CN102516301B - Wogonin derivant for treatment - Google Patents

Abstract

The present invention provides derivatives of wogonin and their preparation method and application. The derivatives of wogonin are obtained by chemically modifying the hydroxyl groups of their structures. The derivatives of wogonin involved can be used in pharmaceutical compositions. It is used in the production of drugs for the treatment of hepatitis B and leukemia.

Description

Wogonin Derivatives for Therapy

Technical field:

The present invention relates to wogonin derivatives for treatment, their preparation method and application.

technical background:

Wogonin (wogonin) is a flavonoid component extracted from Scutellaria baicalensis Georgi. It not only has antibacterial, diuretic and antispasmodic effects, but also has strong antiviral effects. Studies in recent years have shown that wogonin can reduce the amount of serum hepatitis B surface antigen in transgenic mice and alleviate liver inflammation in duck hepatitis B virus models. Can prolong the survival time of leukemia mice induced by NB4 cells. In the further study of wogonin, we found that it has a strong anti-tumor effect and can induce the apoptosis of tumor cells, and the further mechanism of action is under study. About the therapeutic effect of wogonin.

Therefore, wogonin, as a new anti-hepatitis B virus drug, plays a very important role in alleviating the pain of cancer patients and improving the quality of life of patients, and has a broad market and a large demand at home and abroad.

However, due to its very low oral bioavailability, commercially available formulations of wogonin are generally considered unsuitable for other modes of administration than parenteral administration, and generally must be intravenously injected or infused. When wogonin is administered intravenously in a clinical setting, it is suggested that other non-oral routes can be used for certain indications.

Through the research on wogonin derivatives, we found that through derivation, a variety of derivatives with curative effects can be obtained. These derivatives are different from wogonin in chemical properties to a certain extent, which makes them have better It is a compound with further development value due to its stability and preparation formability.

Invention content:

The object of the present invention is to provide a new wogonin derivative.

Its structure is:

n stands for 0, 1, 2

Another object of the present invention is to provide a preparation method of wogonin derivatives.

R1 and R2 may be residues of amino acid, phosphoric acid, carboxylic acid or a composite structure having the above two residues at the same time. Preparation of A can be carried out according to the following process:

Process A

The serine-protected groups Pg1 and Pg2 in Scheme A are removable under specific conditions. Non-limiting examples of useful protecting groups for the nitrogen atom include butoxycarbonyl (Boc), benzyloxycarbonyl (CBz) and 9-fluorenylmethoxycarbonyl (Fmoc) moieties, protecting groups for the carboxyl group include t-butyl, benzyl and 9-fluorenylmethyl ester.

During preparation, further reaction with wogonin can also be carried out by partially modifying the derivative residues in advance, such as the preparation in process B.

Process B

In process B, step 1 uses 2-bromoethanol, N,N-dimethylaminopyridine, dicyclohexylcarbodiimide, the reaction temperature is 0-25°C, and the reaction time is 6-48 hours; step 2 uses chloroiodomethane, carbonic acid Sodium hydrogen, reaction temperature 0-50°C, reaction time 1-18 hours; 3 steps 1, 3-dichloropropane, N,N-dimethylaminopyridine, dicyclohexylcarbodiimide, reaction temperature 0-25°C , The reaction time is 18-60 hours.

The amino acids used in the present invention include those with chiral amino acids, and these chiral amino acids are included in the present invention.

The compounds described in the present invention can be prepared into corresponding pharmaceutically acceptable salts as needed. For example, salts with the amino part of the compound, such as inorganic acid sulfuric acid, hydrochloric acid, phosphoric acid, or organic acids such as citric acid, maleic acid, etc.; salts formed with the carboxyl part of the compound, such as sodium salt, potassium salt, magnesium salt or organic bases .

Typical compound of the present invention is:

The compound of the present invention has an effective dosage range of 80 mg to 5000 mg, can be used for treating and preventing hepatitis B, and can also be used for treating leukemia.

The present invention will be further described in detail below in conjunction with the examples, but it should be understood that the scope of the present invention is not limited to the scope of these examples.

Embodiment 1: the preparation of compound A

Dissolve 1.78g of wogonin in 20ml of acetonitrile, slowly add 3ml of phosphorus oxychloride solution dropwise at room temperature, after the drop is complete, stir and react at room temperature for 5 hours, slowly add ice water and stir for hydrolysis reaction for 2 hours, add ethyl acetate Extract 10ml x 3 times, and evaporate the water layer to dryness to obtain 0.63g of Compound A.

Embodiment 2: the preparation of compound B

Dissolve 2g of wogonin in 20ml of tetrahydrofuran, add 20ml of chloroiodomethane at 30°C, stir for 16 hours, evaporate to dryness at 60°C under reduced pressure, add 20ml of acetonitrile to dissolve the residue as an intermediate, and take another 9ml of triethylamine to dissolve In 10ml of acetonitrile, add 3.6ml of phosphoric acid dropwise, after the dropwise addition, slowly drop in the intermediate under stirring, continue to stir and react at 60°C for 12 hours, evaporate the solvent, add 20ml of water to the residue to dissolve, add ethyl acetate 10ml×3 The aqueous layer was washed once, clarified by filtration, and lyophilized to obtain 0.31 g of Compound B.

Embodiment 3: the preparation of compound C

Dissolve 3 g of BOC-protected serine, 2.5 g of 2-bromoethanol, 3 g of N, N-dimethylaminopyridine, and 3 g of dicyclohexylcarbodiimide in tetrahydrofuran, stir at room temperature for 10 hours, concentrate in vacuo, and purify by chromatography (Elute with n-hexane to 30% ethyl acetate/n-hexane) the crude product, combine the filtrates, and evaporate to dryness to obtain intermediate A; dissolve wogonin 2g and intermediate A 2.5g in tetrahydrofuran 30ml, add triphenyl Phosphorus 2g, slowly add 2ml of diethyl azodicarboxylate solution dropwise, and react at room temperature for 5 hours. 10% ethyl acetate/n-hexane elution) crude product, combined filtrate, evaporated to dryness, then dissolved with dichloromethane 20ml, passed into hydrogen chloride gas to saturation, stirred for 5 hours, filtered to obtain the hydrochloride of compound D , dissolved in distilled water and adjusted the pH to about 8, and the aqueous layer was lyophilized to obtain 0.27 g of compound C.

Embodiment 4: the preparation of compound D

Prepared according to the method of Embodiment 2 except that malonic acid is used instead of phosphoric acid.

Embodiment 5: the preparation of compound E

Take 1.3g of compound B, dissolve it in 30ml of acetonitrile, add 5g of Boc-Ser-OBZL in batches, stir the reaction at 50°C, monitor the reaction by HPLC until the compound B is less than 5%, add 10ml of 0.1M hydrochloric acid solution, heat and hydrolyze at 60°C for 5 hours, adjust pH to 7, evaporate to dryness under reduced pressure, add 20ml of water to dissolve the residue, add 10ml of ethyl acetate in batches × 3 washes, evaporate the water layer to dryness, add absolute ethanol to dissolve, filter to remove insoluble matter, evaporate to dryness under reduced pressure, add Dissolved in 5ml of water, filtered, and freeze-dried to obtain 0.18g of compound E

Embodiment 6: Compound A injection preparation

prescription

Take the prescription amount of excipients and raw materials, add to 90000ml of water for injection, add 10g of activated carbon, stir and decolorize at 80°C for 30 minutes, filter through a 0.22μm filter membrane to remove the activated carbon, measure the content of intermediates, add water for injection, fill, and sterilize the obtained compound A injection.

Example 7: Preparation of Compound B for Injection

prescription:

Take the prescribed amount of raw materials and various excipients, add 100ml of water for injection, stir, add 0.2g of activated carbon, stir at 60°C for 30 minutes, filter and clarify through a 0.22μm filter membrane, detect the content of intermediates, and distribute to the control center according to the specification of 35mg per bottle In the vial, freeze at -50°C for 4 hours, vacuumize, heat up and freeze-dry, and control the water content to not more than 5%, and compound B for injection is obtained.

Example 8: Metabolism study of Compound B in animals

40 HBV transgenic mice were divided into 5 groups according to the level of serum HbsAg concentration, and compound B was administered respectively, high-dose group 50mg/kg, middle-dose group 25mg/kg, low-dose group 12.5mg/kg, positive control group (Lami Vudine 100mg/kg), blank experimental group (physiological saline). Eight rats in each group were administered for 10 days. The high, middle and low dose groups were administered via tail vein every 2 days; the lamivudine group was intragastrically administered every day. Serum samples were collected before administration, 5 days after administration, 10 days after administration and 5 days after withdrawal. 24 hours after the last drug administration, 3 mice in each group were randomly killed, and liver, kidney and other organ samples were taken; the rest of the mice were killed after the last drug administration, and liver, kidney and other organ samples were taken.

Serum HBsAg was tested using the Hepatitis B Antigen Surface Quantitative Kit, and the operation was performed according to the kit instructions. Annotated curves were constructed using recombinant HBsAg. Serum samples were diluted 20 times with normal saline for detection.

On the 10th day of treatment, the serum HBsAg content of the mice in the high-dose group decreased significantly, by about 27.1% compared with before treatment, and there was a significant difference compared with the normal saline control group and before administration; the serum HBsAg content of the mice in the middle-dose group decreased About 41.7%. After 5 days of drug withdrawal, the serum HBsAg content of the mice in the high-dose group and the middle-dose group remained stable, and no rebound phenomenon occurred. On the contrary, the serum HBsAg content of the mice in the lamivudine group increased compared with the 10-day administration.

Conclusion: Treating hepatitis B transgenic mice for 10 days can significantly reduce the amount of serum hepatitis B surface antigen, which is significant compared with the control group and the treatment group.

Example 9: Compound B induces differentiation of leukemia cells in vivo.

The SCID mice were randomly divided into four groups, 8 in each group, and the blank control group was given normal saline (NS) 20ml/kg.

The concentration of the compound B high-dose group was 50 mg/kg, the concentration of the middle-dose group was 25 mg/kg, the concentration of the low-dose group was 12.5 mg/kg, and the positive control group was given retinoic acid 15 mg/kg. 100 μl of NB4 cells (10 ⁶ ) were inoculated into SCID mice by intraperitoneal injection, and the administration began on the second day. Each group was administered for two weeks, and the survival days of the mice were observed.

The test results showed that, compared with the blank control group, high and medium doses could significantly prolong the survival time of SCID mice.

Claims (6)

1. Compounds of general formula (I) and their pharmaceutically acceptable salts: n stands for 1, 2 R ₁ is an amino acid or a phosphate residue, and R ₂ is an amino acid or a phosphate residue. 2. The compound of claim ₁ , characterized in that R1 is an amino acid residue and R2 is a phosphoric _acid residue. 3. The compound of claim ₁ , characterized in that R1 is a phosphate residue and R2 is an amino _acid residue. 4. The compound of claim 1, characterized in that R ₁ and R ₂ are amino acid residues. 5. The compound of claim 1, characterized in that R ₁ and R ₂ are phosphoric acid residues. 6. A pharmaceutical composition, characterized in that it contains a therapeutically effective dose of the compound described in claims 1-5 as an active ingredient and a pharmaceutically acceptable carrier.