CN102600129A - Application of andrographolide C15 substitution derivative in manufacturing anti-hepatitis drug - Google Patents

Abstract

The invention belongs to the technical field of medicinal chemistry, and discloses the application of andrographolide C15-position substituted series derivatives in the preparation of anti-hepatitis C drugs. The present invention utilizes Madara's bovine kidney cells (MDBK) and bovine viral diarrhea virus (BVDV) in vitro screening models to study the inhibitory effect of compounds on cytopathic changes caused by viruses. Through the screening of a large number of andrographolide derivatives, it was found that the compound with the general formula 1 structure can significantly inhibit the MDBK cell pathology caused by BVDV, and has high efficiency and low toxicity. Therefore, its application in the preparation of drugs for the treatment and prevention of hepatitis C has good development and application prospects. Formula 1

Description

Application of andrographolide C15-position substituted derivatives in the preparation of anti-hepatitis C drugs

technical field

The invention relates to the medicinal use of andrographolide derivatives, in particular to the application of andrographolide C15-position substituted series derivatives in the preparation of anti-hepatitis C drugs, and belongs to the technical field of medicinal chemistry.

Background technique

Andrographolide (AD) is a diterpene lactone compound extracted from Andrographis paniculata (Burm. f) Nees. It is one of the main active ingredients of the traditional Chinese medicine Andrographis paniculata, and it is mainly used clinically. Treatment of upper respiratory tract infection, bacillary dysentery and other diseases. In recent years, studies have found that andrographolide also has anti-tumor, liver-protecting and gallbladder-protecting, anti-viral and other effects. Andrographolide and its derivatives have good prospects in anti-flavivirus, pestilence or hepatitis C virus (CN: 200580046253.1) and anti-SARS virus (CN: 03129127.9). U.S. Patent No. 5,833,994 discloses the use of aryl hydrocarbon receptor ligands in combination with andrographolide for the treatment of viral infections. Andrographolide monosuccinate inhibits HIV by interfering with virus-cell association and subsequent to the virus-cell-association phase of the viral replication cycle. Methanol extract of Andrographis paniculata can inhibit HIV-1 replication in vitro by inhibiting c-Mos.

The main causative agent of non-A non-B hepatitis is persistent infection with the human hepatitis C virus HCV, which is closely related to liver diseases such as chronic hepatitis, cirrhosis or hepatocellular carcinoma. The development of these liver diseases is a major public health concern. The pathogenesis of hepatitis C is still not very clear. When HCV replicates in liver cells, it causes changes in the structure and function of liver cells or interferes with protein synthesis in liver cells, which can cause degeneration and necrosis of liver cells. However, most scholars believe that the pathological response of cellular immunity may play an important role in the occurrence and development of chronic hepatitis, liver cirrhosis or hepatocellular carcinoma. Studies have found that hepatitis C is the same as hepatitis B, and its tissue infiltrating cells are mainly CD3+, and cytotoxic T cells (TCL) specifically attack HCV-infected target cells, which can cause liver cell damage. The current effective anti-HCV therapy is limited to interferon therapy or the combination therapy of interferon and ribavirin (ribavirin), and a vaccine that can effectively prevent HCV has not yet been developed. However, since about half of the patients treated with these known drugs are not cleared of HCV virus, there is still a strong need for alternative anti-HCV drugs.

HCV is the only member of the genus hepadnaviruses of the Flaviviridae family. BVDV belongs to the Flavivirus genus of the Flaviviridae family, and has a common antigen with HCV. HCV cannot be stably cultured in vitro, but BVDV is easy to grow in MDBK cells, and its gene-encoded products are easy to obtain, which is more convenient for genome research. BVDV AV69 strain can form lesions in cells, therefore, the cytopathic protection rate can be used to evaluate the antiviral activity of drugs. Drugs that can inhibit the replication of BVDV in cells can generally be used to treat HCV infection. Therefore, BVDV has been widely used as a mock strain of HCV to screen anti-HCV drugs.

The 15-benzylidene-substituted andrographolide derivative synthesized by using andrographolide (AD) as the lead compound has been reported in the patent CN: 200510107247.4, which is the applicant's previous research results. Subsequently, it was found that 15-p-chlorobenzylidene-3,19-nicotinate-14-deoxy-11,12-dehydroandrographolide (compound B of the present invention) and some other andrographolide C15-substituted derivatives It has significant anti-HBV effect, which have been reported in patent CN: 200810231375.3. and patent CN: CN201110184294.4 respectively, which are the applicant's previous research results. It is of great significance to develop the application of such compounds and protect our country's independent intellectual property rights to continue to develop research in this field and explore whether the C15-substituted compounds of andrographolide have anti-hepatitis C activity.

Contents of the invention

On the basis of the previous research results, the inventors used the commonly used anti-HCV drug screening model to screen the synthesized compounds and found that the andrographolide derivatives of the general formula 1 structure can significantly inhibit the MDBK cell pathology caused by BVDV, It has high efficiency and low toxicity, and has the potential to be developed as an anti-hepatitis C drug. The purpose of the present invention is to provide its application in the preparation of anti-hepatitis C medicine.

The structural formula of the compound of the present invention is:

Formula 1

R ₁ is hydrogen; R ₂ is phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl; R ₃ and R ₄ are hydrogen, COR ₅ ; R ₅ is 3-pyridyl or R ₅ = _{CH2CH2COOH ;}

Preferred compounds are: R ₁ is hydrogen; R ₂ is phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl; R ₃ and R ₄ are hydrogen.

Preferred compounds are: R ₁ is hydrogen; R ₂ is phenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl; R ₃ , R ₄ are COR _5, R ₅ = 3-pyridyl or R ₅ =CH ₂ CH ₂ COOH.

More preferred compounds are:

A: R ₁ =H, R ₂ =4-Cl-C ₆ H ₄ , R ₃ = R ₄ =H;

B: R ₁ =H, R ₂ =4-Cl-C ₆ H ₄ , R ₃ = R ₄ =COR ₅ , R ₅ =3-C ₅ H ₄ N;

C: R ₁ =H, R ₂ =4-Cl-C ₆ H ₄ , R ₃ = R ₄ =COR ₅ , R ₅ = CH ₂ CH ₂ COOH;

The preparation method of the above-mentioned compound proposed by the present invention has been disclosed in the invention patent CN: 200510107247.4 applied in the early stage of this research. Its synthesis method is as follows: one of 14-deoxy-11,12-dehydroandrographolide or 14-deoxy-11,12-dehydro-3,19-esterified andrographolide and different aldehydes are dissolved in The andrographolide derivative represented by the general formula 1 can be obtained by heating in methanol, ethanol or tetrahydrofuran at a temperature of 15-70°C under base catalysis. Wherein the used alkali is a kind of in sodium carbonate, salt of wormwood, potassium bicarbonate, sodium bicarbonate, triethylamine, pyridine, N, N-dimethylaminopyridine; Its consumption is 0.2～5% mole. the

The aldehyde used is preferably benzaldehyde, halogenated benzaldehyde, etc., and the halogenated benzaldehyde is preferably p-fluorobenzaldehyde, p-chlorobenzaldehyde, and p-bromobenzaldehyde; the 14-deoxy-11,12-dehydrogenated-3,19-esterified Andrographolide corresponds to formula 1 when R ₃ and R ₄ are COR ₅ , R ₅ is 3-pyridyl or R ₅ =CH ₂ CH ₂ COOH.

In order to realize the purpose of the present invention, adopt MDBK (NBL-1) cell, bovine viral diarrhea virus strain (BVDV-AV69) to study the therapeutic effect of C15 substituted andrographolide derivatives of the present invention on the cytopathic effect that BVDV infection causes; MTT method was used to study the cytotoxicity of the compound of the present invention to MDBK. Studies have confirmed that the C15 substituted andrographolide derivatives of the present invention can significantly inhibit the MDBK cell pathology caused by BVDV, and have the potential to be used in the preparation of anti-HCV drugs. With this type of compound as an effective medicinal ingredient or in combination with other drugs, according to the current conventional pharmaceutical methods and process requirements, after mixing with pharmaceutically acceptable auxiliary and/or additive ingredients, it is made into an oral drug for anti-HCV Type preparations, injection preparations and other drugs. Oral preparations are tablets, pills, capsules, granules or syrups, etc.; injection preparations include injections or freeze-dried powder injections, etc.

The advantages and innovations of the present invention: through activity screening, it is determined that the C15 substituted andrographolide derivatives of the present invention have clear anti-BVDV activity in vitro, high efficiency and low toxicity. Therefore, it has the potential to be used in the preparation of medicines for treating and preventing hepatitis C, and provides a new drug approach for the development of anti-hepatitis C virus medicines.

Description of drawings

Fig. 1 is the result figure of the cytotoxic effect of derivatives A～C of the present invention to MDBK;

Fig. 2 is the half maximal inhibitory concentration result figure of derivatives A～C of the present invention to the cytopathic disease that virus causes;

Figure 3 is a graph showing the cytotoxic effect of the positive drug ribavirin on MDBK.

Detailed ways

Through pharmacological tests, taking compounds A to C of the present invention as examples, their anti-BVDV activities are described in detail.

Example 1 In vitro anti-BVDV activity experiment of C15-substituted andrographolide derivatives

1. Cell culture and drug treatment

Adopt MDBK (NBL-1) cells (purchased from Shanghai Typical Culture Collection Center, Chinese Academy of Sciences), bovine viral diarrhea virus (BVDV-AV69) (purchased from China Veterinary Drug Inspection Institute), study the drug of the present invention on the inoculation of BVDV virus Therapeutic effect on cytopathic diseases. Ribavirin (R; Henan Runhong Pharmaceutical Co., Ltd., batch number: 1102261) was used as a positive drug control. The suspension (1×10 ⁵ /mL) of MDBK cells prepared with RPMI1640 culture medium was inoculated in a 96-well plate (Costar, USA), 200 μL/well, and the culture medium contained 10% fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd., batch number: 100524), 100 μg/mL streptomycin (Shenzhen Huayao Nanfang Pharmaceutical Co., Ltd., batch number: N09081), 100 IU/mL penicillin (produced by North China Pharmaceutical Co., Ltd., batch number: E0909604), Place the cells in a 5% CO ₂ incubator (Binder, Germany) at 37°C for 24 hours to grow into a monolayer, add 100 TCID ₅₀ virus solution (TCID ₅₀ =10 ^-2.5 ) and incubate for 1.5 hours. After washing three times with PBS, 200 μL/well of drug-containing maintenance solution was added.

2. Determination of cytotoxicity by MTT method

The well-growing MDBK cells were trypsinized and counted, and then added to 96-well plates at 7×10 ⁴ /mL, 200 μL/well, at 37 °C ^. C, culture in 5% CO ₂ incubator for 24 hours, add drug-containing medium, continue to cultivate for 48 hours, add MTT (5 mg/mL), 20 μL/well, incubate for 4 hours, discard supernatant, add 150 μL DMSO, shake for 10 minutes, use A microplate reader was used to measure the absorbance. The measurement wavelength is 570nm, and the reference wavelength is 450nm. Calculate the cell survival rate after the action of the compound, survival rate (%) = drug group A value / cell control group A value × 100, the results are shown in Figure 1. Cell proliferation inhibition rate (%) = 100-survival rate, according to the Reed-Muench method to calculate the half toxic concentration (TC ₅₀ ).

3. CPE method to observe the therapeutic effect of drugs on cell lesions

MDBK cells were trypsinized, seeded in a 96-well culture plate at a concentration of 1×10 ⁵ /mL, 200 μL/well, and cultured in a 37°C, 5% CO ₂ incubator for 24 hours. After the cells grew into a monolayer, 100TCID50 virus Challenge, incubate for 1.5h, discard the virus-containing maintenance solution, wash with PBS, add 200 μL of different concentrations of drug-containing maintenance solution, and culture at 37°C and 5% CO ₂ . Set normal cell control group, virus control group, drug treatment group, ribavirin treatment group. Observe under an inverted microscope every day, and record the occurrence of cytopathic changes. The minimum concentration (IC ₅₀ ) at which the lesion inhibition rate of the compounds of the present invention and positive drugs is about 50% is shown in Figure 2. The therapeutic index (TC ₅₀ /IC ₅₀ ) was calculated, and the results are shown in Table 1.

Table 1 Compound of the present invention and positive drug TI value

compound TI value AD 14.4 Compound A of the present invention >127.6 Compound B of the present invention >127.6 Compound C of the present invention >250 Ribavirin (R) 4

4. Results and Conclusions

Before conducting the antiviral experiment of the compound to be tested, it is necessary to determine the minimum non-toxic concentration of the compound to be tested on MDBK cells to ensure that the compound does not affect the normal viability of MDBK cells when it acts on the BVDV virus. Compounds A to C of the present invention have no obvious cytotoxicity after acting on MDBK cells in the concentration range of 0-7.5 μmol/L for 48 hours, and compound C has no cytotoxicity when the concentration increases to 30 μmol/L. Compounds A～C showed better antiviral effect than AD, especially compound C. The results of TI values showed that AD and compounds A~C had higher safety index than positive drugs, and compound C had the highest safety index.

In conclusion, compounds A to C of the present invention have stronger therapeutic effects on cytopathic diseases caused by viruses than ribavirin, and have a higher safety index.

Claims (6)

1. the application of andrographolide C15 position substituted derivatives shown in general formula 1 in the preparation of medicines, characterized in that, it is used as an active ingredient for the preparation of anti-hepatitis C drugs,                                                                              General Formula 1 R ₁ is hydrogen; R ₂ is phenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl; R ₃ and R ₄ are hydrogen or COR ₅ ; R ₅ =3-pyridyl or R ₅ = _{CH2CH2COOH .} 2. the application of andrographolide C15 position substituted derivatives in the preparation of medicine as claimed in claim 1, is characterized in that, described compound is: R ₁ is hydrogen; R ₂ phenyl, 4-chlorophenyl, 4-bromophenyl, 4-fluorophenyl; R ₃ , R ₄ are hydrogen. 3. the application of andrographolide C15 position substituted derivatives in the preparation of medicine as claimed in claim 1, is characterized in that, described compound is: R ₁ is hydrogen; R ₂ is phenyl, 4-chlorophenyl , 4-bromophenyl, 4-fluorophenyl; R ₃ , R ₄ are COR _5, R ₅ =3-pyridyl or R ₅ =CH ₂ CH ₂ COOH. 4. the application of andrographolide C15 position substituted derivatives in the preparation of medicine as claimed in claim 1, is characterized in that, described compound is one of the following: A: R ₁ =H, R ₂ =4-Cl-C ₆ H ₄ , R ₃ = R ₄ =H; B: R ₁ =H, R ₂ =4-Cl-C ₆ H ₄ , R ₃ = R ₄ =COR ₅ , R ₅ =3-C ₅ H ₄ N; C: R ₁ =H, R ₂ =4-Cl-C ₆ H ₄ , R ₃ =R ₄ =COR ₅ , R ₅ =CH ₂ CH ₂ COOH. 5. The application of the C15-position substituted derivative of andrographolide according to one of claims 1-4 in the preparation of medicines, characterized in that, it is used as an active ingredient or in combination with other medicines, and the pharmaceutically acceptable After the auxiliary and/or added ingredients are mixed, according to conventional pharmaceutical methods and technical requirements, oral preparations and injection preparations for anti-hepatitis C virus are prepared. 6. the application of andrographolide C15 position substituted derivatives in the preparation of medicine as claimed in claim 5, is characterized in that, oral preparation is tablet, pill, capsule, electuary or syrup; Injectable preparation is injection or Freeze-dried powder dosage form.

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