CN112694507B - Tetrahydro anthraquinone glycoside compound and application thereof in preparation of antitumor drugs - Google Patents

Abstract

The invention provides a novel tetrahydroanthraquinone glycoside compound and application thereof in preparation of antitumor drugs, the novel tetrahydroanthraquinone glycoside compound has a good inhibition effect on growth and proliferation of lung cancer, breast cancer, colon cancer and liver cancer cells, can induce tumor cells to generate morphological change, promotes expression of tumor cell apoptosis-related protein to induce tumor cells to generate apoptosis, shows excellent antitumor characteristics, and provides a new selection and approach for research and development of antitumor drugs.

Description

Tetrahydro anthraquinone glycoside compound and application thereof in preparation of antitumor drugs

Technical Field

The invention relates to the technical field of medicines, in particular to a tetrahydroanthraquinone glycoside compound and application thereof in preparing antitumor medicines.

Background

Cancer is a major public health problem worldwide, and can affect everyone's life. Cancer causes mortality and disability in the global population and affects the quality of life of everyone. International agency for research on cancer (IARC) under the World Health Organization (WHO) in CA: journal of Cancer Journal for Clinicians issued the latest estimate report of the global Cancer burden in 2020. The world's cancer situation is also severe: with the aging and rapid growth of the population, the global number of cancer attacks and deaths is also rapidly increasing. Cancer will become the leading cause of death in the 21 st century and will be the most important obstacle to improving life expectancy in countries around the world. At present, the main means for treating the tumor comprise chemotherapy, radiotherapy and immunotherapy. Although chemotherapeutic drugs have certain curative effects on most of tumors, the limitations of low efficiency, poor selectivity, large toxic and side effects, tumor resistance and the like still exist, so that the search for high-efficiency, low-toxicity and specific antitumor drugs is still the struggle target of antitumor drug treatment. With the development of cell molecular biology, cancer can be regarded as a disorder of key molecules regulating cell cycle or apoptosis from a cellular level, so that cancer cells are in an unlimited proliferation state. Therefore, the development of new apoptosis inducers is a new anti-tumor therapeutic strategy.

Anthraquinone and its derivatives are natural organic compounds widely existing in nature, 9, 10-anthraquinone is the most common natural anthraquinone, and C-9 and C-10 are in the highest oxidation state, and are relatively stable. The anthraquinone components in Chinese medicinal materials are mainly hydroxy, carboxymethyl, methoxy and carboxyl derivatives of anthraquinone, and exist in free form or nucleoside. Anthraquinone compounds have been reported to have hemostatic, antibacterial, purgative and diuretic effects, so anthraquinone compounds have been an important source of drug lead compounds. The tetrahydroanthraquinone compounds are rare natural structures, contain more secondary metabolites of microorganisms, are derived from plants in a small amount, and have biological activities such as cytotoxic activity, antibacterial activity, plasmodium resistance and the like. Patent CN102134188B provides a tetrahydro anthraquinone compound Prisconnatacin separated from radix et rhizoma Rhei, which can be used for developing drugs for treating hepatocarcinoma, lung cancer, breast cancer and leukemia. However, no studies on the antitumor aspect of the tetrahydroanthraquinone glycoside compounds exist at present.

Disclosure of Invention

The invention aims to provide a tetrahydroanthraquinone glycoside compound and application thereof in preparing antitumor drugs, and the invention provides the tetrahydroanthraquinone glycoside compound.

The invention aims to provide a tetrahydroanthraquinone glycoside compound.

The invention also aims to provide the application of the tetrahydroanthraquinone glycoside compound, or the pharmaceutically acceptable salt, or the stereoisomer, or the prodrug thereof in preparing antitumor drugs.

The invention further aims to provide application of the tetrahydroanthraquinone glycoside compound or the pharmaceutically acceptable salt, the stereoisomer or the prodrug thereof in preparing tumor cell apoptosis inducers.

Still another object of the present invention is to provide an anti-tumor drug.

The above object of the present invention is achieved by the following technical solutions:

the invention provides a tetrahydroanthraquinone glycoside compound, which has the following chemical structural formula:

wherein R is selected from the following groups:

in an in vitro anti-tumor cell growth model, the tetrahydroanthraquinone glycoside compounds with different concentrations are detected to have concentration dependence and time dependence on tumor cells by a thiazole blue (MTT) colorimetric method, and show excellent characteristics as anti-tumor drugs.

In addition, the research on the anti-tumor molecular mechanism of the tetrahydroanthraquinone glycoside compounds shows that the anthraquinone glycoside series compounds can induce cells to undergo apoptosis by influencing the expression quantity of apoptosis protein PARP to realize the anti-tumor activity, thereby further increasing the possibility of becoming candidate anti-tumor compounds and laying a theoretical foundation for further clinical tests.

Therefore, the following applications should be within the scope of the present invention:

the tetrahydroanthraquinone glycoside compound, or pharmaceutically acceptable salt, stereoisomer or prodrug thereof is applied to the preparation of antitumor drugs; and the application of the tetrahydroanthraquinone glycoside compound or the pharmaceutically acceptable salt, the stereoisomer or the prodrug thereof in preparing tumor cell apoptosis inducers.

Preferably, the anti-tumor is the inhibition of tumor cell growth, the inhibition of tumor cell proliferation and the induction of tumor cell apoptosis.

Preferably, the tumor is one or more of lung cancer, breast cancer, colon cancer or liver cancer.

Preferably, the pharmaceutically acceptable salt of the tetrahydroanthraquinone glycoside compound is an inorganic base salt or a metal complex thereof.

The inorganic base is sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, ammonium hydroxide or lithium hydroxide.

The metal complex is a complex of a tetrahydroanthraquinone glycoside compound and metal ions such as iron ions, platinum ions, calcium ions, barium ions, sodium ions, potassium ions and the like.

Preferably, the tetrahydroanthraquinone glycoside compound prodrug refers to a substance that can be converted into the tetrahydroanthraquinone glycoside compound or a salt thereof in vivo.

The invention also claims an anti-tumor medicament containing the tetrahydroanthraquinone glycoside compound or the pharmaceutically acceptable salt or the stereoisomer or the prodrug thereof.

Preferably, the medicament also comprises a medicinal carrier and/or a diluent, and is prepared into different dosage forms.

The pharmaceutically acceptable carrier refers to inactive ingredients in the drug such as, but not limited to: calcium carbonate, calcium phosphate, various sugars (lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic or methacrylic polymers, gelatin (gelatin), water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated or ethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil, etc.

The diluent is starch (such as corn starch, wheat starch, potato starch, etc.), lactose, dextrin, sucrose, pregelatinized starch, microcrystalline cellulose, inorganic salts (such as calcium bicarbonate, calcium sulfate, calcium residue, etc.), mannitol, etc.

Preferably, the medicament is in the form of powder, tablets, granules, capsules, solutions, syrups, suspensions, injections, powder injections, water injections, aerosols, ointments, eye drops or suppositories.

The dosage of the compound to be used will vary depending on the specific dosage form, mode of application and specific site, patient and tumor disease to be treated, and other factors such as age, body weight, sex, diet, administration time, excretion rate, patient's symptoms, drug binding force, reaction sensitivity and disease severity may be continuously or periodically administered within the maximum allowable dose.

In addition, the tetrahydroanthraquinone glycoside compound can be used for combined medication with other medicines.

The administration of the tetrahydroanthraquinone glycosides of the present invention or combinations thereof may be carried out by any suitable method. Preferably, the drug is administered by gastrointestinal administration, injection, respiratory administration, dermal administration, mucosal administration or luminal administration.

As a preferred mode of administration, intravenous infusion as in administration by injection, the infusion time is up to 48 hours.

As an alternative, compounds 1-5 of the present invention may be synthesized by the synthetic route shown in FIG. 3, as follows:

synthesis of Compound 1: weighing compound C (0.1mmol), D-arabinoside imidate a (0.33mmol, 3eq), adding dried dichloromethane (2mL) as solvent, adding dried molecular sieve

MS 0.4g, stirring at room temperature for 1h, adding 4mg AuCl₃(0.1eq), stirring was continued for 1 h. After the reaction was complete, it was filtered, concentrated, and column-packed on silica gel [ V (dichloromethane): v (acetone) ═ 40:1]To obtain a red solid product 1.

Synthesis of Compound 2: weighing compound 1(0.1mmol) in a reaction bottle, adding 3mL 10% methanol/sodium methoxide, stirring at normal temperature for 15min, tracking by TLC until the reaction is complete, and adding resin

Stirring at 50WX8 until the solution turns red again, terminating the reaction, filtering, concentrating, and passing through silica gel column [ V (dichloromethane): V (ethyl acetate): V (methanol): 20:1]To obtain the compound 2.

Synthesis of Compound 3: compound 2(0.056mmol) was weighed into a round-bottomed flask, 1mL of acetone and 0.5mL of 2, 2-dimethoxypropane were sequentially added thereto, 0.05mL of concentrated sulfuric acid was added dropwise, and the mixture was stirred at normal temperature for 2 hours, extracted, concentrated, and subjected to silica gel column chromatography [ V (ethyl acetate): V (petroleum ether) ═ 1: 5], to obtain a compound 3.

Synthesis of Compound 4: weighing compound 3(0.055mmol), L-mannosaminide ester (0.165mmol, 3eq), adding 2mL dry dichloromethane as solvent, adding dried molecular sieve

MS 0.4g, stirring at room temperature for 1h, adding 4mg AuCl₃(0.1eq), stirring was continued for 1 h. After the reaction was complete, it was filtered, concentrated and column-packed with silica gel [ V (ethyl acetate): v (petroleum ether) ═ 1: 2]To obtain red solid compound 4.

Synthesis of Compound 5: balanceTaking compound 4(0.025mmol) to a 5mL round-bottom flask, adding 1mL methanol as a solvent, dropwise adding 1mL 0.1M HCl under stirring at normal temperature, reacting for 6h until the reaction is complete, and reacting with CH₂Cl₂Extracting, drying the organic phase with anhydrous magnesium sulfate, concentrating, and separating with silica gel column [ V (dichloromethane): V (methanol) ═ 40: 1%]To obtain a red solid compound 5.

The synthesis method of the compound 6 is the same as that of the compound 1, and only D-arabinoside imino ester needs to be changed into D-2-deoxyribose imino ester; the method for synthesizing the compound 7 is the same as that for the compound 2; the method for synthesizing the compound 8 is the same as that for the compound 3; the synthesis of compound 9 was performed using compound 1 by changing D-arabinose to D-mannosaminide.

The synthesis method of the compound 10 comprises the following steps: weighing compound 9(0.1mmol) in a reaction bottle, adding 3mL 10% methanol/sodium methoxide, stirring at normal temperature for 15min, tracking point plate by TLC, adding after the reaction is completed

Stirring 50WX8 hydrogen form until the solution turns red again, terminating the reaction, filtering, concentrating, passing through a modified silica gel column [ V (dichloromethane): V (ethyl acetate): V (methanol): 20:1]To obtain an intermediate product D. Weighing D (0.056mmol) and a round-bottom flask, adding 1mL of acetone and 0.5mL of 2, 2-dimethoxypropane in sequence, dripping 0.05mL of concentrated sulfuric acid, stirring at normal temperature for 2h, extracting, and concentrating, [ V (ethyl acetate): V (petroleum ether) ═ 1: 5]And (5) passing through a column to obtain a compound 10.

The synthesis method of the compound 11 is the same as that of the compound 1, and only needs to change D-arabinoside imino ester into D-glucose imino ester; the synthesis method of the compound 12 is the same as that of the compound 2, and only D-arabinoimido ester needs to be changed into D-xyloimido ester; the synthesis method of the compound 13 is the same as that of the compound 2, and only D-arabinoimido ester is changed into D-rhamnose imido ester; the synthesis method of compound 14 is similar to that of compound 3, except that the reaction substrate is changed to compound 13.

Compared with the prior art, the invention has the beneficial effects that:

the tetrahydroanthraquinone glycoside compound provided by the invention has a good inhibition effect on the growth and proliferation of lung cancer, breast cancer, colon cancer and liver cancer cells, can induce the morphological change of tumor cells, promotes the expression of tumor cell apoptosis-related protein to induce the apoptosis of the tumor cells, shows excellent anti-tumor characteristics, and provides a new choice and way for research and development of anti-tumor drugs.

Drawings

FIG. 1 shows the structure of the mother nucleus of an anthraquinone compound.

FIG. 2 is a structural formula of anthraquinone glycoside compounds 1-14.

FIG. 3 is a synthetic route of anthraquinone glycoside compounds 1-5.

FIG. 4 shows the change of cell morphology of A549 cells treated with anthraquinone glycoside compound 1 at different concentrations and different time points.

FIG. 5 shows the change of cell morphology of A549 cells treated with anthraquinone glycoside compound 2 at different concentrations and different time points.

FIG. 6 shows the change of cell morphology of A549 cells treated with anthraquinone glycoside compound 3 at different concentrations and different time points.

FIG. 7 shows the change of cell morphology of A549 cells treated with anthraquinone glycoside compound 13 at different concentrations and different time points.

FIG. 8 shows the effect of anthraquinone glycoside compound 1 on the proliferation of A549 cells.

FIG. 9 shows the result of the statistics of EdU positive cells after the anthraquinone glycoside compound 1 treats A549 cells.

FIG. 10 shows the expression of apoptosis-related proteins after treatment with anthraquinone glycoside compound 1.

FIG. 11 shows the experimental end-point ablated tumors of different tumor-bearing mouse groups.

Figure 12 is the post-tumor-ablation weighing of different tumor-bearing mouse groups (. P <0.05,. P < 0.01).

Detailed Description

The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples in any way. The starting reagents employed in the examples of the present invention are, unless otherwise specified, those that are conventionally purchased.

Tumor cell sources used in the following examples:

human lung carcinoma cell a549 was purchased from the cell bank of chinese academy of sciences, shanghai.

Human lung cancer cells NCI-H460 were purchased from American Type Culture Collection (ATCC).

Human breast cancer cells BT549 were purchased from the chinese academy of sciences cell bank, shanghai.

Human colon cancer cell HCT116 was purchased from cell bank, shanghai, of chinese academy of sciences.

Human hepatoma cells HepG2 were purchased from cell banks, shanghai, from chinese academy of sciences.

Human melanoma cell MDA-MB-435 was purchased from cell bank of Chinese academy of sciences, Shanghai.

For the preparation of compound C, reference is made to the prior art: drugs 2012,10,932 and 952; doi:10.3390/md 10040932.

EXAMPLE 1 Synthesis and characterization of Tetrahydroanthraquinone glycosides

The following compounds 1-14 are obtained by chemical synthesis with the parent nucleus structure of the anthraquinone compound shown in figure 1, and the structures of the compounds 1-14 are shown in figure 2.

1. Synthesis of Compounds

The synthetic routes of compounds 1-5 are shown in FIG. 3.

Synthesis of Compound 1: weighing compound C (0.1mmol), D-arabinoside imidate a (0.33mmol, 3eq), adding dried dichloromethane (2mL) as solvent, adding dried molecular sieve

MS 0.4g, stirring at room temperature for 1h, adding 4mg AuCl₃(0.1eq), stirring was continued for 1 h. After the reaction was complete, it was filtered, concentrated, and column-packed on silica gel [ V (dichloromethane): v (acetone) ═ 40:1]To obtain a red solid product 1.

Synthesis of Compound 2: weighing compound 1(0.1mmol) in a reaction bottle, adding 3mL 10% methanol/sodium methoxide, stirring at normal temperature for 15min, tracking by TLC until the reaction is complete, and adding resin

Stirring at 50WX8 until the solution turns red again, terminating the reaction, filtering, concentrating, and passing through silica gel column [ V (dichloromethane): V (ethyl acetate): V (methanol): 20:1]To obtain the compound 2.

Synthesis of Compound 3: compound 2(0.056mmol) was weighed into a round-bottomed flask, 1mL of acetone and 0.5mL of 2, 2-dimethoxypropane were sequentially added thereto, 0.05mL of concentrated sulfuric acid was added dropwise, and the mixture was stirred at normal temperature for 2 hours, extracted, concentrated, and subjected to silica gel column chromatography [ V (ethyl acetate): V (petroleum ether) ═ 1: 5], to obtain a compound 3.

Synthesis of Compound 4: weighing compound 3(0.055mmol), L-mannosaminide ester (0.165mmol, 3eq), adding 2mL dry dichloromethane as solvent, adding dried molecular sieve

MS 0.4g, stirring at room temperature for 1h, adding 4mg AuCl₃(0.1eq), stirring was continued for 1 h. After the reaction was complete, it was filtered, concentrated and column-packed with silica gel [ V (ethyl acetate): v (petroleum ether) ═ 1: 2]To obtain red solid compound 4.

Synthesis of Compound 5: weighing compound 4(0.025mmol) in a 5mL round-bottom flask, adding 1mL methanol as solvent, dropwise adding 1mL 0.1M HCl under stirring at room temperature, reacting for 6h until the reaction is complete, and reacting with CH₂Cl₂Extracting, drying the organic phase with anhydrous magnesium sulfate, concentrating, and separating with silica gel column [ V (dichloromethane): V (methanol) ═ 40: 1%]To obtain a red solid compound 5.

The synthesis method of the compound 6 is the same as that of the compound 1, and only D-arabinoside imino ester needs to be changed into D-2-deoxyribose imino ester; the method for synthesizing the compound 7 is the same as that for the compound 2; the method for synthesizing the compound 8 is the same as that for the compound 3; the synthesis of compound 9 was performed using compound 1 by changing D-arabinose to D-mannosaminide.

The synthesis method of the compound 10 comprises the following steps: weighing compound 9(0.1mmol) in a reaction bottle, adding 3mL 10% methanol/sodium methoxide, stirring at normal temperature for 15min, tracking point plate by TLC, adding after the reaction is completed

Stirring 50WX8 hydrogen form until the solution turns red again, terminating the reaction, filtering, concentrating, passing through a modified silica gel column [ V (dichloromethane): V (ethyl acetate): V (methanol): 20:1]To obtain an intermediate product D. Weighing D (0.056mmol) and a round-bottom flask, adding 1mL of acetone and 0.5mL of 2, 2-dimethoxypropane in sequence, dripping 0.05mL of concentrated sulfuric acid, stirring at normal temperature for 2h, extracting, and concentrating, [ V (ethyl acetate): V (petroleum ether) ═ 1: 5]And (5) passing through a column to obtain a compound 10.

The synthesis method of the compound 11 is the same as that of the compound 1, and only needs to change D-arabinoside imino ester into D-glucose imino ester; the synthesis method of the compound 12 is the same as that of the compound 2, and only needs to change the D-arabinoimido ester in the previous step reaction into D-xyloimido ester; the synthesis method of the compound 13 is the same as that of the compound 2, and only needs to change the D-arabinoimido ester in the previous step reaction into D-rhamnose imido ester; the synthesis method of compound 14 is similar to that of compound 3, except that the reaction substrate is changed to compound 13.

2. Identification of Compounds

(1) Identification method

The structure of the tetrahydroanthraquinone glycosides was determined by HR-ESI-MS and NMR.

(2) Identification results

Compound 1: red crystal, m.p.115-120 deg.c,¹H NMR(500MHz，CDCl3)δ13.09(s，1H)，12.49(s，1H)，6.21(s，1H)，5.65(d，J＝3.1Hz，1H)，5.25(s，1H)，5.08(m，1H)，4.95(m，1H)，4.52(d，J＝3.1Hz，1H)，4.42–4.10(m，2H)，3.94(m，3H)，3.68(m，1H)，3.49(d，J＝16.1Hz，1H)，2.32(d，J＝16.1Hz，1H)，2.15(s，3H)，1.97(s，3H)，1.79(s，3H)，1.62(s，3H)，1.33(s，3H)，0.88(s，3H)。¹³C NMR(126MHz，CDCl₃)δ187.66(s)，182.14(s)，170.30(s)，169.96(s)，169.17(s)，160.86(s)，156.82(s)，156.14(s)，138.57(s)，135.81(s)，111.24(s)，110.24(s)，108.82(d，J＝17.8Hz)，108.75(s)，99.57(s)，81.27(s)，80.24(s)，70.12(s)，69.08(s)，68.15(s)，67.81(s)，64.10(s)，56.85(s)，32.69(s)，29.42(s)，27.47(s)，26.67(s)，20.94(s)，20.53(d，J＝15.9Hz)，20.47(s) MS (ESI, M-H: 633.04). In summary, the chemical structure is identified as:

compound 2: red solid, m.p.138-140 deg.C,¹H NMR(500MHz，CDCl3)δ13.06(s，1H)，12.45(s，1H)，6.12(s，1H)，5.53(d，J＝2.8Hz，1H)，4.54(d，J＝2.8Hz，1H)，4.27(d，J＝6.9Hz，1H)，4.02(d，J＝12.7Hz，1H)，3.90(s，3H)，3.86(s，1H)，3.68(q，J＝7.0Hz，1H)，3.48-3.59(m，3H)，3.43(d，J＝16.2Hz，1H)，2.52(d，J＝16.2Hz，1H)，1.60(s，3H)，1.31(s，3H)，0.86(s，3H)。¹³C NMR(126MHz，CDCl₃) δ 187.21(s), 181.37(s), 160.78(s), 157.50(s), 156.73(s), 138.99(s), 136.04(s), 111.10(s), 110.02(s), 108.75(s), 108.66(s), 102.44(s), 81.39(s), 80.37(s), 73.14(s), 71.57(s), 69.21(s), 68.33(s), 66.24(s), 56.85(s), 32.77(s), 29.34(s), 27.54(s), 26.90(s). In summary, the chemical structure is identified as:

compound 3: a red solid; melting point 126-;¹H NMR(600MHz，CDCl₃)δ13.10(s，1H)，12.51(s，1H)，6.16(s，1H)，5.59(d，J＝2.9Hz，1H)，4.54(d，J＝2.9Hz，1H)，4.33(d，J＝7.6Hz，1H)，4.23(t，J＝3.0Hz，1H)，4.22(dd，J＝13.1，2.9Hz，1H)，4.00(dd，J＝7.2，5.9Hz，1H)，3.92(s，3H)，3.88(dd，J＝12.9，3.1Hz，1H)，3.54(t，J＝7.6Hz，1H)，3.50(d，J＝16.3Hz，1H)，2.50(d，J＝16.2Hz，1H)，1.64(s，3H)，1.53(s，3H)，1.35(s，3H)，1.34(s，3H)，0.90(s，3H)。¹³C NMR(150MHz，CDCl3)δ187.41(s)，181.70(s)，160.95(s)，157.63(s)，156.73(s)，138.82(s)，136.23(s)，111.31(s)，110.37(s)，110.22(s)，108.95(s)，108.89(s)，101.46(s)，81.64(s)，80.41(s)，78.30(s)，73.73(s)，73.14(s)，68.71(s)，63.52(s)，56.95(s)，33.09(s)，

29.64(s)，28.11(s)，27.69(s)，27.00(s)，26.06(s).HRESIMS m/z 547.18270[M-H]^-(calcd.for C₂₇H₃₁O₁₂,547.18210). In summary, the chemical structure is identified as:

compound 4: a red solid; melting point 135-;¹H NMR(600MHz，CDCl₃)δ13.09(s，1H)，12.49(s，1H)，6.18(s，1H)，5.56(d，J＝2.8Hz，1H)，5.11(dd，J＝10.2，3.5Hz，1H)，4.94(t，J＝10.0Hz，1H)，4.80(d，J＝2.0Hz，1H)，4.70(s，1H)，4.48(d，J＝2.8Hz，1H)，4.39(d，J＝8.1Hz，1H)，4.27–4.18(m，2H)，4.12(dq，J＝12.3，6.1Hz，1H)，4.02(d，J＝6.0Hz，1H)，3.92(s，3H)，3.84(dd，J＝13.4，2.6Hz，1H)，3.58(t，J＝7.8Hz，1H)，3.46(d，J＝16.4Hz，1H)，2.26(d，J＝16.3Hz，1H)，2.01(s，3H)，1.99(s，3H)，1.86(s，3H)，1.56(s，3H)，1.51(s，3H)，1.35(s，3H)，1.31(s，3H)，1.14(d，J＝6.2Hz，3H)，0.87(s，3H)。¹³C NMR(150MHz，CDCl₃)δ187.41(s)，181.78(s)，170.02(s)，169.92(s)，169.14(s)，160.81(s)，157.32(s)，156.19(s)，138.04(s)，135.59(s)，111.51(s)，110.13(s)，109.93(s)，109.14(s)，108.64(s)，101.78(s)，97.12(s)，81.63(s)，80.14(s)，77.34(s)，76.43(s)，73.49(s)，70.98(s)，68.99(s)，68.89(s)，68.27(s)，66.21(s)，63.39(s)，56.76(s)，32.71(s)，29.53(s)，28.04(s)，27.54(s)，26.87(s)，26.09(s)，20.80(s)，20.68(s)，20.56(s)，17.22(s).HRESIMS m/z 821.28644[M+H]⁺(calcd.for C₃₉H₄₉O₁₉,821.28626). In summary, the chemical structure is identified as:

compound 5: a red solid; melting point 140-142 ℃;¹H NMR(600MHz，CDCl3)δ13.10(s，1H)，12.50(s，1H)，6.19(s，1H)，5.59(d，J＝2.9Hz，1H)，5.07(dd，J＝10.2，3.4Hz，1H)，4.95(t，J＝10.0Hz，1H)，4.76(dd，J＝3.4，1.8Hz，1H)，4.72(d，J＝1.5Hz，1H)，4.52(d，J＝2.9Hz，1H)，4.46(d，J＝7.4Hz，1H)，4.08–4.13(m，1H)，4.09(dd，J＝12.9，2.2Hz，1H)，3.94(s，4H)，3.64–3.57(m，2H)，3.56–3.51(m，1H)，3.49(d，J＝16.4Hz，1H)，2.37(d，J＝16.3Hz，1H)，2.04(s，3H)，1.99(s，3H)，1.88(s，3H)，1.61(s，3H)，1.33(s，3H)，1.16(d，J＝6.3Hz，3H)，0.89(s，3H)。¹³C NMR(150MHz，CDCl₃)δ187.47(s)，181.88(s)，169.89(s)，169.28(s)，160.81(s)，157.32(s)，156.16(s)，138.09(s)，135.64(s)，111.59(s)，110.15(s)，109.22(s)，108.71(s)，101.78(s)，98.21(s)，81.66(s)，80.25(s)，78.71(s)，72.12(s)，70.81(s)，69.35(s)，68.71(s)，68.49(s)，68.46(s)，67.06(s)，65.76(s)，56.75(s)，32.79(s)，29.55(s)，27.56(s)，26.90(s)，20.76(s)，20.64(s)，20.60(s)，17.29(s).HRESIMS m/z 781.25541[M+H]⁺(calcd.for C₃₆H₄₅O₁₉,781.25496). In summary, the chemical structure is identified as:

compound 6: a red solid; melting point 93-96 deg.C;¹H NMR(600MHz，CDCl₃)δ13.13(s，1H)，12.59(s，1H)，6.18(s，1H)，5.45(d，J＝2.9Hz，1H)，5.32(s，1H)，5.11–4.92(m，2H)，4.44(d，J＝2.9Hz，1H)，3.92(s，3H)，3.53(d，J＝16.2Hz，1H)，3.50–3.45(m，2H)，2.51(d，J＝16.1Hz，1H)，2.17–2.10(m，2H)，2.07(s，3H)，1.98(s，3H)，1.63(s，3H)，1.33(s，4H)，0.88(s，3H)。¹³C NMR(150MHz，CDCl₃)δ186.94(s)，181.29(s)，170.38(s)，170.08(s)，160.76(s)，157.73(s)，156.73(s)，137.83(s)，137.32(s)，111.03(s)，110.18(s)，108.95(s)，108.65(s)，97.97(s)，80.82(s)，80.51(s)，68.10(s)，67.49(s)，65.42(s)，61.40(s)，56.79(s)，32.90(s)，30.89(s)，29.88(s)，27.52(s)，26.83(s)，20.99(s)，20.94(s).HRESIMS m/z 575.17753[M-H]^-(calcd.for C₂₈H₃₁O₁₃,575.17701). In summary, the chemical structure is identified as:

compound 7: a red solid; melting point 138-140 ℃;¹H NMR(600MHz，CDCl₃)δ13.13(s，1H)，12.62(s，1H)，6.19(s，1H)，5.44(d，J＝3.1Hz，1H)，5.25(s，1H)，4.45(d，J＝3.1Hz，1H)，3.93(s，3H)，3.86–3.80(m，1H)，3.51(d，J＝16.1Hz，1H)，3.49–3.39(m，2H)，2.48(d，J＝16.1Hz，1H)，2.19(s，1H)，2.04(s，1H)，1.93–1.81(m，2H)，1.63(s，3H)，1.34(s，3H)，0.89(s，3H)。¹³C NMR(150MHz，CDCl₃)δ186.83(s)，181.17(s)，160.74(s)，157.89(s)，156.95(s)，137.79(s)，137.72(s)，110.98(s)，110.20(s)，108.93(s)，108.60(s)，98.21(s)，80.81(s)，80.48(s)，77.25(s)，77.04(s)，76.83(s)，68.12(s)，67.92(s)，64.74(s)，63.30(s)，56.80(s)，33.91(s)，32.90(s)，29.87(s)，27.51(s)，26.78(s).HRESIMS m/z491.15616[M-H]^-(calcd.for C₂₄H₂₇O₁₁,491.15588). In summary, the chemical structure is identified as:

compound 8: a red solid; melting point 131-;¹H NMR(600MHz，CDCl₃)δ13.15(s，1H)，12.67(s，1H)，6.21(s，1H)，5.51(d，J＝3.1Hz，1H)，5.21(dd，J＝6.3，4.4Hz，1H)，4.46(d，J＝3.1Hz，1H)，4.37(dt，J＝6.2，4.7Hz，1H)，4.19(tt，J＝11.8，5.7Hz，1H)，4.06–4.02(m，1H)，3.95(s，3H)，3.60–3.52(m，2H)，3.51(d，J＝16.0Hz，2H)，2.52(d，J＝16.0Hz，1H)，2.21–2.13(m，1H)，1.79–1.70(m，2H)，1.64(s，3H)，1.50(s，3H)，1.36(s，3H)，1.32(s，3H)，0.92(s，3H)。¹³C NMR(150MHz，CDCl₃)δ186.39(s)，180.69(s)，160.66(s)，158.45(s)，157.51(s)，138.24(s)，137.91(s)，110.15(s)，110.13(s)，109.35(s)，108.80(s)，108.79(s)，96.78(s)，80.84(s)，80.49(s)，71.55(s)，69.95(s)，67.82(s)，61.77(s)，56.73(s)，32.85(s)，31.71(s)，29.79(s)，27.50(s)，27.34(s)，26.78(s)，25.50(s).HRESIMS m/z 531.18730[M-H]^-(calcd.for C₂₇H₃₁O₁₁,521.18718). In summary, the chemical structure is identified as:

compound 9: a red solid; melting point 120-124 ℃;¹H NMR(500MHz，CDCl₃)δ13.06(s，1H)，12.49(s，1H)，6.17(s，1H)，5.59(d，J＝3.0Hz，1H)，5.30(t，J＝10.0Hz，1H)，5.24(dd，J＝10.1，3.1Hz，1H)，5.01(dd，J＝3.0，1.8Hz，1H)，4.81(s，1H)，4.46(d，J＝2.9Hz，1H)，4.40(dd，J＝12.2，5.5Hz，1H)，4.19(dd，J＝12.2，2.1Hz，1H)，4.15–4.07(m，1H)，3.93(s，3H)，3.56(d，J＝16.3Hz，1H)，2.51(d，J＝16.3Hz，1H)，2.15(s，3H)，2.08(s，3H)，2.07(s，3H)，1.96(s，3H)，1.69(s，3H)，1.36(s，3H)，0.90(s，3H)。¹³C NMR(125MHz，CDCl₃)δ187.59(s)，182.19(s)，170.78(s)，169.97(s)，169.91(s)，169.63(s)，160.81(s)，156.82(s)，156.20(s)，138.80(s)，134.46(s)，111.51(s)，110.22(s)，109.07(s)，108.97(s)，95.66(s)，81.71(s)，80.53(s)，69.45(s)，69.21(s)，68.97(s)，66.66(s)，65.86(s)，62.43(s)，56.81(s)，32.90(s)，29.79(s)，27.53(s)，26.86(s)，20.82(s)，20.79(s)，20.72(s)，20.63(s).HRESIMS m/z 705.20418[M-H]^-(calcd.for C₃₃H₃₇O₁₇,705.20362). To sum up, it is made intoThe chemical structural formula is determined as:

compound 10: a red solid; melting point 136-;¹H NMR(600MHz，CDCl₃)δ13.24(s，1H)，12.53(s，1H)，6.19(s，1H)，5.60(d，J＝2.9Hz，1H)，4.90(s，1H)，4.44(d，J＝2.9Hz，1H)，4.15–4.07(m，2H)，4.00(dd，J＝11.8，2.9Hz，1H)，3.94(s，3H)，3.92(d，J＝5.7Hz，1H)，3.84(dd，J＝11.8，6.0Hz，1H)，3.76(ddd，J＝9.2，5.9，3.1Hz，1H)，3.70–3.65(m，1H)，3.55(d，J＝16.1Hz，1H)，2.46(d，J＝16.1Hz，1H)，2.04(s，1H)，1.63(s，3H)，1.47(s，3H)，1.35(s，3H)，1.30(s，3H)，0.88(s，3H)。¹³C NMR(150MHz，)δ188.14(s)，182.67(s)，161.45(s)，157.72(s)，157.30(s)，139.61(s)，135.84(s)，112.07(s)，110.87(s)，110.51(s)，109.71(s)，109.58(s)，95.55(s)，82.53(s)，81.21(s)，79.23(s)，76.41(s)，71.07(s)，70.76(s)，66.70(s)，63.26(s)，57.52(s)，33.63(s)，30.46(s)，28.68(s)，28.20(s)，27.50(s)，26.92(s).HRESIMS m/z 577.19313[M-H]^-(calcd.for C₂₈H₃₃O₁₃,577.19266). In summary, the chemical structure is identified as:

compound 11: red crystals; melting point 117-;¹H NMR(400MHz，CDCl₃)δ13.17(s，1H)，12.59(s，1H)，6.18(s，1H)，5.37(d，J＝3.0Hz，1H)，5.17(t，J＝9.4Hz，1H)，4.95(t，J＝10.0Hz，1H)，4.90(dd，J＝9.4，8.1Hz，1H)，4.80(d，J＝8.0Hz，1H)，4.32(d，J＝3.0Hz，1H)，3.95–3.90(m，1H)，3.92(s，3H)，3.75(dd，J＝12.3，2.5Hz，1H)，3.61–3.53(m，1H)，3.47(d，J＝16.2Hz，1H)，2.49(d，J＝16.1Hz，1H)，2.07(s，3H)，1.97(s，3H)，1.97(s，3H)，1.92(s，3H)，1.56(s，3H)，1.32(s，3H)，0.87(s，3H)。¹³C NMR(100MHz，CDCl₃)δ186.55(s)，180.71(s)，170.35(s)，170.15(s)，169.33(s)，169.11(s)，160.75(s)，158.23(s)，157.23(s)，138.26(s)，136.90(s)，110.94(s)，109.97(s)，108.96(s)，108.36(s)，101.76(s)，81.49(s)，80.49(s)，72.63(s)，71.79(s)，71.16(s)，71.09(s)，68.17(s)，61.23(s)，56.75(s)，32.62(s)，29.37(s)，27.46(s)，26.82(s)，20.68(s)，20.56(s)，20.52(s)，20.48(s).HRESIMS m/z 705.20430[M-H]^-(calcd.for C₃₃H₃₇O₁₇,705.20362). In summary, the chemical structure is identified as:

compound 12: a red solid; melting point 128-;¹H NMR(400MHz，CDCl₃)δ13.16(s，1H)，12.57(s，1H)，6.17(s，1H)，5.42(d，J＝2.5Hz，1H)，4.57(d，J＝7.2Hz，1H)，4.50(d，J＝2.5Hz，1H)，3.95(s，3H)，3.91(s，3H)，3.74–3.63(m，2H)，3.58–3.39(m，2H)，3.46(d，J＝16.0Hz，1H)，3.30(t，J＝7.7Hz，1H)，3.17–3.05(m，1H)，2.49(d，J＝16.0Hz，1H)，1.60(s，3H)，1.31(s，3H)，0.86(s，3H)。¹³C NMR(100MHz，CDCl₃)δ186.20(s)，180.34(s)，160.72(s)，158.61(s)，157.49(s)，138.53(s)，137.06(s)，111.06(s)，109.97(s)，108.92(s)，108.48(s)，103.37(s)，81.12(s)，80.59(s)，77.34(s)，77.02(s)，76.70(s)，76.13(s)，72.82(s)，70.11(s)，69.49(s)，65.30(s)，56.76(s)，32.58(s)，29.55(s)，27.53(s)，26.95(s).HRESIMS m/z 507.15148[M-H]^-(calcd.for C₂₄H₂₇O₁₂,507.1508). In summary, the chemical structure is identified as:

compound 13: a red solid; melting point 203-206℃；¹H NMR(400MHz，CDCl₃)δ13.12(s，1H)，12.57(s，1H)，6.17(s，1H)，5.42(d，J＝2.8Hz，1H)，5.07(s，1H)，4.42(d，J＝2.8Hz，1H)，3.92(s，2H)，3.83(s，1H)，3.54-3.74(m，3H)，3.47(d，J＝16.2Hz，2H)，3.28(t，J＝9.3Hz，1H)，3.17–3.00(m，1H)，2.42(d，J＝16.2Hz，1H)，1.57(s，3H)，1.32(s，3H)，0.87(d，J＝4.1Hz，3H)，0.86(s，3H)。¹³C NMR(100MHz，CDCl₃)δ186.68(s)，181.01(s)，160.74(s)，157.81(s)，156.88(s)，137.87(s)，137.13(s)，110.93(s)，110.12(s)，108.97(s)，108.56(s)，100.01(s)，80.68(s)，80.38(s)，77.35(s)，77.03(s)，76.71(s)，72.51(s)，71.57(s)，70.82(s)，68.79(s)，68.19(s)，56.77(s)，32.83(s)，29.80(s)，27.48(s)，26.79(s)，16.97(s).HRESIMS m/z 521.16707[M-H]^-(calcd.for C₂₅H₂₉O₁₂,521.16645). In summary, the chemical structure is identified as:

compound 14: a red solid; melting point 171-;¹H NMR(600MHz，CDCl₃)δ13.16(s，1H)，12.63(s，1H)，6.21(s，1H)，5.56(d，J＝3.0Hz，1H)，5.36(s，1H)，4.51(d，J＝3.0Hz，1H)，4.09(d，J＝5.6Hz，1H)，3.96(s，3H)，3.95–3.93(m，1H)，3.74(q，J＝7.0Hz，1H)，3.55(d，J＝16.2Hz，1H)，3.30(ddd，J＝15.8，9.7，6.7Hz，2H)，2.51(d，J＝16.1Hz，1H)，1.66(s，3H)，1.52(s，3H)，1.38(s，3H)，1.37(s，3H)，0.94(d，J＝6.0Hz，3H)，0.92(s，3H)。¹³C NMR(150MHz，CDCl₃)δ186.91(s)，181.40(s)，160.71(s)，157.69(s)，156.85(s)，137.50(s)，129.94(s)，110.91(s)，110.26(s)，109.67(s)，109.01(s)，108.61(s)，97.47(s)，80.67(s)，80.51(s)，78.29(s)，75.88(s)，74.46(s)，68.08(s)，66.28(s)，56.80(s)，32.85(s)，29.89(s)，29.34(s)，28.07(s)，27.52(s)，26.82(s)，16.59(s).HRESIMS m/z561.19816[M-H]^-(calcd.for C₂₈H₃₃O₁₂,561.19775). In summary, the chemical structure is identified as:

example 2 Effect of Tetrahydroanthraquinone glycosides on growth viability of different tumor cells

1, a detection method:

counting after digesting the tumor cells by pancreatin, paving the cells by using a 96-well plate according to 8000-10000 cells/well, placing the cells at 37 ℃ and 5% CO after paving the cells₂Culturing in incubator, replacing culture medium containing tetrahydroanthraquinone glycoside compounds with different concentrations (0, 1.563, 3.125, 6.25, 12.5, 25 μ M or 0, 5, 10, 20, 40, 80 μ M) after 12h, continuing culturing for 48h, adding 25 μ L of thiazole blue (MTT), incubating for 4-6 h, discarding supernatant, adding 160 μ L of dimethyl sulfoxide (DMSO) into each well, shaking by placing shaking table until the blue-purple crystals are completely dissolved, detecting with 490/570nm wavelength, analyzing data, calculating half inhibitory concentration IC₅₀(μM)。

2. The result of the detection

Semi-inhibitory concentration IC of tetrahydroanthraquinone glycoside compounds on different tumor cells₅₀The test results are shown in table 1.

TABLE 1 MTT results for tetrahydroanthraquinone glycosides

The results in table 1 show that the anthraquinone glycoside series compounds have obvious inhibition effect on the growth of different tumor cells.

Example 3 Effect of anthraquinone glycoside series Compounds on tumor cell morphology

1. Experimental methods

Tumor cells were digested with pancreatin and counted at 2.0X 10⁶The cells were inoculated into P60 culture dishes, and placed at 37 ℃ with 5% CO₂Culturing in incubator, changing culture medium containing anthraquinone compounds 1, 2, 3, 13 with different concentrations (2.5 μ M, 5 μ M, 10 μ M) after 12h, continuing culturing tumor cells, adding medicine, observing cell morphology change, and taking pictures of cell morphology using anthraquinone compounds with different concentrations and time points.

2. Results of the experiment

Fig. 4-7 are the changes in cell morphology after treatment of a549 cells with compounds 1, 2, 3, 13 at different concentrations and at different time points, respectively. As can be seen, the number of the dead tumor cells is gradually increased along with the increase of the concentration of the anthraquinone compound, the cell density is further reduced, and typical apoptosis morphological changes can be observed under an optical microscope, the cell nucleus is shrunk, and the cells become round and bright.

Example 4 Effect of anthraquinone glycoside series Compounds on tumor cell proliferation and Positive cell count

1. Experimental methods

EdU (5-ethyl-2' -deoxyuridine) is a thymidine analogue that can be incorporated into replicating DNA molecules during cell proliferation instead of thymine (T) by basing the EdU on the same

The specific reaction of the fluorescent dye can quickly detect the DNA replication activity of cells, and is suitable for cell proliferation, cell differentiation, growth and development, DNA repair, virus replication, cell marking and tracing and the like.

Tumor cells A549 are arranged in a 2X 10 mode⁴Inoculating each cell into a 24-hole plate paved with a cover glass, treating with anthraquinone compound 1 with different concentrations after 12h, removing the culture medium after 24h, adding 4% paraformaldehyde for fixing for 30 min, removing the fixing liquid, adding 50 mu L of 2mg/ml glycine into each hole, incubating for 5min by a decoloring shaker, removing the glycine, adding 100 mu L of PBS into each hole, cleaning for 5min by the decoloring shaker, and removing the PBS; adding 100 μ L of penetrant (0.5% TrxitonX-100 PBS) into each well, decolorizing and shaking for 10 min, washing with PBS for 1 time and 5 min; adding 100 mu L of 1 XApollo staining reaction solution into each hole, keeping out of the sun, incubating for 30 minutes at room temperature by a decoloring shaker, and discarding the staining reaction solution; each timeAdding 100 mu L of penetrating agent (0.5% TrxitonX-100 PBS) into the hole, decoloring and shaking the hole for 2-3 times, washing the hole for 10 minutes each time, and discarding the penetrating agent; PBS wash was performed 1 time for 5 minutes each.

DNA staining: adding 1 Xhoechst 33342 reaction solution into each hole, keeping out of the sun at room temperature, incubating for 30 minutes by a decoloring shaker, and then removing the staining solution; add 100. mu.L PBS to each well and wash 1-3 times. And (5) cleaning the dried tablet, sealing the tablet by using an anti-quenching fluorescent sealing tablet, and storing the tablet in a dark place.

2. Results of the experiment

Fig. 8 shows the effect of the anthraquinone glycoside compound 1 on the proliferation of a549 cells, and fig. 9 shows the statistical result of EdU positive cells after the anthraquinone glycoside compound 1 treats the a549 cells.

As can be seen from the figure, as the treatment concentration of compound 1 increases, the a549 cell proliferation is obviously inhibited, and has a dose-dependent relationship, and the a549 proliferation inhibition effect is enhanced as the dose of compound 1 increases.

Example 5 Effect of anthraquinone glycoside series Compounds on apoptosis-related protein expression

1. Experimental methods

The expression of apoptosis-related proteins was detected by immunoblotting (Western Blotting). Tumor cells A549 were digested with pancreatin and counted at 2.0X 10⁶The cells were inoculated into P60 culture dishes, and placed at 37 ℃ with 5% CO₂Culturing in an incubator, adding anthraquinone compounds with different concentrations for 1 hour after 12 hours, collecting cells and supernatant after 24 hours, washing with PBS for 2-3 times, adding cell lysis solution to treat the cells, and determining the protein content by using a BCA kit. Protein electrophoresis was performed, gel 80V was concentrated, and gel 100V was isolated. The PVDF membrane can be transferred after electrophoresis until bromophenol blue reaches the bottom of the gel, and the transfer is carried out for 2 hours at 300 mA. After 5% skim milk was sealed at room temperature for 1h, 1: 500 human PARP antibody was added and incubated overnight at 4 ℃. The antibody was recovered the next day and washed 3 times with PBST for 15 minutes each. And (3) incubating the horseradish peroxidase-labeled secondary antibody for 1h at room temperature, tabletting and clamping the tablet for 30 minutes, and exposing and developing the tablet by a developing machine.

The precursor and the cleavage zone of the anthraquinone compound induced tumor cell apoptosis related protein PARP protein are further detected by an immunoblotting method.

2. Results of the experiment

FIG. 10 shows the expression of apoptosis-related protein after treatment of A549 cells with anthraquinone compound 1, and the immunoblotting method is used to detect the precursor and cleavage zone of the anthraquinone compound 1-induced tumor cell apoptosis-related protein PARP, and the cleavage zone is gradually increased with the increase of the compound concentration.

Example 6 antitumor Activity test of anthraquinone glycoside series Compounds in mice

1. Experimental methods

The immunodeficient mice (BALB/c-nu/nu) are inoculated with MDA-MB-435 cells subcutaneously until the tumor grows to 80-100 mm³Dosing experiments were performed in random groups. Each group of 6 mice was divided into a solvent control group, a positive control group (cisplatin, DDP, 5mg/kg), an administration group (Compound 1, administered at 30mg/kg and 60mg/kg, respectively), and intraperitoneal injection, administered 1 time every 3 days. The experimental period is about 30 days generally, and the tumor of the solvent control group grows to 3000-3500 mm³The experiment was terminated right and left. Mice tumors were stripped, weighed, measured and photographed.

2. Results of the experiment

FIG. 11 shows the tumor stripped at the experimental end point of different tumor-bearing mouse groups, and FIG. 12 shows the weighing results after the tumor stripping of different tumor-bearing mouse groups. It can be seen that the tumor suppression rate of the high dose group mice was 44.89% (P < 0.05). The in vivo anti-tumor activity experiment of a mouse proves that the anthraquinone glycoside compound 1 has better anti-tumor activity in the mouse.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (9)

1. A tetrahydroanthraquinone glycoside compound is characterized in that the chemical structural formula is as follows:

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2. The use of the tetrahydroanthraquinone glycoside compound of claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of an antitumor medicament.

3. The use of claim 2, wherein the anti-tumor agent is one selected from the group consisting of inhibiting tumor cell growth, inhibiting tumor cell proliferation, and inducing tumor cell apoptosis.

4. Use of the tetrahydroanthraquinone glycoside compound of claim 1, or a pharmaceutically acceptable salt thereof, for the preparation of an inducer of apoptosis in tumor cells.

5. The use of claim 2 or 4, wherein the tumor is one or more of lung cancer, breast cancer, colon cancer or liver cancer.

6. The use as claimed in claim 2 or 4, wherein the pharmaceutically acceptable salt of the tetrahydroanthraquinone glycoside compound is an inorganic base salt or a metal complex thereof.

7. An antitumor agent comprising the tetrahydroanthraquinone glycoside compound according to claim 1 or a pharmaceutically acceptable salt thereof.

8. The medicament of claim 7, further comprising a pharmaceutically acceptable carrier and/or diluent, and being prepared into different dosage forms.

9. The medicament of claim 8, wherein the dosage form is powder, tablet, granule, capsule, solution, syrup, suspension, injection, powder injection, water injection, aerosol, ointment, eye drop or suppository.

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