Anti-cancer active ingredients, compositions, and methods of making and using the same

Abstract

The application discloses an anticancer active ingredient, a composition and preparation and application thereof, belongs to the technical field of biological medicine, and particularly relates to the technical field of anticancer drugs. Specifically, the application relates to a preparation method of a compound shown in formula 1 and a pharmaceutical composition containing the compound and application of the compound to anticancer drugs. The formula 1 can significantly inhibit breast cancer cell proliferation and can be used for preventing and / or treating breast cancer. Further, the application verifies that, compared with treatment of tumor cells by using a chemotherapy drug alone, combination of the formula 1 and the chemotherapy drug can more effectively inhibit breast cancer cell proliferation and avoid the defects of poor treatment effect or large toxic and side effects of single drug treatment. Therefore, the compound and the pharmaceutical composition containing the compound have the biological activity of inhibiting breast cancer cell proliferation and can achieve the technical effect of treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the field of anticancer drug technology. Background Technology

[0002] Malignant tumors are one of the major public health problems worldwide. They are diseases caused by the malignant proliferation, abnormal invasion, and metastasis of cancer cells, seriously threatening human life and health. Among these cancers, breast cancer, due to its high incidence and mortality rates, has become a major threat to women's health and a critical issue that urgently needs to be addressed globally.

[0003] Currently, the main treatments for breast cancer include surgery, neoadjuvant chemoradiotherapy before surgery, adjuvant chemoradiotherapy after surgery, endocrine therapy, molecular targeted therapy, and immunotherapy. Although related diagnostic and treatment technologies have developed rapidly in recent years, the overall prognosis for breast cancer is poor due to its high heterogeneity among patients and the fact that most patients are diagnosed at an intermediate or advanced stage. Therefore, developing novel anti-tumor drugs has become an important research direction for breast cancer treatment.

[0004] Chemotherapy is the first-line standard clinical treatment for breast cancer, and the main drugs include cyclophosphamide, doxorubicin, cisplatin, and paclitaxel. However, chemotherapy drugs have significant toxic side effects, and most patients easily develop resistance to chemotherapy.

[0005] Furthermore, with continuous technological development, existing technologies have also provided some further improvement methods. For example, Chinese patent document CN115813919A discloses the use of indole-3-pyruvic acid or its pharmaceutical salt in the preparation of drugs for treating breast cancer. Chinese patent document CN116036070A discloses the use of 9''-tanshinone B monomethyl ester in the preparation of drugs for treating breast cancer tumor cell proliferation, migration, and invasion. US20190262281A1 discloses a pharmaceutical composition for treating breast cancer with THA as the active ingredient.

[0006] In conclusion, although numerous treatment options exist for breast cancer, the efficacy and selectivity of tumor-specific drugs still need improvement. Summary of the Invention

[0007] In view of the problems existing in the prior art, the purpose of this invention is to provide an anticancer active ingredient, aiming to provide a new compound with excellent anticancer activity and low toxicity and side effects.

[0008] The second objective of this invention is to provide a method for preparing the aforementioned anticancer active ingredient and its application in the preparation of anticancer drugs.

[0009] A third objective of this invention is to provide an anticancer active composition comprising the aforementioned anticancer active ingredient and an anticancer drug.

[0010] An anticancer active ingredient is a compound of formula 1 and its pharmaceutically acceptable salt, solvate, hydrate, or prodrug.

[0011]

[0012] In Formula 1, R is a C1 to C3 alkyl group, and X is a C6 to C3 alkyl group. 10 Alkyl or oxanealkyl.

[0013] This invention provides a novel small molecule compound with excellent tumor-targeting activity, exhibiting superior antitumor activity and selectivity. Furthermore, the compound shows virtually no inhibitory activity against normal cells and demonstrates good biocompatibility in vivo.

[0014] The anticancer active ingredient of this invention, wherein R is methyl or ethyl; and X is... The n mentioned is an integer from 2 to 4;

[0015] A typical anticancer active ingredient of the present invention is a compound having the structural formula 1-A;

[0016]

[0017] The present invention also provides a method for preparing the aforementioned anticancer active ingredient, which is obtained by cyclizing the compound of formula 2 and the compound of formula 3;

[0018]

[0019] Formula 2

[0020]

[0021] Formula 3.

[0022] In this invention, the cyclization conditions are: the molar ratio of Formula 2 and Formula 3 is 1:0.9 to 1.1; further, it is 1:1 to 1.02.

[0023] In this invention, a divalent copper catalyst and a reducing agent are added during the cyclization process.

[0024] Preferably, the divalent copper catalyst is at least one of copper sulfate, copper nitrate, copper acetate, and copper citrate.

[0025] Preferably, the divalent copper catalyst is 0.1 to 0.5 times the molar amount of Formula 2, and more preferably 0.1 to 0.3 times.

[0026] Preferably, the reducing agent is a component capable of reducing divalent copper, and is preferably ascorbic acid and its salts.

[0027] Preferably, the reducing agent is 1 to 5 times the molar amount of Formula 2, and more preferably 2 to 4 times.

[0028] In this invention, during the cyclization reaction, Formulas 2 and 3 are dissolved in an organic solvent (such as DMF) beforehand, and then mixed with an aqueous solution containing the reducing agent and divalent copper catalyst to carry out the cyclization reaction.

[0029] In this invention, the cyclization reaction temperature is below 4°C.

[0030] After the cyclization reaction is completed, the reaction is quenched with water, followed by extraction with a hydrophobic organic solvent (such as DCM, EA, etc.) and chromatographic purification to obtain the final product.

[0031] Formulas 2 and 3 described in this invention can be prepared based on known processes (the synthesis routes can be found in the reference). Figure 1 ).

[0032] The present invention also provides the use of the aforementioned anticancer active ingredient in the preparation of antitumor drugs.

[0033] The application described in this invention combines it with pharmaceutically acceptable excipients to prepare a pharmaceutically acceptable antitumor drug dosage form.

[0034] The application described in this invention refers to an antitumor drug that is effective against at least one of the following tumors: breast cancer, ovarian cancer, lung cancer, melanoma, and glioma.

[0035] The present invention also provides an anticancer active composition comprising the aforementioned anticancer active ingredient and other anticancer active ingredients.

[0036] The anticancer active composition, wherein the other anticancer active ingredients include one or more of paclitaxel, cisplatin, sulfasalazine, cyclophosphamide, 5-fluorouracil, doxorubicin, and mitomycin.

[0037] This invention has found that Formula 1 can enhance the activity of other anticancer active ingredients. Combining it with other anticancer active ingredients can achieve synergy and obtain better performance.

[0038] In this invention, the content of the anticancer active ingredient in the anticancer active composition is 10 wt.% or more, preferably 20 to 90 wt.%.

[0039] The present invention also provides an anticancer drug comprising a pharmaceutically effective amount of the said anticancer active ingredient or the said anticancer active composition;

[0040] The anticancer drug of the present invention also allows the inclusion of pharmaceutically acceptable excipients. Such excipients include, for example, at least one of colorants, flavoring agents, stabilizers, and thickeners;

[0041] The anticancer drug of the present invention also has a pharmaceutically acceptable dosage form. Such dosage forms include, for example, tablets, capsules, aqueous or oily solutions, suspensions, emulsions, creams, ointments, gels, nasal sprays, suppositories, finely dispersed powders, aerosols, or sprays for inhalation, and sterile aqueous or oily solutions, suspensions, or sterile emulsions for parenteral use.

[0042] Beneficial effects:

[0043] This invention provides a novel anticancer active ingredient with excellent anticancer activity and selectivity. Furthermore, combining it with other existing anticancer active ingredients can further achieve synergistic anticancer effects. Attached Figure Description

[0044] Figure 1 This is the total synthesis route diagram for Equation 1-A.

[0045] Figure 2 This is the high-resolution mass spectrum of Equation 1-A.

[0046] Figure 3 The image shows the proton NMR spectrum of Equation 1-A.

[0047] Figure 4 The image shows the carbon NMR spectrum of Equation 1-A.

[0048] Figure 5 This is a schematic diagram illustrating the effect of different concentrations of Formula 1-A on the proliferation of normal cells using CCK-8 assay.

[0049] Figure 6 This diagram illustrates the effects of Formula 1-A on the proliferation of breast cancer cells and organoids. A: Trend of CCK-8 assay on the anti-tumor cell proliferation capacity of Formula 1-A at different concentrations; B: Trend of CCK-8 assay on the anti-tumor cell proliferation capacity of Formula 1-A at different treatment times; C: Trend of Clonogenic assay on the anti-tumor cell proliferation capacity of Formula 1-A at different concentrations; D: Trend of EdU staining on the anti-tumor cell proliferation capacity of Formula 1-A; E: Trend of different concentrations of Formula 1-A on the anti-tumor organoid proliferation capacity.

[0050] Figure 7Figure 1 shows the results of detecting the antitumor effect of Formula 1-A in vivo. A: Comparison of solid tumor volume and tumor weight differences in the in vivo antitumor activity of Formula 1-A in a breast cancer xenograft model; B: Trend of tumor volume changes in the in vivo antitumor activity of Formula 1-A in a breast cancer xenograft model; C: Trend of animal body weight changes in the in vivo antitumor activity of Formula 1-A in a breast cancer xenograft model; D: Comparison of serum biochemical indicators in animals in the in vivo antitumor activity of Formula 1-A in a breast cancer xenograft model; E: Comparison of the weight of the heart, liver, spleen, lung, and kidney in animals in the in vivo antitumor activity of Formula 1-A in a breast cancer xenograft model; F: Morphology of the heart, liver, spleen, lung, and kidney detected by HE staining.

[0051] Figure 8 This diagram illustrates the effect of Formula 1-A on the sensitivity of breast cancer cells to chemotherapy drugs. AB represents the trend of CCK-8 assay in detecting the anti-tumor cell proliferation capacity of Formula 1-A combined with cisplatin or paclitaxel; CD represents the trend of colony formation assay in detecting the anti-tumor cell proliferation capacity of different concentrations of Formula 1-A combined with cisplatin or paclitaxel.

[0052] Figure 9 This diagram illustrates the effect of Formula 1-A on the sensitivity of breast cancer cells to sulfasalazine (labeled SAS). A: Trend graph of the anti-tumor cell proliferation capacity of Formula 1-A combined with sulfasalazine as detected by CCK-8 assay; B: Trend graph of the anti-tumor cell proliferation capacity of Formula 1-A combined with sulfasalazine as detected by clonogenic assay; CD: Trend graph of the anti-tumor organoid proliferation capacity of Formula 1-A combined with sulfasalazine as detected by organoid model; E: Solid tumor volume control and tumor weight difference graph in the in vivo anti-tumor activity of Formula 1-A combined with sulfasalazine as detected by breast cancer xenograft tumor model; F: Breast cancer xenograft... G: Trend of tumor volume change in the in vivo antitumor activity of Formula 1-A combined with sulfasalazine in a breast cancer xenograft tumor model; H: Morphological analysis of heart, liver, spleen, lung, and kidney by HE staining; I: Comparison of serum biochemical indicators in animals in the in vivo antitumor activity of Formula 1-A combined with sulfasalazine in a breast cancer xenograft tumor model; J: Comparison of heart, liver, spleen, lung, and kidney weight in animals in the in vivo antitumor activity of Formula 1-A combined with sulfasalazine in a breast cancer xenograft tumor model. Detailed Implementation

[0053] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. The technical and scientific terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. The basic raw materials and reagents are obtained commercially and have a purity of 95% or higher. The room temperature described in this invention is 25-30°C. The present invention provides a general and specific description of the materials and experimental methods used in the experiments. Although many materials and methods of operation used to achieve the objectives of this invention are well known in the art, they are still described herein as comprehensively as possible.

[0054] Example 1: Synthesis and structural confirmation of Formula 1-A.

[0055] The synthesis route of Equation 1-A is as follows: Figure 1 As shown:

[0056] The cyclization conditions were as follows: Compound 3 (154 mg, 0.25 mmol, 1 equiv.) was dissolved in N,N-dimethylformamide (1.5 mL, 0.16 M) in a 100 mL round-bottom flask equipped with a magnetic stirrer. Compound 2 (124.2 mg, 0.25 mmol, 1 equiv.) was added to the reaction system, followed by a 1 mL aqueous solution of sodium D-isoascorbate (147.3 mg, 0.74 mmol, 3 equiv.) and copper sulfate pentahydrate (18.6 mg, 0.074 mmol, 0.3 equiv.) in an ice-water bath. After stirring the reaction at 0 °C for 24 hours, the reaction system was quenched with water and extracted with dichloromethane (3 x 10 mL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered, and concentrated. The compound was separated and purified by silica gel column chromatography (3-6% methanol / dichloromethane) to finally obtain compound 1 (204 mg, 65%).

[0057] The high-resolution mass spectrum of Formula 1-A is as follows: Figure 2 The mass spectrometry results are analyzed as follows: ESI HRMS (m / z): [M+Na] + Calculated for C 50 H 55 Cl2FN 12 NaO9S2, 1143.29097; Found of 1143.29044. The 1H NMR spectrum of Formula 1-A is shown below. Figure 3 Its spectral data are as follows: 1H NMR (600MHz, DMSO-d6): 10.66 (s, 1H), 8.41 (d, J = 12Hz, 1H), 8.37 (m, 1H), 8.2 (s, 1H), 8.07 (t, J =6Hz,1H),7.41(d,J=6Hz,1H),7.14-7.26(m,11H),6.84-6.85(m,1H),6.81(m,1H),6.54(s,1H) 6.24(s, 1H), 5.17(s, 2H), 5.09(s, 2H), 4.55(t, J=6Hz, 2H), 4.49-4.50(m, 1H), 3.95-4.05(m, 2H), 3.82(t, J=6Hz, 2H), 3.50-3.52(m, 2H), 3.42-3.47(m, 7H), 3.37-3.39(4H, overlapped with H2O), 3.25-3.29(m, 2H), 3.17-3.19(m, 2H), 1.84(s, 3H). The carbon NMR spectrum of Equation 1-A is shown below. Figure 4 Its spectral data are as follows: 13 C NMR (151MHz, DMSO-d6) δ = 169.85, 169.53, 168.61, 165.63, 162.40, 160.79, 155.77, 153.78, 153. 41,152.84,142.30,141.80,139.37,136.03,132.67,131.18,129.86,129.13,129.07,128.76,12 8.37,128.11,126.47,126.15,125.37,116.70,115.66,115.51,113.28,69.77,69.70,69.65,69.61,68.81,68.75,62.25,59.20,52.34,49.56,44.71,43.18,40.69,38.77,35.00,34.87,22.63.

[0058] Example 2: In vitro and in vivo antitumor activity experiment of Formula 1-A.

[0059] The MDA-MB-231 cell line of this invention was obtained from the Kunming Institute of Zoology, Chinese Academy of Sciences; the MCF7 cell line was purchased from the Peking Union Medical College Cell Bank, Chinese Academy of Sciences; and the HEK293T cell line was purchased from the Shanghai Cell Bank, Chinese Academy of Sciences.

[0060] All experiments in this study were repeated three times or more. The error bar represents the standard error (±SD) of three samples. Statistical analysis was performed using GraphPad Prism (version 6.01). Two-tailed independent samples t-tests were used for comparisons between groups, and two-way ANOVA was used for comparisons among multiple groups. All experimental data in this study are expressed as mean ± standard deviation. ns, *, **, *** represent no statistical significance, p < 0.05, p < 0.01, and p < 0.001, respectively.

[0061] This invention uses CCK-8 assays to detect the proliferation capacity of different concentrations of 1-A against tumor cells. The specific steps are as follows: HEK293T cells were cultured at 5 × 10⁻⁶ cells / mL. 3 Seeds were seeded per well in a 96-well plate with 3 replicates. The plates were treated with 0, 5, 10, 20, and 40 μM 1-A for 72 h. 10 μL of CCK-8 solution was added to each well and the plates were incubated in the dark for 1-2 h. When the solution in the plate turned a distinct orange color, the absorbance was measured at 450 nm using a microplate reader and the data was saved. Figure 5 The results showed that Formula 1-A had virtually no inhibitory activity on normal cells.

[0062] This invention uses CCK-8 assay to detect the proliferation ability of different concentrations of 1-A against tumor cells. The specific steps are as follows: MDA-MB-231 and MCF-7 cells were respectively inoculated at 2×10⁻⁶... 3 and 5×10 3 Seeds were seeded per well in a 96-well plate with 3 replicates. The plates were treated with 0, 5, 10, 20, and 40 μM 1-A for 72 h. 10 μL of CCK-8 solution was added to each well and the plates were incubated in the dark for 1-2 h. When the solution in the plate turned a distinct orange color, the absorbance was measured at 450 nm using a microplate reader and the data was saved. Figure 6 The results showed that Equation 1-A could inhibit tumor cell viability in a concentration-dependent manner, and the difference was statistically significant.

[0063] This invention uses CCK-8 assay to detect the proliferation ability of 1-A anti-tumor cells treated for different durations. The specific steps are as follows: MDA-MB-231 and MCF-7 cells were cultured at 1×10⁻⁶ cells / mL. 3 Tumor cells were seeded per well in a 96-well plate with 3 replicates. They were treated with 20 μM 1-A for 0, 24, 48, 72, and 96 hours, and tumor cell viability was detected using the CCK-8 assay. Figure 6 The results in B showed that Equation 1-A could inhibit tumor cell viability in a time-dependent manner, and the difference was statistically significant.

[0064] This invention uses a colony formation assay to detect the proliferative capacity of different concentrations of 1-A anti-tumor cells. The specific steps are as follows: MDA-MB-231 and MCF-7 cells were cultured at 1×10⁻⁶...3 Cells were seeded in 6-well plates with 3 replicates. 0, 5, and 10 μM of 1-A were added to each well for 24 hours, followed by changing the culture medium every 3 days for approximately 12 days. When cell colonies reached a visually identifiable single-clone size, the cells were washed twice with PBS, fixed with 4% paraformaldehyde solution, and stained with 0.1% crystal violet solution. The number of colonies was then imaged and statistically analyzed. Figure 6 The results showed that Equation 1-A could inhibit the ability of tumor cells to form colonies in a concentration-dependent manner, and the difference was statistically significant.

[0065] This invention uses EdU staining to detect the proliferation ability of Formula 1-A anti-tumor cells. The specific steps are as follows: MDA-MB-231 and MCF-7 cells were respectively inoculated at 2×10⁻⁶... 3 and 5×10 3 Seeds were planted in 96-well plates with 3 replicates per well. Each well was treated with 20 μM Formula 1-A for 72 h, incubated with 50 μM EdU for 2 h, fixed with 4% paraformaldehyde solution, permeabilized with 0.1% Triton X100, and stained with 5 μg / mL Hoechst 33342. The nuclei were observed and photographed under a fluorescence microscope. Figure 6 The results showed that Formula 1-A significantly reduced the proportion of EdU-positive cells and inhibited tumor cell proliferation, with statistically significant differences.

[0066] This invention uses a breast cancer organoid model to detect the proliferative capacity of antitumor organoids of different concentrations of Formula 1-A. The specific steps are as follows: Breast cancer tissue was collected from surgically removed breast cancer patients (the breast surgery department of Xiangya No. 2 Hospital diagnosed breast cancer, and the clinician took the cancer tissue from the patient during the mastectomy). The tissue blocks were cleaned, minced, digested, filtered, and the cell suspension was filtered. The cells were then resuspended and cultured in three dimensions to prepare breast cancer organoids. The organoids were seeded in 96-well plates with three replicates. 0, 5, 10, 20, and 40 μM Formula 1-A were added for 5 days, and cell viability was detected. Figure 6 The results from the study showed that Equation 1-A inhibited the growth of breast cancer organoids in a dose-dependent manner, and the difference was statistically significant.

[0067] This invention uses a subcutaneous tumorigenesis assay in nude mice to detect the effect of formula 1-A on breast cancer growth. The specific steps are as follows: MDA-MB-231 cells in good growth condition were digested with trypsin, counted, centrifuged to collect the cells, redispersed and diluted with serum-free culture medium, and stored in an ice box for later use. The tumor cell suspension, evenly dispersed in serum-free culture medium, was inoculated into the subcutaneous tissue of 4-6 week old BALB / c female nude mice (5 × 10⁻⁶ cells / mL). 6 To establish a tumor-bearing mouse model, a sample of 100 μL of cells was used. Subcutaneous solid tumors in nude mice reached 50-100 mm in size.3 Eighteen mice were randomly divided into three groups of six each. The mice were administered (1) solvent (DMSO, intraperitoneal injection), (2) formula 1-A (10 mg / kg, intraperitoneal injection), and (3) formula 1-A (20 mg / kg, intraperitoneal injection), respectively. The drugs were administered every two days for 12 consecutive days. Tumor volume and mouse weight were recorded every other day to plot tumor growth curves and body weight change curves. On day 12, mice were anesthetized, and 1 mL of whole blood was collected for analysis. Serum biochemical indicators selected included aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and urea (UREA). Subsequently, the tumor was surgically removed, photographed, and weighed. Mouse organs were weighed, cut into pieces, and fixed with 4% paraformaldehyde. HE staining was used to detect the morphology of the heart, liver, spleen, lungs, and kidneys. The tumor volume was calculated as: Tumor volume = (long axis × short axis × short axis) / 2.

[0068] Subcutaneous tumorigenesis experiments in nude mice demonstrated that Formula 1-A exhibited a strong antitumor effect even at low concentrations (10 mg / kg). Figure 7 (AB). During the treatment, the mice's body weight did not change significantly ( Figure 7 (C). Biochemical indicators were detected in the serum of mice. Figure 7 The results from the D assay showed no statistically significant differences among the groups. Mouse organs were compared by weight and stained with hematoxylin and eosin (HE). Figure 7 The results of the EF study showed no statistically significant differences or morphological changes among the groups, indicating that Formula 1-A has good biosafety.

[0069] Example 3: Effect of Formula 1-A on drug therapy sensitivity.

[0070] This invention uses CCK-8 assay to detect the anti-tumor cell proliferation ability of α1-A combined with chemotherapy drugs cisplatin or paclitaxel (PTX). The specific steps are as follows: MDA-MB-231 cells were cultured at 1×10⁻⁶ cells / cells. 3 Each cell was seeded in a 96-well plate with three replicates. The cells were treated with 20 μM 1-A, 10 nMP acryltaxel, or 1 μM Cisplatin for 48 h, respectively. The tumor cell viability was then assessed using the CCK-8 assay. Figure 8 The results of the AB study showed that, compared with cisplatin or paclitaxel alone, the combined application of Formula 1-A enhanced the sensitivity of breast cancer cells to paclitaxel and cisplatin, respectively, and the differences were statistically significant.

[0071] This invention uses a colony formation assay to detect the effect of Formula 1-A combined with the chemotherapy drugs cisplatin or paclitaxel on the anti-tumor cell proliferation ability. The specific steps are as follows: MDA-MB-231 cells were cultured at 1×10⁻⁶ cells / year. 3 / wells were seeded in 6-well plates with 3 replicates. Each well was treated with 10 μM of 1-A, 10 nM Paclitaxel, or 1 μM Cisplatin for 24 h. The proliferation of tumor cells was detected by a colony formation assay. Figure 8 The results of the CD study showed that, compared with cisplatin or paclitaxel alone, the combined application of Formula 1-A enhanced the sensitivity of breast cancer cells to paclitaxel and cisplatin, respectively, and the differences were statistically significant.

[0072] This invention uses the CCK-8 assay to detect the anti-tumor cell proliferation ability of formula 1-A combined with sulfasalazine. The specific steps are as follows: MDA-MB-231 and MCF-7 cells were respectively inoculated at 2×10⁻⁶... 3 and 5×10 3 Tumor cells were seeded in 96-well plates with 3 replicates. The cells were treated with 20 μM 1-A combined with 0, 0, 125, 0.25, 0.5, and 1 mM sulfasalazine for 48 h, respectively. The activity of the tumor cells was detected by the CCK-8 assay. Figure 9 The results showed that the combined application of formula 1-A significantly enhanced the sensitivity of breast cancer cells to sulfasalazine, and the difference was statistically significant.

[0073] This invention employs a colony formation assay to detect the anti-tumor cell proliferation ability of formula 1-A combined with sulfasalazine. The specific steps are as follows: MDA-MB-231 and MCF-7 cells were cultured at 1×10⁻⁶ cells / year. 3 / wells were seeded in 6-well plates with 3 replicates. The cells were treated with 10 μM 1-A or 0.5 mM sulfasalazine for 24 h. The proliferation of tumor cells was detected by colony formation assay. Figure 9 The results from the study showed that, compared with cisplatin or paclitaxel alone, the combined application of formula 1-A significantly enhanced the sensitivity of breast cancer cells to sulfasalazine, and the difference was statistically significant.

[0074] This invention uses a breast cancer organoid model to detect the anti-tumor organoid proliferation ability of formula 1-A combined with sulfasalazine. The specific steps are as follows: breast cancer tissue surgically removed from breast cancer patients is collected, breast cancer organoids are prepared through organoid culture, the organoids are seeded in 96-well plates with 3 replicates, and 10 μM formula 1-A or 0.5 mM sulfasalazine is added for 5 days for treatment, and cell viability is detected. Figure 9 The results of the CD study showed that Formula 1-A combined with sulfasalazine significantly inhibited the growth of breast cancer organoids, and the difference was statistically significant.

[0075] This invention uses a subcutaneous tumorigenesis assay in nude mice to detect the effect of formula 1-A combined with sulfasalazine on breast cancer growth. First, a tumor-bearing mouse model is established. When the size of the subcutaneous solid tumor in the nude mice reaches 50-100 mm... 3Twenty-four mice were randomly divided into four groups of six each. The mice were administered (1) solvent (DMSO, intraperitoneal injection), (2) formula 1-A (10 mg / kg, intraperitoneal injection), (3) sulfasalazine (100 mg / kg, intraperitoneal injection), and (4) formula 1-A + sulfasalazine (intraperitoneal injection), respectively. The drugs were administered every two days for 12 consecutive days. Tumor volume and mouse weight were recorded every other day to plot tumor growth curves and body weight change curves. On day 12, mice were anesthetized, and 1 mL of whole blood was collected for analysis. Serum biochemical indicators included aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and urea (UREA). Subsequently, the tumor was surgically removed, photographed, and weighed.

[0076] Subcutaneous tumorigenesis experiments in nude mice demonstrated that formula 1-A combined with sulfasalazine exhibited a strong antitumor effect. Figure 9 (EF). During the treatment, the mice's body weight did not change significantly ( Figure 9 (G). Biochemical indicators were detected in the serum of mice. Figure 9 The results of the I test showed no statistically significant differences among the groups. Mouse organs were compared by weight and stained with hematoxylin and eosin (HE). Figure 9 The H / J results showed no statistically significant differences or morphological changes among the groups, indicating that the combined application of formula 1-A and sulfasalazine has good biocompatibility.

Claims

1. An anticancer active ingredient, characterized in that, For compounds of formula 1 and their pharmaceutically acceptable salts; ； Formula 1 In Formula 1, R is a C1-C3 alkyl group, and X is a C6-C3 alkyl group. 10 Oxyalkyl groups.

2. The anticancer active ingredient as described in claim 1, characterized in that, R is methyl or ethyl; X is The n mentioned is an integer from 2 to 4.

3. The anticancer active ingredient as described in claim 1, characterized in that, The anticancer active ingredient is a compound having the structural formula 1-A; Formula 1-A.

4. A method for preparing the anticancer active ingredient according to any one of claims 1 to 3, characterized in that, It was obtained by cyclization of the compound of formula 2 and the compound of formula 3; ； Formula 2 ； Formula 3.

5. The method for preparing the anticancer active ingredient as described in claim 4, characterized in that, The cyclization condition is that the molar ratio of Equation 2 and Equation 3 is 1:0.9~1.

1.

6. The method for preparing the anticancer active ingredient as described in claim 5, characterized in that, The cyclization condition is that the molar ratio of Equation 2 and Equation 3 is 1:1 to 1.

02.

7. The method for preparing the anticancer active ingredient as described in claim 4, characterized in that, The cyclization process involves the addition of a divalent copper catalyst and a reducing agent; The divalent copper catalyst is at least one of copper sulfate, copper nitrate, copper acetate, and copper citrate. The divalent copper catalyst is 0.1 to 0.5 times the molar amount of Formula 2; The reducing agent is a component capable of reducing divalent copper; The reducing agent is 1 to 5 times the molar amount of Formula 2.

8. The method for preparing the anticancer active ingredient as described in claim 7, characterized in that, The divalent copper catalyst is 0.1 to 0.3 times the molar amount of Formula 2; The reducing agent is ascorbic acid and its salt; The reducing agent is 2 to 4 times the molar amount of Formula 2.

9. The method for preparing the anticancer active ingredient as described in claim 7, characterized in that, In the cyclization reaction, Formulas 2 and 3 are dissolved in an organic solvent beforehand, and then mixed with an aqueous solution containing the reducing agent and divalent copper catalyst to carry out the cyclization reaction; The cyclization reaction occurs at temperatures below 4°C.

10. The use of the anticancer active ingredient according to any one of claims 1 to 3 in the preparation of an antitumor drug, characterized in that, The anti-tumor drug mentioned is an anti-breast cancer drug.

11. The application as described in claim 10, characterized in that, By combining it with pharmaceutically acceptable excipients, a pharmaceutically acceptable antitumor drug formulation can be prepared.

12. An anticancer active composition, characterized in that, It includes the anticancer active ingredient as described in any one of claims 1 to 3, as well as other anticancer active ingredients; The other anticancer active ingredients mentioned are one or more of paclitaxel, cisplatin, and sulfasalazine.

13. The anticancer active composition according to claim 12, characterized in that, In the aforementioned anticancer active composition, the content of the anticancer active ingredient is above 10 wt.%.

14. The anticancer active composition according to claim 13, characterized in that, In the aforementioned anticancer active composition, the content of the anticancer active ingredient is 20-90 wt.%.

15. An anticancer drug, characterized in that, The anticancer active ingredient comprising a pharmaceutically effective amount as described in any one of claims 1 to 3.

16. An anticancer drug, characterized in that, The anticancer active composition comprising a pharmaceutically effective amount according to any one of claims 12 to 14.

17. The anticancer drug as described in claim 15 or 16, characterized in that, It also contains pharmaceutically acceptable excipients.

18. The anticancer drug as described in claim 17, characterized in that, The excipients include at least one of coloring agents, flavoring agents, stabilizers, and thickeners.

19. The anticancer drug as described in claim 15 or 16, characterized in that, It has a pharmaceutically acceptable dosage form; wherein the dosage form is a tablet, capsule, aqueous or oily solution, suspension, emulsion, cream, ointment, gel, nasal spray or suppository.

20. The anticancer drug as described in claim 15 or 16, characterized in that, The anticancer drug is in the form of a finely dispersed powder, aerosol, or spray for inhalation.

21. The anticancer drug as described in claim 15 or 16, characterized in that, The anticancer drug is in the form of a sterile aqueous or oily solution, suspension, or sterile emulsion for use outside the gastrointestinal tract.

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