阅读说明：本技术 一种盐酸小檗碱二聚物及其制备方法和应用 (Berberine hydrochloride dimer and preparation method and application thereof ) 是由 陈健 陶成 王庆文 林献 张苗苗 何莲花 张楚楚 于 2021-07-13 设计创作，主要内容包括：本发明提供一种盐酸小檗碱二聚物,所述盐酸小檗碱二聚物具有如下所示的化学结构：本发明盐酸小檗碱二聚物的抗肿瘤效果显著优于阳性对照盐酸小檗碱,具有很好的抗肿瘤活性和很强的成药性,该类化合物作为新型抗肿瘤化合物具有很大的开发价值。本发明还提供所述盐酸小檗碱二聚物的制备方法。(The invention provides a berberine hydrochloride dimer, which has the following chemical structure: the berberine hydrochloride dimer has an anti-tumor effect obviously superior to that of positive control berberine hydrochloride, has good anti-tumor activity and strong drug forming property, and has great development value as a novel anti-tumor compound. The invention also provides a preparation method of the berberine hydrochloride dimer.)

1. A berberine hydrochloride dimer, characterized in that the berberine hydrochloride dimer has a chemical structure as shown in the following:

2. an anticancer agent comprising the berberine hydrochloride dimer according to claim 1 or a pharmaceutically acceptable salt thereof.

3. The anticancer agent as claimed in claim 2, wherein said anticancer agent further comprises pharmaceutically acceptable auxiliary agents;

preferably, the anticancer drug is a tablet, capsule powder, injection or injection.

4. The use of the berberine hydrochloride dimer of claim 1 in the preparation of an anti-cancer medicament.

5. The use according to claim 4, characterized in that said berberine hydrochloride dimers are used as raw material for the preparation of anticancer drugs in the form of compositions, or in the form of any pharmaceutically acceptable salts.

6. The process for preparing berberine hydrochloride dimers of claim 1, wherein said process comprises obtaining said berberine hydrochloride dimers by the reaction of:

7. the process for preparing berberine hydrochloride dimers of claim 6, wherein the molar ratio of berberine hydrochloride to initiator is 1: 1-1.5: 1, preferably 1: 1;

preferably, the initiator is [ bis (trifluoroacetoxy) iodo ] benzene or iodobenzene acetate;

the molar ratio of the berberine hydrochloride to the boron trifluoride diethyl etherate is 1: 1-1: 2, preferably 1: 2.

8. the method for preparing berberine hydrochloride dimers of claim 6, wherein the berberine hydrochloride is mixed with an organic solvent to form a mixture, and the molar concentration of the berberine hydrochloride in the mixture is 0.05-0.2 mol/L, preferably 0.1 mol/L.

9. The method for producing berberine hydrochloride dimers of claim 8, wherein the organic solvent is selected from the group consisting of dichloromethane, tetrahydrofuran, 1, 4 dioxane, chloroform and carbon tetrachloride.

10. The process for preparing berberine hydrochloride dimers of claim 6, wherein said process comprises the steps of:

dissolving berberine hydrochloride in dichloromethane, adding [ bis (trifluoroacetoxy) iodine ] benzene and boron trifluoride diethyl etherate at the temperature of-78 to-30 ℃, and reacting to obtain reaction liquid;

extracting the reaction solution with ethyl acetate, collecting an organic phase, drying the organic phase with sodium sulfate, filtering, concentrating the organic phase, and separating by a silica gel column to obtain berberine hydrochloride dimer;

preferably, the eluent used for passing through the silica gel column is a mixed solution of petroleum ether and ethyl acetate;

preferably, the volume ratio of the petroleum ether to the ethyl acetate is 3: 1-1: 1.

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a berberine hydrochloride dimer and a preparation method and application thereof.

Background

The research on medicinal chemistry using natural products as lead compounds plays an increasingly important role in promoting the development of modern medicine, and medicines related to natural products account for a considerable proportion of various approved medicines on the market, and according to statistics, the medicines related to natural products account for more than 50% of the approved medicines on the market worldwide.

Therefore, structural modification of natural product monomeric compounds by means of medicinal chemistry research is one of important means in the development of new drugs.

Berberine hydrochloride is one of the main effective components of the alkaloid extract of Chinese medicinal coptis for clearing heat. As an important traditional Chinese medicine monomer compound, berberine hydrochloride has very important medicinal value in the aspects of anti-inflammation, bacteriostasis, amebiasis resistance and the like. In recent years, research has shown that berberine hydrochloride has anti-tumor activity (Cancer Manag Res.2020,12: 695-containing 702.), and research on synthesis and medicinal chemistry of berberine hydrochloride and derivatives thereof has become a hotspot (chem. Eur. J.2016,22: 7084-containing 7089.).

However, the research on the dimer of berberine compounds is very rare at present, mainly because the dimer of berberine compounds is difficult to synthesize, which also limits the further research on the compounds.

Therefore, the research on how to prepare the dimer of the berberine compounds by adopting a medicinal chemical method has important significance.

Disclosure of Invention

In order to solve the technical problems, the invention provides a berberine hydrochloride dimer with a novel structure and a preparation method thereof, and the berberine hydrochloride dimer has higher anticancer activity and great development value as a novel antitumor compound.

The invention provides a berberine hydrochloride dimer, which has the following chemical structure:

the invention provides an anticancer drug, which comprises the berberine hydrochloride dimer or pharmaceutically acceptable salt of the berberine hydrochloride dimer.

As an example, the anticancer drug further comprises a pharmaceutically acceptable auxiliary agent.

As an example, the anticancer drug is a tablet, capsule powder, injection or injection.

The invention also relates to the application of the berberine hydrochloride dimer in preparing anticancer drugs.

As an example, the berberine hydrochloride dimer is used as a raw material to prepare a composition type anticancer drug, or to prepare any pharmaceutically acceptable salt.

The invention provides a preparation method of the berberine hydrochloride dimer, which comprises the following steps of:

as an example, the molar ratio of berberine hydrochloride to initiator is 1: 1-1.5: 1, preferably 1: 1.

as an example, the initiator is [ bis (trifluoroacetoxy) iodo ] benzene or iodobenzene acetate.

As an example, the molar ratio of berberine hydrochloride to boron trifluoride etherate is 1: 1-1: 2, preferably 1: 2.

as an example, the berberine hydrochloride and the organic solvent form a mixture, and the molar concentration of the berberine hydrochloride in the mixture is 0.05-0.2 mol/L, and preferably 0.1 mol/L.

As an example, the organic solvent is selected from dichloromethane, tetrahydrofuran, 1, 4 dioxane, chloroform or carbon tetrachloride.

As an example, the preparation method comprises the following steps:

dissolving berberine hydrochloride in dichloromethane, adding [ bis (trifluoroacetoxy) iodine ] benzene and boron trifluoride diethyl etherate at the temperature of-78 to-30 ℃, and reacting to obtain reaction liquid;

extracting the reaction solution with ethyl acetate, collecting an organic phase, drying the organic phase with sodium sulfate, filtering, concentrating the organic phase, and separating by a silica gel column to obtain berberine hydrochloride dimer;

preferably, the eluent used for passing through the silica gel column is a mixed solution of petroleum ether and ethyl acetate;

preferably, the volume ratio of the petroleum ether to the ethyl acetate is 3: 1-1: 1.

compared with the prior art, the invention has the following beneficial effects:

1. the invention provides a berberine hydrochloride derivative (dimer CT4-1 of berberine hydrochloride) with a novel structure.

2. The dimer CT4-1 of the berberine hydrochloride of the invention is biologically evaluated, the anti-tumor effect is obviously better than that of positive control berberine hydrochloride, the dimer CT4-1 has good anti-tumor activity and strong drug forming property, and the dimer CT4-1 has great development value as a novel anti-tumor compound.

3. The design idea of the CT4-1 molecules provides a new idea and way for developing novel antitumor drugs.

4. The invention adopts a novel and efficient chemical synthesis method to obtain the berberine hydrochloride dimer (CT4-1) with a novel structure for the first time.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and not to limit the invention.

FIG. 1 shows the chemical reaction principle involved in the method for preparing berberine hydrochloride derivative CT4-1 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention are clearly and completely described below. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the described embodiments of the invention without any inventive step, are within the scope of protection of the invention.

Example 1

Dissolving 371mg and 1mmol of berberine hydrochloride in 10mL of dichloromethane, adding 430mg and 1mmol of [ bis (trifluoroacetoxy) iodine ] benzene and 282mg and 2mmol of boron trifluoride diethyl etherate at the temperature of-78 ℃, and reacting for 4 hours to obtain a reaction solution;

extracting the reaction solution by using 60mL of ethyl acetate, collecting an organic phase, drying the organic phase by using sodium sulfate, filtering, concentrating the organic phase, and separating by using a silica gel column, wherein the volume ratio of eluent is 3: 1-1: 1 was mixed with petroleum ether and ethyl acetate and isolated to give 420mg of the compound as a pale yellow solid in 80 wt% yield.

Example 2

The difference from example 1 is in the reaction parameters.

Dissolving 371mg and 1mmol of berberine hydrochloride in 10mL of tetrahydrofuran, adding 430mg and 1mmol of [ bis (trifluoroacetoxy) iodine ] benzene and 282mg and 2mmol of boron trifluoride diethyl etherate at-78 ℃, and reacting for 4 hours to obtain a reaction solution;

extracting the reaction solution by using 60mL of ethyl acetate, collecting an organic phase, drying the organic phase by using sodium sulfate, filtering, concentrating the organic phase, and separating by using a silica gel column, wherein the volume ratio of eluent is 3: 1-1: 1 was mixed with petroleum ether and ethyl acetate and isolated to give 420mg of the compound as a pale yellow solid in 60 wt% yield.

Example 3

The difference from example 1 is in the reaction parameters.

Dissolving 371mg and 1mmol of berberine hydrochloride in 10mL of dichloromethane, adding 430mg and 1mmol of [ bis (trifluoroacetoxy) iodine ] benzene and 282mg and 2mmol of boron trifluoride diethyl etherate at the temperature of-30 ℃, and reacting for 4 hours to obtain a reaction solution;

extracting the reaction solution by using 60mL of ethyl acetate, collecting an organic phase, drying the organic phase by using sodium sulfate, filtering, concentrating the organic phase, and separating by using a silica gel column, wherein the volume ratio of eluent is 3: 1-1: 1 was mixed with petroleum ether and ethyl acetate and isolated to give 420mg of the compound as a pale yellow solid in 50 wt% yield.

Example 4

The difference from example 1 is in the reaction parameters.

Dissolving 371mg and 1mmol of berberine hydrochloride in 10mL of dichloromethane, adding 640mg and 1.5mmol of [ bis (trifluoroacetoxy) iodine ] benzene and 282mg and 2mmol of boron trifluoride diethyl etherate at the temperature of-78 ℃, and reacting for 4 hours to obtain a reaction solution;

extracting the reaction solution by using 60mL of ethyl acetate, collecting an organic phase, drying the organic phase by using sodium sulfate, filtering, concentrating the organic phase, and separating by using a silica gel column, wherein the volume ratio of eluent is 3: 1-1: 1 was mixed with petroleum ether and ethyl acetate and isolated to give 420mg of the compound as a pale yellow solid in 40 wt% yield.

Example 5

Dissolving 371mg and 1mmol of berberine hydrochloride in 10mL of tetrahydrofuran, adding 640mg,1.5mmol of iodobenzene acetate, 282mg and 1mmol of boron trifluoride diethyl etherate at the temperature of-78 ℃, and reacting for 4 hours to obtain a reaction solution;

extracting the reaction solution by using 60mL of ethyl acetate, collecting an organic phase, drying the organic phase by using sodium sulfate, filtering, concentrating the organic phase, and separating by using a silica gel column, wherein the volume ratio of eluent is 3: 1-1: 1 was mixed with petroleum ether and ethyl acetate and isolated to give 420mg of the compound as a pale yellow solid in 30 wt% yield.

The chemical characterization results for the final products prepared in examples 1-5 above are as follows:

R_f＝0.2(petroleum ether/EtOAc,1:1)；¹H NMR(500MHz,Chloroform-d)δ7.19(d,J＝9.0Hz,2H),7.04(d,J＝8.9Hz,2H),6.40(s,2H),6.17(s,2H),5.92–5.60(m,4H),5.08(dd,J＝13.3,3.3Hz,2H),4.09(s,6H),3.92(s,6H),3.12(d,J＝2.7Hz,2H),2.50(dt,J＝15.3,2.8Hz,2H),0.94(t,J＝7.4Hz,2H).¹³C NMR(126MHz,CDCl₃)δ167.66,159.72,151.75,149.43,147.30,145.18,135.89,133.30,133.12,130.87,128.79,123.58,122.20,119.67,118.45,110.53,108.36,106.70,100.98,65.52,61.66,56.45,41.10,29.65,29.47,19.14,13.70.HRMS(ESI):m/zcalcd for C₄₀H₃₂N₂NaO₁₀[M+Na]⁺:723.1955,found:723.1950.

biological evaluation

Pharmacological and pharmacodynamic test of example 1

Experiment one effect of CT4-1 on the proliferation of human pancreatic cancer cell line SW1990

Purpose of the experiment: study on the Effect of CT4-1 on the proliferation of human pancreatic cancer cell line SW1990

1. Experimental Material

1.1 cell line human pancreatic cancer cell line SW1990

Reagent and instruments fetal bovine serum (FBS, Hyclone), L-15 medium (Gibco), DMSO (Sigma), phosphate buffered saline (PBS, BI), CCK8 kit (Nippon colleague chemical research institute), trypsin (Gibco), BioTek Synergy full-function microplate reader (Molecular Devices), low-speed centrifuge (Anhui Zhongjia scientific instruments Co., Ltd.), constant temperature water bath (Shanghai Jinghong Experimental facilities Co., Ltd.), clean bench (Singapore Tech high tech Co., Ltd.), cell culture box (Singapore Tech high tech Co., Ltd.), inverted microscope (Leica).

2. Experimental methods

2.1 culture of SW1990 cells, after growth to logarithmic phase, digestion with 0.25% trypsin, centrifugation at 1000rpm for 5min, resuspension of the cell pellet in L-15 medium containing 10% FBS, inoculation at 5000/well density in 96-well plates, then setting at 37 ℃ and 5% CO₂And culturing overnight under saturated humidity conditions.

2.2 each group is provided with 6 multiple holes, each hole is added with medicine according to the following conditions, and then the culture is continued for 24 h.

Control group: namely, no medicine group is added.

Positive control group: the berberine hydrochloride components are divided into 6 groups:

positive control group 1: adding berberine hydrochloride to give final concentration of 20 μ M.

Positive control group 2: adding berberine hydrochloride to give final concentration of 40 μ M.

Positive control group 3: adding berberine hydrochloride to give final concentration of 80 μ M.

CT4-1 group: the CT4-1 component was divided into 3 groups:

CT4-1 group 1: CT4-1 was added to a final concentration of 20. mu.M.

CT4-1 group 2: CT4-1 was added to a final concentration of 40. mu.M.

CT4-1 group 3: CT4-1 was added to a final concentration of 80. mu.M.

After the cells are intervened for 24 hours, the CCK8 kit is used for detecting the influence of the medicine on the proliferation of the tumor cells. The specific operation is as follows: adding 10 mu L of CCK8 reagent into each well, continuing culturing for 2h, measuring the absorbance (OD) value at the wavelength of 450nm of the microplate reader, and calculating the cell survival rate of other groups by taking the cell survival rate of the control group as 100%.

3. Results of the experiment

TABLE 1 Effect of CT4-1 on the proliferation of human pancreatic cancer cell line SW1990

The results are shown in Table 1, CT4-1 can obviously inhibit the proliferation of human pancreatic cancer cell line SW1990, the inhibition effect increases with increasing dosage, and the inhibition effect is obviously superior to that of positive control medicament berberine hydrochloride.

Experiment II CT4-1 influence on proliferation of human oral squamous cell carcinoma cell strain SCC15

Purpose of the experiment: study on the effect of CT4-1 on the proliferation of human oral squamous cell carcinoma cell line SCC15

1. Experimental Material

1.1 cell line human oral squamous carcinoma cell line SCC15

Reagent and instruments fetal bovine serum (FBS, Hyclone), DMEM high-sugar medium (Gibco), DMSO (Sigma), phosphate buffered saline (PBS, BI), CCK8 kit (Nippon colleague chemical research institute), trypsin (Gibco), BioTek Synergy full-function microplate reader (Molecular Devices), low-speed centrifuge (Anhui Zhongjia scientific instruments Co., Ltd.), constant-temperature water bath (Shanghai sperm macro laboratory Equipment Co., Ltd.), clean bench (Singapore Tech high-tech Co., Ltd.), cell culture chamber (Singapore Tech high-tech Co., Ltd.), inverted microscope (Leica).

2. Experimental methods

2.1 culturing SCC15 cells, after growth to logarithmic phase, digesting with 0.25% trypsin, centrifuging at 1000rpm for 5min, resuspending the cell pellet in 10% FBS-containing DMEM high-sugar medium, inoculating the pellet in 96-well plate at 5000/well density, placing the plate at 37 ℃ and 5% CO₂And culturing overnight under saturated humidity conditions.

2.2 each group is provided with 6 multiple holes, each hole is added with medicine according to the following conditions, and then the culture is continued for 24 h.

Positive control group: the berberine hydrochloride components are divided into 3 groups:

positive control group 1: adding berberine hydrochloride to give final concentration of 20 μ M.

Positive control group 2: adding berberine hydrochloride to give final concentration of 40 μ M.

Positive control group 3: adding berberine hydrochloride to give final concentration of 80 μ M.

CT4-1 group: the CT4-1 component was divided into 3 groups:

CT4-1 group 1: CT4-1 was added to a final concentration of 20. mu.M.

CT4-1 group 2: CT4-1 was added to a final concentration of 40. mu.M.

CT4-1 group 3: CT4-1 was added to a final concentration of 80. mu.M.

After the cells are intervened for 24 hours, the CCK8 kit is used for detecting the influence of the medicine on the proliferation of the tumor cells. The specific operation is as follows: adding 10 mu L of CCK8 reagent into each well, continuing culturing for 2h, measuring the absorbance (OD) value at the wavelength of 450nm of the microplate reader, and calculating the cell survival rate of other groups by taking the cell survival rate of the control group as 100%.

3. Results of the experiment

TABLE 2 Effect of CT4-1 on proliferation of human oral squamous cell carcinoma cell line SCC15

The results are shown in table 2, CT4-1 can obviously inhibit the proliferation of human oral squamous cell carcinoma cell line SCC15, the inhibition effect increases with increasing dosage, and the inhibition effect is obviously better than that of positive control drug berberine hydrochloride.

Experiment on the Effect of three CT4-1 on the proliferation of human colorectal cancer cell line SW480

Purpose of the experiment: study on the Effect of CT4-1 on the proliferation of human colorectal cancer cell line SW480

1. Experimental Material

1.1 cell line human colorectal carcinoma cell line SW480

1.2 reagents and instruments fetal bovine serum (FBS, Hyclone), RPMI-1640 medium (Gibco), DMSO (Sigma), phosphate buffered saline (PBS, BI), CCK8 kit (Nippon colleague chemical research institute), trypsin (Gibco), BioTek Synergy full-function microplate reader (Molecular Devices), Low-speed centrifuge (Anhui Zhongjia scientific instruments Co., Ltd.), constant temperature water bath (Shanghai sperm macro laboratory Equipment Co., Ltd.), clean bench (Singapore Tech high tech Co., Ltd.), cell culture box (Singapore Tech high tech Co., Ltd.), inverted microscope (Leica).

2. Experimental methods

2.1 culture of SW480 cells until they grow to logarithmic phase, i.e., they are digested with 0.25% trypsin, centrifuged at 1000rpm for 5min, the cell pellet is resuspended in 10% FBS-containing RPMI-1640 medium, seeded at 5000/well density in 96-well plates, and then placed at 37 ℃ in a 5% CO medium₂And culturing overnight under saturated humidity conditions.

2.2 each group is provided with 6 multiple holes, each hole is added with medicine according to the following conditions, and then the culture is continued for 24 h.

Control group: namely, no medicine group is added.

Positive control group: the berberine hydrochloride components are divided into 3 groups:

positive control group 1: adding berberine hydrochloride to give final concentration of 20 μ M.

Positive control group 2: adding berberine hydrochloride to give final concentration of 40 μ M.

Positive control group 3: adding berberine hydrochloride to give final concentration of 80 μ M.

CT4-1 group: the CT4-1 component was divided into 3 groups:

CT4-1 group 1: CT4-1 was added to a final concentration of 20. mu.M.

CT4-1 group 2: CT4-1 was added to a final concentration of 40. mu.M.

CT4-1 group 3: CT4-1 was added to a final concentration of 80. mu.M.

After the cells are intervened for 24 hours, the CCK8 kit is used for detecting the influence of the medicine on the proliferation of the tumor cells. The specific operation is as follows: adding 10 mu L of CCK8 reagent into each well, continuing culturing for 2h, measuring the absorbance (OD) value at the wavelength of 450nm of the microplate reader, and calculating the cell survival rate of other groups by taking the cell survival rate of the control group as 100%.

3. Results of the experiment

TABLE 3 Effect of CT4-1 on the proliferation of human colorectal cancer cell line SW480

As shown in Table 3, CT4-1 can significantly inhibit the proliferation of human colorectal cancer cell line SW480, the inhibition effect increases with increasing dosage, and the inhibition effect is significantly better than that of the positive control drug berberine hydrochloride.

In conclusion, CT4-1 can inhibit the proliferation of various tumor cells, has antitumor activity obviously superior to that of berberine hydrochloride, is an antitumor compound with development potential, and can be directly used for treating related diseases and preparing related medicaments.

Although the embodiments of the present invention have been described above, the above description is only for the convenience of understanding the present invention, and is not intended to limit the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.