CN115813892A - Drug combination of protocatechualdehyde and dacarbazine and its use in the treatment of malignant melanoma - Google Patents

Abstract

The invention relates to a drug combination of protocatechualdehyde and dacarbazine and its application in treating malignant melanoma. The drug combination comprises: protocatechualdehyde or a composition containing protocatechualdehyde as a first active ingredient; and dacarbazine as a second active ingredient. The drug combination according to the embodiment of the present invention can increase the number of DNA double-strand breaks in tumor cells caused by dacarbazine, promote tumor cell apoptosis, and then achieve the purpose of treating and controlling cancer, especially malignant melanoma.

Description

Drug Combination of Protocatechualdehyde and Dacarbazine and Its Application in the Treatment of Malignant Melanoma the use of

technical field

The invention belongs to the technical field of biopharmaceuticals. Specifically, the invention relates to a drug combination of protocatechualdehyde and dacarbazine and its application in treating malignant melanoma.

Background technique

Cancer is a disease that seriously threatens human life and health. In recent years, the incidence and mortality of cancer worldwide have been increasing, which is a problem that plagues human life and health. Currently available cancer treatments include surgical resection, radiotherapy, chemotherapy, small molecule targeted therapy, antibody targeted therapy, and macromolecule immunotherapy.

There are many types of cancer, such as thyroid cancer, nasopharyngeal cancer, adenocarcinoma, liver cancer, skin cancer, etc. Among them, malignant melanoma is the most invasive and fatal skin cancer. The current treatment methods for malignant melanoma mainly include surgery and adjuvant therapy, radiotherapy, chemotherapy, biological immunotherapy, targeted therapy, etc., but for the economic Chemotherapy is still an important means of treatment for melanoma in patients with poor condition, drug resistance to targeted therapy, and immunosuppressant unresponsiveness. Dacarbazine (DTIC) is currently a commonly used chemotherapeutic drug, but the effective rate of DTIC alone is about 8% to 12%, which is relatively low.

Therefore, there is an urgent need to develop an effective drug for the treatment of cancer.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent. Therefore, the present invention provides a drug combination of protocatechualdehyde and DNA alkylating agent, which can promote the apoptosis of tumor cells and effectively treat cancers such as melanoma.

The present invention has been accomplished based on the following findings of the inventors:

Dacarbazine was approved by the US FDA in 1975 for the treatment of malignant melanoma, and it is still the gold standard of chemotherapy drugs for the clinical treatment of advanced malignant melanoma. As a DNA alkylating agent, dacarbazine works by metabolizing into the active compound 5-(3-methyltriazin-1-yl)imidazol-4-amide (MTIC), and further by making the Guanine (Guanine, G) is methylated to form O ⁶ -methylguanine. The formation of O ⁶ -methylguanine will make the GC pairing in the DNA replication process mismatch and form the GT pairing form. Once GT pairing is formed, the intracellular DNA damage repair system will repeatedly cut at the mismatch, resulting in DNA double-strand breaks. The formation of a large number of DNA double-strand breaks will lead to the apoptosis of tumor cells, so as to achieve the purpose of treating and controlling tumors.

However, the effective rate of DTIC single drug use is about 8% to 12%, and its efficiency is not high, and tumor patients are prone to drug resistance. In order to solve this problem, people try to use various drugs in combination with DTIC to improve the curative effect. Survival benefit of melanoma patients, such as cisplatin, recombinant human endostatin (endostar), doxorubicin, vincristine, etc. However, the commonly used chemotherapeutic drugs combined with dacarbazine can not improve the response level of melanoma to the drug and the survival of melanoma patients (Chapman PB, Einhorn LH, Meyers ML, et al: Phase III multicenter randomized trial of the Dartmouth regimen versus dacarbazine in patients with metastatic melanoma. J Clin Oncol 17:2745-2751, 1999). Therefore, there is an urgent need to develop a drug with low toxicity and side effects, which can increase the efficacy of DTIC and inhibit or reverse its drug resistance.

Protocatechuic aldehyde (PA), whose chemical name is 3,4-dihydroxybenzaldehyde, is a natural phenolic acid compound, which is used as a compound in traditional Chinese medicines such as salvia miltiorrhiza, clary sage, gastrodia elata, wolfberry, black fern, Sijiqing, grass One of the effective active ingredients of fruit, cedar, white scorpion, iron amaranth, begonia, etc. According to research reports, it has various pharmacological activities such as antibacterial, anti-inflammatory, anti-tumor, and anti-oxidative stress. Wherein, the structural formula of protocatechualdehyde is shown in formula (II):

Based on this, in the first aspect of the present invention, the present invention proposes a compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt thereof or a derivative thereof Use in the preparation of reagents for reducing the expression or activity of MGMT protein,

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

或者，

与其所连接的C连接形成5～10元杂环烷基，其中，5～10元杂环烷基任选被一个或多个R₇取代；R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ;

R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH;

R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxy.

The inventors have found through experiments that protocatechuic aldehyde or drugs containing protocatechuic aldehyde can effectively reduce the expression or activity of MGMT protein. The expression or activity can be used for scientific research on cells in vitro.

It should be noted that, as used herein, "reducing the expression or activity of MGMT protein" includes reducing the expression or activity of MGMT protein.

Exemplarily, reducing the expression of MGMT protein includes but is not limited to the following methods: reducing the mRNA level of MGMT protein, inhibiting the translation of MGMT protein mRNA, and promoting the degradation of MGMT protein.

Exemplarily, reducing the activity of the MGMT protein may refer to changing the spatial structure of the MGMT protein to reduce the activity of the MGMT protein. According to an embodiment of the present invention, the reduction of the expression of MGMT protein is achieved by promoting the degradation of MGMT protein. The inventors have found through experiments that contacting the reagent of the present invention with cells can effectively promote the degradation of MGMT protein.

According to an embodiment of the present invention, the compound represented by the formula (I) or its salt or its derivative or the composition containing the compound represented by the formula (I) or its salt or its derivative comprises a compound selected from Salvia miltiorrhiza or its Extract, Clary sage or its extract, Gastrodia elata or its extract, wolfberry or its extract, black fern or its extract, Sijiqing or its extract, Tsaoguo or its extract, cedar or its extract, At least one of white scorpion or its extracts, amaranth or its extracts, begonia or its extracts, thorny buttercup or its extracts, dog ridge or its extracts, palm and its extracts, and persimmon leaves and its extracts one.

According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or its salt or its derivatives includes compounds selected from Compound Danshen Tablets, Compound Danshen Dripping Pills, Compound Danshen Injection, Danshen Tablets, Xinkeshu Tablets, Xiaojian Tongluo Tablets, Anshen Buxin Pills, Tianwang Buxin Pills, Wenxin Granules, Shuangdan Oral Liquid, Xintong Oral Liquid, Danhong Huayu Oral Liquid, Danshen Water Extract, Prunella Oral Liquid, Fu Cong Mixture, Xiangdan Injection, Yinaoxin Granules, Shenpu Penyan Granules, Huoluoxiaotong Tablets, Guyanxiaofang, Guanxinning Injection, Danhong Injection, Dingxi Yatong Capsules, Compound Qilin Papua . Huayu Oral Liquid, Danyi Tablets, Xinnaoning Capsules, Xinnaokang Tablets, Vitiligo Capsules, Fuyankang Tablets, Qidong Yixin Oral Liquid, Lian Shen Tong Lin Tablets, Bubai Granules, Kunning Oral Liquid, Shen Kang Ning Tablets, Ru Ning Granules, Rukuaixiao Granules, Jizhi Syrup, Vigorous Su Oral Liquid, Naoxinqing Tablets, Xiaoxuan Zhiyun Tablets, Tiaojing Huoxue Capsules, Qingnao Jiangya Tablets, Qingnao Jiangya Capsules, Qingnao Jiang At least one of compressed granules and Tangmaikang granules.

In the second aspect of the present invention, the present invention provides a single dose inhibitor of MGMT protein. According to an embodiment of the present invention, the single-dose MGMT protein degradation agent includes 20-200 μM of the compound represented by formula (I) or a salt or derivative thereof,

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

或者，

与其所连接的C连接形成5～10元杂环烷基，其中，5～10元杂环烷基任选被一个或多个R₇取代；R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ;

R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxyl. The single-dose MGMT protein degradation agent of the present invention can effectively inhibit the expression and activity of intracellular MGMT protein, for example, promote the degradation of intracellular MGMT protein, and can especially be used for scientific research on cells in vitro.

In a third aspect of the present invention, the present invention proposes a kit. According to an embodiment of the present invention, the kit includes a compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt or derivative thereof,

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

或者，

与其所连接的C连接形成5～10元杂环烷基，其中，5～10元杂环烷基任选被一个或多个R₇取代；R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ;

R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxyl. The kit of the invention can be used to promote the degradation of intracellular MGMT protein.

Exemplarily, the kit of the present invention is used to control the level of MGMT protein in cells, so as to obtain cells with a specific level of MGMT protein.

According to an embodiment of the present invention, the working concentration of the compound represented by formula (I) or its salt or derivative thereof is 20-200 μM, preferably 40-150 μM, more preferably 40-100 μM. Adopting the above-mentioned working concentration can further reduce the intracellular MGMT protein level, especially can effectively promote the degradation of MGMT protein.

According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or a salt thereof or a derivative thereof includes a composition selected from the group consisting of Salvia miltiorrhiza or its extract, Clary sage or its extract, Gastrodia elata or its extract, Lycium barbarum or its extracts, black fern or its extracts, Sijiqing or its extracts, Caoguo or its extracts, cedar or its extracts, white pomegranates or its extracts, amaranth or its extracts, begonias or their extract, buttercup thorn or its extract, dog ridge or its extract, palm and its extract, and persimmon leaf and its extract.

In the fourth aspect of the present invention, the present invention provides a method for reducing the expression or activity of MGMT protein. According to an embodiment of the present invention, the method includes: contacting the cell with a compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt thereof or a derivative thereof , the cells express MGMT;

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

或者，

与其所连接的C连接形成5～10元杂环烷基，其中，5～10元杂环烷基任选被一个或多个R₇取代；R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ;

R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxyl. The method according to the embodiment of the present invention can effectively promote the degradation of MGMT protein, and can especially be used for scientific research of cells in vitro. For example, it can be used to degrade MGMT protein in cells in vitro, so as to obtain cells with a specific level of MGMT protein.

According to an embodiment of the present invention, the working concentration of the compound represented by formula (I) or its salt or derivative thereof is 20-200 μM, preferably 40-150 μM, more preferably 40-100 μM. Adopting the above-mentioned working concentration can further reduce the intracellular MGMT protein level, especially can effectively promote the degradation of MGMT protein.

According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or a salt thereof or a derivative thereof includes a composition selected from the group consisting of Salvia miltiorrhiza or its extract, Clary sage or its extract, Gastrodia elata or its extract, Lycium barbarum or its extracts, black fern or its extracts, Sijiqing or its extracts, Caoguo or its extracts, cedar or its extracts, white pomegranates or its extracts, amaranth or its extracts, begonias or their extract, buttercup thorn or its extract, dog ridge or its extract, palm and its extract, and persimmon leaf and its extract.

In the fifth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: a compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt thereof or a derivative thereof; and DNA Alkylating agent;

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

或者，

与其所连接的C连接形成5～10元杂环烷基，其中，5～10元杂环烷基任选被一个或多个R₇取代；R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ;

R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxyl. The pharmaceutical composition according to the embodiment of the present invention can increase the number of DNA double-strand breaks in tumor cells caused by DNA alkylating agents, promote tumor cell apoptosis, and further achieve the purpose of treating and controlling cancer.

The inventor also found through experiments that the pharmaceutical composition can effectively reduce the level of MGMT protein. Further, the inventors detected the mRNA transcription level of genes related to DNA damage repair in cells, and found that, compared with the pharmaceutical composition, it can reduce the mRNA level of MGMT protein and inhibit the translation of MGMT protein mRNA. Reducing the level of MGMT protein is mainly achieved by promoting the degradation of MGMT protein.

According to an embodiment of the present invention, the DNA alkylating agent includes dacarbazine, temozolomide, busulfan, streptozotocin, carmustine, lomustine, melphalan, procarbazine hydrochloride, Triosulfan, Thiotepa, Methylmustine and its derivatives, Oxymustine hydrochloride and its derivatives, Chlorambucil derivatives, Phenylalanine mustard and its derivatives, Benzoambucil, Monosubstituted aryl mustard derivatives, polysubstituted aryl mustards, oligopeptide aryl mustards, macrocyclic polyamine mustards, cyclophosphamide derivatives, straight-chain phosphoramide derivatives, metal complex nitrogen mustards, At least one of uracil mustard and solanesyl amine derivatives.

According to an embodiment of the present invention, the DNA alkylating agent is dacarbazine. The inventors have found through experiments that, compared with other DNA alkylating agents, the combined use of protocatechuic aldehyde and dacarbazine is better.

According to an embodiment of the present invention, the DNA alkylating agent is temozolomide.

According to an embodiment of the present invention, the compound represented by formula (I) or its salt or derivative thereof or the composition containing the compound represented by formula (I) includes Salvia miltiorrhiza or its extract, Clary sage or its extract Gastrodia elata or its extracts, wolfberry or its extracts, black fern or its extracts, Sijiqing or its extracts, Caoguo or its extracts, cedar or its extracts, white pomegranates or its extracts, iron amaranth or an extract thereof, begonia or an extract thereof, buttercup thorn or an extract thereof, dog ridge or an extract thereof, palm and an extract thereof, and persimmon leaves or an extract thereof.

According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or its salt or its derivatives includes compounds selected from Compound Danshen Tablets, Compound Danshen Dripping Pills, Compound Danshen Injection, Danshen Tablets, Xinkeshu Tablets, Xiaojian Tongluo Tablets, Anshen Buxin Pills, Tianwang Buxin Pills, Wenxin Granules, Shuangdan Oral Liquid, Xintong Oral Liquid, Danhong Huayu Oral Liquid, Danshen Water Extract, Prunella Oral Liquid, Fu Cong Mixture, Xiangdan Injection, Yinaoxin Granules, Shenpu Penyan Granules, Huoluoxiaotong Tablets, Guyanxiaofang, Guanxinning Injection, Danhong Injection, Dingxi Yatong Capsules, Compound Qilin Papua . Huayu Oral Liquid, Danyi Tablets, Xinnaoning Capsules, Xinnaokang Tablets, Vitiligo Capsules, Fuyankang Tablets, Qidong Yixin Oral Liquid, Lian Shen Tong Lin Tablets, Bubai Granules, Kunning Oral Liquid, Shen Kang Ning Tablets, Ru Ning Granules, Rukuaixiao Granules, Jizhi Syrup, Vigorous Su Oral Liquid, Naoxinqing Tablets, Xiaoxuan Zhiyun Tablets, Tiaojing Huoxue Capsules, Qingnao Jiangya Tablets, Qingnao Jiangya Capsules, Qingnao Jiang At least one of compressed granules and Tangmaikang granules.

According to an embodiment of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

According to an embodiment of the present invention, the molar ratio of the compound represented by formula (I) or its salt or derivative thereof to the DNA alkylating agent is (1-100): (100-1). The inventors have found through experiments that adopting the above ratio can further promote tumor cell apoptosis and improve the therapeutic effect on cancer.

In a sixth aspect of the present invention, the present invention proposes a drug combination or kit. According to an embodiment of the present invention, the drug combination or kit includes: a compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt thereof or a derivative thereof as a first active ingredient; and a DNA alkylating agent as a second active ingredient;

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

或者，

与其所连接的C连接形成5～10元杂环烷基，其中，5～10元杂环烷基任选被一个或多个R₇取代；R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ;

R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxyl. The drug combination or kit according to the embodiments of the present invention can increase the number of DNA double-strand breaks in tumor cells caused by DNA alkylating agents, promote tumor cell apoptosis, and then achieve the purpose of treating and controlling cancer.

The inventor also found through experiments that the drug combination or kit can effectively reduce the level of MGMT protein. Further, the inventors detected the mRNA transcription level of genes related to DNA damage repair in cells, and found that, compared with drug combinations or kits, it can reduce the mRNA level of MGMT protein and inhibit the translation of MGMT protein mRNA. Combination or kit reduces the level of MGMT protein mainly by promoting the degradation of MGMT protein.

According to an embodiment of the present invention, the DNA alkylating agent includes dacarbazine, temozolomide, busulfan, streptozotocin, carmustine, lomustine, melphalan, procarbazine hydrochloride, Triosulfan, Thiotepa, Methylmustine and its derivatives, Oxymustine hydrochloride and its derivatives, Chlorambucil derivatives, Phenylalanine mustard and its derivatives, Benzoambucil, Monosubstituted aryl mustard derivatives, polysubstituted aryl mustards, oligopeptide aryl mustards, macrocyclic polyamine mustards, cyclophosphamide derivatives, straight-chain phosphoramide derivatives, metal complex nitrogen mustards, At least one of uracil mustard and solanesyl amine derivatives.

According to an embodiment of the present invention, the DNA alkylating agent is dacarbazine. The inventors have found through experiments that, compared with other DNA alkylating agents, the combined use of protocatechuic aldehyde and dacarbazine is better.

According to an embodiment of the present invention, the DNA alkylating agent is temozolomide.

According to an embodiment of the present invention, the compound represented by formula (I) or its salt or derivative thereof or the composition containing the compound represented by formula (I) or its salt or derivative thereof includes Salvia miltiorrhiza or its extract, Taiga or its extracts, Gastrodia elata or its extracts, wolfberry or its extracts, black fern or its extracts, Sijiqing or its extracts, Tsaoguo or its extracts, cedar or its extracts, white scorpion or its At least one of extracts, amaranth or its extracts, begonia or its extracts, thorny buttercups or its extracts, dog spines or its extracts, palm and its extracts, and persimmon leaves and its extracts.

According to an embodiment of the present invention, the composition containing the compound represented by formula (I) or its salt or its derivatives includes compounds selected from Compound Danshen Tablets, Compound Danshen Dripping Pills, Compound Danshen Injection, Danshen Tablets, Xinkeshu Tablets, Xiaojian Tongluo Tablets, Anshen Buxin Pills, Tianwang Buxin Pills, Wenxin Granules, Shuangdan Oral Liquid, Xintong Oral Liquid, Danhong Huayu Oral Liquid, Danshen Water Extract, Prunella Oral Liquid, Fu Cong Mixture, Xiangdan Injection, Yinaoxin Granules, Shenpu Penyan Granules, Huoluoxiaotong Tablets, Guyanxiaofang, Guanxinning Injection, Danhong Injection, Dingxi Yatong Capsules, Compound Qilin Papua . Huayu Oral Liquid, Danyi Tablets, Xinnaoning Capsules, Xinnaokang Tablets, Vitiligo Capsules, Fuyankang Tablets, Qidong Yixin Oral Liquid, Lian Shen Tong Lin Tablets, Bubai Granules, Kunning Oral Liquid, Shen Kang Ning Tablets, Ru Ning Granules, Rukuaixiao Granules, Jizhi Syrup, Vigorous Su Oral Liquid, Naoxinqing Tablets, Xiaoxuan Zhiyun Tablets, Tiaojing Huoxue Capsules, Qingnao Jiangya Tablets, Qingnao Jiangya Capsules, Qingnao Jiang At least one of compressed granules and Tangmaikang granules.

According to an embodiment of the present invention, the molar ratio of the compound represented by formula (I) or its salt or derivative thereof to the DNA alkylating agent is (1-100): (100-1). The inventors have found through experiments that adopting the above ratio can further promote the apoptosis of tumor cells and improve the therapeutic effect on cancer.

According to an embodiment of the present invention, the compound represented by formula (I) or a salt thereof or a derivative thereof is prepared together with or separately from a DNA alkylating agent.

According to an embodiment of the present invention, the compound represented by the formula (I) or its salt or its derivative is used simultaneously or separately with the DNA alkylating agent.

In a seventh aspect of the invention, the present invention proposes a single dosage form. According to an embodiment of the present invention, the single dosage form comprises: a compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt thereof or a derivative thereof as the first active agent composition; and a DNA alkylating agent as the second active ingredient; wherein, the molar ratio of the compound represented by the formula (I) or its salt or derivative thereof to the DNA alkylating agent is (1～100): (100～1 );

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

或者，

与其所连接的C连接形成5～10元杂环烷基，其中，5～10元杂环烷基任选被一个或多个R₇取代；R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ;

R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxyl. The single dosage form according to the embodiment of the present invention can increase the number of DNA double-strand breaks in tumor cells caused by DNA alkylating agents, promote apoptosis of tumor cells, and further achieve the purpose of treating and controlling cancer.

According to the embodiments of the present invention, the DNA alkylating agent includes dacarbazine, temozolomide, thiotepa, methyl mustard and its derivatives, oxambucil hydrochloride and its derivatives, chlorambucil derivatives, Phenylalanine mustard and its derivatives, benzoic acid mustard, monosubstituted aryl mustard derivatives, polysubstituted aryl mustard, oligopeptide aryl mustard, macrocyclic polyamine mustard, cyclophosphamide At least one of derivatives, straight-chain phosphoramide derivatives, metal complex nitrogen mustards, uracil nitrogen mustards and solanesyl amine derivatives.

According to an embodiment of the present invention, the DNA alkylating agent is dacarbazine. The inventors have found through experiments that, compared with other DNA alkylating agents, the combined use of protocatechuic aldehyde and dacarbazine is better.

According to an embodiment of the present invention, the DNA alkylating agent is temozolomide.

According to an embodiment of the present invention, the compound represented by formula (I) or its salt or derivative thereof or the composition containing the compound represented by formula (I) or its salt or derivative thereof includes Salvia miltiorrhiza or its extract, Taiga or its extracts, Gastrodia elata or its extracts, wolfberry or its extracts, black fern or its extracts, Sijiqing or its extracts, Tsaoguo or its extracts, cedar or its extracts, white scorpion or its At least one of extracts, amaranth or its extracts, begonia or its extracts, thorny buttercups or its extracts, dog spines or its extracts, palm and its extracts, and persimmon leaves and its extracts.

Further, the use described in the first aspect above, the single-dose MGMT protein degradation agent described in the second aspect, the kit described in the third aspect, the method described in the fourth aspect, and the drug combination described in the fifth aspect The drug combination or kit described in the sixth aspect or the single dosage form described in the seventh aspect may further include at least one of the following additional technical features:

或者，

与其所连接的C连接形成5元杂环烷基，其中，5元杂环烷基任选被两个R₇取代，其中，R₇选自-CH₃或-CH₂CH₂OH。According to an embodiment of the present invention, R ₂ and R ₃ are independently selected from H, -CH ₃ , -CH ₂ (CH ₃ ) ₂ , -CH ₂ CH ₂ CH ₂ CH ₃ , -COCH ₃ , -CH ₂ OCH _3. -CH ₂ CH ₂ OH, -CH=CH-CH ₃ , -CF ₃ , -CHF ₂ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-membered heterocycloalkyl group, wherein the 5-membered heterocycloalkyl group is optionally substituted by two R _{7 s} , wherein R ₇ is selected from —CH ₃ or —CH ₂ CH ₂ OH.

According to an embodiment of the present invention, the compound represented by the formula (I) has the following structure:

In the eighth aspect of the present invention, the present invention proposes a use of the aforementioned pharmaceutical composition, the aforementioned drug combination or kit, or the aforementioned single dosage form in the preparation of medicines for the prevention and/or treatment of cancer .

According to an embodiment of the present invention, the cancers include breast cancer, ovarian cancer, breast cancer, testicular cancer, pancreatic cancer, liver cancer, colon cancer, colorectal cancer, thyroid cancer, lung cancer, prostate cancer, kidney cancer, melanoma, squamous Cell carcinoma, gastrointestinal adenocarcinoma, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, promyelocytic leukemia, meningeal leukemia, multiple myeloma, lymphosarcoma, lymphoma (TLX5 lymphoma lymphoma, malignant lymphoma, B-cell lymphoma), bladder cancer, head and neck cancer, esophageal cancer, brain cancer, pharyngeal cancer, tongue cancer, synovial cell carcinoma, neuroblastoma, uterine cancer, fibrosarcoma, myxosarcoma, fat Sarcomas, soft tissue tumors, osteosarcomas (chondrosarcoma, osteosarcoma), chordomas, angiosarcomas, endothelial cell sarcomas, lymphangiosarcomas, synovial tumors, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, basal cell Carcinoma, epidermoid carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver tumor, cholangiocarcinoma, choriocarcinoma, seminal carcinoma Primary cell tumor, embryonal carcinoma, Wilms tumor, cervical cancer, cervical cancer, small cell lung cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependyma tumor, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, brain or intrathecal tumor, glioma, and at least one of retinoblastoma; preferably, The cancer includes at least one of melanoma, glioma, small cell lung cancer, osteosarcoma, and lymphoma.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

Fig. 1 is the result graph of the survival rate of malignant melanoma cells in each group in Example 1 of the present invention;

Fig. 2 is the result graph of the survival rate of malignant melanoma and glioma cells of each group in Example 2 of the present invention;

Fig. 3 is the result graph of the survival rate of malignant melanoma cells of each group in Example 3 of the present invention;

Fig. 4 is the synergistic score of PA and DTIC by ZIP model and Bliss model in the embodiment of the present invention 3;

Fig. 5 is the detection result of Western Blotting in the embodiment of the present invention 4;

Fig. 6 is the detection result of Western Blotting in the embodiment of the present invention 5;

Fig. 7 is the alkaline comet experiment result in the embodiment of the present invention 6;

FIG. 8 is a standard curve for PA content detection in Example 1 of the present invention.

Detailed ways

Embodiments of the present invention are described in detail below. The embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Thus, a feature defined as "first" and "second" may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

Neither the endpoints nor any values of the ranges disclosed herein are limited to such precise ranges or values, and these ranges or values are understood to include values approaching these ranges or values. For numerical ranges, between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, these values Ranges should be considered as specifically disclosed herein.

In this article, the term "comprising" or "comprising" is an open expression, that is, it includes the content specified in the present invention, but does not exclude other aspects of the content.

As used herein, the terms "optionally", "optionally" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes the circumstances in which it occurs, and The circumstances in which the event or condition did not occur.

As used herein, the term "substitution" means that a hydrogen atom in a molecule is replaced by another different atom or group.

In this article, the term "optionally substituted" means that "substitution" may or may not occur, that is, the hydrogen atoms in the molecule or group are replaced by other same or different atoms or groups.

As used herein, the minimum and maximum carbon atom content in a hydrocarbon group is indicated by a prefix, for example, the prefix C _a-b alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Thus, for example, C _1-6 alkyl refers to an alkyl group containing 1-6 carbon atoms.

As used herein, the term "alkyl" refers to a saturated hydrocarbon chain having the indicated number of member atoms. Alkyl groups can be straight or branched. Representative branched alkyl groups have one, two or three branches. Alkyl groups may be optionally substituted with one or more substituents as defined herein. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl base) and hexyl. Alkyl groups may also be part of other groups such as -O(C _1-6 alkyl).

As used herein, the terms "heterocycloalkyl", "heterocycle" and "heterocycloalkane" all refer to a saturated ring or a non-aromatic unsaturated ring containing at least one heteroatom; atoms, sulfur atoms, etc. As used herein, generally refers to a monovalent saturated or partially unsaturated monocyclic ring of multiple ring atoms comprising 1, 2 or 3 ring heteroatoms selected from O, the remaining ring atoms being carbon.

Herein, the term "5-10 membered heterocycloalkyl" refers to 5, 6, 7, 8, 9 or 10-membered saturated or unsaturated heterocycloalkyl, preferably 5-6 membered saturated heterocycloalkyl. The unsaturated in the present invention refers to the group or molecule containing carbon-carbon double bond, carbon-carbon triple bond, carbon-oxygen double bond, carbon-sulfur double bond, carbon-nitrogen triple bond, etc.; the unsaturated carbocycle of the present invention The group includes or does not include an aromatic ring group, and the unsaturated heterocyclic group includes or does not include a heteroaryl group, those skilled in the art can freely choose.

As used herein, the term "halogen" refers to fluorine, chlorine, bromine or iodine. Wherein, "halogen-substituted alkyl" means that one or more hydrogen atoms in the alkyl group are replaced by halogen; for example, trifluoromethyl, difluoromethyl, monofluoromethyl and the like.

As used herein, the term "-OR" means that the R group is attached to an oxygen atom with a single bond.

As used herein, the oxygen atom in the term "-C(O)R" is double bonded to a carbon atom.

Herein, the term "single dose" refers to a single dose; Exemplarily, "single dose of MGMT protein activity inhibitor" means that a single addition of the MGMT protein activity inhibitor can achieve inhibition of MGMT protein activity in cells dosage.

As used herein, the term "single dosage form" refers to a dosage form in which a single administration of a preparation is used up.

In this article, the term "drug combination" refers to the simultaneous or sequential application of two or more drugs to achieve therapeutic purposes, the result of which is mainly to increase the efficacy of the drug or to reduce the side effects of the drug.

As used herein, the term "nitrogen mustards" refers to a class of cytotoxic chemotherapeutic drugs similar in structure to mustard gas, such as cyclophosphamide, chlorambucil, melphalan, bendamustine, ifosfamide and estramustine etc.

As used herein, the term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms, and/or therapeutic in terms of partial or complete cure of the disease and/or adverse effects caused by the disease. "Treatment" as used herein encompasses disease in mammals, especially humans, including: (a) preventing the disease or condition in a predisposed but undiagnosed individual; (b) inhibiting the disease, e.g., arresting its progression; Or (c) ameliorating the disease, eg, alleviating symptoms associated with the disease. "Treatment" as used herein encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, alleviate or inhibit a disease in that individual, including but not limited to administering a drug comprising a compound described herein to an individual in need thereof.

Compounds mentioned herein include, but are not limited to, the compounds themselves and deuterated compounds. The "deuterated compound" of the present invention means that one or more hydrogen atoms in a molecule or group are replaced by deuterium atoms, wherein the proportion of deuterium atoms is greater than the abundance of deuterium in nature.

It should be noted that the "compound represented by formula (I)" herein can be used directly as an active ingredient, or as a prodrug of the active ingredient, and is not specifically limited. When taken as a prodrug, it can be taken after pretreatment in vitro, or taken directly, and then converted into active ingredients in the body.

Herein, the term " _γH2AX " generally refers to one of the members of the chromosomal histone _H2A family. Under the stimulation of various physical and chemical factors, the DNA double strand of the cell breaks, and phosphatidylinositol 3-kinases such as ATM and ATR phosphorylate the 139th serine on _H2AX to form phosphorylated _H2AX , namely γH ₂ AX. γH ₂ AX produced by phosphorylation of H ₂ AX as a biomarker can clearly reflect the degree of DNA damage and repair.

In this paper, the term "O ⁶ -methylguanine-DNA methyltransferase (MGMT for short)" is a DNA repair protein that can remove alkyl adducts in DNA to eliminate DNA alkylating agents. cytotoxicity. Studies have shown that the expression level of MGMT is significantly increased in many malignant tumors such as melanoma and glioma, which is an important factor for its resistance to chemotherapy drugs (such as DTIC and TMZ).

In this article, the term "Temozolomide (TMZ)" is a DNA alkylating agent, and its pharmacology is the same as that of DTIC, the difference is that TMZ can pass through the blood-brain barrier and can be degraded spontaneously and quickly in the body to produce active metabolites MTIC produces anti-tumor effects and is used as a therapeutic drug for adult malignant glioma and malignant melanoma.

Herein, the term "Cicloheximide (CHX)" is an organic compound with a chemical formula of C ₁₅ H ₂₃ NO ₄ , capable of inhibiting protein synthesis in eukaryotes. Wherein, the chemical structural formula of cycloheximide is:

In this paper, the term "Protocatechuic aldehyde (PA)", the chemical name is 3,4-dihydroxybenzaldehyde, is a natural phenolic acid compound, which is used for the traditional Chinese medicines Salvia miltiorrhiza, Sage, Gastrodia elata, Lycium barbarum, One of the effective active ingredients of black fern, perennial green, grass fruit, cedar, white scorpion, amaranth, begonia, thorny buttercup, dog spine, palm, persimmon leaf, etc.

The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1: Effects of Salvia Miltiorrhiza Extract and TMZ on the Survival Rate of Malignant Melanoma Cells

The inventor investigated the effect of combined use of Salvia miltiorrhiza extract and TMZ on the survival rate of malignant melanoma cells, and the specific test steps are as follows:

1. Materials and methods

1.1 Experimental materials

1.1.1 Source of cells

Human melanoma cells A375 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, and A2058 cells were obtained from ATCC.

1.1.2 Salvia miltiorrhiza extract and TMZ solution preparation

For the convenience of experimental investigation, the commercially available Compound Danshen Tablets (Shanghai Leiyunshang Pharmaceutical Co., Ltd.) was dissolved in PBS to prepare a stock solution with a concentration of 100 mg/mL for future use. TMZ (S1237) was purchased from Selleck Company, and was dissolved in DMSO to prepare a stock solution with a concentration of 200 mM for future use.

1.1.3 Cell culture reagents and culture conditions

The medium used for cell culture is a complete medium, which is prepared as follows: 89% DMEM high-glucose medium, 10% fetal bovine serum, and 1% penicillin-streptomycin double antibody solution. The cell culture environment is: a cell culture incubator at 37°C and 5% CO ₂ .

1.1.4 Main reagents

Complete medium, dimethyl sulfoxide (DMSO), PBS, trypsin, CCK8 reagent.

1.1.5 Main instruments and consumables

Ultra-clean bench, cell incubator, microplate reader, low-speed centrifuge, freeze dryer, high performance liquid chromatography, cell culture dish, 96-well cell culture plate, centrifuge tube, EP tube, microporous membrane.

2. Experimental method

1) CCK8 measures cell viability

A3755 and A2058 were respectively cultured with complete medium, and cells with good growth were selected (the cell density grew to more than 80%) for plating operation in a sterile environment. After washing the surface of the above-mentioned cells with sterile PBS, digest them with trypsin to make the adherent cells fall off, and fully pipette and mix to prepare A375 and A2058 melanoma cell suspensions.

The above-mentioned A375, A2058 melanoma cell suspensions were inoculated on a 96-well plate at a quantity of 3000 per well, and cultured with complete medium. After 24 hours of cell attachment, the control group, Danshen tablet group, and TMZ group were respectively set up. group and Danshen Tablets+TMZ group, discard the original medium and re-add the medium containing the corresponding Danshen extract and/or TMZ to culture in the cell incubator for 48-72h. Complete medium with the same concentration of DMSO (without Danshen extract and TMZ), the Danshen tablet group was added with different concentrations of Danshen extract alone, the TMZ group was separately added with different concentrations of TMZ, and the Danshen tablet+TMZ group was added with different concentrations of Danshen extract Mixed with different concentrations of TMZ, see Figure 1 for details. Then discard the original medium, use CCK8 reagent (Shanghai Biyuntian Biotechnology Co., Ltd., C0039) according to the instructions, incubate in a cell culture incubator at 37°C for 1 hour, and then use a microplate reader to measure the absorbance value of each well at a wavelength of 450nm. , each assay was repeated 3 times.

The detection results are shown in Figure 1, wherein, compared with the TMZ group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

The results showed that in the A375 and A2058 melanoma cell lines, compared with the Danshen extract and TMZ alone, the survival rate of the melanoma cells in the Danshen tablet + TMZ group was significantly reduced.

The above experimental results showed that: Salvia miltiorrhiza extract can significantly enhance the lethality of TMZ on melanoma cells, and reduce the survival rate of melanoma cells.

2) Determination of PA content in Danshen Tablets

Take 10 tablets (300mg/tablet) of Danshen tablets and grind them to powder, add 60ml of water, reflux in a water bath at 100°C for 1 hour, filter, lyophilize and weigh, and the weight is 1651mg. Accurately weigh 1.00 mg of the water extract, add 1 ml of 50% methanol, ultrasonically dissolve it, and filter it with a 0.22-micron microporous membrane to be tested as the test product. Accurately weigh 1.00mg of PA, add 50% methanol, ultrasonically dissolve, filter with a 0.22 micron microporous membrane, and prepare standard solutions with concentrations of 0.0625, 0.125, 0.20, 0.25, 0.5, and 1mg/ml respectively . Set chromatographic conditions: chromatographic column is Shim-pack GIST (C18, 4.6×250mm, particle size 5μm); mobile phase A is 0.1% formic acid aqueous solution, B is acetonitrile, 25% acetonitrile isocratic elution; flow rate is 1ml/min; The detection wavelength is 280nm, and the injection volume is 5ul. After the detection, the chromatographic peak response areas of different concentrations of PA were calculated respectively, with the concentration as the abscissa and the response area as the ordinate, a standard curve was drawn, and a linear regression was performed on it to obtain a regression equation. Subsequently, the water extract chromatogram is compared with the PA standard chromatogram, and the corresponding chromatographic peak area is calculated, and the calculated chromatographic peak area is brought into the regression equation to calculate that the PA content in the water extract is 2.3%, that is, the PA content in Danshen Tablets is 3.8% mg/tablet, the standard curve is shown in Figure 8.

Example 2: Effects of PA and TMZ on Survival Rates of Malignant Melanoma Cells and Glioma

The inventor further analyzed the active ingredients in the salvia miltiorrhiza extract, speculated that the main effective active ingredient in the salvia miltiorrhiza was protocatechualdehyde (PA), and investigated the effect of the combined use of PA and TMZ on the survival rate of malignant melanoma and glioma cells. The influence of the experimental procedure is shown in Example 1. The difference is that the control group, PA group, TMZ group and PA+TMZ group are set up respectively. The amount of medicine added in each group is shown in Figure 2. The PA (D108405) used in the experiment was purchased from Sigma company, and it was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 200mM for future use.

The specific experimental steps are as follows:

1. Materials and methods

1.1 Experimental materials

1.1.1 Source of cells

Human melanoma cell A375 comes from Shanghai Cell Bank of Chinese Academy of Sciences, A2058 comes from ATCC, human glioma cell U87MG comes from Shanghai Cell Bank of Chinese Academy of Sciences, and HS683 comes from Shanghai Cell Bank of Chinese Academy of Sciences.

1.1.2 Preparation of PA and TMZ solution

PA (D108405) used in the experiment was purchased from Sigma, and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 200 mM for future use. TMZ (S1237) was purchased from Selleck Company, and was dissolved in DMSO to prepare a stock solution with a concentration of 200 mM for future use.

1.1.3 Cell culture reagents and culture conditions

Same as Example 1

1.1.4 Main reagents

Complete medium, dimethyl sulfoxide (DMSO), PBS, trypsin, CCK8 reagent.

1.1.5 Main instruments and consumables

Ultra-clean bench, cell incubator, microplate reader, low-speed centrifuge, cell culture dish, 96-well cell culture plate, centrifuge tube, EP tube.

2. Experimental method

1) CCK8 measures cell viability

Referring to Example 1, the difference is that the U87MG and HS683 human glioma cell suspensions were inoculated on 96-well plates at the number of 2000 and 3000 per well, respectively, and cultured with complete medium. After 24 hours of cell attachment, respectively Set up the control group, PA group, TMZ group and PA+TMZ group, and treat for 72 hours. See Figure 2 for the amount of drug added in each group.

The detection results are shown in Figure 2, wherein, compared with the TMZ group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

The experimental results showed that the combination of PA and TMZ could significantly reduce the survival rate of malignant melanoma cells A375 and A2058 as well as U87MG and HS683 glioma cells.

Example 3: Effects of PA and DTIC on survival rate of malignant melanoma cells and their synergistic effect

1. Materials and methods

1.1 Experimental materials

1.1.1 Source of cells

Human melanoma cells A375 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, and SK-MEL-28 was obtained from ATCC.

1.1.2 Preparation of PA and DTIC solutions

PA (D108405) used in the experiment was purchased from Sigma, and dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 200 mM for future use. DTIC (S1221) was purchased from Selleck Company, and was dissolved in DMSO to prepare a stock solution with a concentration of 30 mM for future use.

1.1.3 Cell culture reagents and culture conditions

The medium used for cell culture is a complete medium, which is prepared as follows: 89% DMEM high-glucose medium, 10% fetal bovine serum, and 1% penicillin-streptomycin double antibody solution. The cell culture environment is: a cell culture incubator at 37°C and 5% CO ₂ .

1.1.4 Main reagents

Complete medium, dimethyl sulfoxide (DMSO), PBS, trypsin, CCK8 reagent.

1.1.5 Main instruments and consumables

Ultra-clean bench, cell incubator, microplate reader, low-speed centrifuge, cell culture dish, 96-well cell culture plate, centrifuge tube, EP tube.

2. Experimental method

1) CCK8 measures cell viability

A375 and SK-MEL-28 were respectively cultured with complete medium, and cells with good growth were selected (cell density grew to more than 80%) and plated in a sterile environment. After washing the surface of the above-mentioned cells with sterile PBS, digest them with trypsin to make the adherent cells fall off, and fully blow and mix to prepare A375 or SK-MEL-28 melanoma cell suspension.

The above-mentioned A375 and SK-MEL-28 melanoma cell suspensions were inoculated on 96-well plates at a quantity of 3000 per well, and cultured with complete medium. After 24 hours of cell attachment, the control group and the PA group were respectively set up. , DTIC group and PA+DTIC group, the original medium was discarded and the medium containing corresponding PA and DTIC was added again and cultured in the cell incubator for 48-72h. Among them, the control group was added with the same concentration of DMSO Complete medium (without PA and DTIC), the PA group was added with different concentrations (20, 40, 60, 80 and 100 μM) of PA alone, and the DTIC group was used with different concentrations (20, 40, 60, 80 and 100 μM) of DTIC Added alone, PA+DTIC group was mixed with different concentrations of PA (20, 40, 60, 80 and 100 μM) and different concentrations of DTIC (20, 40, 60, 80 and 100 μM). Then discard the original medium, use CCK8 reagent (Shanghai Biyuntian Biotechnology Co., Ltd., C0039) according to the instructions, incubate in a cell culture incubator at 37°C for 1 hour, and then use a microplate reader to measure the absorbance value of each well at a wavelength of 450nm. , each assay was repeated 3 times.

The detection results are shown in Figure 3, wherein, compared with the DTIC group, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001.

The results showed that in A375, SK-MEL-28 melanoma cell lines, different concentrations of PA and different concentrations of DTIC alone had less effect on cell viability compared to DTIC alone, melanoma cells in the PA+DTIC group The survival rate of DTIC decreased significantly, which indicated that PA could significantly enhance the lethality of DTIC to melanoma cells and reduce the survival rate of melanoma cells.

2) Determination of synergy

The experimental method is the same as that of CCK8 to measure the cell viability, that is, use 20, 40, 80, 160 μM PA and 5, 10, 20, 40, 80 μM DTIC to treat A375 cells for 48 hours respectively, and use 40, 60, 80, 100μM of PA and 20, 40, 60, 80, 100μM of DTIC were respectively and jointly treated SK-MEL-28 cells for 72 hours, and the cell viability was measured by CCK8 method. Then the obtained absorbance was converted to calculate the cell survival rate, and then the SynergyFinder software (https://synergyfinder.fimm.fi) online collaborative scoring software was used to evaluate the synergy score. The degree of drug combination synergy or antagonism can be quantified by comparing the observed drug combination response with the expected response, calculated using a reference model that assumes no interaction between the drugs. Commonly used reference models include the highest single-dose HSA model, the Bliss model, the Loewe model, and the zero interaction potency model ZIP. According to the different assumptions of different models, the inventors chose the Bliss and ZIP models to quantify the degree of synergy between PA and DTIC. Among them, the Bliss model assumes a stochastic process in which two drugs elicit their effects independently, and the expected combination effect can be calculated from the probabilities of independent events. The zero-interaction potency ZIP model captures drug-drug interaction relationships by comparing potency changes (effects at specific dose levels) in dose-response curves between individual drugs and their combinations. ZIP assumes that there is little change in the dose-response curves of two non-interacting drugs, which utilizes the advantages of the Loewe model additivity and the Bliss independence model, and aims to systematically evaluate various drug interactions that may occur in high-throughput drug combination screening The mode of action, the results are shown in Figure 4.

By definition when the synergy score is less than -10: the interaction between the two drugs is likely to be antagonistic; from -10 to 10: the interaction between the two drugs is likely to be additive; greater than 10: the interaction between the two drugs The interaction between them may be synergistic.

The experimental results are shown in Figure 4: PA and DTIC compositions in the two cell lines, no matter under the ZIP model or the Bliss model, the synergy score is greater than 10 points (the white dashed box area indicates the concentration area with the highest synergy score), It shows that there is a synergistic effect between PA and DTIC.

Furthermore, the inventors explored the synergistic mechanism of PA and DTIC, investigated the protein levels of DNA damage repair-related genes, and unexpectedly found that the drug combination of PA+DTIC can further increase the protein level of the DNA damage protein γH2AX, reduce Protein levels of the DNA repair protein MGMT. Furthermore, the inventors found that the change of MGMT protein level is caused by PA promoting the degradation of MGMT protein.

Example 4: DNA damage repair-related gene protein levels

1. Experimental materials

1.1 Protein lysate

The protein lysate was prepared in the ratio of RIPA150 lysate (150 means that the concentration of sodium chloride is 150 mM): protease inhibitor (PI): sodium glycerophosphate: NaF = 90:10:1:1.

1.2 Preparation of Cycloheximide (CHX) Solution

The CHX (66-81-9) used in the experiment was purchased from Selleck Company. The drug powder was centrifuged and then dissolved in PBS to prepare a stock solution with a final concentration of 15 mg/mL, which was sub-packaged and frozen at -80°C for storage.

1.3 Source of cells

Human melanoma cells A375 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, and SK-MEL-28 was obtained from ATCC.

1.4 Relevant buffer and preparation

10× electrophoresis buffer: Tris base 30g, glycine 144g, SDS 10g, ddH ₂ O to 1L.

1×transfer buffer: glycine 28.8g, Tris base 6g, methanol 400ml, ddH ₂ O to 2L.

10×TBS solution: 88g of sodium chloride, 24g of Tris base, 13ml of concentrated hydrochloric acid, dilute to 1L with ddH ₂ O.

1×TBST solution: Measure 100ml 10×TBS solution and mix with 1ml 20% Tween, dilute to 1L.

1.5 Main instruments and consumables

Vertical electrophoresis tank, multi-function imager system, 24-well plate, platform scale, analytical balance, shaker, PVDF membrane, filter paper.

2 Experimental methods

1) Drug treatment

The A375 and SK-MEL-28 cells were respectively cultured using the complete cell culture medium of Example 3, and the cells with good growth (cell density growing to over 80%) were selected for testing. The above-mentioned two kinds of cells were set into 4 groups respectively, namely the control group, PA group, DTIC group and PA+DTIC group, wherein, the control group was added with the complete medium containing the same concentration of DMSO as the other drug-dosed groups (without PA and DTIC), PA group was added with PA alone (the final concentration of A375 cells was 40 μM, and that of SK-MEL-28 cells was 100 μM), and in the DTIC group, DTIC was added alone (the final concentration of A375 cells was 40 μM, SK-MEL-28 cells were added at a final concentration of 100 μM), PA+DTIC group was simultaneously added with PA (A375 cells were added with a final concentration of 40 μM, SK-MEL-28 cells were added with a final concentration of 100 μM) and DTIC (A375 cells were added The final concentration of SK-MEL-28 cells was 40 μM, and the final concentration of SK-MEL-28 cells was 100 μM). Then put it back into the cell culture incubator at 37°C and 5% CO ₂ to continue culturing for 48-72h.

2) Western blot analysis

Lyse the drug-treated and untreated A375 and SK-MEL-28 cells with protein lysate, add 1/4 of the volume of protein lysate with 5×SDS loading buffer and mix well, heat the protein sample at 100°C for 8 minutes It was completely denatured, and then the obtained protein was subjected to SDS-PAGE polyacrylamide gel electrophoresis to separate the protein samples, then transferred to PVDF membrane (O162-0177, Bio-Rad), and incubated with the corresponding primary antibody (Table 1 Antibodies 1-3) and secondary antibodies (antibodies 4-5 in Table 1) were used to detect protein bands using a chemiluminescence kit (K22030, Abbkine). The detection results are shown in Figure 5.

The results in Figure 5 show that compared with the control group, PA alone can increase the protein level of DNA damage protein γH2AX, and reduce the protein level of DNA repair protein MGMT; and the drug combination of PA+DTIC can further increase the protein level of DNA damage protein γH2AX levels, reducing protein levels of the DNA repair protein MGMT.

The above results suggest that the combined use of PA and DTIC has a certain effect on the DNA damage repair ability of tumor cells, and can be used to improve the ability of malignant tumors such as melanoma, glioma, small cell lung cancer, osteosarcoma and lymphoma to resist DNA alkylation such as DTIC. The level of response to the chemical agent.

Table 1: Antibody Types

Example 5: Investigation of the influence of PA on the level of DNA damage repair-related proteins

Referring to the experimental method in Example 4, the effect of PA on the DNA damage repair related protein MGMT protein was further investigated in the presence of CHX. A375 cells and SK-MEL-28 cells were treated with CHX (40 μg/ml) alone or combined with PA (40 μM, 100 μM) for 0h, 1h, 2h, 4h, and 8h, and protein samples were collected for western blot analysis.

The experimental results are shown in Figure 6. CHX was used to treat melanoma cells at different times to inhibit the synthesis of cellular proteins. At the same time, a certain amount of DMSO was added as a control. It can be clearly seen from Figures (6A, 6C) that the increase in MGMT protein Gradual degradation (ie, reduced protein levels) suggested that CHX played a role. In addition, on the basis of adding CHX and adding a certain concentration of PA, it can be clearly seen from the figure (6B, 6D) that the protein degradation rate of MGMT is significantly faster than that of the DMSO group (that is, the protein level is lower), indicating that PA can promote MGMT protein degradation.

The inventors further verified the synergistic effect of the combined use of PA and DTIC at the cellular level through comet experiments.

Embodiment 6: DNA damage investigation

1. Experimental materials

1.1 Source of cells

Human melanoma cells A375 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, and SK-MEL-28 was obtained from ATCC.

1.2 Main reagents

Green I(SY1020,Solarbio)染液。Sodium hydroxide (NaOH), absolute ethanol, low melting point agarose gel, Comet Aaasy kit (4250-050-K, Trevigen),

Green I (SY1020, Solarbio) staining solution.

1.2 Preparation of main buffer

Alkaline Unwinding Solution: Weigh 0.4 g of NaOH, add 250 μL of 0.2 M EDTA solution, and dilute to 50 mL with ddH ₂ O.

Alkaline electrophoresis buffer: Weigh 8 g of NaOH, add 2 mL of EDTA solution with a concentration of 0.5 M and a pH value of 8.0, and dilute to 1000 mL with ddH ₂ O.

1.5 Main instruments and consumables

Ultra-clean bench, cell counter, horizontal electrophoresis tank, oven, bench scale, fluorescence microscope, glass slide, 24-well plate, low melting point agarose gel.

2. Experimental method

1) Drug treatment

The A375 and SK-MEL-28 cells were respectively cultured using the complete cell culture medium of Example 3, and the cells with good growth (cell density growing to over 80%) were selected for testing. The above two types of cells were divided into 4 groups, namely the control group, the PA group, the DTIC group and the PA+DTIC group, wherein the control group was added with the same concentration of DMSO as the drug-dosed group, and the PA group was added with PA alone ( A375 cells were added at a final concentration of 40 μM, SK-MEL-28 cells were added at a final concentration of 100 μM), and DTIC was added to the DTIC group alone (the final concentration of A375 cells was 40 μM, and the final concentration of SK-MEL-28 cells was 100 μM), PA+DTIC group was added PA (the final concentration of A375 cells was 40 μM, the final concentration of SK-MEL-28 cells was 100 μM) and DTIC (the final concentration of A375 cells was 40 μM, SK-MEL-28 Cells were added at a final concentration of 100 μM). Then put it back into the cell culture incubator at 37°C and 5% CO ₂ to continue culturing, A375 cells for 48 hours and SK-MEL-28 cells for 72 hours.

2) Alkaline comet experiment

Green I对玻片进行染色后使用荧光显微镜对样品进行观察，结果参见图7。The cell samples treated with drugs were digested with trypsin to collect the cell suspension, washed once with ice-cold PBS, and then resuspended with PBS. Count them with a cell counter to ensure that the density of the cell suspension is 1*10 ⁵ cell/mL, mix the cell suspension with low-melting point agarose gel at a volume ratio of 1:10, and spread it evenly on the glass slide , solidified at 4°C for half an hour, then put into pre-cooled Lysis Buffer (included with the kit), and lysed at 4°C for 2 hours. After the lysis was completed, wipe off the excess lysate, immerse it in the newly configured Alkaline Unwinding Solution, and incubate at room temperature in the dark for 40 minutes. Install the electrophoresis device, pour about 850ml pre-cooled alkaline electrophoresis buffer, set the voltage at 21V, and perform electrophoresis for 45 minutes. Then the slides were taken out and immersed in ddH ₂ O twice for 5 min each, and in 70% ethanol twice for 5 min each. After completing the above steps, place the slides in an oven at 37°C, dry the slides, and use

After staining the slides with Green I, the samples were observed with a fluorescence microscope, and the results are shown in Figure 7.

3. Experimental results

As shown in Figure 7, the alkaline comet experiment was performed on the drug-treated cell samples, and the tailing of cells in different groups was observed. The results showed that the comet tail in the PA+DTIC combination group increased significantly and thickened, indicating that its DNA double-strand break The situation is more serious.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims (15)

1. The compound shown in formula (I) or its salt or its derivative or the purposes of the composition containing compound shown in formula (I) or its salt or its derivative in preparation reagent, described reagent is used for reducing MGMT protein expression or activity of

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ; R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxy. 2. purposes according to claim 1, is characterized in that, described reduction MGMT protein expression or activity is realized by promoting the degradation of MGMT protein; Optionally, the compound represented by the formula (I) or its salt or its derivative or the composition containing the compound represented by the formula (I) or its salt or its derivative comprises a compound selected from Salvia miltiorrhiza or its extract, Clary sage or its extracts, Gastrodia elata or its extracts, wolfberry or its extracts, black fern or its extracts, Sijiqing or its extracts, Tsao Kuo or its extracts, cedar or its extracts, white scorpion or At least one of its extract, amaranth or its extract, begonia or its extract, thorny buttercup or its extract, dog ridge or its extract, palm and its extract, and persimmon leaf and its extract; Optionally, the composition containing the compound represented by formula (I) or its salt or its derivatives includes compounds selected from Compound Danshen Tablets, Compound Danshen Dropping Pills, Compound Danshen Injection, Danshen Tablets, Xinkeshu Tablets, and Xiaoshen Tablets. Jiantongluo Tablets, Anshen Buxin Pills, Tianwang Buxin Pills, Wenxin Granules, Shuangdan Oral Liquid, Xintong Oral Liquid, Danhong Huayu Oral Liquid, Danshen Water Extract, Prunella Oral Liquid, Fucong Mixture, Xiangdan Injection, Yinaoxin Granules, Shenpu Penyan Granules, Huoluoxiaotong Tablets, Guyanxiaofang, Guanxinning Injection, Danhong Injection, Dingxi Yatong Capsules, Compound Qilin Babu Medicine, Dog Ji Decoction pieces, Shenmai Granules, Shensong Yangxin Capsules, Danshen Ligustrazine Injection, Shenshuaining Capsules, Tongmai Granules, Dingxinning Tablets, Qimailing Oral Liquid, Yiganning Granules, Sanbao Capsules, Danhong Huayu Oral Liquid, Xinnaoning Capsules, Xinnaokang Tablets, Vitiligo Capsules, Fuyankang Tablets, Qidong Yixin Oral Liquid, Lian Shen Tonglin Tablets, Bubai Granules, Kunning Oral Liquid, Shenkangning Tablets, Runing Granules, Milk Kuaixiao Granules, Jizhi Syrup, Vigorous Su Oral Liquid, Naoxinqing Tablets, Xiaoxuanzhiyun Tablets, Tiaojing Huoxue Capsules, Qingnao Jiangya Tablets, Qingnao Jiangya Capsules, Qingnao Jiangya Granules and Tangmaikang Granules at least one of the . 3. A single-dose MGMT protein inhibitor, characterized in that it comprises 20-200 μM of the compound shown in formula (I) or its salt or derivative thereof,

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ; R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxy. 4. A kit, characterized in that it comprises a compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt thereof or a derivative thereof,

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ; R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxy. 5. A method for inhibiting MGMT protein expression or activity, characterized in that, comprising: Contacting cells expressing MGMT with a compound represented by formula (I) or a salt or derivative thereof or a composition containing a compound represented by formula (I) or a salt or derivative thereof;

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ; R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxy. 6. The use according to any one of claims 1 to 2, the single-dose MGMT protein degradation agent according to claim 3, the kit according to claim 4 or the method according to claim 5, characterized in that, R ₂ and R ₃ are independently selected from H, -CH ₃ , -CH ₂ (CH ₃ ) ₂ , -CH ₂ CH 2 CH _{2 CH 3} , -COCH ₃ , -CH ₂ OCH ₃ , -CH ₂ CH ₂ OH, -CH=CH-CH ₃ , -CF ₃ , -CHF ₂ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is connected forms a 5-membered heterocycloalkyl group, wherein the 5-membered heterocycloalkyl group is optionally substituted by two R ₇ , wherein R ₇ is selected from -CH ₃ or -CH ₂ CH ₂ OH; Optionally, the compound represented by the formula (I) has the following structure:

7. The kit according to claim 4 or the method according to claim 5, wherein the working concentration of the compound represented by the formula (I) or its salt or derivative thereof is 20 μM to 200 μM, preferably 40-150 μM, more preferably 40-100 μM; Optionally, the composition containing the compound represented by formula (I) or its salt or its derivatives includes a compound selected from Salvia miltiorrhiza or its extract, Clary sage or its extract, Gastrodia elata or its extract, Chinese wolfberry or its Extract, black fern or its extract, Four Seasons Green or its extract, Cao Guo or its extract, cedar or its extract, white pomegranate or its extract, iron amaranth or its extract, begonia or its extract, At least one of buttercup thorns or its extracts, dog spines or its extracts, palm and its extracts, and persimmon leaves and its extracts. 8. A pharmaceutical composition, characterized in that, comprising: A compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt or derivative thereof; and DNA alkylating agent;

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ; R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxy. 9. The pharmaceutical composition according to claim 8, wherein the DNA alkylating agent comprises a group selected from dacarbazine, temozolomide, busulfan, streptozotocin, carmustine, lolimus Mephalan, melphalan, procarbazine hydrochloride, trosulfan, thiotepa, methyl mustard and its derivatives, oxambucil hydrochloride and its derivatives, chlorambucil derivatives, phenylalanine mustard And its derivatives, benzoic acid mustard, monosubstituted aryl mustard derivatives, polysubstituted aryl mustard, oligopeptide aryl mustard, macrocyclic polyamine mustard, cyclophosphamide derivatives, linear phosphorus mustard At least one of amide derivatives, metal complex nitrogen mustards, uracil mustards and solanesyl amine derivatives; Preferably, the DNA alkylating agent is dacarbazine; Preferably, the DNA alkylating agent is temozolomide; Optionally, the compound represented by formula (I) or its salt or its derivative or the composition containing the compound represented by formula (I) or its salt or its derivative includes Salvia miltiorrhiza or its extract, rat tail Grass or its extracts, Gastrodia elata or its extracts, wolfberry or its extracts, black fern or its extracts, Sijiqing or its extracts, Tsaoguo or its extracts, cedar or its extracts, white pomegranate or its extracts At least one of extracts, amaranth or its extracts, begonia or its extracts, thorny buttercup or its extracts, dog ridge or its extracts, palm and its extracts, and persimmon leaves and its extracts; Optionally, the composition containing the compound represented by formula (I) or its salt or its derivatives includes compounds selected from Compound Danshen Tablets, Compound Danshen Dropping Pills, Compound Danshen Injection, Danshen Tablets, Xinkeshu Tablets, and Xiaoshen Tablets. Jiantongluo Tablets, Anshen Buxin Pills, Tianwang Buxin Pills, Wenxin Granules, Shuangdan Oral Liquid, Xintong Oral Liquid, Danhong Huayu Oral Liquid, Danshen Water Extract, Prunella Oral Liquid, Fucong Mixture, Xiangdan Injection, Yinaoxin Granules, Shenpu Penyan Granules, Huoluoxiaotong Tablets, Guyanxiaofang, Guanxinning Injection, Danhong Injection, Dingxi Yatong Capsules, Compound Qilin Babu Medicine, Dog Ji Decoction pieces, Shenmai Granules, Shensong Yangxin Capsules, Danshen Ligustrazine Injection, Shenshuaining Capsules, Tongmai Granules, Dingxinning Tablets, Qimailing Oral Liquid, Yiganning Granules, Sanbao Capsules, Danhong Huayu Oral Liquid, Danyi Tablets, Xinnaoning Capsules, Xinnaokang Tablets, Vitiligo Capsules, Fuyankang Tablets, Qidong Yixin Oral Liquid, Lian Shen Tonglin Tablets, Bubai Granules, Kunning Oral Liquid, Shenkangning Tablets, Milk Ning Granules, Rukuaixiao Granules, Jizhi Syrup, Vigorous Su Oral Liquid, Naoxinqing Tablets, Xiaoxuan Zhiyun Tablets, Tiaojing Huoxue Capsules, Qingnao Jiangya Tablets, Qingnao Jiangya Capsules, Qingnao Jiangya Granules and At least one of Tangmaikang granules; Optionally, further comprising pharmaceutically acceptable excipients; Optionally, the molar ratio of the compound represented by formula (I) or its salt or derivative thereof to the DNA alkylating agent in the pharmaceutical composition is (1-100):(100-1). 10. A drug combination or kit, characterized in that it comprises: A compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt or derivative thereof as the first active ingredient; and DNA alkylating agent as the second active ingredient;

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ; R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1-6 alkyl optionally substituted by hydroxy. 11. The drug combination or kit according to claim 10, wherein the DNA alkylating agent comprises a group selected from dacarbazine, temozolomide, busulfan, streptozotocin, carmustine, lox Mostine, melphalan, procarbazine hydrochloride, trosulfan, thiotepa, methyl mustard and its derivatives, oxambucil hydrochloride and its derivatives, chlorambucil derivatives, phenylalanine Nitrogen mustards and their derivatives, benzoic acid mustards, monosubstituted aryl nitrogen mustards, polysubstituted aryl mustards, oligopeptide aryl mustards, macrocyclic polyamine nitrogen mustards, cyclophosphamide derivatives, direct At least one of streptophosphamide derivatives, metal complex nitrogen mustards, uracil mustards and solanesylamine derivatives; Preferably, the DNA alkylating agent is dacarbazine; Preferably, the DNA alkylating agent is temozolomide; Optionally, the compound represented by formula (I) or its salt or its derivative or the composition containing the compound represented by formula (I) or its salt or its derivative includes Salvia miltiorrhiza or its extract, rat tail Grass or its extracts, Gastrodia elata or its extracts, wolfberry or its extracts, black fern or its extracts, Sijiqing or its extracts, Tsaoguo or its extracts, cedar or its extracts, white pomegranate or its extracts At least one of extracts, amaranth or its extracts, begonia or its extracts, thorny buttercup or its extracts, dog ridge or its extracts, palm and its extracts, and persimmon leaves and its extracts; Optionally, the composition containing the compound represented by formula (I) or its salt or its derivatives includes Compound Danshen Tablets, Compound Danshen Dropping Pills, Compound Danshen Injection, Danshen Tablets, Xinkeshu Tablets, Xiaojiantong Luo Tablets, Anshen Buxin Pills, Tianwang Buxin Pills, Wenxin Granules, Shuangdan Oral Liquid, Xintong Oral Liquid, Danhong Huayu Oral Liquid, Danshen Water Extract, Prunella Oral Liquid, Fucong Mixture, Xiangdan Injection, Yinaoxin Granules, Shenpu Penyan Granules, Huoluoxiaotong Tablets, Guyanxiaofang, Guanxinning Injection, Danhong Injection, Dingxi Yatong Capsules, Compound Kylin Babu, Gouji Decoction Pieces, Shenmai Granules, Shensong Yangxin Capsules, Danshen Chuanxiongzine Injection, Shenshuaining Capsules, Tongmai Granules, Dingxinning Tablets, Qimailing Oral Liquid, Yiganning Granules, Sanbao Capsules, Danhong Huayu Oral Liquid, Danyi Tablets, Xinnaoning Capsules, Xinnaokang Tablets, Vitiligo Capsules, Fuyankang Tablets, Qidong Yixin Oral Liquid, Lian Shen Tonglin Tablets, Bubai Granules, Kunning Oral Liquid, Shenkangning Tablets, Runing Granules , Rukuaixiao Granules, Jizhi Syrup, Huoxinsu Oral Liquid, Naoxinqing Tablets, Xiaoxuanzhiyun Tablets, Tiaojing Huoxue Capsules, Qingnao Jiangya Tablets, Qingnao Jiangya Capsules, Qingnao Jiangya Granules and Tangmai at least one of at least one of the health particles; Optionally, the molar ratio of the compound represented by formula (I) or its salt or its derivative to the DNA alkylating agent is (1-100): (100-1); Optionally, the compound represented by the formula (I) or its salt or its derivative is co-formulated or separately formulated with the DNA alkylating agent; Optionally, the compound represented by the formula (I) or its salt or its derivative is used simultaneously or separately with the DNA alkylating agent. 12. A single dosage form, characterized in that it comprises: a compound represented by formula (I) or a salt thereof or a derivative thereof or a composition containing a compound represented by formula (I) or a salt thereof or a derivative thereof as the first active agent ingredients; and DNA alkylating agent as the second active ingredient;

Wherein, R ₁ is selected from -CHO, -COOH, -CH ₂ CH ₂ OH, -CH ₂ OH or

R ₂ and R ₃ are independently selected from H, -C _1~6 alkyl optionally substituted by halogen or hydroxyl, -C _2~6 alkenyl, -C(O)R ₇ , -C _1~4 ethylene Alkyl-OR ₇ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is attached forms a 5-10 membered heterocycloalkyl group, wherein the 5-10 membered heterocycloalkyl group is optionally substituted by one or more _R7 ; R ₄ , R ₅ , and R ₆ are each independently selected from H or -OH; R ₇ is selected from -C _1～6 alkyl optionally substituted by hydroxyl; The molar ratio of the compound represented by the formula (I) or its salt or its derivative to the DNA alkylating agent is (1-100): (100-1). 13. The single-dose form according to claim 12, wherein the DNA alkylating agent comprises selected from dacarbazine, temozolomide, thiotepa, methyl mustard and derivatives thereof, nitrogen mustard hydrochloride and its derivatives. Derivatives, chlorambucil derivatives, phenylalanine mustard and its derivatives, benzoate mustard, monosubstituted aryl mustard derivatives, polysubstituted aryl mustard, oligopeptide aryl mustard, large At least one of cyclic polyamine nitrogen mustards, cyclophosphamide derivatives, linear phosphoramide derivatives, metal complex nitrogen mustards, uracil mustards and solanesylamine derivatives; Preferably, the DNA alkylating agent is dacarbazine; Preferably, the DNA alkylating agent is temozolomide; Optionally, the compound represented by the formula (I) or its salt or its derivative or the composition containing the compound represented by the formula (I) includes Salvia miltiorrhiza or its extract, Clary sage or its extract, Gastrodia elata or its Extract, wolfberry or its extract, black fern or its extract, Sijiqing or its extract, Caoguo or its extract, cedar or its extract, white pomegranate or its extract, iron amaranth or its extract, At least one of begonia or an extract thereof, buttercup thorn or an extract thereof, dog ridge or an extract thereof, palm and an extract thereof, and persimmon leaf or an extract thereof. 14. The pharmaceutical composition according to any one of claims 8 to 9, the pharmaceutical combination or kit according to any one of claims 10 to 11 or the single dosage form according to any one of claims 12 to 13, wherein It is characterized in that R ₂ and R ₃ are independently selected from H, -CH ₃ , -CH ₂ (CH ₃ ) ₂ , -CH ₂ CH ₂ CH ₂ CH ₃ , -COCH ₃ , -CH ₂ OCH ₃ , -CH ₂ CH ₂ OH, -CH=CH-CH ₃ , -CF ₃ , -CHF ₂ , -Si(CH ₃ ) ₃ ,

or,

Linking with the C to which it is connected forms a 5-membered heterocycloalkyl group, wherein the 5-membered heterocycloalkyl group is optionally substituted by two R ₇ , wherein R ₇ is selected from -CH ₃ or -CH ₂ CH ₂ OH; Optionally, the compound represented by the formula (I) has the following structure:

15. The pharmaceutical composition according to any one of claims 8-9, 14, the drug combination or kit according to any one of claims 10-11, 14, or the single drug composition according to any one of claims 12-14. Use of a dosage form in the preparation of a medicament for the prevention and/or treatment of cancer; Optionally, the cancer comprises breast cancer, ovarian cancer, breast cancer, testicular cancer, pancreatic cancer, liver cancer, colon cancer, colorectal cancer, thyroid cancer, lung cancer, prostate cancer, kidney cancer, melanoma, squamous cell carcinoma, Gastrointestinal adenocarcinoma, chronic myeloid leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, promyelocytic leukemia, meningeal leukemia, multiple myeloma, lymphosarcoma, lymphoma, bladder cancer, head and neck cancer , Esophageal Cancer, Brain Cancer, Pharyngeal Cancer, Tongue Cancer, Synovial Cell Carcinoma, Neuroblastoma, Uterine Cancer, Fibrosarcoma, Myxosarcoma, Liposarcoma, Soft Tissue Tumor, Osteosarcoma, Chordoma, Angiosarcoma, Endothelial Cell Sarcoma , lymphangiosarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cyst Adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver tumor, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, uterine cancer, cervical cancer, small cell lung cancer, epithelial carcinoma, Glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, brain or At least one of intrathecal tumor, glioma, and retinoblastoma; Preferably, the cancer comprises at least one of melanoma, glioma, small cell lung cancer, osteosarcoma and lymphoma.

Images