 20-30 minutes. After the completion of the reaction was determined by thin-layer chromatography, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and the system was concentrated to dark yellow. Then add isopropyl ether under stirring, at this time, a large amount of white solid can be precipitated, which is the product. After stirring for 1 hour, the product was collected by suction filtration, washed with isopropyl ether and dried in a vacuum oven for 24 hours.

Compounds I4-I16 were sequentially synthesized according to the above method.

Example 5:

The target compound synthesized by the present invention has been tested for activity on two tumor cell lines of human gastric cancer cells MGC-803, HGC-27, SGC-7901 and human prostate cancer cell PC-3, and the experimental results show that the derivatives have certain anti-tumor activity .

(1) Experimental method:

(1) Preparation of 1640 culture medium: under sterile conditions, take an appropriate amount of serum-free RPMI1640 medium, add it to 10% fetal bovine serum and shake evenly; then add double antibodies (streptomycin 100 μg/mL and penicillin 100 μg/mL) and shake well. Store in the refrigerator at 4°C for later use.

(2) Preparation of PBS buffer salt: Weigh 1.56g Na ₂ HPO ₄ , 0.2g KH ₂ PO ₄ , 0.2g KCl, 8.0g NaCl, dissolve in 950mL ultrapure water after weighing, stir and dissolve with a clean glass rod , and then add ultrapure water to make up to 1000mL. Place it in a clean infusion bottle, insert a needle into the bottle stopper, sterilize at 121°C for 20 minutes under high temperature and high pressure, cool it down, and place it in a refrigerator at 4°C for use.

(3) Plating: preheat 1640 culture medium, trypsin and PBS in a 37°C water bath. Discard the culture medium in the culture flask full of cells, wash it twice with PBS, add 1 mL of trypsin, shake gently to mix, and put it in the incubator for 1 min. After the cells are digested, add 2mL of culture medium in time, use a pipette to make the cells into a suspension, transfer to a centrifuge tube for centrifugation, discard the centrifuged supernatant, add 1mL of 1640 culture medium and mix well, continue to add to 2mL, and mix well . Take a small amount of cell suspension to a counting plate for counting to obtain the total number of cells. Calculate the required number of cells and the volume of the cell suspension based on the number of cells to be plated per well and the number of plates to be plated. Take the cell suspension and add it to a 96-well plate, 100 μL per well. After the addition, shake gently to mix the cells, and put them in an incubator for 24 hours to make the cells adhere to the wall.

(4) Dosing: Prepare the drug to be tested with DMSO to prepare 10 ⁴ μmol/mL of the original drug solution. When adding the drug, take 10 ⁴ μmol/mL of the original drug solution to make 9 concentration gradients, respectively 128, 64, 32 . Place in the incubator for 72h, then add 5mg/mL MTT, 20μL/well, continue to culture for 4h-6h, discard the supernatant in the well plate, add DMSO, 150μL/well, shake in the shaker for 10min, The absorbance value at 490 nm was measured on an enzyme-linked immunosorbent assay instrument, and the IC ₅₀ value was calculated by SPSS statistical software.

(2) Experimental data and results

The present invention is shown in Table 2 to the compound antitumor activity data of above-mentioned synthesis

Table 2 Pharmacological activity (IC ₅₀ ) of a series of target compounds on two kinds of tumor cells

JDAO is 11,20-dicarbonyl oridonin

Oridonin is Oridonin

(3) Conclusion

The above experimental results show that the compounds of the present invention have better antitumor activity in vitro, and are useful for screening esophageal cancer, gastric cancer, primary liver cancer, pancreatic cancer, cardia cancer, colorectal cancer, bladder cancer, breast cancer, and acute myeloid leukemia. Antineoplastic drugs provide the basis. The compound has good stability, and the compound of the present invention is used as an active ingredient to prepare new anticancer drugs, and has potential application value.

Claims (6)

1.11,20- dicarbapentaborane Jiyuan Oridonins, which is characterized in that there is structure as follows:

2.L- amino acid -14- (11,20- dicarbapentaborane Jiyuan Oridonin) ester trifluoroacetic acid salt compound, feature exist In with structure shown in general formula I:

R is to contain the heteroatomic 5-10 membered heterocycloalkyl of 1-2 N, O or S；Or R isWherein R₁For hydrogen, straight chain or branch The C1-C10 alkyl of sulfenyl, amino substitution that the C1-C10 alkyl or C1-C10 linear or branched alkyl group of chain replace, phenyl replace C1-C5 alkyl；R ' is hydrogen or C1-C3 alkyl.

3. l-amino acid -14- (11,20- dicarbapentaborane Jiyuan Oridonin) ester trifluoroacetic acid salt as claimed in claim 2 Compound, which is characterized in that R is to contain the heteroatomic 5-6 membered heterocycloalkyl of 1 N；Or R isWherein R ' be hydrogen or Methyl, R₁For one of hydrogen or following substituent group:

4. l-amino acid -14- (11,20- dicarbapentaborane Jiyuan Oridonin) ester trifluoroacetic acid salt as claimed in claim 2 Compound, which is characterized in that select following compound:

5. preparing l-amino acid -14- as claimed in claim 2 (11,20- dicarbapentaborane Jiyuan Oridonin) ester trifluoroacetic acid The method of salt compounds, which is characterized in that realize by the following method:

It is raw material with compound 1, acetone is solvent, stirring and dissolving, then in the diluted Jones examination of condition of ice bath addition acetone Agent generates 11,20- carbonyl Jiyuan Oridonin (compound 2)；

11,20- dicarbapentaborane Jiyuan Oridonins in methylene chloride, with EDCI (1- (3- dimethylamino-propyl) -3- ethyl carbon Diimmonium salt hydrochlorate), DMAP (4-dimethylaminopyridine) and DIPEA (n,N-diisopropylethylamine) they are catalyst, and N- is added The reaction of Boc-L- amino acid, N-Boc-L- amino acid -14- is made after purification by column chromatography, and (11,20- dicarbapentaborane Jiyuan winters insulted Careless A prime) ester type compound (compound 3)；

N-Boc-L- amino acid -14- (11,20- dicarbapentaborane Jiyuan Oridonin) ester type compound ice bath in methylene chloride, Methylene chloride is added into system: the mixed solution of trifluoroacetic acid 1:1, ice bath stirring reaction；It has been reacted with thin-layer chromatography determination Quan Hou, revolving remove methylene chloride and trifluoroacetic acid, are then added with stirring isopropyl ether, obtain product (compound 4).

6. as described in one of claim 1-4 11,20- dicarbapentaborane Jiyuan Oridonin and l-amino acid -14- (11, 20- dicarbapentaborane Jiyuan Oridonin) application of ester trifluoroacetic acid salt compound in medicine preparation, which is characterized in that with It is active ingredient, preparation treatment cancer of the esophagus, gastric cancer, primary carcinoma of liver, cancer of pancreas, cardia cancer, colorectal cancer, bladder cancer, mammary gland Cancer or acute myeloid leukemia drug.