An antitumor compound, pharmaceutical composition, and preparation method and use thereof

Abstract

The application provides an anti-tumor compound, a pharmaceutical composition and a preparation method and application thereof, and belongs to the field of biological medicines. The compound can effectively activate the endoplasmic reticulum autophagy pathway in cells, and target-induced endoplasmic reticulum degradation. The activity evaluation results show that the compound exhibits a significant endoplasmic reticulum autophagy induction effect at the cell level, and has a good proliferation inhibition effect on tumor cell strains. The structure-activity relationship analysis shows that the series of compounds have the potential for further structure optimization. The novel compound provided by the application is a kind of endoplasmic reticulum autophagy target-induced small molecule with clear structure and clear mechanism, which provides a new candidate entity for anti-tumor drug research and development, and has a wide scientific research and application development prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an antitumor compound, a pharmaceutical composition, its preparation method, and its uses. Background Technology

[0002] In recent years, the high incidence and mortality rates of malignant tumors have made them a major disease posing a serious threat to human health. Current clinical practice primarily employs treatment strategies for tumors, including surgery, radiotherapy, chemotherapy, targeted therapy, and immune checkpoint blockade. While these methods have improved patient prognosis to some extent, their clinical application remains limited by numerous bottlenecks, including systemic toxicity, acquired drug resistance after treatment, and poor efficacy against recurrent, metastatic, or refractory tumors. Therefore, developing novel anti-tumor drugs with well-defined mechanisms of action, based on entirely new pharmacological mechanisms, has become an important direction in the field of new drug development.

[0003] Autophagy, a highly conserved catabolic mechanism, maintains cellular homeostasis by forming autophagosomes with double membranes, encapsulating cytoplasmic contents or damaged organelles, and transporting them to lysosomes for degradation. In recent years, selective autophagy targeting specific organelles has received increasing attention. Among these, endoplasmic reticulophagy mediates the specific recognition and clearance of redundant or damaged endoplasmic reticulum fragments. Evidence suggests that regulating endoplasmic reticulophagy levels in tumor cells can significantly affect the balance between survival and apoptosis; in some tumor models, inducing excessive or abnormally activated endoplasmic reticulophagy has been shown to trigger tumor cell death and potentially reverse resistance to existing therapeutic drugs. Therefore, developing interventions that can specifically trigger endoplasmic reticulophagy represents a novel strategy with significant exploratory value in the field of cancer therapy.

[0004] Although several small molecule compounds have been reported to induce autophagy, such as the classic mTOR pathway inhibitor rapamycin and its derivatives, these compounds primarily induce non-selective macrophagy, with insufficient selective targeting of the endoplasmic reticulum (ER). Furthermore, while some ER stress inducers (such as tunicamycin and carotenoids) can indirectly stimulate ER autophagy to some extent, they are often accompanied by strong stress toxicity, severely limiting their in vivo application prospects. In summary, the current technological field lacks small molecule compounds capable of efficiently and selectively activating the ER autophagy pathway, and no antitumor drugs developed based on such compounds have been identified. Therefore, discovering and optimizing selective ER autophagy activators with novel scaffold structures has significant scientific exploration value and translational application significance for overcoming the shortcomings of existing antitumor strategies and breaking through drug resistance barriers. Summary of the Invention

[0005] The purpose of this invention is to provide an antitumor compound, a pharmaceutical composition, a method for preparing the same, and its uses.

[0006] This invention provides compounds of Formula I, or pharmaceutically acceptable salts thereof, or solvates thereof, or tautomers thereof, or isotopic compounds thereof, or metabolites thereof: Formula I Where 'a' is selected from any integer from 1 to 6.

[0007] Furthermore, the compound is as shown in Formula II or Formula III: Where a is 2, 3, 4 or 5.

[0008] Furthermore, the compound is selected from one of the following structures: .

[0009] The present invention also provides a method for preparing the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, the method comprising the following steps: Where R is hydrogen or Ts; Compound 1a was reacted with p-toluenesulfonyl chloride to give compound 2a; then compound 2a was reacted with compound 3a to give the compound shown in Formula I.

[0010] "Ts" represents the abbreviation for p-toluenesulfonyl, and its structure is represented as: .

[0011] The present invention also provides the use of the above-described compounds, or pharmaceutically acceptable salts thereof, or solvates thereof, or tautomers thereof, or isotopic compounds thereof, or metabolites thereof, in the preparation of medicaments for the prevention and / or treatment of tumors.

[0012] Furthermore, the drug is used as an endoplasmic reticulum autophagy-targeting inducer.

[0013] Furthermore, the tumor is selected from one or more of the following: breast cancer, lung cancer, liver cancer, kidney cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, osteosarcoma, central nervous system tumors, or peripheral nervous system tumors.

[0014] Furthermore, the tumor is breast cancer.

[0015] The present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition is a formulation prepared by adding pharmaceutically acceptable excipients to the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof as the active ingredient.

[0016] The present invention has achieved the following beneficial effects: This invention provides a novel class of compounds that can effectively induce selective autophagic degradation of the endoplasmic reticulum (ER) in tumor cells at the mechanistic level. Specifically, Western blotting experiments showed that in a murine triple-negative breast cancer 4T1 cell model, after 24 hours of treatment with the compounds of this invention, the abundance of the ER marker protein RAMP4 showed a significant decreasing trend, and this degradation effect was significantly stronger than that of the blank control group, indicating that these compounds possess good ER autophagy activation ability. Regarding antitumor activity, CCK-8 proliferation inhibition assays showed that the compounds of this invention exhibited a concentration-dependent growth inhibitory effect on 4T1 tumor cells. Representative compounds such as compounds 7, 8, and 6 have half-maximal inhibitory concentrations (IC50). 50 The concentrations reached 12.6 μM, 12.7 μM, and 13.1 μM, respectively, demonstrating potent in vitro antiproliferative activity, suggesting that this series of compounds has the potential to further enhance efficacy through structural optimization. In summary, the compounds of this invention, as a novel class of small molecules capable of targeting and inducing endoplasmic reticulum autophagy, simultaneously exhibit clear endoplasmic reticulum degradation regulation activity and considerable antitumor proliferation effects at the cellular level. This lays a solid experimental foundation for their subsequent in-depth research and development as candidate molecules for antitumor therapy, possessing significant scientific exploration value and prospects for further application development.

[0017] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0018] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0019] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0020] Example 1: Preparation of compounds 1-8 1. Preparation of compounds 1 and 2 (1) Preparation of compounds 1-2 At 0 °C, p-toluenesulfonyl chloride (3.81 g, 19.98 mmol, 2.10 eq) was added fractionally to a pyridine solution (10 mL) of compound 1-1 (1.00 g, 9.51 mmol, 1.00 eq), and the mixture was stirred for 3 h at the same temperature. After the reaction was complete, 1 N hydrochloric acid solution (50 mL) was slowly poured into the system to terminate the reaction, followed by three extractions with ethyl acetate (50 mL × 3). The resulting organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give crude product of compound 1-2 (3.12 g). This crude product could be used in subsequent reactions without further purification. [M+H] + 414.2.

[0021] (2) Preparation of compounds 1 and 2 Compounds 1-2 (1.30 g, 3.15 mmol, 1.00 eq) and potassium carbonate (435.4 mg, 3.15 mmol, 1.00 eq) were dissolved together in DMF (10 mL), followed by the addition of compound 1-3 (1.00 g, 3.93 mmol, 1.25 eq). The reaction mixture was then heated to 50 °C and reacted for 12 h. After the reaction was complete, the reaction solution was cooled to room temperature, diluted with pure water (10 mL), and extracted three times with ethyl acetate (20 mL × 3). The organic layers were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated, and then passed sequentially through a liquid chromatography column (column model: Welch Ultimate XB-CN 250). 70 10µm; elution system: n-hexane / ethanol; gradient: mobile phase B increased from 30% to 60% within 20 min) and SFC column (column model: DAICEL CHIRALCEL OJ (250 mm)). The mixture was separated and purified by a process involving elution of 30 mm (10 μm); elution system: carbon dioxide-methanol (0.1% ammonia); gradient: mobile phase B at 25%, constant elution, to finally obtain compound 1 and compound 2.

[0022] 2. Preparation of compounds 3-8 The preparation methods for compounds 1 and 2 are the same as those for compounds 1 and 2, with the only difference being that: compound 1-1 is prepared by... Replace with compound 3-1 Compounds 3 and 4 were prepared; compound 1-1 was replaced with compound 5-1. Compounds 5 and 6 were prepared; compound 1-1 was replaced with compound 7-1. Compounds 7 and 8 were prepared.

[0023] The characterization data of compounds 1-8 are shown in Table 1: Table 1. Structural characterization data of compounds 1–8 The structures of compounds 1-8 are as follows: The following experimental examples demonstrate the beneficial effects of the present invention.

[0024] Experimental Example 1: Evaluation of the endoplasmic reticulum autophagy activity induced by the compound of this invention (Western protein immunoblotting) 1. Experimental Methods This experiment used triple-negative breast cancer 4T1 cells as an evaluation model and the endoplasmic reticulum marker protein RAMP4 as the detection target. Western blotting was used to determine the abundance changes of this protein, thereby reflecting the ability of the compound of this invention to promote selective autophagic degradation of the endoplasmic reticulum. The specific operation procedure is as follows: 4T1 cells were seeded in six-well cell culture plates and incubated for 24 h. Subsequently, cells were treated with a specific concentration (2 μM) of the test compound and an equal volume of solvent control (serving as a blank control group), and cultured for another 24 h. After treatment, cells were collected from each well, centrifuged at low speed to obtain cell pellets, and immediately resuspended in pre-chilled RIPA lysis buffer containing protease inhibitors and PMSF. The pellets were then sonicated on ice, and the supernatant was collected after centrifugation. Protein concentrations of each sample were determined using a BCA protein quantification kit. Based on the results, the protein concentrations of each group were adjusted to a consistent level using lysis buffer. SDS-PAGE loading buffer was then added, and the samples were heated in a 95°C water bath to denature the proteins, thus preparing the samples for testing.

[0025] After separation by polyacrylamide gel electrophoresis, the protein bands were transferred to a PVDF blot membrane with a pore size of 0.45 μm. Following transfer, the membrane was blocked at room temperature for 2 hours in TBST blocking buffer containing 5% skim milk powder. After blocking, the membrane strips were incubated overnight at 4°C with primary antibody solution (anti-SERP1 antibody diluted 1:1000) prepared according to the manufacturer's recommended ratio on a shaker. The next day, the membrane was washed three times with TBST buffer for 10 minutes each time. Horseradish peroxidase-labeled secondary antibody dilution was then added, and the membrane was incubated at room temperature with shaking for 2 hours, followed by three more washes with TBST buffer. Finally, equal volumes of solutions A and B of the ECL chemiluminescence detection solution were mixed and uniformly added to the membrane surface under light-protected conditions. The membrane was then placed in a fully automated chemiluminescence imaging system for signal acquisition and exposure imaging. The obtained band images were analyzed by optical density scanning using gel image analysis software. The ratio of the gray value of the target protein RAMP4 band to the gray value of the internal reference β-actin band was used as the relative expression level of the target protein to assess the differences in the degree of endoplasmic reticulum autophagy degradation among different treatment groups.

[0026] 2. Experimental Results Table 2. Effects of relative RAMP4 protein expression levels in each group of cells The experimental results are shown in Table 2. Compared with the control group, the expression level of RAMP4 protein in 4T1 cells decreased significantly after treatment with the compounds of this invention, indicating that the compounds of this invention can effectively induce endoplasmic reticulum autophagy degradation. Among them, compound 7 had the strongest effect.

[0027] Experimental Example 2: Evaluation of the inhibitory effect of the compounds of this invention on tumor cell proliferation 1. Experimental Methods The in vitro antiproliferative effect of the target compound on the triple-negative breast cancer cell line 4T1 was detected using the CCK-8 assay. The specific procedure is as follows: 4T1 cells were seeded into 96-well cell culture plates and cultured adherently for 24 h. The test compound of this invention was then added to each well at concentration gradients (0.1, 1, 1.5, 2.5, 5, 10, 25, 50, 100 μM), and incubated for another 24 h. After treatment, the culture medium containing the drug was discarded, and fresh culture medium supplemented with 10% CCK-8 reagent was added. Incubation was continued for 1-2 h. After the reaction, the absorbance value of each well at 450 nm was read using a microplate reader (denoted as A). 样品 The experiment also included a solvent control group (containing only DMSO, with absorbance value denoted as A). 空白 ) and the control group of normal cells without drug treatment (absorbance value denoted as A) 对照Based on the obtained absorbance data, the cell viability of each treatment group was calculated using the following formula: Viability (%) = (A 样品 –A 空白 ) / (A 对照 –A 空白 The half-maximal inhibitory concentration (IC50) of each compound was calculated using Graphpad Prism software, representing 100% of the total concentration. 50 ).

[0028] 2. Experimental Results Table 3. Inhibitory activity of compounds against the proliferation of 4T1 tumor cells The experimental results are shown in Table 3. Most compounds exhibited moderate to excellent antitumor activity. Among them, compounds 7, 8, and 6 showed strong inhibitory activity, with IC50 values ​​of [missing value]. 50 The values ​​were 12.6 μM, 12.7 μM, and 13.1 μM, respectively; the compounds of the present invention show good potential as antitumor active substances.

Claims

1. The compound of formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof: Formula I in, a is any integer from 1 to 6.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, characterized in that: The compound is shown as in Formula II or Formula III: Where a is 2, 3, 4 or 5.

3. The compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, characterized in that: The compound is selected from one of the following structures: 。 4. A method for preparing the compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, characterized in that: The method includes the following steps: Where R is hydrogen or Ts; Compound 1a was reacted with p-toluenesulfonyl chloride to give compound 2a; then compound 2a was reacted with compound 3a to give the compound shown in Formula I.

5. Use of the compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof, in the preparation of a medicament for the prevention and / or treatment of tumors.

6. The use according to claim 5, characterized in that: The drug is used as an endoplasmic reticulum autophagy-targeting inducer.

7. The use according to claim 5, characterized in that: The tumor is selected from one or more of the following: breast cancer, lung cancer, liver cancer, kidney cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, bladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, osteosarcoma, central nervous system tumors, or peripheral nervous system tumors.

8. The use according to claim 7, characterized in that: The tumor is breast cancer.

9. A pharmaceutical composition, characterized in that: The pharmaceutical composition is a formulation prepared by adding pharmaceutically acceptable excipients to a compound as described in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a tautomer thereof, or an isotopic compound thereof, or a metabolite thereof as the active ingredient.

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