CN111393278A - A kind of Usnea longiflora derivative and its use in preparing medicine for treating gallbladder cancer - Google Patents

Abstract

The invention provides a usnea derivative and application thereof in preparing a medicine for treating gallbladder cancer, wherein the usnea derivative has a chemical name of 3, 6-diacetyl-2, 7, 9-trihydroxy-8, 9 b-dimethyl-1 (9bH) -dibenzofuranone, has an effect of inhibiting the activity of human gallbladder cancer cells, and has small toxic effect on normal cells. The compounds can be used alone or in combination with other drugs in the treatment of gallbladder cancer. The inventor unexpectedly finds that the compound provided by the invention can achieve better effect of inhibiting gallbladder cancer cells and effectively relieve the side effect of cisplatin when being used together with the cisplatin. Is a medicine with great application potential for treating gallbladder cancer.

Description

A kind of Usnea longiflora derivative and its use in preparing medicine for treating gallbladder cancer

technical field

The invention belongs to the technical field of medicine, and in particular relates to a Usnea chinensis derivative and its use, especially the application in preparing a drug for treating gallbladder cancer.

Background technique

Gallbladder cancer is a rare malignant tumor and ranks first among the malignant tumors of the gallbladder. Gallbladder cancer is the most common malignant tumor of the biliary system, among which adenocarcinoma is the main one, accounting for about 82%. The incidence of gallbladder cancer has increased rapidly in recent years, ranking fifth among digestive tract tumors. Gallbladder cancer is considered to be one of the most aggressive and lethal malignancies. The incidence of gallbladder cancer is relatively high in my country, and it has increasingly become a malignant disease that seriously threatens the lives of the general public. According to Global Cancer Statistics (2012), in developed regions, the age-standardized (ASR, age-standardized rate per 100,000) incidence rate of gallbladder cancer is 2.0, while the age-standardized mortality rate reaches 1.4; in underdeveloped regions, the age-standardized incidence rate is 1.4. The mortality rate is 2.4, and the age-standardized fatality rate is as high as 2.0, which means that as long as the disease occurs, the final outcome of most patients is death. The onset of gallbladder cancer is insidious, and it often coexists with benign gallbladder diseases. The most common is the coexistence of gallbladder stones and cholecystitis. Chronic stimulation of stones is an important pathogenic factor. There are no specific clinical symptoms and signs in the early stage of gallbladder cancer, and the clinical manifestations of gallbladder cancer are often masked by the symptoms of other diseases such as gallbladder stones. The 5-year survival rate is less than 5%.

At present, the pathogenesis of gallbladder cancer is still unclear. Epidemiological investigations have shown that gallstones, gallbladder adenoma, gallbladder adenomyosis, biliary bacterial infection, bile acid metabolism disorder, gastrectomy, high-fat diet, smoking and Alcoholism is a risk factor for gallbladder cancer.

Because gallbladder cancer has the characteristics of high malignancy, easy early metastasis, difficult early detection, and insensitivity to chemotherapy drugs, most patients are already in the middle and late stages when diagnosed. At present, the treatment methods of gallbladder cancer include surgery, chemotherapy, radiotherapy, interventional therapy and gene therapy. At present, the commonly used chemotherapeutic drugs for gallbladder cancer include mitomycin, fluorouracil, paclitaxel, cisplatin, etc. These drugs have their own advantages and disadvantages. Cisplatin has the advantages of broad anti-cancer spectrum and strong effect, but it is easy to cause renal toxicity. In view of the above situation, it is of great clinical significance to find a drug or a drug composition with a therapeutic effect on gallbladder cancer.

Usnea is a lichen type Usnea Usnea Usnea longissi ma Ach, also known as cloud grass, Laojunxu, dragon mustard grass, canopy grass, canopy grass, centipede usnea, tree cinnamon, female dill, snow Wind vines, mountain noodles, and tree hair seven, all in a linear shape, are distributed in the Daxing'an Mountains, Changbai Mountains, Xinjiang, Tibet, Hubei, Anhui, Zhejiang and other places in China. Its various known derivatives, such as substituted monobenzene, dep, anthraquinone, dibenzofuran, etc., have antibacterial, anti-inflammatory, antioxidant, antitumor and other biological activities. It is the current research hotspot of natural products. At present, a variety of compounds have been isolated and identified from Usnea longa, such as ethyl erythromycin, cork, β-amyrinol, β-sitosterol, 2,4-dihydroxy-3,6-dimethyl Methyl benzoate, baba acid, terpene, ethyl mossate, 3β-hydroxy-myxo-5-ene, oleanolic acid, usnic acid, methyl mossate, and 4-methyl -2,6-Dihydroxybenzaldehyde. The inventors of the present application have unexpectedly found that one of uscone and its derivatives has the function of inhibiting the activity of gallbladder cancer cells, and is expected to be used in clinic as a drug for the treatment of gallbladder cancer.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a compound and its derivatives with the activity of inhibiting gallbladder cancer cells, which not only have obvious inhibitory effect on gallbladder cancer cells, but also have low cytotoxicity.

In a first aspect, the present invention provides a compound of general formula I, and a pharmaceutically acceptable salt thereof,

Wherein, R ₁ and R ₂ are independently selected from H, -OH, -CN, C _1-6 straight/branched chain alkyl, -OC _1-6 alkyl, -N(C _0-10 alkyl) ( C _0-10 alkyl), -CO (C _0-10 alkyl), -CON (C _0-10 alkyl) (C _0-10 alkyl).

Preferably, R ₁ and R ₂ are independently selected from H, -OH, -CH ₃ , -C ₂ H ₅ , -COCH ₃ .

Most preferably, R ₁ is selected from -OH, -OCH ₃ , -OC ₂ H ₅ , and R ₂ is selected from -COOH, -COOCH ₃ , -COOC ₂ H ₅

In the most preferred embodiment of the present invention, the R ₁ is -OH, R ₂ is selected from -COCH ₃ , and the compound structure is shown in formula (II):

The chemical name of the compound is 3,6-diacetyl-2,7,9-trihydroxy-8,9b-dimethyl-1(9bH)-dibenzofuranone, which is obtained from the natural medicine Usnea longiflora. It was extracted, and it was named as long usnicotine.

The pharmaceutically acceptable salts of the present invention include potassium, sodium or ammonium salts, or salts formed from methylamine, ethylamine and ethanolamine. The salts may be in the form of mono-, di-, or tri-salts. The present invention is preferably the monosodium, disodium and trisodium salts of the compounds of formula (II).

In a second aspect, the present invention provides a pharmaceutical composition in which a compound represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof is used as a pharmaceutical active ingredient.

Preferably, the pharmaceutical composition comprises a therapeutically effective amount of the compound represented by formula (II). The pharmaceutical composition can be formulated into the following dosage forms: tablet, capsule, injection, powder injection, suspension, emulsion. The pharmaceutical preparation is preferably a unit preparation, and the unit preparation contains an effective dose of the active compound, which is preferably changed or adjusted between 0.1 mg and 10 mg, and can be specifically selected from 0.1 mg, 0.5 mg, 1 mg, 2 mg, 4 mg, 5 mg, 10mg.

Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable adjuvant or carrier, and the adjuvant or carrier depends on the specific administration method of the composition. In the practice of the present invention, the pharmaceutical compositions can be administered parenterally (intravenous, intramuscular, intradermal and subcutaneous routes), parenterally (oral, nasal, sublingual routes) ) and intraperitoneal administration.

In a specific mode of administration of the present invention, the composition is administered intravenously, therefore, the pharmaceutical composition also includes antioxidants, buffers, bacteriostatic agents, and solutes that make the preparation isotonic with blood. More preferably, the pharmaceutical composition may further include a suspending agent, a solubilizer, a thickener, and a stabilizer.

In the dosing environment of the present invention, the dose to a subject will be determined by the potency of the particular active compound and the condition of the subject, as well as the body weight or body surface area of the subject to be treated, to produce a beneficial therapeutic response in the subject. whichever occurs, depending on the presence, nature and extent of any adverse side effects concomitant with administration in a particular subject.

In certain embodiments, the pharmaceutical compositions may be used alone or in combination with other classes of pharmaceutical formulations and/or methods of treatment.

The other kinds of pharmaceutical preparations and/or treatment methods include, but are not limited to: immunosuppressants, targeted anti-tumor drugs, non-steroidal anti-inflammatory drugs, anti-tumor vaccines, EGFR tyrosine kinase inhibitors, adoptive cellular immunity therapy or radiation therapy.

Specifically, the drugs that can be used in combination with the pharmaceutical composition of the present invention include: one or a combination of two or more of mitomycin, fluorouracil, paclitaxel, cisplatin, carboplatin, and oxaliplatin. In the present invention, the inventor unexpectedly found that the compound of formula (I) or formula (II) provided by the present invention in combination with cisplatin can not only achieve better inhibition of gallbladder cancer cells, but also effectively slows down the side effects of cisplatin.

Cisplatin is currently the first-line drug for the treatment of various solid tumors, and it is also one of the commonly used clinical chemotherapy drugs for gallbladder cancer at this stage. Cisplatin is a non-specific drug in the cell cycle, which can inhibit the DNA replication process of cancer cells, damage the structure on the membrane of cancer cells, and has a broad-spectrum anti-cancer effect. Therefore, cisplatin has high cytotoxicity and large side effects in clinical application, especially nephrotoxicity.

In a third aspect, the present invention provides a compound pharmaceutical composition, the pharmaceutical composition comprising a compound represented by formula (I) or formula (II) or a pharmaceutically acceptable salt thereof as a first active pharmaceutical ingredient; the Cisplatin is also included in the pharmaceutical composition as a second active pharmaceutical ingredient.

The mass ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient in the compound pharmaceutical composition is 1:1-10; more preferably, the mass ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient is 1:1 -3.

The second active pharmaceutical ingredient is selected from one or a combination of two or more selected from mitomycin, fluorouracil, paclitaxel, cisplatin, carboplatin, and oxaliplatin, preferably cisplatin.

Preferably, the compound pharmaceutical composition further includes a pharmaceutically acceptable adjuvant or carrier, and the selection of the specific adjuvant or carrier is as described above.

The active pharmaceutical ingredients in the compound pharmaceutical composition can be combined with pharmaceutically acceptable adjuvants or carriers to form a pharmaceutical preparation. Preferably, the compound pharmaceutical composition is in the form of sterile powder, solution for injection or suspension. The term "pharmaceutically acceptable carrier" refers to one or more compatible solids, liquids, diluents, etc., suitable for administration to humans which do not destroy the therapeutic effect of the active compound.

The administration mode and effective dose of the compound pharmaceutical composition are determined according to the specific stage of the treatment disease, the age and physical condition of the patient, and the duration of the disease, and can also be selected according to actual needs within the knowledge and time range of those skilled in the art. In general, an effective dose is one sufficient to induce beneficial phenotypic changes, such as alleviation and elimination of symptoms, permanent arrest of the progression of the disease, or delay in the onset of the disease, and the like.

In a fourth aspect, the present invention provides a compound represented by formula (I) or (II) or a pharmaceutically acceptable salt thereof, or a compound represented by formula (I) or (II) or a pharmaceutically acceptable salt thereof. The use of a pharmaceutical composition whose salt is an active ingredient in the preparation of a medicament for treating gallbladder cancer.

The term C _0-10 alkyl described in the present invention, C ₀ alkyl refers to H, therefore, C _0-10 alkyl includes H, C ₁ alkyl, C ₂ alkyl, C ₃ alkyl, C ₄ Alkyl, _C5 alkyl, _C6 alkyl, _C7 alkyl, _C8 alkyl, _C9 alkyl, _C10 alkyl.

The term "unit dosage" as used in the present invention refers to the smallest subpackage of each pharmaceutical preparation containing an appropriate amount of active compound, eg, a unit dosage in a capsule refers to a capsule, a unit dosage in a tablet refers to a tablet, an injection The unit preparation in liquid refers to a single injection, and so on for other types of preparations.

The compound represented by the formula (I) or (II) of the present invention and the pharmaceutically acceptable salt thereof have the following advantages as a drug for the treatment of gallbladder cancer:

(1) The compound represented by formula (I) or (II) as an active ingredient has a significant inhibitory effect on human gallbladder cancer cells, indicating that the compound has the potential for clinical application;

(2) Compared with the commonly used clinical chemotherapy drugs, the compounds represented by formula (I) or (II) have less cytotoxicity, stronger safety, and less side effects produced by the drugs in the mouse test;

(3) In the present application, the combined use of the compound represented by formula (I) or (II) and cisplatin has a synergistic effect, which can significantly reduce the side effects caused by cisplatin.

Description of drawings

Fig. 1 Inhibitory effects of the compounds represented by formula (II) uscine and cisplatin on human gallbladder cancer cells.

Fig. 2 Inhibitory effect of the compounds represented by formula (II) uscone and fluorouracil on human gallbladder cancer cells.

Figure 3 The cytotoxic effects of the compounds represented by formula (II) uscone and cisplatin on macrophages.

Fig. 4 Cytotoxicity of the compounds represented by formula (II) uscone and fluorouracil on macrophages.

Figure 5. Change curve of body weight of nude mice under the action of drugs.

Fig. 6 Survival curve of nude mice under the action of drugs.

Figure 7. Tumor weight distribution map under the action of drugs.

Figure 8. Distribution of tumor volume in nude mice under the action of drugs.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1 The preparation of usnepinephrine injection

S1: Dosing, taking 10 mg of the compound usnicotene shown in formula II and dissolving it in 20 mL of sodium chloride injection to prepare a drug solution with a drug concentration of 0.5 mg/mL;

S2: Activated carbon treatment, adding 0.2% of the total solution to the medicinal solution for 0.5 hour adsorption treatment with activated carbon for needles;

S3: primary filtration, filter the medicinal liquid with filter paper;

S4: filter sterilization, use a microporous filter membrane with a pore size of 0.22 μm to sterilize the medicinal solution and remove the heat source;

S5: The medicinal solution is sub-packed in 2 mL units, and then filled and sealed to obtain an injection solution.

Example 2 Preparation of uscine-cisplatin compound injection

S1: Preparation of solution, take 10 mg of usnicotine compound represented by formula II and 30 mg of cisplatin and dissolve them together in 20 mL of sodium chloride injection, the concentration of solubilin is 0.5 mg/mL, and the concentration of cisplatin is 1.5 mg /mL of compound medicine;

S2: Activated carbon treatment, adding 0.2% of the total solution to the medicinal solution for 0.5 hour adsorption treatment with activated carbon for needles;

S3: primary filtration, filter the medicinal liquid with filter paper;

S4: filter sterilization, use a microporous filter membrane with a pore size of 0.22 μm to sterilize the medicinal solution and remove the heat source;

S5: The medicinal solution is sub-packed in 2 mL units, and then filled and sealed to obtain an injection solution.

The applicant needs to stress here that the mass ratio of uscine and cisplatin can be any ratio between 1:1 and 10. The specific examples of this application only reflect the mass ratio of 1:3, and those skilled in the art can also Preparation of compound medicinal liquid with the mass ratios of uscine and cisplatin of 1:1, 1:2, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10 .

Efficacy Example 1 Usneine has an inhibitory effect on gallbladder cancer cells

The GBC-SD cell lines used in all experiments in this application are human gallbladder cancer cells, purchased from ATCC (American Type Culture Collection, Manassas, VA). Culture conditions of GBC-SD cell line: cultured in 1640 medium containing 10% fetal bovine serum, 100U/mL penicillin, and 100 μg/mL streptomycin at 37°C and 5% CO ₂ . Specific experimental operations Take logarithmic growth phase cells.

GBC-SD cell processing steps: The human gallbladder cancer cells that were frozen in liquid nitrogen were taken out and immediately placed in a 37°C water bath to recover for 10 minutes; the recovered cell suspension was transferred to a clean centrifuge tube, and 10 mL of Serum 1640 medium, mix gently, centrifuge at 4000 rpm for 5 minutes, discard the supernatant, repeat three times; add 1 mL of 10% FBS-1640 medium, mix well, place in a 37°C cell incubator, and incubate with 5% carbon dioxide; The medium was replaced with fresh medium every 2 days.

OBJECTIVE: Using cisplatin and fluorouracil as controls, to detect whether uscone has a significant inhibitory effect on GBC-SD human gallbladder cancer cells.

Experimental steps:

S1: Gently wash twice with PBS buffer, then count the cells and evenly spread them into a 96-well plate at a density of 8000 cells/well, with a volume of 200 μL per well, put them at 37°C, containing 5% CO ₂ and saturated humidity Incubate for 24h in the incubator of 96-well plate, design the position and number of control wells and zero-adjustment wells, in order to avoid affecting the experimental results after concentration changes due to water evaporation, add 200 μL of sterile PBS to each well of the 96-well plate for one week;

S2: The usnicotine was prepared into four gradient concentrations of 0.5 μM, 1.0 μM, 2.0 μM and 5.0 μM with sodium chloride injection as solvent. In addition, the control drugs cisplatin and fluorouracil were injected with sodium chloride The solution was also prepared into four gradient concentrations of 0.5 μM, 1.0 μM, 2.0 μM, and 5.0 μM. The two combinations of the control drug and the experimental drug in this experiment were carried out simultaneously in two 96-well plates;

S3: After culturing the cells for 24 hours, suck out the old medium from each well with a pipette, add the corresponding concentrations of the experimental drug and the control drug, respectively, add the same amount of sodium chloride injection to the control well and the zero-adjusting well, and continue to culture for 24 hours.

S4: color, carefully weigh the MTT with sterile PBS to prepare a solution with a concentration of 5 mg/mL, take out the 96-well plate after 24 hours of drug action, add 20 μL of MTT solution to each well, and incubate in the incubator for 4 hours to avoid sucking the cells. Remove the old medium with a sterile syringe, add 150 μL DMSO to each well, and incubate for 10 min.

S5: Colorimetric, select the wavelength of 490 nm, measure the light absorption value of each well on an enzyme-linked immunosorbent assay instrument, and record the results.

Statistical analysis: The data of each group in this experiment are the data detected by the instrument. The statistical analysis software SPSS 17.0 was used for statistical analysis. One-way ANOVA and t test were used for statistical analysis. P<0.05 indicated that the difference was statistically significant.

Experimental results: Fig. 1 is a graph showing the inhibitory effect of usnicotine and cisplatin on the growth of gallbladder cells, and Fig. 2 is a graph showing the inhibitory effect of usnicone and fluorouracil on the growth of gallbladder cancer cells. As shown in Figure 1 and Figure 2, no matter it is uscine, cisplatin or fluorouracil, the proliferation and growth of each cell line shows a downward trend with the increase of drug concentration. Compared with the inhibitory trend of cisplatin and fluorouracil on gallbladder cancer cells, it can be seen that when the concentration of uscone was 2.0 μM, the inhibitory effect on cancer cells was significantly different from that of the control group. Although there is no significant difference in the inhibitory effect of the experimental drug and the control drug on gallbladder cancer cells at other concentrations (0.5, 1.0, 5.0 μM), it can be seen from the figure that the inhibitory effect of uscone on gallbladder cancer cells The inhibitory effect with cisplatin and fluorouracil was stronger at drug concentrations of 2.0-5.0 μM.

Effect Example 2 Toxic effect of uscone on normal cells

In this experiment, RAW264 macrophages were used to detect the toxicity of the active compound uscone in this application to normal cells. RAW264 macrophages were purchased from ATCC. RAW264 cells were cultured in DMEM low-glucose medium containing 10% fetal bovine serum (FBS). Take the cryopreservation tube out of the liquid nitrogen tank and put it into a 37°C water bath to shake it quickly to dissolve it. Put it into a centrifuge tube with fresh medium added in advance, put it in a centrifuge at 1000rpm/min for 5 minutes, take out and discard the supernatant, transfer the cells to a culture flask, add fresh medium, shake well, at 37°C, Routine culture in an incubator with 5% CO ₂ .

Observe the growth of the cells under the microscope, and prepare for cell passage when the cells are denser under the microscope. In a sterile operating table, pour out the culture medium in the culture flask, quickly add sterile PBS to rinse it once, then add 0.25% trypsin (containing 0.02% EDTA) digestion solution, add fresh medium after incubation for 5 minutes, use glue Pipette with the head dropper to blow down the cells on the bottle wall and add a part to the new culture bottle to continue culturing, and the remaining cells are used for this experiment.

OBJECTIVE: Using cisplatin and fluorouracil as controls, to detect the toxic effect of uscone on RAW264 macrophages.

Experimental steps:

S1: Inoculate cells, design the position and number of control wells and zero-adjustment wells, in order to avoid affecting the experimental results after concentration changes due to water evaporation, add 200 μL of sterile PBS to each well on the outer edge of the 96-well plate for one week. The macrophages were dispersed in DMEM medium to make a single cell suspension, and the cells were counted under a microscope. The macrophages were seeded on a 96-well plate with about 5,000 cells per well, and the volume of each well was 200 μL. ℃, incubator with 5% CO ₂ and saturated humidity for 24h;

S2: The usnicotine was prepared into four gradient concentrations of 1.0 μM, 2.0 μM, 5.0 μM and 10.0 μM with sodium chloride injection as solvent. In addition, the control drugs cisplatin and fluorouracil were injected with sodium chloride The solution was also prepared into four gradient concentrations of 1.0 μM, 2.0 μM, 5.0 μM, and 10.0 μM. The two combinations of the control drug and the experimental drug in this experiment were carried out simultaneously in two 96-well plates; S3 drug effect, S4 color rendering, S5 colorimetric operation is the same as the effect embodiment 1.

Experimental results: Figures 3 and 4 show the cytotoxic effects of uscine and cisplatin, and uscine and fluorouracil on RAW264 macrophages, respectively. As shown in Fig. 3 and Fig. 4, compared with cisplatin and fluorouracil, the toxic effect of uscone on macrophages was significantly reduced, and the effect of reducing the toxicity was more obvious when the drug concentration was higher, which was sufficient It shows that the cytotoxicity of uscone is smaller, the safety is better, and it has great potential for clinical application.

Efficacy Example 3 Usnepinephrine has inhibitory effect on tumor-bearing nude mice

Objective: To test the inhibitory effect of usnicotine on tumor-bearing nude mice with cisplatin and fluorouracil as controls, and also to detect the inhibitory effect of the compound injection of longustin and cisplatin on tumor-bearing nude mice.

Experimental steps:

S1: Establishment of human gallbladder carcinoma xenograft model in nude mice: The experimental animals were 50 SPF female nude mice, aged 7-8 weeks and weighing 18-20 g. The GBC-SD cells in the logarithmic growth phase were taken, prepared with sodium chloride injection to a concentration of 1×10 ⁶ , and subcutaneously injected into nude mice with a total cell volume of 0.5 mL/mouse, and were raised under normal conditions. Model establishment was completed after 2-3 weeks when the tumor volume reached approximately 200 ^mm3 .

S2: Nude mice were randomly divided into five groups, all of which were administered by tail vein. The experimental group 1 was given the usnicotine injection prepared in Example 1 (specification 1 mg/2mL, diluted 100 times, taking 600 μL), and the experimental group 2 was given. To the usnicotine-cisplatin compound injection prepared in Example 2 (specification 1mg: 3mg/2mL, diluted 100 times, take 600 μL), the control group 1 was given commercially available cisplatin injection (specification 10mg/2mL, diluted 100 times , take 600μL), the control group 2 was given fluorouracil injection (specification 0.25g/10mL, diluted 100 times, take 600μL), and the nude mice in the blank control group were injected with 600μL sodium chloride injection through the tail vein. The number of treatments was twice a week, on Monday and Wednesday, respectively, and the rest of the time was fed normally. The survival rate of the five groups of nude mice was counted every week, and the weight changes of the nude mice were measured every week, and the activity status of the nude mice was observed for six weeks. After that, the surviving nude mice were sacrificed by cervical dislocation, and the tumor was excised to measure the weight and volume of the tumor.

Experimental results:

Body weight: As shown in Figure 5, the body weight of the mice in the uscone and compound preparation treatment group showed a slow upward trend, indicating that the growth of the tumor was effectively inhibited, while the body weight of the nude mice in the cisplatin and fluorouracil treatment group was significantly reduced in the later stage. , it can also be visually observed that as the tumor grows larger, the nude mice become thinner and thinner, especially the nude mice treated with cisplatin, not only the mortality rate is higher, but the overall condition of the nude mice is the worst. It can be seen that the weight loss of nude mice is obviously slowed down after the combination of uscine on the basis of cisplatin, indicating that the uscine can effectively alleviate the side effects of cisplatin treatment.

Survival rate: As shown in Figure 6, among all drug treatment groups, the nude mice treated with uscone had the highest survival rate after 6 weeks of treatment, and the survival rate reached 100%, followed by the uscone-cisplatin compound In the preparation treatment group, 2 died after 6 weeks of treatment, and the survival rate was 80%. The highest mortality rate was in the cisplatin-treated group. The mortality rate of nude mice was as high as 60%. The number of dead mice after 6 weeks of treatment was comparable to that of the blank control group, and the condition of the remaining surviving nude mice after 6 weeks of treatment was also very poor. From the survival rate curve, it can be seen that compared with the current clinical first-line drugs cisplatin and fluorouracil, the uscone and the compound preparation containing uscine provided in this application are safer, and have further clinical application. potential.

Observation of adverse reactions: The mice in the drug treatment group provided by the present invention did not show symptoms such as decreased activity and listlessness. During the experiment, the mice had no obvious reduction in food intake, no obvious weight loss, no mouse death, and no skin ulceration or erosion at the transplanted tumor. .

Tumor volume and weight: The liquefied tumors were removed, and then the weight and volume distribution of tumors in each group were counted, as shown in Figure 7 and Figure 8, the volume and weight distribution trends of each group of tumors were basically the same, and the blank control group of nude mice The tumor weight and volume were the largest. Although the tumor growth of the nude mice in the cisplatin and fluorouracil treatment group was inhibited, the tumor weight and volume were relatively reduced. The compound injection, especially the compound injection treatment group, had the smallest tumor weight and volume, indicating that the compound injection had the best tumor inhibitory effect. The tumor liquefaction phenomenon in the cisplatin and fluorouracil treatment groups was relatively obvious, and the probability of tumor liquefaction was about 30-40%; it was unexpectedly found that the nude mice in the long usnicotine treatment group basically had no liquefaction phenomenon. At the same time, the tumor size and volume distribution of the surviving nude mice in the cisplatin and fluorouracil treatment groups are relatively discrete, indicating that the therapeutic effects of cisplatin and fluorouracil on some individuals are not obvious. The tumor size distribution of the nude mice in the treatment group was relatively concentrated, indicating that the therapeutic effect was relatively uniform and the individual differences were not large.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.

Claims (10)

1. An usnea longissima derivative or a pharmaceutically acceptable salt thereof, wherein the usnea longissima derivative has a structure shown as a general formula (I):

wherein R is₁And R₂Independently selected from H, -OH, -CN, C_1-6Straight chain/branched alkyl, -OC_1-6Alkyl, -N (C)_0-10Alkyl) (C_0-10Alkyl), -CO (C)_0-10Alkyl), -CON (C)_0-10Alkyl) (C_0-10Alkyl groups).

2. The usnea derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is R₁is-OH, R₂Is selected from-COCH₃The chemical name of the compound is 3, 6-diacetyl-2, 7, 9-trihydroxy-8, 9 b-dimethyl-1 (9bH) -dibenzofuranone, and the structure is shown as the formula (II):

3. the pharmaceutically acceptable salt of usnea longissima derivative of claim 1 or 2, wherein the pharmaceutically acceptable salt includes potassium salt, sodium salt or ammonium salt, and the salt can be in the form of mono-salt, di-salt or tri-salt.

4. A pharmaceutical composition comprising the usnea longissima derivative or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 as a pharmaceutically active ingredient, and further comprising a pharmaceutically acceptable adjuvant and/or carrier.

5. The pharmaceutical composition of claim 4, wherein the excipients comprise antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with blood.

6. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition comprises a unit preparation of the usnea longissima derivative or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the dosage of the pharmaceutically active ingredient in the unit preparation is 0.1mg to 10 mg.

7. The pharmaceutical composition according to any one of claims 4 to 6, wherein the pharmaceutical composition is used alone or in combination with other kinds of pharmaceutical preparations and/or methods of treatment, in particular, other kinds of pharmaceutical preparations including immunosuppressants, targeted antitumor drugs, non-steroidal anti-inflammatory drugs, antitumor vaccines, EGFR tyrosine kinase inhibitors; such other treatment methods include adoptive cellular immunotherapy or radiotherapy.

8. The pharmaceutical composition according to claim 7, wherein the drug which can be used in combination with the pharmaceutical composition is one or a combination of two or more selected from the group consisting of mitomycin, fluorouracil, paclitaxel, cisplatin, carboplatin and oxaliplatin.

9. A combination pharmaceutical composition comprising the usnea longissima derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 as a first active pharmaceutical ingredient; the compound pharmaceutical composition further comprises a second active pharmaceutical ingredient, the mass ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient in the compound pharmaceutical composition is 1:1-10, the mass ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient is preferably 1:1-3, and the second active pharmaceutical ingredient is one or a combination of more than two of mitomycin, fluorouracil, paclitaxel, cisplatin, carboplatin and oxaliplatin, preferably cisplatin.

10. Use of the usnea longissima derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, or the pharmaceutical composition according to any one of claims 4 to 6, or the combination pharmaceutical composition according to claim 9 in the preparation of a medicament for treating gallbladder cancer.

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