Application of spermidine alkaloid in preparation of anti-tumor drugs and / or reversal of drug resistance

Abstract

The application belongs to the technical field of natural medicine, and particularly relates to application of spermidine alkaloids in preparation of anti-tumor drugs and / or reversal of drug resistance. The application provides application of macrocyclic spermidine alkaloids in preparation of anti-tumor drugs and / or reversal of drug resistance, wherein the macrocyclic spermidine alkaloids include one or more than two of 13-oxo-furanic Rauwolfia serpentina, furanic Rauwolfia serpentina, benzyl Rauwolfia serpentina and Rauwolfia serpentina cinnamylamide base. The macrocyclic spermidine alkaloids have the characteristics of wide spectrum of anti-tumor effect, good anti-tumor effect, low toxic and side effect, reversal of 5-fluorouracil drug resistance, high water solubility, easy source, simple structure, only one chiral carbon in the structure, easy artificial synthesis, intervention of cell autophagy mechanism and the like, so that the overall treatment cost is low, and the macrocyclic spermidine alkaloids have potential important clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to the application of spermidine alkaloids in the preparation of antitumor drugs and / or reversal of drug resistance. Background Technology

[0002] Cancer, or malignant tumor, is one of the major diseases threatening human health worldwide. Cancer causes serious harm to the physical and mental health of patients and their families, and imposes a heavy socioeconomic burden.

[0003] Currently, chemotherapy remains one of the most important treatments for cancer. However, most chemotherapy drugs have significant side effects, and as chemotherapy progresses, cancer develops resistance, sometimes rendering the drugs completely ineffective. For example, 5-fluorouracil is one of the most widely used antimetabolites in clinical practice, showing good efficacy against gastrointestinal cancers and other solid tumors. It plays an important role in medical oncology and is a first-line treatment for colorectal cancer. However, 5-fluorouracil has serious side effects, such as bone marrow suppression (mainly leukopenia and thrombocytopenia), loss of appetite, nausea, vomiting, stomatitis, gastritis, abdominal pain, and diarrhea, as well as hair loss, erythematous dermatitis, skin pigmentation, hand-foot syndrome, and transient cerebellar ataxia. Although 5-fluorouracil, alone or in combination, is still used as a first-line treatment for gastrointestinal malignancies, the overall effectiveness is greatly reduced due to patients developing resistance, sometimes even leading to chemotherapy failure. Reversing chemotherapy resistance is of great significance for improving the clinical efficacy of drug treatment for cancer and improving patient survival rates, and has become one of the hot topics in chemotherapy research.

[0004] Paclitaxel is a broad-spectrum natural anticancer drug widely used clinically to treat breast cancer, ovarian cancer, and some head and neck cancers and lung cancers. However, paclitaxel has significant side effects, mainly including hematological toxicity, allergic reactions, and neurological abnormalities. Hematological toxicity mainly manifests as decreased white blood cell count, decreased hemoglobin, and decreased platelet count. Allergic reactions mainly manifest as decreased blood pressure, angioedema, dyspnea, and generalized urticaria. The most common adverse reaction of paclitaxel is neurotoxicity, manifesting as numbness in the fingers and toes, and in rare cases, grand mal seizures during infusion. Literature reports that paclitaxel has extremely low water solubility, classifying it as a Class IV drug in the Biopharmaceutics Classification System (BCS) (low solubility and low permeability), meaning oral absorption is almost ineffective; currently, it can only be effective through injection in clinical practice. The solubility of paclitaxel significantly impacts its clinical application. Due to its poor water solubility and inability to form salts with acid radicals to overcome this issue, paclitaxel injection solutions typically require specific organic solvents for dilution to ensure drug concentration, stability, and efficacy. Common adverse reactions to paclitaxel are caused by the hydrophobicity of the drug's molecular structure and hypersensitivity reactions induced by excipients such as polyoxyethylene castor oil. In other words, the poor water solubility of paclitaxel and the co-solvents contribute to adverse reactions; therefore, pretreatment is necessary before use to reduce the occurrence of adverse reactions. Furthermore, due to its highly functionalized C6-C8-C6 ring skeleton structure and complex chiral center (with 11 chiral carbons), the synthesis of paclitaxel is often considered one of the most challenging aspects for chemists. Currently, although successful total synthesis of paclitaxel in the laboratory has been reported, the synthesis involves numerous steps, extremely low overall yields, and very high costs, making it difficult to meet the needs of industrial production. The chemical synthesis of paclitaxel remains far from industrialization and commercialization. The difficulty in obtaining natural sources of paclitaxel and its extremely high synthesis costs have become a bottleneck restricting its further research and application. Similarly, paclitaxel as a single chemotherapy drug is difficult to effectively inhibit cancer development, and drug resistance frequently occurs in the later stages of treatment.

[0005] In summary, severe toxic side effects, high drug resistance, extremely low water solubility, difficulty in sourcing, and the high cost of treatment due to the complexity of their structures are common drawbacks of current antitumor chemotherapeutic drugs, and also major challenges facing cancer treatment today. Therefore, developing antitumor drugs with novel structures and mechanisms to address these shortcomings has significant research value and broad application prospects. Summary of the Invention

[0006] The purpose of this invention is to provide the application of spermidine alkaloids in the preparation of antitumor drugs and / or the reversal of drug resistance. Speridine alkaloids have broad-spectrum antitumor effects and drug resistance reversal effects, and have good application effects.

[0007] To address the aforementioned technical problems, the present invention proposes the following technical solution:

[0008] This invention provides the application of macrocyclic spermidine alkaloids in the preparation of drugs for treating antitumor diseases and / or reversing drug resistance, wherein the macrocyclic spermidine alkaloids include one or more of 13-oxo-furano-celabenzine, celacarfurine, celabenzine, and celacinnine.

[0009] Preferably, the antitumor includes at least one of the following:

[0010] 1) It promotes the necrosis of tumor cells, forming vacuoles and inhibiting the growth of cancer cells;

[0011] 2) Induces autophagy in tumor cells.

[0012] Preferably, the tumor includes one or more of the following: colorectal cancer, breast cancer, lung cancer, ovarian cancer, stomach cancer, liver cancer, endometrial cancer, and pancreatic cancer.

[0013] Preferably, the reversal of drug resistance includes reversing resistance to antitumor drugs.

[0014] Preferably, the antitumor drug includes 5-fluorouracil.

[0015] This invention provides a drug for treating antitumor diseases and / or reversing drug resistance, comprising macrocyclic spermidine alkaloids and pharmaceutically acceptable excipients; said macrocyclic spermidine alkaloids include one or more of 13-oxo-furanoside, furanoside, benzooside, and cinnamamide.

[0016] Preferably, the reversal of drug resistance includes reversing resistance to antitumor drugs; the antitumor drugs include 5-fluorouracil.

[0017] Preferably, the dosage form of the drug includes one or more of oral dosage forms, injections, and suppositories.

[0018] The beneficial effects of the present invention: The macrocyclic spermidine alkaloid of the present invention has the following beneficial effects:

[0019] First, it has a broad spectrum of anti-tumor effects, with macrocyclic spermidine alkaloids inhibiting the growth of 14 tumor cell lines, including colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, liver cancer, endometrial cancer, and pancreatic cancer.

[0020] Secondly, it has good anti-tumor effects, and its macrocyclic spermidine alkaloids are comparable to those of paclitaxel;

[0021] Third, macrocyclic spermidine alkaloids have lower toxicity and side effects, and the animal mortality rate is lower than that of paclitaxel. Paclitaxel has greater toxicity and side effects. The most common adverse reaction of paclitaxel is neurotoxicity, which manifests as symptoms such as numbness in the fingers and toes. Some patients may experience grand mal seizures during infusion. Paclitaxel also has the toxic side effect of hypersensitivity reactions.

[0022] Fourth, macrocyclic spermidine alkaloids can reverse 5-fluorouracil resistance, while paclitaxel itself has a resistance rate of over 50%.

[0023] Fifth, it has high water solubility. Some macrocyclic spermidine alkaloids have an amino group in their structure, which makes them easily soluble in water (greater than 100g / L) after forming hydrochloride. However, paclitaxel has extremely low water solubility (<0.34mg / L) and cannot form salts with acid radicals, thus solving the problem of low water solubility.

[0024] Sixth, the source is relatively easy. Macrocyclic spermidine alkaloids are found in the roots, stems and leaves of Tripterygium wilfordii. Tripterygium wilfordii has been cultivated on a large scale. However, paclitaxel is rare. It is derived from the bark of Taxus chinensis, has a low content, and is difficult to cultivate and mature. It takes more than ten years for the medicinal material to mature.

[0025] Seventh, the structure is simple. Macrocyclic spermidine alkaloids have only one chiral carbon in their structure, making them relatively easy to synthesize artificially. In contrast, paclitaxel has a complex structure with 11 chiral carbons, and industrial production has not been achieved. Total synthesis remains a challenge, and artificial synthesis is very difficult.

[0026] Eighth, macrocyclic spermidine alkaloids can intervene in the mechanism of cell autophagy, increase the level of Beclin-1, and decrease the level of LC3-I, while the mechanism of action of paclitaxel is to stabilize microtubule structure, etc.

[0027] In summary, the macrocyclic spermidine alkaloids of the present invention have a low overall treatment cost and have significant potential for clinical application. Attached Figure Description

[0028] Figure 1 Western blot was used to detect the expression of Beclin-1 and LC3-I in the SH-SY5Y injury model. In the figure, a shows the effect of celabenzine on Beclin-1 expression in SH-SY5Y cells; b shows the effect of celabenzine on LC3-I expression in SH-SY5Y cells.

[0029] Figure 2 The graphs show tumor volume versus day growth curves for each group of animals. A represents the model group, B represents the positive control group (paclitaxel), C represents the low-dose celacinnine group, and D represents the high-dose celacinnine group.

[0030] Figure 3Images of ex vivo tumors in mice for each group are shown. A represents the model group, B represents the positive control group (paclitaxel), C represents the low-dose celacinnine group, and D represents the high-dose celacinnine group.

[0031] Figure 4 HE staining of tumor tissue (magnification: ×200), where A represents the model group, B represents the positive drug (paclitaxel) group, C represents the low-dose celacinnine group, and D represents the high-dose celacinnine group. Detailed Implementation

[0032] This invention provides the application of spermidine alkaloids in the preparation of antitumor drugs, wherein the macrocyclic spermidine alkaloids include one or more of 13-oxo-furanohornine, furanohornine, benzohornine, and cinnamamide. The molecular formulas of the four macrocyclic spermidine alkaloids—13-oxo-furanohornine, furanohornine, benzohornine, and cinnamamide—are C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 21 H 25 N3O4, C 21 H 27 N3O3, C 23 H 29 N3O2 and C 25 H 31 N3O2.

[0033] The spermidine alkaloids described in this invention were all extracted and isolated from Tripterygium wilfordii. The four macrocyclic spermidine alkaloids extracted and isolated were celacarfurine, celafurine, celabenzine, and celacinnine, respectively. Their chemical structures are shown in Formulas I to IV below. The chemical structure of celacarfurine is shown in Formula I below, the chemical structure of celafurine is shown in Formula II below, the chemical structure of celabenzine is shown in Formula III below, and the chemical structure of celacinnine is shown in Formula IV below.

[0034]

[0035] Formula I

[0036]

[0037] Formula II

[0038]

[0039] Formula III

[0040]

[0041] Formula IV

[0042] The present invention describes the inhibitory effects of four macrocyclic spermidine alkaloids—13-oxo-furano-erythrine, furano-erythrine, benzo-erythrine, and cinnamamide alkaloid—on the growth of 14 tumor cell lines, including intestinal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, liver cancer, endometrial cancer, and pancreatic cancer.

[0043] The antitumor effect of this invention includes at least one of the following: 1) inducing tumor cell necrosis and vacuolation to inhibit cancer cell growth; 2) inducing tumor cell autophagy; more preferably, inducing tumor cell necrosis and vacuolation to inhibit cancer cell growth and induce tumor cell autophagy. Results from the examples show that celacinnine can upregulate the autophagy level of β-amyloid 1-42-induced human neuroblastoma SH-SY5Y cells. Further in vivo animal experiments showed that a dose of 3.5 μmol / kg of celacinnine inhibited the growth of xenografts in a C57BL / 6J mouse axillary lung cancer model by 45.04%, comparable to the antitumor effect of an equimolar dose of paclitaxel.

[0044] The tumors described in this invention include one or more of the following: colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, liver cancer, endometrial cancer, and pancreatic cancer; more preferably, colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, liver cancer, endometrial cancer, and pancreatic cancer. The results of the examples show that the 13-oxo-celacarfurine, celacarfurine, benzocelacarfurine, and celacarfurine cinnamamide of this invention have inhibitory effects on the growth of 14 tumor cell lines of colorectal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, liver cancer, endometrial cancer, and pancreatic cancer. Among them, 13-oxo-celacarfurine exhibits stronger antitumor activity, with an IC50 of 72 h against the 14 cancer cell lines. 50 The lowest concentration reached 9.40 μM. Celafurine and celabenzine showed certain antitumor activity, with an IC50 value of 72 h against 14 cancer cell lines. 50 The lowest values ​​were 91.07 μM and 90.46 μM, respectively. Celacinnine, a cinnamic acid alkaloid from Celastrus orbiculatus, exhibited strong antitumor activity, with an IC50 of 91.07 μM and 90.46 μM against 14 cancer cell lines over 72 hours. 50 The lowest value reached 31.17 μM.

[0045] This invention provides the application of spermidine alkaloids in reversing drug resistance. The macrocyclic spermidine alkaloids include one or more of 13-oxo-furanoside, furanoside, benzoateranoside, and cinnamamide, more preferably 13-oxo-furanoside and furanoside. Drug resistance reversal experiments revealed that 13-oxo-furanoside and furanoside exhibited strong resistance reversal effects against 5-fluorouracil in human colon cancer HCT116 cells, with resistance reversal indices (CI) of 0.50 and 0.66, respectively.

[0046] The source, chemical structure, and molecular formula of the spermidine alkaloid described in this invention have been discussed above and will not be repeated here.

[0047] The reversal of drug resistance described in this invention includes reversing resistance to antitumor drugs, including 5-fluorouracil.

[0048] This invention provides an antitumor drug and / or a drug for reversing drug resistance, comprising a macrocyclic spermidine alkaloid and pharmaceutically acceptable excipients; the macrocyclic spermidine alkaloid includes one or more of 13-oxo-furanoside, furanoside, benzooside, and cinnamamide. This invention does not have specific limitations on the pharmaceutically acceptable excipients; conventional methods can be used.

[0049] The reversal of drug resistance described in this invention includes reversing resistance to antitumor drugs; the antitumor drugs include 5-fluorouracil. The dosage forms of the drugs described in this invention include one or more of oral dosage forms, injections, and suppositories.

[0050] Traditional Chinese medicine and natural drug resources are a natural treasure trove containing compounds with antitumor activity and an important source for discovering next-generation antitumor lead structures. In particular, naturally derived alkaloids, as an important class of nitrogen-containing natural products widely distributed in nature, provide valuable clues for discovering new drug lead structures due to their rich structural diversity, activity diversity, and specific receptor binding functions. They have become the best guides for finding active compounds, leading and inspiring many important new drug inventions. For example, paclitaxel, camptothecin, and maytinine are all natural nitrogen-containing alkaloids with significant antitumor activity, and some have become first-line anticancer drugs.

[0051] Macrocyclic spermidine alkaloids are distributed in plants, bacteria, and marine organisms. Belonging to the macrocyclic polyamine alkaloid family, they are special alkaloids containing two or more amino groups in their macrocyclic core. They possess various pharmacological activities, including antiparasitic activity, anti-plasmid DNA breakage, and anti-inflammatory activity, and show great promise for medicinal research. Tripterygium wilfordii Hook. f., a plant belonging to the genus Tripterygium in the Celastraceae family, is clinically used to treat refractory immunodeficiency diseases such as rheumatoid arthritis, chronic nephritis, and lupus erythematosus, and is a key medicine for treating intractable rheumatic diseases. This invention isolated and identified four macrocyclic spermidine alkaloids from Tripterygium wilfordii. These are thirteen-membered ring spermidine alkaloids containing three nitrogen atoms in their macrocycles: 13-oxo-celacarfurine, celafurine, celabenzine, and celacinnine. Through antitumor activity screening studies, this invention for the first time discovered and disclosed that this type of thirteen-membered cyclic spermidine alkaloid possesses broad-spectrum antitumor activity, significantly inhibiting the growth of tumor cells from colon cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, liver cancer, endometrial cancer, and pancreatic cancer, with a minimum IC50 value. 50 The concentration was 9.40 μM (13-oxo-celacarfurine), meeting the requirements for antitumor lead compounds. Among them, compounds 13-oxo-celacarfurine and celafurine showed strong resistance reversal effects against 5-fluorouracil in human colon cancer HCT-116 cells, with resistance reversal indices (CIs) of 0.50 and 0.66, respectively. Furthermore, celabenzine was found to upregulate autophagy levels in β-amyloid 1-42-induced human neuroblastoma SH-SY5Y cells. These findings indicate that these thirteen-membered ring spermine alkaloids possess broad-spectrum antitumor and resistance reversal effects. Their antitumor mechanism may be related to intervention in cellular autophagy, representing a newly disclosed class of distinctive novel antitumor natural drugs with significant potential for clinical application.

[0052] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1: Study on the inhibitory effect of macrocyclic spermidine alkaloids on tumor cell growth

[0054] 1. Fourteen tumor cell lines used in the experiment: human colon cancer HCT-116 cells and SW-837 cells, breast cancer BT-549 cells and MCF-7 cells, lung cancer PC-9 cells and HCC-827 cells, ovarian cancer OVCAR-3 cells, gastric cancer AGS cells and MKN-74 cells, liver cancer HepG2 cells and Huh-7 cells, endometrial cancer RL95-2 cells and AN3 CA cells, and pancreatic cancer SW1990 cells.

[0055] Experimental Methods: Treatment 1: Cell viability was determined using the sulforhodamine B (SRB) method. When tumor cells were in the logarithmic growth phase, they were seeded at a volume of 135 μL per well in 96-well plates and incubated overnight. After incubation, 15 μL of medium containing 13-oxo-furanoside was added, with the final drug concentration diluted to 200 μM, 40 μM, 8 μM, 1.6 μM, and 0.32 μM, respectively. After 72 h of treatment, the medium was aspirated, and 50 μL of cold 10% (w / v) trichloroacetic acid was added. Cells were fixed at 4 °C for 1 h, followed by staining with 0.4% (w / v) SRB for 30 min at room temperature. Then, 200 μL of 10 mM unbuffered Tris base solution was added, and the OD value was measured at 515 nm using a SpectraMax iD5 multi-functional fluorescent microplate reader. The inhibition rate was calculated, and the IC50 of the compound was fitted using Graphpad software. 50 value.

[0056] Treatment 2: Same as Treatment 1, except that “13-oxo-furano-serodin” is replaced with “furano-serodin”;

[0057] Treatment 3: Same as treatment 1, except that “13-oxo-furanohornine” is replaced with “benzohornine”;

[0058] Treatment 4: Same as treatment 1, except that “13-oxo-furano-nephrine” is replaced with “nephrine cinnamamide”.

[0059] The experimental results are shown in Table 1. All four macrocyclic spermidine alkaloids exhibited varying degrees of inhibitory effects on different tumor cell types. Among them, 13-oxo-furanoside (celacarfurine) showed stronger antitumor activity, with an IC50 of 72 h against gastric cancer AGS cells, endometrial cancer RL95-2 cells, and breast cancer MCF-7 cells. 50 The concentrations reached 9.40 μM, 11.97 μM, and 14.33 μM, respectively. Celafurine exhibited certain antitumor activity, with IC50 values ​​against human breast cancer MCF-7 cells, lung cancer PC-9 cells, and endometrial cancer RL95-2 cells. 50The concentrations reached 91.07 μM, 92.24 μM, and 110.30 μM, respectively. Celabenzine exhibited certain antitumor activity, with IC50 values ​​against human colon cancer SW-837 cells, lung cancer PC-9 cells, gastric cancer AGS cells, and liver cancer Huh-7 cells. 50 The values ​​reached 104.00 μM, 105.00 μM, 90.46 μM, and 111.60 μM, respectively. Celacinnine, derived from *Celastrus orbiculatus*, exhibited strong antitumor activity, with IC50 values ​​against human colon cancer SW-837, breast cancer BT-549 and MCF-7 cells, lung cancer PC-9 cells, ovarian cancer OVCAR-3 cells, liver cancer Huh-7 cells, and endometrial cancer RL95-2 cells. 50 The values ​​can reach 55.87 μM, 58.36 μM, 38.80 μM, 31.17 μM, 61.44 μM, 53.54 μM and 56.95 μM, respectively.

[0060] Table 1. IC50 of macrocyclic spermidine alkaloids on different tumor cells after 72 hours. 50 (μM)

[0061]

[0062] Example 2: Experimental Study on the Reversal of 5-Fluorouracil Resistance by Macrocyclic Spermine Alkaloids

[0063] Although 5-fluorouracil, whether used alone or in combination, remains a first-line treatment for gastrointestinal malignancies, its overall effectiveness is significantly reduced due to patients' tendency to develop resistance, sometimes even leading to chemotherapy failure. Reports indicate that 5-fluorouracil monotherapy is only 10%–15% effective in treating advanced colorectal cancer. Therefore, reversing 5-fluorouracil resistance is crucial.

[0064] Experimental Methods: Treatment 5: The SRB method was used to determine the reversal of drug resistance. When HCT-116 cells resistant to 5-fluorouracil were in the logarithmic growth phase, cells were seeded at a volume of 135 μL per well in 96-well plates and incubated overnight. After incubation, medium containing 15 μL of 5-fluorouracil (15 μM) was added. After 72 h of treatment, the medium was aspirated, 50 μL of cold 10% (w / v) trichloroacetic acid was added, and the cells were fixed at 4 °C for 1 h. Subsequently, the cells were stained with 0.4% (w / v) SRB for 30 min at room temperature. After adding 200 μL of 10 mM unbuffered Tris base solution, the OD value was measured at 515 nm using a SpectraMax iD5 multi-functional fluorescent microplate reader, and the inhibition rate was calculated. The drug resistance reversal index (CI) was further calculated based on the inhibition rate. The drug resistance reversal index CI was calculated using the following formula: CI = (E a +E b +E a ×E b ) / E ab E a E b and E ab The values ​​represent the inhibition rates of 5-fluorouracil, the drug, and a mixture of 5-fluorouracil and the drug on HCT-116 cells over 72 hours, respectively. CI < 1 indicates synergistic effect, and CI > 1 indicates antagonistic effect.

[0065] Treatment 6: Same as Treatment 5, except that after incubation, medium containing 15 μL of celacarfurine, 15 μL of celacarfurine, 15 μL of celabenzine, and 15 μL of celacinnine were added respectively. The concentrations of celacarfurine in the medium were 80 μM, 40 μM, 20 μM, and 10 μM, respectively, and the concentrations of celacarfurine, celabenzine, and celacinnine were 250 μM, 200 μM, 150 μM, and 100 μM, respectively.

[0066] Treatment 7: Same as Treatment 5, except that after incubation, the following media were added: 15 μL of medium containing 5-fluorouracil (15 μM) and celacarfurine; 15 μL of medium containing 5-fluorouracil (15 μM) and celacarfurine; 15 μL of medium containing 5-fluorouracil (15 μM) and celacarfurine; and 15 μL of medium containing 5-fluorouracil (15 μM) and celacarfurine. The concentrations of celacarfurine in the media were 80 μM, 40 μM, 20 μM, and 10 μM, respectively, and the concentrations of celacarfurine, celacarfurine, and celacarfurine in the media were 250 μM, 200 μM, 150 μM, and 100 μM, respectively.

[0067] Experimental Results: Celacarfurine and celafurine showed strong resistance reversal effects against 5-fluorouracil in human colon cancer HCT-116 cells. The resistance reversal indices (CIs) of 80, 40, 20, and 10 μM celacarfurine against 5-fluorouracil in human colon cancer HCT-116 cells were 1.27, 0.50, 0.97, and 0.88, respectively. The resistance reversal CIs of 250, 200, 150, and 100 μM celafurine against 5-fluorouracil in human colon cancer HCT-116 cells were 1.93, 0.66, 0.77, and 0.88, respectively. Celabenzine and celafurine did not show significant resistance reversal effects (CI > 1).

[0068] Example 3: Study on the effect of macrocyclic spermidine alkaloids on autophagy levels in human neuroblastoma SH-SY5Y cells.

[0069] 1. Experimental Methods

[0070] (1) Cell model: by using different doses of β Human neuroblastoma SH-SY5Y cells were induced with amyloid 1-42 (0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 80 μM) for 24 h, and a cell damage model was established at a modeling concentration of 40 μM after screening. A blank control group (C), a model group (M), and an experimental group were set up. The blank control group contained only culture medium and CCK 8. Untreated human neuroblastoma SH-SY5Y cells were used as the control group. SH-SY5Y cells incubated with 40 μM β-amyloid 1-42 served as the model group. SH-SY5Y cells incubated with β-amyloid 1-42 and macrocyclic spermidine alkaloids served as the experimental group.

[0071] (2) Immunoblotting: To assess the expression levels of Beclin-1 and LC3-I proteins in human neuroblastoma SH-SY5Y cells obtained from each group, Western blotting was performed using Proteintech's Beclin-1 antibody and LC-3 I / II antibody as follows: SH-SY5Y cells in logarithmic growth phase obtained from the blank group (C), model group (M), and experimental group were added to 60 mm disposable culture dishes and cultured in an incubator at 37 ℃ and 5% CO2 saturated humidity. RIPA lysis solution and PMSF were mixed at a ratio of 100:1 to obtain lysis buffer, which was then placed on ice for use. The following operations were all performed on ice: the culture medium was discarded from the culture dish, the cells were washed 3 times with pre-cooled PBS, 120 μL of lysis buffer was added to each culture dish, and the cells were evenly dropped onto the cells. After 3 min, the cells were scraped off with a cell scraper. The solution was collected in a clean 1.5 mL EP tube and placed on ice for 30 min. The lysate was centrifuged at 16099 × g for 10 min at 4 °C. The supernatant was then carefully transferred into the EP tube, and approximately 1 / 5 of the sample buffer was added. The EP tube was then boiled in water for 10 min, removed, slightly cooled, and stored at -20 °C. The SDS-PAGE electrophoresis program was initially set to 55 V for 50 min, followed by 95 V for 90 min. The membrane was transferred using a wet membrane transfer method with a transfer current of 200 mA. The membrane transfer time depended on the protein molecular weight. The primary antibody was cultured overnight at 4 °C. The secondary antibody was incubated in the dark at room temperature for 2 h.

[0072] (3) Statistical Analysis: Data were analyzed using GraphPad Prism 9.5. Data are expressed as mean ± standard deviation (SD). Statistical differences were assessed using one-way ANOVA with or without Tukey-Kramer multiple comparison (post-hoc) tests. In all cases, p A value <0.05 is considered statistically significant. .

[0073] 2. Experimental Results

[0074] Expression levels of Beclin-1 and LC3-I: The levels of recombinant human autophagy effector protein (Beclin-1) and microtubule-associated protein light chain 3-I (LC3-I) in the SH-SY5Y injury model were detected by Western blot. Figure 1As shown in the figure, compared with the control group, administration of 5 μM or 20 μM celabenzine had no significant effect on the expression of Beclin-1 and LC3-I. However, administration of 40 μM celabenzine significantly increased the level of Beclin-1 and decreased the level of LC3-I, indicating that celabenzine can upregulate the expression of Beclin-1 and LC3-I. β Autophagy level in SH-SY5Y cells induced by amyloid 1-42.

[0075] Example 4: In vivo anti-lung cancer efficacy study of celacinnine in tumor-bearing C57BL / 6J mice.

[0076] 1. Experimental Methods

[0077] (1) Preparation of drugs

[0078] Preparation of positive control solution: Accurately pipette 0.5 ml of paclitaxel injection (Hainan Quanxing Pharmaceutical Co., Ltd., batch number H20084032, containing 6 mg / ml of paclitaxel) and dilute it with 0.9% sodium chloride injection to prepare a 0.5 mg / ml stock solution for later use.

[0079] Preparation of celacinnine solution: Accurately weigh 5 mg of celacinnine and dissolve it in an equimolar amount of hydrochloric acid. Prepare a 1 mg / ml celacinnine stock solution with 0.9% sodium chloride injection. Then dilute with 0.9% sodium chloride injection to the concentration required for each dosage group. Prepare fresh solution each time.

[0080] (2) Preparation of Lewis lung cancer cell suspension

[0081] Lewis lung cancer cells were cultured in DMEM medium containing 10% FBS until they reached the exponential growth phase. Eighteen culture dishes containing Lewis lung cancer cells in the logarithmic growth phase were used. The culture medium was discarded, and the dishes were washed twice with PBS buffer. Then, 2 mL of trypsin-EDTA digestion solution was added to each dish, and the dishes were placed in a CO2 cell culture incubator for 1 min. Immediately after 1 min, the dishes were removed, and 2 mL of complete culture medium (10% fetal bovine serum + 90% DMEM high-glucose medium with antibiotics) was added to stop the digestion (DMEM high-glucose medium with antibiotics, purchased from Jiangsu Kaiji Biotechnology Co., Ltd., product number: KGL1206-500, batch number: 20240820). The cells were pipetted to ensure even distribution and transferred to 15 mL centrifuge tubes. The cells were centrifuged (1000 rpm, 5 min), the supernatant was discarded, and the cells were reconstituted with the aforementioned complete culture medium to a concentration of 1.5 × 10⁻⁶. 7 ~2×10 7The dose was 1.5 × 10⁶ cells / mL, with each mouse inoculated with 0.1 mL. 6 ~2×10 6 Lewis lung cancer cells.

[0082] 2. Subcutaneous inoculation of Lewis lung cancer cells into C57BL / 6J mice:

[0083] C57BL / 6J mice were acclimatized for one week before inoculation. The skin at the inoculation site was disinfected before inoculation. A 1 mL syringe was used to precisely aspirate a suspension of Lewis lung cancer cells (concentration 1.5 × 10⁻⁶) from a centrifuge tube. 7 ~2×10 7 0.1 mL (cells / mL) Hold the mouse with your left hand and gently lift the skin under the mouse's right armpit to subcutaneously inoculate tumor cells. Inject slowly. After injection, a distinct wheal will appear. Press the injection site with a sterile cotton swab for 5 seconds to prevent the cell suspension from flowing out.

[0084] 3. Animal grouping and administration:

[0085] The growth of subcutaneous xenografts in mice was observed daily. Seven days after subcutaneous inoculation with Lewis lung cancer cells, the average volume at the inoculation site was approximately 70 mm. 3 The small, hard nodules, oval or round in shape, no longer shrinking, indicate successful tumor inoculation. Successfully inoculated C57BL / 6J mice were randomly divided into four groups of 12 mice each: a positive control group (paclitaxel, dose 3.50 μmol / kg based on mouse body weight); a low-dose celacinnine group (dose 3.5 μmol / kg based on mouse body weight); a high-dose celacinnine group (dose 7.0 μmol / kg based on mouse body weight); and a model group. Additionally, 10 C57BL / 6J mice not inoculated with Lewis lung cancer cells served as a blank control group.

[0086] Before drug administration, there was no significant difference in the volume of subcutaneous xenografts among the groups of mice. Mice were administered drugs starting on day 7 after inoculation with Lewis lung cancer cells. The positive control group (paclitaxel) received 3.5 μmol / kg, the low-dose celacinnine group received 3.5 μmol / kg, and the high-dose celacinnine group received 7.0 μmol / kg, via tail vein injection, once daily for 7 consecutive days. The maximum injection volume per mouse was 200 μl. The model group and blank control group did not receive any drug. During the drug administration period, mice were housed in a 12-hour cyclic light environment at a temperature of (22±2℃), and were fed pure water and maintenance diet (sterilized by cobalt-60 irradiation). The diet was purchased from Spiefol (Beijing) Biotechnology Co., Ltd., and the bedding was changed every 2 days. Mice were allowed free movement. All mice in each group were euthanized by cervical dislocation and dissected on day 1 after drug withdrawal.

[0087] Statistical analysis:

[0088] Data were processed using IBM SPSS Statistics 21 software. Normally distributed continuous data are expressed as mean ± s. One-way ANOVA analysis was used for inter-group comparisons. P <0.05 indicates a statistically significant difference. All images were plotted using GraphPad Prism 9. Experimental methods and results are as follows:

[0089] 1. Recording the survival status of mice and plotting their weight change curves.

[0090] From the initial inoculation of mice with Lewis lung cancer cells, we observed the mice's survival, diet, water intake, mental state, etc., every day, and weighed the mice before each administration and recorded the weight change curve to complete the data analysis.

[0091] Table 2 shows the survival status of mice in each group after inoculation with Lewis lung cancer cells. The dosage is expressed in μmol / kg.

[0092] Table 2. Survival status of mice during the experiment.

[0093]

[0094] 2. Plotting growth curves of mouse lung cancer xenografts:

[0095] Three days after mice were inoculated with Lewis lung cancer cells, the same person measured the major and minor diameters of the tumors daily using calipers and calculated the tumor volume to create a tumor volume-day growth curve. Figure 2 The differences in tumor growth among the mouse groups were statistically analyzed. The calculation formula is as follows:

[0096] Tumor volume (mm) 3= 0.5 × tumor long diameter (mm) × tumor short diameter (mm) × tumor short diameter (mm)

[0097] Effects of the drug on the growth of lung cancer xenografts in mice: Changes in tumor volume in each group from day 3 to day 14 after modeling are shown in the figure. Figure 2 .Depend on Figure 2 It can be seen that as the experimental time increased, the volume of the transplanted tumors in each group of mice gradually increased, but the average volume of the tumors in the three drug administration groups on the same day was smaller than that in the model group. Compared with the model group, the tumor volume growth in each drug administration group was slower. Drug administration was stopped on the 7th day, and the next day (i.e., the 14th day of the experiment), the animals in all four groups were sacrificed, and the tumor tissue was dissected, obtaining ex vivo tumors from 7 mice in each group. These tumors were arranged in descending order of volume and photographed to obtain images of the ex vivo tumors. Figure 3 The volume of ex vivo tumors was measured, and the results showed that the volumes of ex vivo tumors in the positive control group, the low-dose celacinnine group, and the high-dose celacinnine group were significantly smaller than those in the model group, with statistically significant differences. P <0.05)( Figure 3 (Day 14).

[0098] The results in summary indicate that celacinnine has a significant inhibitory effect on the growth of lung cancer xenografts in mice.

[0099] 3. Tumor weight and tumor inhibition rate of xenograft lung cancer in mice:

[0100] Mice in all groups were euthanized by cervical dislocation the day after drug withdrawal. Tumor tissue was dissected, and residual blood was gently blotted dry with filter paper. The tumor weight of each group was weighed and recorded. The differences in tumor weight among the groups were statistically analyzed, the tumor inhibition rate was calculated, and images of the ex vivo tumor tissue were obtained. Figure 3 ).

[0101] The calculation formula is as follows:

[0102] Tumor inhibition rate (%) = (Average tumor weight in the model group - Average tumor weight in the treatment group) / Average tumor weight in the model group × 100%

[0103] Effects of the drug on tumor weight and growth inhibition rate of xenograft lung cancer in mice:

[0104] The results are shown in Table 3: Compared with the model group, the tumor weight of mice in the positive drug (paclitaxel) control group, the low-dose celacinnine group, and the high-dose celacinnine group was significantly reduced, and all differences were statistically significant. P<0.05). The positive control drug paclitaxel (3.5 μmol / kg), low-dose celacinnine (3.5 μmol / kg), and high-dose celacinnine (7.0 μmol / kg) inhibited the growth of xenografts in mouse lung cancer by 48.84%, 45.04%, and 45.85%, respectively. There was no statistically significant difference between the celacinnine group and the positive control drug paclitaxel group. P >0.05). The results showed that the macrocyclic spermidine alkaloid celacinnine had a significant inhibitory effect on the growth of xenografts in mouse lung cancer, with an inhibitory effect comparable to that of the positive control drug paclitaxel. The unit of dosage is μmol / kg.

[0105] Table 3. Tumor inhibition rate of mouse lung cancer cell xenografts ( n =7)

[0106]

[0107] 4. HE staining to observe pathological changes in mouse tumor tissue:

[0108] Weighed and photographed tumor tissues were fixed in 4% paraformaldehyde fixative for at least 24 hours, then washed with pH 7.4 PBS, and subjected to routine paraffin embedding. The tissues were then cut into thin sections (5 μm). Hematoxylin-eosin (HE) staining was subsequently performed on the sections. The pathological structure of the tumor tissues was observed using an electron microscope. Figure 4 .

[0109] It was observed that, through HE staining of tumor tissue sections, the tumor cells in the model group were densely packed, with large, deeply stained nuclei, fewer and smaller necrotic areas, and few morphological changes such as nuclear pyknosis. In contrast, the tumor cells in the drug-treated group were sparsely packed, lightly stained, and showed vacuolation of multiple areas of necrotic tumor cells. Most tumor cells exhibited various morphological changes such as nuclear pyknosis and karyorrhization. This indicates that drug treatment can induce necrosis of tumor tissue in mice, thereby inhibiting tumor growth.

[0110] To overcome the shortcomings of current anti-tumor chemotherapy drugs, such as severe toxic side effects, high drug resistance, extremely low water solubility, difficulty in sourcing, difficulty in artificial synthesis due to the ultra-complex structure, and high treatment costs, relevant studies show that about 30% of breast cancer patients develop drug resistance after receiving initial chemotherapy, and the resistance rate is as high as 50% after multiple chemotherapy sessions. Paclitaxel is a first-line drug for chemotherapy of lung adenocarcinoma, but the incidence of drug resistance in clinical application of paclitaxel is as high as 55%.

[0111] This invention proposes macrocyclic spermidine alkaloids. The antitumor effects of the macrocyclic spermidine alkaloids of this invention are compared with those of the first-line antitumor drug paclitaxel, as shown in Table 4 below.

[0112] Table 4 Comparison of the antitumor characteristics of macrocyclic spermine alkaloids and paclitaxel disclosed in this invention.

[0113]

[0114] Example 5: Determination of the solubility of macrocyclic spermidine alkaloids

[0115] The four macrocyclic spermidine alkaloids mentioned above are generally poorly soluble in water. However, furanosine, benzosine, and cinnamamide contain secondary amines in their structures, which can form salts with hydrochloric acid, thus increasing their water solubility. The determination is as follows:

[0116] 10 mg of furanohornine was added to 1 mL of 3M hydrochloric acid and completely dissolved. The solution was then freeze-dried or evaporated to dryness to completely remove the solvent and excess hydrochloric acid, yielding furanohornine hydrochloride solid. This solid was then dissolved in 100 μL of pure water at room temperature to obtain a completely clear solution, thus demonstrating that the solubility of furanohornine hydrochloride is greater than 100 g / L. The solubility of p-benzohornine and cinnamamide hydrochloride in water at room temperature was also determined using the same method, both showing a solubility greater than 100 g / L.

[0117] In summary, the four macrocyclic spermidine alkaloids of this invention—celacarfurine, celafurine, celabenzine, and celacinnine—exhibit strong in vitro and in vivo antitumor activity, and some of them show good reversal of 5-fluorouracil resistance. Their antitumor mechanism may be related to the induction of autophagy, making them a new class of antitumor lead compounds with promising potential applications in clinical practice.

[0118] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of macrocyclic spermidine alkaloids in the preparation of antitumor drugs, characterized in that, The macrocyclic spermidine alkaloid is one or more of 13-oxo-furanoside, furanoside, benzooside, and cinnamamide, and the tumor is one or more of intestinal cancer, breast cancer, lung cancer, ovarian cancer, gastric cancer, liver cancer, endometrial cancer, and pancreatic cancer.

2. The application of macrocyclic spermidine alkaloids in the preparation of drugs that reverse resistance to antitumor drugs, characterized in that, The macrocyclic spermidine alkaloid is one or both of 13-oxo-furanoside and furanoside, the antitumor drug is 5-fluorouracil, and the tumor is colorectal cancer.

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