A c-Myc protein degradation agent as well as a preparation method and application thereof, and an antitumor drug
By preparing a c-Myc protein degrader with a formula I structure, the problem of difficulty in inhibiting c-Myc protein in existing technologies has been solved, thus achieving both effectiveness and safety in tumor treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- LONGHUA HOSPITAL SHANGHAI UNIV OF TRADITIONAL CHINESE MEDICINE
- Filing Date
- 2023-09-15
- Publication Date
- 2026-07-21
AI Technical Summary
Existing technologies struggle to effectively inhibit c-Myc protein at the protein level, making tumor treatment difficult, especially due to the lack of specific degrading agents.
A c-Myc protein degrader with Formula I was developed. 4-hydroxyacetophenone and derivatives of substituted benzaldehyde were prepared by condensation reaction for specific degradation of c-Myc protein, thus preparing an anti-tumor drug.
It achieves stable degradation of c-Myc protein, effectively inhibits tumor cell proliferation, and causes no significant damage to the liver and kidneys, making it applicable to the treatment of various tumors.
Smart Images
Figure CN117229135B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical application technology, specifically relating to a c-Myc protein degrader, its preparation method and application, and an anti-tumor drug. Background Technology
[0002] c-Myc is a transcription factor responsible for regulating fundamental cellular processes, including proliferation, metabolism, biosynthesis, and apoptosis. Studies have shown that abnormal c-Myc expression is a driving factor in tumor development and maintenance. Therefore, inhibiting c-Myc function is an important approach to achieving tumor therapy.
[0003] In recent years, researchers have attempted to inhibit c-Myc function directly or indirectly at multiple levels, achieving some results. However, targeting c-Myc at the protein level with clinical-grade small molecules remains a significant challenge. The fact that important functional domains of c-Myc are inherently disordered and lack enzymatic active sites has ruled out structure-guided drug design. Furthermore, the high affinity and interaction between c-Myc and its specific partner MAX, as well as the partial functional redundancy and nuclear localization of different Myc family members, pose persistent obstacles to designing effective c-Myc inhibitors. Therefore, developing degradative agents that specifically degrade c-Myc proteins has very broad application value. Summary of the Invention
[0004] The purpose of this invention is to provide a c-Myc protein degrading agent, its preparation method and application, and an anti-tumor drug. The c-Myc protein degrading agent provided by this invention is stable and can specifically degrade c-Myc protein, thereby achieving tumor treatment.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides a c-Myc protein degrader having the structure shown in Formula I, or an optical isomer thereof, hydrate, organic solvate, or pharmaceutically acceptable salt thereof;
[0007]
[0008] In Equation I, R is either -F or -CF3.
[0009] Preferably, the c-Myc protein degrader has the structure shown in Formula II or Formula III:
[0010]
[0011] This invention provides a method for preparing the c-Myc protein degrading agent, comprising the following steps:
[0012] 4-hydroxyacetophenone, substituted benzaldehyde, a catalyst, and an organic solvent are mixed and subjected to a condensation reaction to obtain a c-Myc protein degrading agent; the substituted benzaldehyde has the structure shown in Formula IV:
[0013]
[0014] In formula IV, R is either -F or -CF3.
[0015] Preferably, the ratio of 4-hydroxyacetophenone to substituted benzaldehyde is 0.1-10 g: 0.1-10 mL.
[0016] This invention provides the use of the c-Myc protein degrader or its optical isomer, hydrate, organic solvate or pharmaceutically acceptable salt in the preparation of antitumor drugs.
[0017] Preferably, the tumor includes liver cancer, lung cancer, kidney cancer, esophageal cancer, or leukemia.
[0018] Preferably, the antitumor drug is administered orally, intravenously, or subcutaneously.
[0019] The present invention provides an antitumor drug comprising a first active pharmaceutical ingredient and a pharmaceutically acceptable excipient; the first active pharmaceutical ingredient is the c-Myc protein degrader or its optical isomer, hydrate, organic solvate or pharmaceutically acceptable salt described in the above technical solution.
[0020] Preferably, the antitumor drug further includes a second active pharmaceutical ingredient; the second active pharmaceutical ingredient includes at least one of cytarabine, daunorubicin, demethoxydaunorubicin, doxorubicin, 5-fluorouracil, docetaxel, paclitaxel, cisplatin, carboplatin, gemcitabine, capecitabine and sorafenib.
[0021] This invention provides the use of the c-Myc protein degrader or its optical isomer, hydrate, organic solvate or pharmaceutically acceptable salt in the treatment of c-Myc upregulation-induced diseases; the c-Myc upregulation-induced diseases include cardiovascular and cerebrovascular diseases or viral infection-related diseases.
[0022] The c-Myc protein degrading agent provided by this invention is a derivative of 4-hydroxychalcone, which is stable and can specifically degrade c-Myc protein. Antitumor drugs prepared based on this agent can treat various tumors and have good practicality and application prospects. Examples show that the c-Myc protein degrading agent provided by this invention can effectively inhibit the proliferation of various tumors and has no significant damage to the liver and kidneys. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0024] Figure 1 The 1H NMR spectrum of the c-Myc protein degrader obtained in Example 2;
[0025] Figure 2 The image shows the carbon NMR spectrum of the c-Myc protein degrader obtained in Example 2.
[0026] Figure 3 The graph shows the effect of different concentrations of c-Myc protein degrader on the proliferation of 786-O cells (human clear cell adenocarcinoma cells) in Examples 1 and 2.
[0027] Figure 4 The graph shows the effect of different concentrations of c-Myc protein degrading agents on c-Myc protein expression in 786-O cells (human clear cell adenocarcinoma cells) in Examples 1 and 2.
[0028] Figure 5 The figure shows the effect of different concentrations of c-Myc protein degrading agents on the proliferation of different tumor cells;
[0029] Figure 6 Inhibition of IC50 by different concentrations of c-Myc protein degrading agent 50 And bar charts showing c-Myc protein expression in different renal cells;
[0030] Figure 7 Inhibiting IC50 at different concentrations of c-Myc protein degrading agent 50 Correlation diagram with c-Myc protein expression;
[0031] Figure 8 Growth curves of subcutaneous tumors in nude mice treated with different concentrations of c-Myc protein degrading agent;
[0032] Figure 9 Morphological images of subcutaneous tumors in nude mice after treatment with different concentrations of c-Myc protein degrading agents;
[0033] Figure 10 A comparison of the weight of subcutaneous tumors in nude mice treated with different concentrations of c-Myc protein degrading agent;
[0034] Figure 11 Comparison of HE staining results of liver and kidney cells at different concentrations of c-Myc protein degrading agent. Detailed Implementation
[0035] This invention provides a c-Myc protein degrader having the structure shown in Formula I, or an optical isomer thereof, hydrate, organic solvate, or pharmaceutically acceptable salt thereof;
[0036]
[0037] In Equation I, R is either -F or -CF3.
[0038] The c-Myc protein degrader of the present invention preferably has the structure shown in Formula II or Formula III:
[0039]
[0040] The present invention also provides a method for preparing the c-Myc protein degrading agent, comprising the following steps:
[0041] 4-hydroxyacetophenone, substituted benzaldehyde, a catalyst, and an organic solvent are mixed and subjected to a condensation reaction to obtain a c-Myc protein degrading agent; the substituted benzaldehyde has the structure shown in Formula IV:
[0042]
[0043] In formula IV, R is either -F or -CF3.
[0044] In this invention, unless otherwise specified, all raw materials required for preparation are commercially available products well known to those skilled in the art.
[0045] In this invention, the preferred ratio of 4-hydroxyacetophenone to substituted benzaldehyde is 0.5-2g:0.5-1mL, more preferably 1g:1mL.
[0046] In this invention, the catalyst is preferably an aqueous solution of potassium hydroxide and / or an aqueous solution of sodium hydroxide, more preferably an aqueous solution of sodium hydroxide. The mass concentration of the catalyst is preferably 5-20%, more preferably 10%. The ratio of the catalyst to 4-hydroxyacetophenone is preferably 2-10:0.5-2, more preferably 5 mL:1.36 g.
[0047] In this invention, the organic solvent is preferably ethanol, and more preferably anhydrous ethanol.
[0048] In this invention, 4-hydroxyacetophenone, substituted benzaldehyde, a catalyst, and an organic solvent are preferably mixed under ice bath conditions; the temperature of the ice bath is preferably -10 to 8°C, more preferably 0°C. In this invention, the temperature of the condensation reaction is preferably 20 to 30°C, more preferably 22 to 25°C; the time is preferably 1 to 4 hours, more preferably 2 to 3 hours. The condensation reaction of this invention is preferably carried out under stirring conditions. This invention does not have a particular limitation on the stirring method; any stirring method well known to those skilled in the art can be used. In this invention, 4-hydroxyacetophenone and substituted benzaldehyde are used as raw materials for a condensation reaction under the action of a catalyst and an organic solvent.
[0049] In this invention, the pH value of the mixture obtained after the condensation reaction is preferably adjusted to 5-6, followed by solid-liquid separation to collect the solid material. The solid material is then purified to obtain the c-Myc protein degrading agent. This invention does not have a specific limitation on the method of pH adjustment; any method well-known to those skilled in the art can be used. In an embodiment of this invention, specifically, 10% hydrochloric acid is used to adjust the pH value of the mixture obtained after the condensation reaction to 5-6. In this invention, the solid-liquid separation method is preferably filtration. The purification method of this invention is preferably recrystallization; the reagent used for recrystallization is preferably an organic solvent, preferably ethanol, specifically anhydrous ethanol; the recrystallization is preferably carried out under stirring conditions.
[0050] This invention also provides the use of the c-Myc protein degrader or its optical isomer, hydrate, organic solvate, or pharmaceutically acceptable salt in the preparation of antitumor drugs. In this invention, the tumor preferably includes liver cancer, lung cancer, kidney cancer, esophageal cancer, or leukemia; more preferably, liver cancer, kidney cancer, or esophageal cancer. The clinical use range of the antitumor drug of this invention is preferably 5–100 mg / kg, more preferably 20–50 mg / kg. The administration method of the antitumor drug of this invention preferably includes oral, intravenous, or subcutaneous injection, more preferably intravenous or subcutaneous injection. The dosage form of the antitumor drug of this invention is preferably granules, pills, tablets, capsules, or injections, more preferably granules.
[0051] This invention also provides an antitumor drug comprising a first active pharmaceutical ingredient and pharmaceutically acceptable excipients; the first active pharmaceutical ingredient is the c-Myc protein degrader or its optical isomer, hydrate, organic solvate, or pharmaceutically acceptable salt described in the above-mentioned technical solution. In this invention, the antitumor drug preferably further comprises a second active pharmaceutical ingredient; the second active pharmaceutical ingredient preferably comprises at least one of cytarabine, daunorubicin, demethoxydaunorubicin, doxorubicin, 5-fluorouracil, docetaxel, paclitaxel, cisplatin, carboplatin, gemcitabine, capecitabine, and sorafenib. In this invention, the mass ratio of the first active pharmaceutical ingredient to the second active pharmaceutical ingredient is preferably 0.5 to 2:1, more preferably 1:1.
[0052] This invention also provides the use of the c-Myc protein degrader or its optical isomers, hydrates, organic solvates, or pharmaceutically acceptable salts in the treatment of c-Myc upregulation-induced diseases. In this invention, the c-Myc upregulation-induced diseases preferably include cardiovascular and cerebrovascular diseases or viral infection-related diseases.
[0053] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0054] Example 1
[0055] Under ice bath conditions at 0°C, 5 mL of sodium hydroxide aqueous solution (10% by mass), 1.36 g of 4-hydroxyacetophenone (10 mmol), 1.36 mL of 4-fluorobenzaldehyde (10 mmol), and anhydrous ethanol were mixed and stirred. The mixture was reacted at 22°C for 2 h. After the reaction was completed, dilute hydrochloric acid (10% by mass) was added to the resulting mixture to adjust the pH to 5-6. The mixture was then filtered to obtain a solid material. The solid material was dissolved in anhydrous ethanol under stirring, recrystallized, and the precipitated material was collected. This solid material is the c-Myc protein degrading agent.
[0056] Example 2
[0057] Under ice bath conditions at 0°C, 5 mL of sodium hydroxide aqueous solution (10% by mass), 1.36 g of 4-hydroxyacetophenone (10 mmol), 1.36 mL of 4-(trifluoromethyl)benzaldehyde (10 mmol), and anhydrous ethanol were mixed and stirred. The mixture was reacted at 22°C for 2 h. After the reaction was completed, dilute hydrochloric acid (10% by mass) was added to the resulting mixture to adjust the pH to 5-6. The mixture was then filtered to obtain a solid material. The solid material was dissolved in anhydrous ethanol under stirring, recrystallized, and the precipitated material was collected. This solid material is the c-Myc protein degrading agent.
[0058] Figure 1 and Figure 2 The images show the 1H and 1C NMR spectra of the c-Myc protein degrading agent obtained in Example 2 of this invention.
[0059] The proton NMR spectrum shows: 1 H NMR(600MHz,Chloroform-d)δ8.07–8.02(m,2H),7.82(d,J=15.7Hz,1H),7.76(d ,J=8.1Hz,2H),7.70(d,J=8.1Hz,2H),7.62(d,J=15.7Hz,1H),7.00–6.94(m,2H).
[0060] The carbon NMR spectrum shows: 13 C NMR (101MHz, Chloroform-d) δ188.25,160.18,142.11,131.27,130.92,128.45,125.92,125.89,124.04,115.58.
[0061] Depend on Figure 1 and Figure 2 It can be seen that the synthesized compound has the structure shown in Formula III:
[0062]
[0063] Application Example 1
[0064] The c-Myc protein degrading agents obtained in Examples 1 and 2 were dissolved in dimethyl sulfoxide to prepare 10 mmol / L solutions, which were designated as c-Myc protein degrading agent stock solutions and stored at -80°C. They were diluted as needed before use.
[0065] 786-O cells (human clear cell adenocarcinoma of the kidney) were seeded at a density of 1000 cells / well in 96-well cell culture plates containing serum-free medium. After the 786-O cells adhered, c-Myc protein degrading agent solution at a concentration of 0–100 μmol / L was added, and the cells were incubated at 37°C for 72 h. Then, the medium was discarded, 100 μL of CCK-8 reaction solution was added, and the cells were incubated at 37°C for another 0.5–4 h. Subsequently, the cells were transferred to a microplate reader to measure the absorbance at 450 nm to test the effect of c-Myc protein degrading agent on the proliferation of 786-O cells.
[0066] Figure 3 The graph shows the effect of different concentrations of c-Myc protein degrader on the proliferation of 786-O cells in Examples 1 and 2 (Example 1 is on the left, and Example 2 is on the right).
[0067] Application Example 2
[0068] The c-Myc protein degrading agents obtained in Examples 1 and 2 were dissolved in dimethyl sulfoxide to prepare 10 mmol / L solutions, which were designated as c-Myc protein degrading agent stock solutions and stored at -80°C. They were diluted as needed before use.
[0069] 786-O cells (human clear cell renal adenocarcinoma cells) were seeded at a density of 20,000 cells / well in 6-well cell culture plates containing serum-free medium. After the 786-O cells adhered, a c-Myc protein degrading agent solution with a concentration of 0–20 μmol / L was added, and the cells were incubated at 37°C for 72 h. Cells were collected, lysed, and proteins were extracted. Western blotting was used to detect c-Myc protein expression, and the effect of the c-Myc protein degrading agent on c-Myc protein expression in 786-O cells was determined.
[0070] Figure 4 The graph shows the effect of different concentrations of c-Myc protein degrading agents on c-Myc protein expression in Examples 1 and 2 (left side is Example 1, right side is Example 2). Figure 4 It can be seen that the different concentrations of c-Myc protein degrading agents in Examples 1 and 2 all had different degrees of inhibitory effect on c-Myc protein expression in 786-O cells.
[0071] Application Example 3
[0072] The c-Myc protein degrading agent obtained in Example 2 was dissolved in dimethyl sulfoxide to prepare a 10 mmol / L solution, which was designated as the c-Myc protein degrading agent stock solution. It was stored at -80°C and diluted as needed before use.
[0073] Different tumor cell lines (786-O cells (human clear cell adenocarcinoma of the kidney), KYSE-150 cells (human esophageal squamous cell carcinoma cells), U937 cells (human histiocytic lymphoma cells), HUH-7 cells (human hepatocellular carcinoma cells), and A549 cells (human non-small cell lung cancer cells)) were seeded at a density of 1000 cells / well in 96-well cell culture plates containing serum-free medium. After the tumor cells adhered, c-Myc protein degrading agent solution at a concentration of 0.5–20 μmol / L was added, and the cells were incubated at 37°C for 72 h. The medium was then discarded, and 100 μL of CCK-8 (Cell Counting Kit-8) reaction solution was added. The cells were then incubated at 37°C for 0.5–4 h, and the absorbance at 450 nm was measured using a microplate reader to test the effect of the c-Myc protein degrading agent on the proliferation of different tumor cell lines.
[0074] Figure 5 This figure shows the effects of different concentrations of c-Myc protein degrading agents on the proliferation of different tumor cells. Figure 5 It can be seen that as the concentration of c-Myc protein degrader increases, the proliferation of different tumor cells (786-O cells (human clear cell adenocarcinoma cells of the kidney), KYSE-150 cells (human esophageal squamous cell carcinoma cells), U937 cells (human histiocytic lymphoma cells), HUH-7 cells (human liver cancer cells), and A549 cells (human non-small cell lung cancer cells)) is significantly inhibited.
[0075] Application Example 4
[0076] The c-Myc protein degrading agent obtained in Example 2 was dissolved in dimethyl sulfoxide to prepare a 10 mmol / L solution, which was designated as the c-Myc protein degrading agent stock solution. It was stored at -80°C and diluted as needed before use.
[0077] Different tumor cell lines (769-P cells (human renal cell adenocarcinoma cells), 786-O cells (human renal clear cell adenocarcinoma cells), ACHN cells (human renal cell adenocarcinoma cells), Caki-1 cells (human renal clear cell carcinoma skin metastases), and RPTEC / TERT1 cells (renal proximal tubular epithelial cells)) were seeded at a density of 1000 cells / well in 96-well cell culture plates containing serum-free medium. After the tumor cells adhered, c-Myc protein degrading agent solution at a concentration of 0–15 μmol / L was added, and the cells were incubated at 37°C for 72 h. The medium was then discarded, and 100 μL of CCK-8 (Cell Counting Kit-8) reaction solution was added. The cells were then incubated at 37°C for 0.5–4 h, and the absorbance at 450 nm was measured using a microplate reader to test the effect of c-Myc protein degrading agent on the proliferation of 786-O cells.
[0078] Renal cancer cell lines (769-P, 786-O, ACHN, Caki-1) and RPTEC renal epithelial cells were seeded at a density of 20,000 cells / well in 6-well plates and cultured at 37°C for 72 h. Cells were collected, lysed, and proteins were extracted. Western blotting was used to detect the expression level of c-Myc protein in different renal cell lines.
[0079] Figure 6 Inhibiting IC50 at different concentrations of c-Myc protein degrading agent 50 And a bar chart showing the expression of c-Myc protein in different renal cells, by Figure 6 It is known that renal cells with high expression of c-Myc protein are more sensitive to degradation agents.
[0080] Figure 7 Inhibit IC by c-Myc protein degraders 50 A graph showing the correlation between c-Myc protein expression and other proteins. Figure 7 It can be seen that the inhibitory effect of the degrading agent on cell proliferation is negatively correlated with the expression of c-Myc protein in cells.
[0081] Application Example 5
[0082] Inhibitory effect of c-Myc protein degraders on subcutaneous tumor growth
[0083] Drug preparation: The c-Myc protein degrading agent obtained in Example 2 was dissolved in polyethylene glycol 8000 (PEG8000-NaCl solution (30%)) to prepare a solution with a concentration of 10 mg / mL, which was designated as c-Myc protein degrading agent stock solution and stored at -80℃.
[0084] Prepare nude mice: Place 4-6 week old nude mice in an SPF-grade animal laboratory for breeding.
[0085] Prepare ACHN tumor cell suspensions separately: Take logarithmically growing ACHN tumor cells, digest them, centrifuge them, and then count them.
[0086] Subcutaneous xenograft inoculation: Select nude mice aged 4–6 weeks and inoculate ACHN tumor cells into the hind leg of the abdomen of each mouse. Inoculate 100 μL (1–10 × 10⁻⁶) per mouse. 6 indivual).
[0087] Drug administration: Seven days after inoculation with ACHN tumor cells, nude mice were injected with the drug intraperitoneally, with injections every two days. Specifically, the nude mice were divided into three groups. Group 1 mice were injected with c-Myc protein degrading agent solution at a standard dose of 20 mg / kg, and Group 2 mice were injected with c-Myc protein degrading agent solution at a standard dose of 50 mg / kg. The control group was injected with the same volume of polyethylene glycol 8000 (PEG8000-NaCl solution (30%)). The administration continued until differences in the size of subcutaneous tumors in Groups 1 and 2 and the control group were observed at three time points.
[0088] Collect subcutaneous tumors: Sacrifice nude mice, remove subcutaneous tumors, photograph and weigh them.
[0089] Figure 8 The growth curves of subcutaneous tumors in nude mice after treatment with c-Myc protein degrading agent solutions of different concentrations according to the present invention are shown. Figure 9 The morphology of subcutaneous tumors in nude mice after treatment with c-Myc protein degrading agent solutions of different concentrations according to the present invention; Figure 10 The weight of subcutaneous tumors in nude mice after treatment with different concentrations of c-Myc protein degrading agent solutions according to the present invention.
[0090] Depend on Figure 8 It can be seen that, with the passage of time, the growth rate of subcutaneous tumors in the control group (injected with polyethylene glycol 8000) gradually accelerated, while the growth curve of subcutaneous tumors in Experiment 1 (injected with c-Myc protein degrading agent solution at a standard rate of 20 mg / kg) showed a slow upward trend. The growth curve of subcutaneous tumors in Experiment 2 (injected with c-Myc protein degrading agent solution at a standard rate of 50 mg / kg) showed a trend of first decreasing, then slowly increasing, and finally stabilizing. The rate of increase in the growth curve of subcutaneous tumors in Experiments 1 and 2 was much smaller than that in the control group. Figures 9-10 It was found that the subcutaneous tumors in the control group of nude mice injected with polyethylene glycol 8000 were larger, while the subcutaneous tumors in experimental group 1 nude mice injected with c-Myc protein degrading agent solution at standard rates of 20 mg / kg and 50 mg / kg were significantly smaller than those in the control group. This demonstrates that the c-Myc protein degrading agent provided by this invention can effectively inhibit tumor proliferation.
[0091] Liver and kidney tissues were harvested from dead nude mice, sectioned, and stained with hematoxylin and eosin (HE). The HE staining results are as follows: Figure 11 As shown. By Figure 11 It can be seen that the hepatocytes and kidney cells remained morphologically intact, and their nuclei did not show significant enlargement. Therefore, the c-Myc protein degrading agent provided by this invention does not cause significant damage to the liver and kidneys.
[0092] 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 use of a compound having the structure shown in Formula I or a pharmaceutically acceptable salt thereof in the preparation of an anti-hepatocellular carcinoma, lung cancer, kidney cancer, esophageal cancer, or leukemia drug; wherein the anti-hepatocellular carcinoma, lung cancer, kidney cancer, esophageal cancer, or leukemia drug inhibits tumor cell proliferation by degrading c-Myc protein; ； In Equation I, R is either -F or -CF3.
2. The application according to claim 1, characterized in that, The compound has the structure shown in Formula II or Formula III: ； 。 3. The application according to claim 1, characterized in that, The drug can be administered orally, intravenously, or subcutaneously.