Application of double baicalein compound in preparation of antitumor drugs
阅读说明：本技术 一种双黄芩素化合物在制备抗肿瘤药物中的用途 (Application of double baicalein compound in preparation of antitumor drugs ) 是由 袁崇均 陈帅 罗森 余梦瑶 许晓燕 汤依娜 王笳 于 2021-09-08 设计创作，主要内容包括：本发明提供了一种双黄芩素化合物在制备抗肿瘤药物中的用途,属于制药领域。该双黄芩素化合物的结构如式A所示。体外抗肿瘤活性实验表明,本发明化合物结肠癌、肺癌、乳腺癌、肝癌细胞株均有很好的抑制作用；与黄芩素相比,本发明化合物对肿瘤细胞的抑制活性均明显提高。体内抗肿瘤活性实验表明,本发明化合物对H22荷瘤小鼠的肿瘤增殖有明显的抑制作用；与相同剂量的黄芩素组相比,本发明双黄芩素化合物组的肿瘤增殖抑制效果更佳。本发明化合物能够用于制备预防和/或治疗肿瘤的药物,其制备方法简单,成本较低,产品收率和纯度高,适合工业化大生产。(The invention provides an application of a double baicalein compound in preparing an anti-tumor medicament, belonging to the field of pharmacy. The structure of the double baicalein compound is shown as a formula A. In-vitro anti-tumor activity experiments show that the compound has good inhibition effect on colon cancer, lung cancer, breast cancer and liver cancer cell strains; compared with baicalein, the compound pair of the inventionThe inhibition activity of the tumor cells is obviously improved. The in vivo anti-tumor activity experiment shows that the compound has obvious inhibition effect on the tumor proliferation of H22 tumor-bearing mice; compared with baicalein group with the same dosage, the double baicalein compound group has better tumor proliferation inhibition effect. The compound can be used for preparing medicaments for preventing and/or treating tumors, has simple preparation method, lower cost and high product yield and purity, and is suitable for industrial mass production.)
1. The application of the compound shown in the formula A in preparing a medicament for preventing and/or treating tumors:
wherein M is 0-2 methylene groups;
R1selected from hydrogen, C1～4Alkyl radical, C1～4Alkoxy, halogen;
R2selected from hydrogen, C1～4Alkyl radical, C1～4Alkoxy, halogen.
2. Use according to claim 1, characterized in that: the compound shown in the formula A is a compound 1:
3. use according to claim 1 or 2, characterized in that: the tumor is colon cancer.
4. Use according to claim 1 or 2, characterized in that: the tumor is lung cancer.
5. Use according to claim 1 or 2, characterized in that: the tumor is breast cancer.
6. Use according to claim 1 or 2, characterized in that: the tumor is liver cancer.
7. Use according to claim 1 or 2, characterized in that: the medicine is a preparation prepared by taking the compound as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
8. Use according to claim 7, characterized in that: the preparation is an oral preparation or an injection preparation.
9. Use according to claim 8, characterized in that: the oral preparation is tablets, powder, granules, capsules, pills or oral solution;
the injection preparation is a freeze-dried powder injection or an injection;
preferably, the tablet is a common compressed tablet, a chewable tablet, an effervescent tablet, a multi-layer tablet, a sustained release tablet, a controlled release tablet, a coated tablet, a dispersible tablet, a buccal tablet or a sublingual tablet;
the pill is a dripping pill, a buccal dripping pill or a pellet;
the capsule is hard capsule, soft capsule, enteric capsule, sustained release capsule or microcapsule.
10. A process for preparing compound 1, characterized by: the method comprises the following steps: heating and refluxing baicalein and formaldehyde serving as raw materials in absolute ethyl alcohol for 4-12 hours to obtain a compound 1;
preferably, the reaction is carried out in the presence of an acid, the acid is phosphoric acid, and the mass-to-volume ratio of baicalein to phosphoric acid is 1: 2 g/mL;
or, the reaction is carried out in the presence of a base, the base is triethylamine, and the mass-to-volume ratio of the baicalein to the triethylamine is 1: 1 g/mL.
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to application of a double baicalein compound in preparation of an anti-tumor medicament.
Background
Baicalein (baicalein, 5,6, 7-trihydroxyflavone) is a flavonoid monomer compound separated from root of Scutellaria baicalensis Georgi (Scutellaria baicalensis Georgi) belonging to Labiatae, and is the main drug effect substance basis of Scutellaria baicalensis. It is reported that baicalein has antibacterial, antiviral, antiallergic, antitumor, free radical scavenging, cardiovascular and cerebrovascular protecting, and immunoregulatory effects. The baicalein has certain anti-tumor activity and wide anti-tumor spectrum, and the action mechanism of the baicalein has close relation with a plurality of links of tumor development. However, researches show that the anti-tumor effect of baicalein is obviously lower than that of clinical cancer drugs, such as 5-FU, cisplatin, adriamycin and the like. Therefore, the application of baicalein as an anti-tumor medicament in clinic is limited.
The development of baicalein derivatives with better antitumor activity is of great significance.
Disclosure of Invention
The invention aims to provide application of a double baicalein compound in preparing an anti-tumor medicament.
The invention provides an application of a compound shown as a formula A in preparing a medicament for preventing and/or treating tumors, which comprises the following steps:
wherein M is 0-2 methylene groups;
R1selected from hydrogen, C1～4Alkyl radical, C1～4Alkoxy, halogen;
R2selected from hydrogen, C1～4Alkyl radical, C1～4Alkoxy, halogen.
Further, the compound represented by formula a is compound 1:
further, the tumor is colon cancer.
Further, the tumor is lung cancer.
Further, the tumor is breast cancer.
Further, the tumor is liver cancer.
Furthermore, the medicine is a preparation prepared by taking the compound as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
Further, the preparation is an oral preparation or an injection preparation.
Further, the oral preparation is tablet, powder, granule, capsule, pill or oral solution;
the injection preparation is freeze-dried powder or injection.
Further, the tablet is a common compressed tablet, a chewable tablet, an effervescent tablet, a multi-layer tablet, a sustained release tablet, a controlled release tablet, a coated tablet, a dispersible tablet, a buccal tablet or a sublingual tablet;
the pill is a dripping pill, a buccal dripping pill or a pellet;
the capsule is hard capsule, soft capsule, enteric capsule, sustained release capsule or microcapsule.
The present invention also provides a process for preparing compound 1, comprising the steps of: heating and refluxing baicalein and formaldehyde serving as raw materials in absolute ethyl alcohol for 4-12 hours to obtain a compound 1;
further, the reaction is carried out in the presence of acid, the acid is phosphoric acid, and the mass-volume ratio of the baicalein to the phosphoric acid is 1: 2 g/mL;
or, the reaction is carried out in the presence of a base, the base is triethylamine, and the mass-to-volume ratio of the baicalein to the triethylamine is 1: 1 g/mL.
Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.
The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by a prefix, e.g. prefix Ca～bAlkyl represents any alkyl group containing from "a" to "b" carbon atoms. E.g. C1～4The alkyl group is a straight-chain or branched alkyl group having 1 to 4 carbon atoms. C1～4The alkoxy group means a straight chain or branched chain alkoxy group having 1 to 4 carbon atoms.
Halogen is fluorine, chlorine, bromine or iodine.
In-vitro anti-tumor activity experiments show that the compound of the invention has good inhibition effect on colon cancer, lung cancer, breast cancer and liver cancer cell strains, and IC thereof50Are all lower than 15 mg/L; with yellowCompared with scutellarin, the compound of the invention has obviously improved inhibitory activity on colon cancer, lung cancer, breast cancer and liver cancer cells.
The in vivo anti-tumor activity experiment shows that the compound has obvious inhibition effect on the tumor proliferation of H22 tumor-bearing mice; compared with the baicalein group with the same dose, the double-baicalein compound group has better tumor proliferation inhibition effect on H22 tumor-bearing mice.
The compound can be used for preparing medicaments for preventing and/or treating tumors, has simple preparation method, lower cost and high product yield and purity, and is suitable for industrial mass production.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is an HPLC chart of sample 1.
FIG. 2 is an HPLC chart of sample 2.
FIG. 3 is a UV spectrum of sample 2.
FIG. 4 is an infrared spectrum of sample 2.
FIG. 5 is a mass spectrum of sample 2.
Fig. 6 is a nuclear magnetic hydrogen spectrum of sample 2.
FIG. 7 is a nuclear magnetic carbon spectrum of sample 2.
Detailed Description
The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.
Wherein, baicalein is purchased from Shaanxi Lvqing bioengineering GmbH, the content is more than 98%.
Method for measuring content of baicalein compound (HPLC area normalization method):
chromatographic conditions are as follows: a chromatographic column: kromasil C18(4.6 mm. times.150 mm, 5 μm); mobile phase: carbonitrile (A) -0.02mol/L sodium dihydrogen phosphate buffer (B), gradient elution (0min, 30% A; 30min, 70% A); flow rate: 1.0 mL/min; column temperature: 35 ℃; detection wavelength: 280 nm; the sample was dissolved in methanol and the amount of the sample was 10. mu.L.
Preparation of sodium dihydrogen phosphate buffer: 3.12g NaH was weighed2PO4.2H2Adding appropriate amount of water into a 1000mL volumetric flask, performing ultrasonic treatment until the mixture is completely dissolved, adding water to scale, shaking uniformly, and adding H3PO4Adjusting pH to 3.0, and shaking.
Example 1: preparation method of Bisbeianin compound 1 (method without catalyst)
Step 1.1: adding baicalein 50.0g into anhydrous ethanol 2500ml, heating to dissolve, adding formaldehyde solution 800ml, mixing, filtering, and heating under reflux for 12 hr (silica gel GF)254The reaction was followed by TLC, the development system was chloroform-methanol-formic acid 10:2: 0.1). Standing overnight to separate out light yellow precipitate, filtering, washing the precipitate with 100ml anhydrous ethanol, and drying at 60 deg.C under reduced pressure to obtain double baicalein compound. Is light yellow dry loose powder, the yield is 46.2g, the yield is 90 percent, and the content of a sample by an HPLC area normalization method is 96.54 percent.
Step 1.2: taking 40.0g (the content is 96.54%) of the double baicalein compound prepared in the step 1.1, adding 100ml of absolute ethyl alcohol into a triangular flask, carrying out ultrasonic treatment for 30 minutes, and filtering; treating for 2 times by the same method, filtering, and drying under reduced pressure at 60 deg.C to obtain sample 1 of baicalein compound with yield of 35.6g, yield of 89%, and content of 98.12% by HPLC area normalization method. The HPLC profile of sample 1 is shown in FIG. 1.
Example 2: preparation method of double baicalein compound 2 (method using acid as catalyst)
Step 2.1: adding baicalein 50.0g into anhydrous ethanol 2500ml, heating to dissolve, adding formaldehyde solution 800ml, adding phosphoric acid 100ml, mixing, filtering, and heating under reflux for 6 hr (silica gel GF)254The reaction was followed by TLC, the development system was chloroform-methanol-formic acid 10:2: 0.1). Standing overnight to precipitate a pale yellow precipitate, filtering, washing with 100ml of absolute ethanolWashing the precipitate, and drying at 60 deg.C under reduced pressure to obtain the double baicalein compound. Is light yellow dry loose powder, the yield is 46.1g, the yield is 90 percent, and the content of a sample by an HPLC area normalization method is 96.43 percent.
Step 2.2: taking 40.0g (the content is 96.43%) of the double baicalein compound prepared in the step 2.1, adding 200ml of absolute ethyl alcohol, heating and refluxing for 2 hours, standing overnight, filtering, washing with 50ml of absolute ethyl alcohol, and drying under reduced pressure at 60 ℃ to obtain a double baicalein compound sample 2, wherein the obtained amount is 37.1g, the yield is 92.75%, and the content of the sample is 98.94% by an HPLC area normalization method. The HPLC profile of sample 2 is shown in FIG. 2.
Structural identification of the double baicalein compound:
the bis-baicalein compound obtained in step 2.2 of example 2 (i.e. sample 2, content 98.94%) was used as a test sample, and UV, IR and MS were performed respectively,1H NMR、1c NMR measurements, the results are as follows:
UVλmaxnm:280nm；ESI-MS m/z553.11224[M+H]+(theoretical value 552.48), UV, IR, MS,1H NMR、1the C NMR is respectively shown in figures 3-7, and the double baicalein compound is identified to be 8, 8' -methylene-double baicalein, and the structural formula is as follows:
example 3: preparation method of double baicalein compound 3 (method using alkali as catalyst)
Dissolving baicalein 50.0g in 2500ml absolute ethanol under heating, adding formaldehyde solution 800ml, adding triethanolamine 50ml, adding sodium bisulfite 5.0g as antioxidant, mixing, dissolving, filtering, and refluxing under heating for 4 hr (silica gel GF)254The reaction was followed by TLC, the development system was chloroform-methanol-formic acid 10:2: 0.1). Standing overnight to separate out light yellow precipitate, filtering, washing the precipitate with 100ml anhydrous ethanol, and drying at 60 deg.C under reduced pressure. Is light yellow dry loose powder, the yield is 43.1g, the yield is 84 percent, and the content of a sample by an HPLC area normalization method is 95.16 percent.
Example 4: the preparation of the double baicalein compound pharmaceutical preparation
The double baicalein compound can be prepared into different medicinal preparations such as injection, freeze-dried powder injection, tablets, powder, granules, capsules, pills, oral solution and the like by adding a pharmaceutically acceptable carrier, wherein the tablets comprise: common compressed tablets, chewable tablets, effervescent tablets, multilayer tablets, sustained release tablets, controlled release tablets, coated tablets, dispersible tablets, buccal tablets, sublingual tablets and the like; the pill includes dripping pill, buccal dripping pill, pellet, etc. The capsule comprises: hard capsule, soft capsule, enteric capsule, sustained release capsule, microcapsule, etc.
Preparation of injection: is prepared by conventional injection preparation process, and adjuvants can be polyethylene glycol 400, antioxidant (sodium bisulfite), etc. Each 2mL of the tablets contains 100mg of the double baicalein compound.
Preparation of the powder: adding magnesium stearate as adjuvant to increase fluidity, and packaging.
Preparation of granules: the double baicalein compound and the auxiliary materials are fully mixed after being crushed, and then proper adhesive, wetting agent and the like are added for granulation, drying, size stabilization and split charging, thus obtaining the double baicalein compound.
Preparation of capsules: capsules are divided into soft capsules and hard capsules, wherein the hard capsules are prepared by encapsulating powder or granules of a medicament, and the soft capsules are prepared by sealing liquid medicine in spherical or soft capsule materials. According to the dry plasticizer (the plasticizer is glycerol, sorbitol or a mixture of the two): dry gelatin: water is 0.4-0.6: 1.0: 1.0, mixing evenly, preparing the mixture to be used as the capsule wall of the soft capsule, dissolving the medicine in a solvent prepared by polyethylene glycol 400 and polyethylene glycol 6000 according to a proper proportion, and then preparing the anti-tumor medicine soft capsule by adopting a dropping method or a pressing method. Meanwhile, the enteric-coated capsule can be prepared. In addition, according to the preparation method of the conventional sustained-release capsule, different auxiliary materials are selected as follows: ethyl Cellulose (EC), Cellulose Acetate (CA), polyacrylic resin, etc. as the slow-release film-forming material to make the slow-release and controlled-release preparation skeleton. The double baicalein compound is prepared into micro-capsules, and then the micro-capsules are filled into common empty capsules to prepare the slow release/controlled release capsules.
Preparing a dripping pill or a pellet: taking a drug matrix, such as: heating polyethylene glycol and stearic acid, mixing the medicinal liquid with matrix, placing in dripping pill machine, keeping temperature, dripping into appropriate condensing agent, placing into bowl, washing to remove condensing agent, drying, inspecting quality, and packaging.
Preparation of tablets: comprises common compressed tablets, chewable tablets, effervescent tablets, multilayer tablets, sustained-release tablets, controlled-release tablets, coated tablets (sugar-coated tablets, film-coated tablets and enteric-coated tablets), dispersible tablets, buccal tablets, sublingual tablets and the like. The medicine and the auxiliary materials are fully mixed after being crushed, and then proper adhesive, wetting agent and the like are added for granulation and tabletting, thus obtaining the common pressed tablet. The medicine of the present invention is mixed with supplementary material for dispersing tablet, and the mixture is pelletized and tabletted to obtain the dispersing tablet. Coating the obtained tablet with sugar coating, film coating, and enteric coating, and making into coated tablet. Can also be made into chewable tablet, effervescent tablet, multilayer tablet, buccal tablet, sublingual tablet, etc. by conventional method.
The beneficial effects of the present invention are demonstrated by the following experimental examples.
Experimental example 1: in vitro antitumor Activity test of Compounds
1 test Material
1.1 cell lines
HCT116 and A549 tumor cell strains are purchased from cell stocks of Chinese academy of sciences. Culturing in RPMI-1640 medium (containing penicillin 100U/ml and streptomycin 100. mu.g/ml) containing 10% fetal calf serum at 37 deg.C under 5% CO2And a saturated humidity cell culture box. The passage was digested with 0.05% trypsin-0.53 mM EDTA every 2-3 d.
MCF-7 and HepG2 tumor cell lines were purchased from the cell stock of Chinese academy of sciences. Culturing in RPMI-1640 medium (containing penicillin 100U/ml, streptomycin 100. mu.g/ml, recombinant human insulin 0.4U/ml) containing 10% fetal calf serum at 37 deg.C under 5% CO2And a saturated humidity cell culture box. The passage was digested with 0.05% trypsin-0.53 mM EDTA every 4-5 days.
1.2 samples
Example 2 the bis-baicalein compound obtained in step 2.2 (i.e., sample 2, content 98.94%). Dissolved in DMSO to prepare a 100mg/ml solution/suspension, and stored at-20 ℃ for later use.
Commercial baicalein and 5-FU were used as controls.
1.3 reagents
Fetal bovine serum was purchased from Bovogen, RPMI-1640 medium, MTT, trypsin, EDTA, DMSO from Sigma, and the remaining reagents were all home-made analytical grade.
2 test method
Respectively taking tumor cells in logarithmic growth phase to carry out tests, and carrying out tests on HCT-116, A549, MCF-7 and HepG2 cells according to 6 x 103The cells were added to a 96-well plate at 180. mu.l per well and cultured overnight. Each sample was added to a 96-well plate at the designed concentration and incubated for 72 hours. 4h before the end of the incubation, the medium was aspirated and 100. mu.l of Earle's BSS was added. Mu.l of MTT solution (5mg/ml) was added, and after incubation for 4 hours, 100. mu.l of 10% SDS solution (prepared in 0.01M HCl) was added to each well, and the mixture was incubated overnight in a cell incubator, and the OD value was measured at 570nm using a microplate reader. The inhibition rate of each sample concentration was calculated, and the half-Inhibitory Concentration (IC) was calculated using the Curve Expert50) The value is obtained. The test results are shown in Table 1.
3 results of the test
TABLE 1 in vitro antitumor Activity of Compounds on various human cancer cells
As can be seen from the above table, the double baicalein compound of the invention has good inhibition effect on 4 cancer cells, namely colon cancer, lung cancer, breast cancer and liver cancer cell strains, and IC thereof50Are all lower than 15 mg/L; compared with baicalein, the double baicalein compound of the invention has obviously improved inhibitory activity on colon cancer, lung cancer, breast cancer and liver cancer cells.
Experimental example 2: in vivo antitumor Activity test of Compounds
1 test Material
1.1 Experimental animals
18-22g of KM mice, 70 male and female, purchased from the laboratory animal center of Sichuan academy of traditional Chinese medicine, and bred in the SPF barrier of the laboratory animal center of Sichuan academy of traditional Chinese medicine.
1.2 cell lines
H22 tumor cell lines were purchased from the cell bank of the department of chinese academy of sciences. Culturing in RPMI-1640 medium (containing penicillin 100U/ml and streptomycin 100. mu.g/ml) containing 10% fetal calf serum at 37 deg.C under 5% CO2And a saturated humidity cell culture box. The passage was digested with 0.05% trypsin-0.53 mM EDTA every 2-3 d.
Before the experiment, 5X 10 is taken6The individual cells were inoculated in the abdominal cavity of KM mice and ascites formed in about 7 days. The ascites of 0.5ml and physiological saline of 0.5ml are mixed in the mouse of the previous generation every week, inoculated in the abdominal cavity of the mouse of the next generation, preserved and transferred.
1.3 samples
Example 2 the bis-baicalein compound obtained in step 2.2 (i.e., sample 2, content 98.94%). Immediately before daily use, 5mg/ml and 2.5mg/ml solutions are prepared by using 5% DMSO, 15% Tween-80 and 80% physiological saline.
Commercial baicalein was used as a control.
1.4 reagents
Fetal bovine serum was purchased from Bovogen, RPMI-1640 medium, MTT, trypsin, EDTA, DMSO from Sigma, and the remaining reagents were all home-made analytical grade.
2 test method
Taking the H22 ascites cells after 7d of the seed transformation, and diluting the cells to 1X 10 by using normal saline7One cell/ml, inoculated subcutaneously in the right underarm of mice, 0.2ml each. After 24h, the mice were randomly divided into 5 groups of 10 mice each. In the control group, a solvent (5% DMSO, 15% Tween-80, 80% physiological saline) was intraperitoneally injected daily at an injection volume of 0.1ml/10 g. High and low dose groups of the double baicalein compound and the baicalein sample are respectively set, wherein the dose of the high dose group is 50mg/kg, and the dose of the low dose group is 25 mg/kg. Each administration group was intraperitoneally injected with the corresponding drug solution daily, with an injection volume of 0.1ml/10 g. Is administered once dailyAnd 9d of continuous administration. After 1h of the last dose, mice were sacrificed by cervical dislocation, tumor tissue dissected and weighed. The experimental data are expressed by mean +/-standard deviation, the experimental difference is analyzed by adopting one-factor variance, and the difference between groups is compared by adopting an LSD method. The test results are shown in Table 2.
3 results of the test
TABLE 2 Effect of samples on tumor growth in H22 tumor-bearing mice
Note: p <0.05, p <0.01, as compared to control group
The test result shows that compared with a control group, the tumor weight of the double baicalein compound high-dose group has significant difference (p is less than 0.05), and the double baicalein compound is proved to have obvious inhibition effect on the tumor proliferation of H22 tumor-bearing mice. In addition, compared with the baicalein group with the same dose, the double-baicalein compound group has better tumor proliferation inhibition effect on H22 tumor-bearing mice.