CN107814801A - New matrine compound and preparation method and application - Google Patents

Abstract

The invention relates to a new class of matrine compounds and their salts, which belong to the technical field of medicine. The matrine compounds have the following general chemical structure In the general formula, R is selected from hydrogen, alkyl, halogen, cyano, nitro, alkoxy, and haloalkyl, and can be located at the ortho, meta, or para positions of the benzene ring, and can be mono-substituted or multi-substituted. Its salts include hydrochloride, sulfate, bisulfate, hydrobromide, oxalate, citrate, methanesulfonate. The present invention also provides the preparation method of the above compounds and their salts, and the in vitro inhibitory activity of these compounds on human liver cancer cell line (HCC‑LM3), which can be used to prepare drugs for treating liver cancer and other related cancers.

Description

Novel matrine compound and its preparation method and application

technical field

The present invention relates to the field of medical technology, specifically, a new class of matrine compounds and their pharmaceutically acceptable salts, their preparation methods, and their antitumor activity, which can be used as medicines for treating liver cancer and other related cancers .

Background technique

Sophora flavescens, also known as Sophora flavescens Ait., also known as Sophora flavescens Ait., is a traditional Chinese medicine with a long history and is widely distributed in various places in my country. Jing He. Medicinal Botany. Shanghai: Shanghai Science and Technology Press, 1990:149.). The medicinal value of Sophora flavescens is closely related to the types of alkaloids it contains. In recent years, a lot of research has been done on the extraction, separation, and pharmacological effects of alkaloids in Sophora flavescens, and certain research results have been obtained. Matrine (Matrine) is a representative of Sophora flavescens alkaloids, a kind of alkaloids of Sophora flavescens, belongs to quinolizidine derivatives, has a wide range of pharmacological effects, such as anti-inflammatory, anti-viral, anti- Rheumatism, anti-hepatic fibrosis, anti-tumor, antibacterial, anti-allergy, anti-parasite, anti-arrhythmia, swelling and diuresis, immune and biological response regulation, etc. (Jiang Hezhong, Matrine and oxymatrine pharmacological effects and Research progress on preparation methods, Practical Integrative Medicine, 2007, 7(1): 89). Matrine has inhibitory effects on a variety of tumors, and has a variety of anti-cancer effects, such as anti-liver cancer, anti-lung cancer, anti-gastric cancer, anti-breast cancer, anti-nasopharyngeal cancer, anti-leukocyte cancer, etc. (Xiao Shuo. Matrine is various Research progress on anticancer efficacy. Journal of Practical Medicine, 2010,26(24):4605-4606.). Matrine has a direct killing effect on tumor cells, and can also induce tumor cell apoptosis, and matrine hardly affects the life process of normal cells, and can even increase the number of white blood cells in the blood, and promote the improvement of the immune function of the body ( Yu Xishui, Sun Wenping, Zhu Fei, et al. Traditional application of Sophora flavescens and development of new drugs. Heilongjiang Medicine, 1995, 8(1): 314-316. Xu Xiangru, Jiang Jikai. Research progress of Sophora flavescens and its alkaloid antitumor activity. Chinese Journal of Integrative Medicine, 1998,18(5):314-316.).

Contents of the invention

The object of the present invention is to provide a class of matrine compounds and pharmaceutically acceptable salts thereof to address the deficiencies in the prior art.

The second object of the present invention is to provide a preparation method of a class of matrine compounds and pharmaceutically acceptable salts thereof.

The third object of the present invention is to provide the application of a class of matrine compounds and pharmaceutically acceptable salts thereof.

In order to achieve the above object, the technical solution adopted by the present invention is: a class of matrine compounds and pharmaceutically acceptable salts thereof, the general structural formula of which is:

R: selected from hydrogen, alkyl, halogen, cyano, nitro, alkoxy, haloalkyl, can be located in the ortho, meta, para position of the benzene ring, can be mono-substituted or multi-substituted;

Wherein, the alkyl group is an alkyl group with 1-4 carbon atoms;

Halogen is selected from F, Cl, Br, I;

Alkoxy is selected from methoxy, ethoxy, tert-butyloxy;

Haloalkyl is selected from trifluoromethyl, monochloromethyl, dichloromethyl, trichloromethyl.

The pharmaceutically acceptable salts are hydrochloride, sulfate, hydrogensulfate, hydrobromide, oxalate, citrate, methanesulfonate and the like.

In order to achieve the above-mentioned second purpose, the technical solution adopted by the present invention is: a preparation method of matrine compounds, the preparation method being:

Compound (I) undergoes Michael addition reaction with substituted thiophenol and triethylamine in anhydrous dichloromethane under the protection of argon at room temperature to obtain compound (II).

The synthesis of its pharmaceutically acceptable salts is based on the above-mentioned reactions, and further reacts as follows:

Compound (II) reacts with acid to obtain its corresponding salt (III), HA is hydrochloric acid, sulfuric acid, hydrogen sulfate, hydrobromic acid, oxalic acid, citric acid or methanesulfonic acid.

In order to achieve the above third objective, the technical solution adopted by the present invention is: the application of the matrine compound and its pharmaceutically acceptable salts in the preparation of anti-liver cancer drugs.

The chemical structures, yields, NMR and mass spectrum data of some preferred compounds synthesized by the present invention are shown in Table 1.

The structure, yield, mass spectrum, NMR and molecular formula of some preferred compounds in Table 1

The present invention has the advantage that:

The present invention synthesizes the above compound for the first time, and the compound of the present invention has good inhibitory activity on human liver cancer cells in vitro, and in addition, the compound of the present invention also has the advantages of low toxicity and the like.

Detailed ways

The specific implementation modes provided by the present invention will be described in detail below in conjunction with the examples.

Embodiment 1: Preparation of 13-(2'-bromophenylene sulfide)matrine (compound 1 in the table)

Weigh 0.246g (1mmol) of sophocarpine and 0.378g (2mmol) of o-bromothiophenol into a 25mL round bottom flask, add 10mL of dry dichloromethane under the protection of argon to dissolve, stir magnetically at room temperature, and slowly add triethyl Add 0.5 mL of amine, and continue the reaction after the dropwise addition is completed. The reaction progress is detected by TLC (dichloromethane/methanol=10:1), and the reaction is complete in about 5 hours. After the reaction solution was filtered with celite, it was concentrated under reduced pressure to remove the solvent, and the crude product obtained was purified by a thin-layer silica gel column with dichloromethane/methanol as the eluent to obtain 0.369 g of a light yellow solid with a yield of 85%.

Embodiment 2: Preparation of 13-(3'-bromophenylene sulfide)matrine (compound 7 in the table)

Weigh 0.246g (1mmol) of sophocarpine and 0.378g (2mmol) of m-bromothiophenol into a 25mL round bottom flask, add 10mL of dry dichloromethane under the protection of argon to dissolve, stir magnetically at room temperature, and slowly add triethyl Add 0.5 mL of amine, and continue the reaction after the dropwise addition is completed. The reaction progress is detected by TLC (dichloromethane/methanol=10:1), and the reaction is complete in about 5 hours. After the reaction solution was filtered with celite, it was concentrated under reduced pressure to remove the solvent, and the crude product obtained was purified by a thin-layer silica gel column with dichloromethane/methanol as the eluent to obtain 0.343 g of a light yellow solid with a yield of 79%.

Embodiment 3: Preparation of 13-(4'-bromophenylene sulfide)matrine (compound 12 in the table)

Weigh 0.246g (1mmol) of sophocarpine and 0.378g (2mmol) of p-bromothiophenol into a 25mL round bottom flask, add 10mL of dry dichloromethane under the protection of argon to dissolve, stir magnetically at room temperature, and slowly add triethyl Add 0.5 mL of amine, and continue the reaction after the dropwise addition is completed. The reaction progress is detected by TLC (dichloromethane/methanol=10:1), and the reaction is complete in about 5 hours. After the reaction solution was filtered with celite, it was concentrated under reduced pressure to remove the solvent, and the crude product obtained was purified by a thin-layer silica gel column with dichloromethane/methanol as the eluent to obtain 0.330 g of a light yellow solid with a yield of 76%.

Embodiment 4: Preparation of 13-(4'-tert-butylphenylsulfide)matrine (compound 13 in the table)

Weigh 0.246g (1mmol) of sophocarpine and 0.332g (2mmol) of p-tert-butylthiophenol into a 25mL round bottom flask, add 10mL of dry dichloromethane under the protection of argon to dissolve, stir magnetically at room temperature, and slowly add Triethylamine 0.5mL, continue to react after the dropwise addition, TLC detects the reaction progress (dichloromethane/methanol=10:1), and the reaction is complete in about 5h. After the reaction solution was filtered with celite, it was concentrated under reduced pressure to remove the solvent, and the crude product obtained was purified by a thin-layer silica gel column with dichloromethane/methanol as the eluent to obtain 0.33 g of a light yellow solid with a yield of 80%.

Embodiment 5: Preparation of 13-(4'-trifluoromethylphenylsulfide)matrine (compound 14 in the table)

Weigh 0.246g (1mmol) of sophocarpine and 0.356g (2mmol) of p-trifluoromethylthiophenol into a 25mL round bottom flask, add 10mL of dry dichloromethane under the protection of argon to dissolve, stir magnetically at room temperature, slowly drop Add 0.5 mL of triethylamine, continue the reaction after the dropwise addition, and detect the reaction progress by TLC (dichloromethane/methanol=10:1), and the reaction is complete in about 5 hours. After the reaction solution was filtered with celite, it was concentrated under reduced pressure to remove the solvent, and the crude product obtained was purified by a thin-layer silica gel column with dichloromethane/methanol as the eluent to obtain 0.369 g of a light yellow solid with a yield of 87%.

Example 6: Preparation of 13-(2',4'-difluorophenylsulfide)matrine (compound 22 in the table)

Weigh 0.246g (1mmol) of sophocarpine and 0.292g (2mmol) of 2',4'-difluorothiophenol into a 25mL round bottom flask, add 10mL of dry dichloromethane under the protection of argon to dissolve, and stir magnetically at room temperature , slowly added 0.5 mL of triethylamine dropwise, and continued the reaction after the dropwise addition was completed. The reaction progress was detected by TLC (dichloromethane/methanol=10:1), and the reaction was complete in about 5 hours. After the reaction solution was filtered with celite, it was concentrated under reduced pressure to remove the solvent, and the crude product obtained was purified by a thin-layer silica gel column with dichloromethane/methanol as the eluent to obtain 0.298 g of a light yellow solid with a yield of 76%.

Example 7: Preparation of 13-(2',4'-difluorophenylene sulfide)matrine hydrochloride (the hydrochloride of compound 22 in the table)

Take 100 mg (0.26 mmol) of compound 22 prepared above, dissolve it in 5 mL CH ₂ Cl ₂ , slowly add 1 mL concentrated hydrochloric acid dropwise, react at room temperature for 4 hours after the dropwise addition, concentrate after the reaction, filter, and recrystallize from isopropanol to obtain white crystals 50mg, yield 45%.

If sulfuric acid, hydrogen sulfate, hydrobromic acid, oxalic acid, citric acid or methanesulfonic acid are selected, the sulfuric acid, hydrogen sulfate, hydrobromic acid, oxalic acid, citric acid or methanesulfonic acid salt of compound 22 can be obtained, and the preparation method is the same.

The implementation of the present invention is not limited to the above examples. When preparing the remaining target compounds, the corresponding R-substituted compounds are used as raw materials, and the method is the same as above.

Embodiment 8: biological activity test

1 Instrument and equipment

Sterile operating table, biological microscope, constant temperature cell incubator, sterilizing ultraviolet lamp, enzyme-linked immunoassay instrument, pipette, cell culture dish, 6-well cell culture plate, 96-well cell culture plate.

2 Experimental materials and preparation

2.1 Test drug:

Matrine Derivatives, Matrine

Appearance: powder

Content: 99%

Preparation method: dissolve in DMSO, and dilute with PBS to obtain the required concentration sample;

2.2 Cell lines:

Human liver cancer cell line (HCC-LM3) was provided by Dongfang Hepatobiliary Hospital of Second Military Medical University.

2.3 Reagents:

Reagents: cell culture medium (DMEM), MTT kit (5mg/mL), PBS phosphate buffer, 1% dimethyl sulfoxide, 0.25% trypsin-EDTA (Trypsin-EDTA), fetal bovine serum (FBS) , double antibody (penicillin-streptomycin).

2.4 Experimental consumables: 96-well cell culture plate, 50ml cell culture flask, purchased from Corning Company.

3 experimental steps

3.1 Preparation of the test drug: Weigh the test drug, each weighing 1.5mg (±20μg), add 200μL of dimethyl sulfoxide to dissolve, then add 800μL of PBS solution to make a 1.5mg/mL sample solution, place Shake well in a shaker, and then sterilize under a sterilizing ultraviolet lamp for later use.

3.2 Take the HCC-LM3 cell line, resuscitate the cells to a 6-well cell culture plate for subculture growth, and grow to the logarithmic growth phase after 24 hours. Digest the cells with trypsin to make a cell suspension, add it to a 96-well cell culture plate with a pipette gun, add 200 μL of the cell suspension (about 5000 cells) to each well, and fill the edge wells with sterile PBS.

3.3 Put the 96-well cell culture plate into a constant temperature cell culture incubator. After about 12 hours, the cells adhere to the wall and grow. Add the prepared test drug, add 20 μL to each well, and make the corresponding dilution to make the drug concentration 0.1 mg/mL. Each drug was tested in triplicate.

3.4 Put the 96-well cell culture plate into a constant temperature cell culture incubator and culture at 37.5°C for 24 hours.

3.5 After the drug effect is over, replace the culture medium with a new one, add 20 μL of MTT to each well under the condition of avoiding light, and place it in a constant temperature cell incubator to continue culturing for 3-4 hours.

3.6 Terminate the culture, carefully absorb the culture medium in the 96-well cell culture plate with a pipette gun, add 150 μL of DMSO to each well, shake it in a shaker at a low speed for 10 minutes, take it out, and measure the absorbance at 490 nm with an enzyme-linked immunosorbent assay (OD value). At the same time, set the zero well and the control well. The zero well is composed of serum-free medium, MTT and DMSO, and the control well is composed of cells, serum-free medium, MTT and DMSO. The OD value is determined.

3.7 Calculate the cell viability (Cell Viability) according to the measured OD value, and obtain the preliminary in vitro anti-tumor activity data of the tested drug.

Cell viability (Cell Viability of Control) = (OD value of drug group - OD value of zero-adjusted well) / (OD value of control well - OD value of zero-adjusted well) × 100%.

3.8 Calculate the inhibition ratio (Inhibition ratio) according to the cell viability. Inhibition rate=(1-cell viability)×100%.

4 Experimental results

The in vitro growth inhibition rate of matrine derivatives (0.1mg/mL) on human liver cancer cell line (HCC-LM3) is shown in the table below.

Table 2 matrine derivatives (0.1mg/mL) to the in vitro growth inhibition rate of human liver cancer cell line (HCC-LM3)

The compound of the present invention has better inhibitory activity on human liver cancer cells in vitro, and in addition, the compound of the present invention also has the advantages of low toxicity and the like.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, some improvements and supplements can also be made, and these improvements and supplements should also be considered Be the protection scope of the present invention.

Claims (9)

1. a kind of matrine compound and its pharmaceutically acceptable salt class, it is characterised in that its structure is as shown in formula：

R is selected from hydrogen in formula, alkyl, halogen, cyano group, nitro, alkoxy, haloalkyl, can be located at o-, m-, the contraposition of phenyl ring, It is monosubstituted or polysubstituted.

2. matrine compound according to claim 1 and its pharmaceutically acceptable salt class, it is characterised in that alkyl For the alkyl of 1-4 carbon atom；Halogen is selected from F, Cl, Br, I；Alkoxy is selected from methoxyl group, ethyoxyl, tert-butyl group epoxide；Halo Alkyl is selected from trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl.

3. matrine compound according to claim 1 and its pharmaceutically acceptable salt class, it is characterised in that R is selected From the alkyl of 1-4 carbon atom, F, Cl, Br, methoxyl group, ethyoxyl, trifluoromethyl.

4. matrine compound according to claim 1 and its pharmaceutically acceptable salt class, it is characterised in that R is It is monosubstituted or disubstituted.

5. matrine compound according to claim 1 and its pharmaceutically acceptable salt class, it is characterised in that described Compound be：

6. matrine compound according to claim 1 and its pharmaceutically acceptable salt class, it is characterised in that pharmacy Upper acceptable salt is hydrochloride, sulfate, disulfate, hydrobromate, oxalates, citrate or mesylate.

A kind of 7. preparation method of matrine compound as claimed in claim 1, it is characterised in that described preparation method For：

With substitution benzenethiol and triethylamine in anhydrous methylene chloride, the lower generation Michael of room temperature argon gas protection adds compound (I) Compound (II) is obtained into reaction.

8. a kind of preparation method of the pharmaceutically acceptable salt class of matrine compound as claimed in claim 1, it is special Sign is that described preparation method is：

With substitution benzenethiol and triethylamine in anhydrous methylene chloride, the lower generation Michael of room temperature argon gas protection adds compound (I) Compound (II) is obtained into reaction,

Compound (II) obtains its corresponding salt (III) with acid reaction, and HA is hydrochloric acid, sulfuric acid, hydrogen sulfate, hydrobromic acid, oxalic acid, lemon Lemon acid or methanesulfonic acid.

9. matrine compound according to claim 1 and its pharmaceutically acceptable salt class are preparing medicines resistant to liver cancer In application.