A curcuminol derivative for treating tumors and its preparation method and application

Abstract

The present invention provides a curcuminol derivative for treating tumors and a preparation method and application thereof, which belongs to the field of medical technology. The present invention obtains a curcuminol derivative, HCL-23, for the first time by structurally modifying curcuminol. The test results show that HCL-23 inhibits cancer cell proliferation by inducing G2 phase cycle arrest, apoptosis and ferroptosis of cancer cells, and can inhibit cancer cell metastasis. In vivo tests show that HCL-23 can inhibit the growth of tumor volume in tumor-bearing mice, reduce the tumor volume and weight of tumor-bearing mice, and thus play an anti-tumor role. The curcuminol derivative HCL-23 of the present invention provides a new idea for the treatment of tumors, especially the research and development of breast cancer drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a curcuminol derivative for treating tumors and a preparation method and application thereof. Background Art

[0002] Tumors are the primary major disease that threatens human health and life, and are a difficult problem that modern medicine is facing. At present, the treatment strategies for malignant tumors include surgical treatment, chemotherapy, radiotherapy, endocrine therapy, targeted therapy, etc. However, for patients with malignant tumors who are in poor general condition, old and weak and cannot tolerate surgery, it is necessary to formulate corresponding chemotherapy and radiotherapy plans for treatment, so the development of innovative drugs that effectively treat malignant tumors is urgent. For decades, compared with many chemical drugs, natural products have the advantages of being non-mutagenic, low toxicity, safe and reliable, and effective for a variety of diseases. Therefore, natural products have always been an important source of new drugs, providing many successful chemical entities and lead compounds for drug discovery.

[0003] Curcumol is a colorless needle-shaped crystal, a sesquiterpenoid compound, and an important monomer component extracted and separated from the volatile oil of Curcuma zedoariae. As an anti-tumor drug discovered in recent years, curcumol has been increasingly studied in tumor treatment due to its effectiveness and low toxicity. However, due to the extremely poor water solubility of curcumol, its anti-tumor activity is greatly affected, which limits its clinical application. Therefore, it is necessary to modify its structure. However, it is not necessary for the curcumol after structural modification to have anti-tumor activity. Therefore, obtaining a curcumol derivative with anti-tumor activity is a technical problem that needs to be solved urgently. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a curcuminol derivative for treating tumors and a preparation method and application thereof.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a curcuminol derivative for treating tumors, and the structural formula of the curcuminol derivative is as follows:

[0007] The present invention also provides an application of the curcuminol derivative in preparing a drug for treating tumors.

[0008] Preferably, the tumor comprises breast cancer.

[0009] Preferably, the curcuminol derivatives induce G2 phase cell cycle arrest, apoptosis and ferroptosis in tumor cells.

[0010] Preferably, the curcuminol derivatives inhibit the proliferation and metastasis of tumor cells.

[0011] The present invention also provides a medicine for treating tumors, wherein the active ingredient of the medicine comprises the above-mentioned curcuminol derivative.

[0012] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0013] The present invention also provides a method for preparing the above-mentioned curcumol derivative, characterized in that it comprises the following steps:

[0014] Mixing curcumol, m-chloroperbenzoic acid and a first organic solvent, and performing an epoxidation reaction to obtain an oily liquid; mixing the oily liquid, a second organic solvent and an inorganic base, and performing a hydrolysis ring-opening and elimination reaction to obtain a compound EZC-17; mixing the compound EZC-17, p-nitrobenzoic acid, dicyclohexylcarbodiimide, a catalyst and the first organic solvent, and performing an esterification reaction to obtain a compound HCL-13; mixing the compound HCL-13, sodium hydride, chloroacetic anhydride and the first organic solvent, and performing an esterification reaction to obtain the curcumol derivative;

[0015] The structural formula of the compound EZC-17 is The structural formula of the compound HCL-13 is

[0016] Preferably, the first organic solvent comprises dichloromethane; and the second organic solvent comprises isopropanol.

[0017] Preferably, the inorganic base includes one of sodium hydroxide and potassium hydroxide; and the catalyst includes 4-dimethylaminopyridine.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a curcuminol derivative for treating tumors, a preparation method and an application thereof. By modifying the structure of curcuminol, a curcuminol derivative, HCL-23, is obtained for the first time. The test results show that HCL-23 inhibits cancer cell proliferation by inducing G2 phase cycle arrest, cell apoptosis and ferroptosis of cancer cells, and can also inhibit cancer cell metastasis. In vivo tests show that HCL-23 can inhibit the growth of tumor volume in tumor-bearing mice, reduce the tumor volume and weight of tumor-bearing mice, and thus play an anti-tumor role. The curcuminol derivative HCL-23 of the present invention provides a new idea for the research and development of drugs for treating tumors, especially breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of different concentrations of HCL-23 on breast cancer cells MDA-MB-231;

[0021] Figure 2 The effect of HCL-23 drug on the morphology of MDA-MB-231 cells;

[0022] Figure 3 The effect of HCL-23 drug on the proliferation of MDA-MB-231 cells;

[0023] Figure 4 The effect of HCL-23 on the cell cycle of MDA-MB-231 cells;

[0024] Figure 5 The effect of HCL-23 on apoptosis of MDA-MB-231 cells;

[0025] Figure 6 A is the effect of HCL-23 on ferroptosis of MDA-MB-231 cells, A is the effect of HCL-23 on the lipid peroxidation level of MDA-MB-231 cells, and B is the effect of HCL-23 on the variable iron pool level of MDA-MB-231 cells;

[0026] Figure 7 The effect of HCL-23 on the metastatic ability of MDA-MB-231 cells, A is the result of the scratch healing assay, B is the result of the Transwell migration assay, and C is the result of the Transwell invasion assay;

[0027] Figure 8 This is an in vivo experiment to evaluate the effect of HCL-23 on breast cancer. A is the effect of HCL-23 on tumor volume growth, B is the effect of HCL-23 on tumor volume, and C is the effect of HCL-23 on tumor weight;

[0028] Fig. 9 is the structural formula of the curcuminol derivative prepared in Example 1. DETAILED DESCRIPTION

[0029] The present invention provides a curcuminol derivative for treating tumors, characterized in that the structural formula of the curcuminol derivative is as follows:

[0030] The present invention also provides an application of the curcuminol derivative in preparing a drug for treating tumors.

[0031] In the present invention, the tumor preferably includes breast cancer, and more preferably includes triple-negative breast cancer. The research results show that the curcuminol derivatives of the present invention induce G2 phase cycle arrest, apoptosis and ferroptosis in tumor cells, thereby inhibiting the proliferation and metastasis of tumor cells.

[0032] The present invention also provides a medicine for treating tumors, wherein the active ingredient of the medicine comprises the above-mentioned curcuminol derivative.

[0033] In the present invention, the drug preferably further comprises a pharmaceutically acceptable adjuvant, and the adjuvant preferably comprises one or more of a binder, an excipient, a diluent, a disintegrant, a wetting agent, a cosolvent and a surfactant. The drug described in the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously or nasally. The dosage form of the drug includes tablets, pills, granules, injections or capsules. The content of the active ingredient curcumol derivative in the present invention is 1 to 99% of the drug. The dosage of the drug of the present invention can be determined by factors such as the type of disease to be treated, the severity of the disease, the route of administration, the age, sex, and health status of the patient. For example, the dosage of the drug of the present invention can be 0.01 μg to 1000 mg / day per patient.

[0034] The present invention also provides a method for preparing the above-mentioned curcumol derivative, characterized in that it comprises the following steps:

[0035] Mixing curcumol, m-chloroperbenzoic acid and a first organic solvent, and performing an epoxidation reaction to obtain an oily liquid; mixing the oily liquid, a second organic solvent and an inorganic base, and performing a hydrolysis ring-opening and elimination reaction to obtain a compound EZC-17; mixing the compound EZC-17, p-nitrobenzoic acid, dicyclohexylcarbodiimide, a catalyst and the first organic solvent, and performing an esterification reaction to obtain a compound HCL-13; mixing the compound HCL-13, sodium hydride, chloroacetic anhydride and the first organic solvent, and performing an esterification reaction to obtain the curcumol derivative;

[0036] The structural formula of the compound EZC-17 is The structural formula of the compound HCL-13 is

[0037] In the present invention, curcumin, meta-chloroperbenzoic acid and a first organic solvent are mixed, and an epoxidation reaction is carried out to obtain an oily liquid. The first organic solvent preferably includes dichloromethane; the molar ratio of curcumin to meta-chloroperbenzoic acid is preferably 1:1.5; the epoxidation reaction is preferably carried out at room temperature until the reaction is complete. In the present invention, TLC is preferably used to track and monitor the progress of the reaction. The mixing method can be a method known in the art, such as stirring. In the present invention, before obtaining the oily liquid, it is also preferably included in the steps of washing with saturated sodium bisulfite, saturated sodium bicarbonate, and saturated saline in sequence, drying with anhydrous sodium sulfate, and concentrating under reduced pressure.

[0038] In the present invention, the oily liquid, the second organic solvent and the inorganic base are mixed, and hydrolysis ring opening and elimination reactions are carried out to obtain compound EZC-17. The second organic solvent preferably includes isopropanol. The inorganic base preferably includes one of sodium hydroxide and potassium hydroxide. The reaction temperature of the hydrolysis ring opening and elimination reaction is preferably 65-75°C. Before obtaining compound EZC-17, it also includes the steps of washing with saturated tartaric acid and saturated salt water in sequence, drying with anhydrous sodium sulfate and concentrating under reduced pressure, and separating and purifying by column chromatography. The column chromatography is preferably a silica gel column (300-400 mesh), and the solvent used for column chromatography separation and purification is preferably a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 3:1. The compound EZC-17 is a white solid.

[0039] In the present invention, the compound EZC-17, p-nitrobenzoic acid, dicyclohexylcarbodiimide, a catalyst and a first organic solvent are mixed and subjected to an esterification reaction to obtain a compound HCL-13. The molar ratio of the compound EZC-17, p-nitrobenzoic acid and dicyclohexylcarbodiimide is preferably 1:1.2:3. The catalyst preferably includes 4-dimethylaminopyridine. The first organic solvent preferably includes dichloromethane. The esterification reaction is carried out at room temperature until the reaction is complete. In the present invention, TLC is preferably used to track and monitor the progress of the reaction. Before obtaining the compound HCL-13, it also includes the steps of washing with saturated sodium bicarbonate, saturated tartaric acid, and saturated salt water in sequence, drying with anhydrous sodium sulfate, concentrating under reduced pressure, and separating and purifying by column chromatography. The column chromatography is preferably a silica gel column (300-400 mesh), and the solvent used for separation and purification by column chromatography is a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is preferably 10:1. The compound HCL-13 is a colorless, transparent, oily liquid.

[0040] In the present invention, the compound HCL-13, sodium hydride, chloroacetic anhydride and the first organic solvent are mixed and subjected to esterification reaction to obtain the curcuminol derivative. The molar ratio of the compound HCL-13, sodium hydride and chloroacetic anhydride is preferably 1:10:2.5. The first organic solvent preferably includes dichloromethane. The esterification reaction is carried out at room temperature until the reaction is complete. In the present invention, TLC is preferably used to track and monitor the reaction process. Before obtaining the curcuminol derivative, it also includes quenching with water, washing with saturated tartaric acid and saturated salt water in turn, drying with anhydrous sodium sulfate and concentrating under reduced pressure, and separating and purifying by column chromatography. The column chromatography is preferably a silica gel column (300-400 mesh), and the solvent used for the column chromatography separation and purification is preferably a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 15:1. The curcuminol derivative is a colorless transparent liquid.

[0041] The synthetic route of the curcumol derivative is as follows:

[0042]

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] In the following examples, the curcuminol derivative is referred to as HCL-23.

[0045] The data processing method of the present invention was that each experiment was repeated three times independently, all data were expressed as mean±SD, and Student's t-test was performed using GraphPad Prism 6. P<0.05 was considered statistically significant, *P<0.05, **P<0.01, ***P<0.001.

[0046] Example 1

[0047] A method for preparing a curcuminol derivative, HCL-23, comprises the following steps:

[0048] Curcuma alcohol (3 g, 1 equiv.) was dissolved in dichloromethane, and meta-chloroperbenzoic acid (3.28 g, 1.5 equiv.) was added. The mixture was stirred at room temperature and monitored by TLC (5% sulfuric acid ethanol for color development) until the reaction was completed. The mixture was washed with saturated sodium bisulfite, saturated sodium bicarbonate, and saturated brine in sequence. After drying over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure to obtain an oily liquid. The oily liquid was dissolved in isopropanol, and sodium hydroxide (1.525 g, 3 equiv.) was added. The mixture was reacted at 70°C and monitored by TLC (5% sulfuric acid ethanol for color development) until the reaction was completed. The mixture was washed with saturated tartaric acid and saturated brine in sequence. After drying over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure. 2.242 g of a white solid intermediate (EZC-17) was separated by column chromatography (petroleum ether: ethyl acetate = 3:1) with a yield of 70%. The intermediate EZC-17 (50 mg, 1 equiv.) was dissolved in dichloromethane, and then p-nitrobenzoic acid (40 mg, 1.2 equiv.), dicyclohexylcarbodiimide (DCC) (123 mg, 3 equiv.) and a catalytic amount of 4-dimethylaminopyridine (DMAP) were added in sequence. The mixture was stirred at room temperature and monitored by TLC (5% sulfuric acid ethanol for color development) until the reaction was complete. The mixture was washed with saturated sodium bicarbonate, saturated tartaric acid and saturated brine in sequence, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 53 mg of colorless transparent oily liquid (HCL-13) was obtained by separation by column chromatography (petroleum ether: ethyl acetate = 10: 1). The yield was 67%.

[0049] The previously prepared compound HCL-13 (100 mg, 1 equiv.) was dissolved in dichloromethane, sodium hydride (60 mg, 10 equiv.) was added, and the mixture was stirred at room temperature for 30 min. Then, chloroacetic anhydride (106 mg, 2.5 equiv.) was added, and the mixture was stirred at room temperature. TLC (5% sulfuric acid ethanol color development) was used to track the reaction until it was complete. The reaction was quenched with water, and the mixture was washed with saturated tartaric acid and saturated brine in turn. After drying over anhydrous sodium sulfate, the mixture was concentrated under reduced pressure. 60 mg of colorless transparent liquid (HCL-23) was obtained by column chromatography (petroleum ether: ethyl acetate = 15: 1), i.e., a curcuminol derivative, with a yield of 49%.

[0050] The structural characterization data of HCL-13 are as follows:

[0051] 1H NMR (600MHz, Acetone-d6) δ8.38-8.40(2H,dt,J=8.8,1.9Hz,H-4′,6′),8.29-8.31(2H,dt,J=8.8,1.9Hz,H-3′,H-7′),6.00(1H,s,H-7),5.35(1H,s ,OH),4.83-4.91(2H,m,H-9),1.00-1.01(3H,d,J=6.6Hz,H-11),0.97-1. 01(3H,d,J=6.6Hz,H-12),0.86-0.88(3H,d,J=6.6Hz,H-13); 13CNMR(150M Hz, Acetone-d6)δ:164.1(C-1′),150.8(C-5′),137.7(C-8),135.6(C-2′ ),130.7(C-3′,7′),128.9(C-7),123.7(C-4′,6′),103.0(C-6),85.9(C-3 α),66.8(C-9),59.1(C-5),49.6(C-8α),40.1(C-3),36.2(C-4),31.3(C-2 ),30.8(C-10),27.5(C-1),22.3(C-11),21.0(C-12),11.2(C-13); ESI-MS m / z:424.1[M+Na]+.

[0052] The structural characterization data of the curcuminol derivative HCL-23 are as follows:

[0053] 1H NMR (600MHz, Acetone-d6) δ8.38-8.40(2H,d,J=8.9Hz,H-4′,6′),8.30-8.32(2H,d,J=8.9Hz,H-3′,H-7′),6.17(1H,s,H-7),4.88-4.94(m,2 13C NMR (150MHz, Acetone-d6) δ164.1(C-1′,1″),150.8(C-5′),136.7(C-8),135.6( C-2′),130.7(C-3′,7′),126.2(C-7),123.7(C-4′,C-6′),106.2(C-6),89.1(C-3 a),66.7(C-9),57.8(C-5),49.4(C-8a),41.3(C-2″),39.8(C-3),34.5(C-4),31 .0(C-2),30.2(C-10),27.4(C-1),21.9(C-11),20.8(C-12),11.1(C-13); ESI-MS m / z:500.0[M+Na]+.

[0054] Example 2

[0055] Effect of the Curcumol Derivative - HCL-23 Prepared in Example 1 on Tumor Activity

[0056] 2.1 Test of HCL-23 on MDA-MB-231 cell viability

[0057] MDA-MB-231 cells were collected at logarithmic phase and cultured at 6×10 3 Cells were planted at a density of 100 μL / well in a 96-well plate and cultured overnight. After the cells adhered to the wall, different concentrations of HCL-23 (1.25, 2.5, 5, 10, 20 μM) were added for drug treatment, and 0.1% DMSO was used as the control group. After 72 hours of HCL-23 drug treatment, 10 μL of 5 mg / mL MTT was added to each well, incubated at 37°C for 4 hours, the supernatant was discarded, and 160 μL of DMSO was added to each well to dissolve. After the blue-purple crystalline formazan was completely dissolved, the absorbance value OD (490 nm) was measured by a microplate reader.

[0058] Calculation method of inhibition rate: inhibition rate = (control group OD 490nm - Treatment group OD 490nm) / control group OD 490nm The half inhibition rate (IC 50 ) calculation method: Use the forecast function to obtain the corresponding IC of the compound 50 .

[0059] like Figure 1 Figure A shows that different concentrations of HCL-23 can significantly inhibit the cell viability of MDA-MB-231 in a concentration-dependent manner, and the IC 50 The value was 7.18±0.41μM.

[0060] At the same time, in this example, the HCL-13 prepared in Example 1 was further tested for the viability of MDA-MB-231 cells.

[0061] MDA-MB-231 cells were collected at logarithmic phase and cultured at 6×10 3 Cells were planted at a density of 1000 cells / well in a 96-well plate and cultured overnight. After the cells adhered to the wall, 20 μM HCL-13 was added for drug treatment, and 0.1% DMSO was used as the control group. After 72 hours of 20 μM HCL-13 drug action, 10 μL of 5 mg / mL MTT was added to each well, incubated at 37°C for 4 hours, the supernatant was discarded, and 160 μL DMSO was added to each well to dissolve. After the blue-purple crystalline formazan was completely dissolved, the absorbance value OD (490 nm) was measured by a microplate reader.

[0062] The calculation method of inhibition rate is: inhibition rate = (control group OD490nm - treatment group OD490nm) / control group OD490nm. Half inhibition rate (IC 50 ) calculation method: Use the forecast function to obtain the corresponding IC of the compound 50 .

[0063] The results showed that the IC of HCL-13 50 The value was greater than 20 μM, and there was no inhibitory effect on MDA-MB-231 cells. Therefore, HCL-23 was subsequently used to study breast cancer.

[0064] 2.2 Effect of HCL-23 on the morphology of MDA-MB-231 cells

[0065] MDA-MB-231 cells were collected at logarithmic phase and cultured at 6×10 3 The cells were seeded at a density of 100 μg / well in a 96-well plate and cultured overnight. After the cells adhered to the wall, different concentrations of HCL-23 (5, 10, 20 μM) were added for treatment, and 0.1% DMSO was used as the control group. The morphological changes of the cells were observed after 12h, 24h, 36h and 48h of HCL-23 treatment.

[0066] like Figure 2 As shown, with the increase of HCL-23 concentration and the extension of action time, the number of MDA-MB-231 cells decreased significantly, and after HCL-23 acted on MDA-MB-231 cells, the cell morphology changed from long spindle to round, indicating that HCL-23 inhibited the proliferation of MDA-MB-231 cells.

[0067] 2.3 Effect of HCL-23 on cell proliferation of MDA-MB-231 cells

[0068] MDA-MB-231 cells were collected at logarithmic phase and cultured at 6×10 3 The cells were seeded at a density of 100 / well in a 96-well plate and cultured overnight. After the cells attached to the wall, high, medium and low (20 μM, 10 μM and 5 μM) concentrations of drugs were added for treatment, and 0.1% DMSO was used as the control group. OD was measured at 0h, 24h, 48h, 72h and 96h. 490nm , and draw a growth curve.

[0069] like Figure 3 As shown, compared with the control group, different concentrations of HCL-23 could inhibit the proliferation of MDA-MB-231 cells, and with the increase of HCL-23 concentration, the growth rate of MDA-MB-231 cells gradually slowed down, indicating that HCL-23 could inhibit the proliferation of MDA-MB-231 in a concentration-dependent manner.

[0070] 2.4 Effect of HCL-23 on the cell cycle of MDA-MB-231 cells

[0071] MDA-MB-231 cells were cultured at 2.0 × 10 5 Cells were planted in 6-well plates for culture. After the cells adhered to the wall, they were treated with high, medium and low concentrations of HCL-23 (20μM, 10μM and 5μM), respectively. 0.1% DMSO was used as the control group. After 12 hours, the cells were collected. Centrifuged at 1000rpm for 5min, washed once with pre-cooled PBS, and resuspended with 70% pre-cooled ethanol, and fixed at -20℃ overnight. Centrifuged at 1000rpm for 5min, discarded the supernatant, washed once with PBS, centrifuged at 1000rpm for 5min, and the cell pellet was collected. Resuspended the cells with 500μL PBS, added 5μL RNaseA, 25μL PI and 0.25μL Triton X-1000, flicked to mix, incubated at 37℃ in the dark for 30min, centrifuged at 1000rpm for 5min, discarded the dye, resuspended with 200μL PBS, and analyzed by flow cytometry.

[0072] like Figure 4As shown, compared with the control group, after 12 hours of HCL-23 treatment, the proportion of G2 phase cells increased from 20.25%±0.66% to 35.80%±1.66% (20μM). The results showed that HCL-23 can significantly increase the proportion of MDA-MB-231 cells in G2 phase and block the transformation of MDA-MB-231 cells from G2 phase to M phase.

[0073] 2.5 Detection of apoptosis of MDA-MB-231 cells by HCL-23

[0074] MDA-MB-231 cells were cultured at 2.0 × 10 5 Cells were planted in 6-well plates at 100 μL / well for culture. After the cells adhered to the wall, they were treated with high, medium and low concentrations of HCL-23 (20 μM, 10 μM and 5 μM) respectively. 0.1% DMSO was used as the control group. After 24 hours, the cells were collected. Centrifuged at 1000 rpm for 5 min, washed once with pre-cooled PBS, and resuspended with 100 μL 1×binding buffer. 5 μL FITC-Annexin V and 5 μL PI dye were added to each tube, flicked to mix, incubated at room temperature in the dark for 15 min, centrifuged at 1000 rpm for 5 min, discarded the dye, resuspended with 100 μL 1×binding buffer, and analyzed by flow cytometry.

[0075] like Figure 5 As shown, compared with the control group, the total apoptosis rate in the HCL-23 treatment group increased from 4.67%±1.00% to 6.88%±0.93% (10 μM) and 24.14%±0.38% (20 μM). These results indicate that HCL-23 can induce apoptosis of MDA-MB-231 cells in a concentration-dependent manner.

[0076] 2.6 Effect of HCL-23 on ferroptosis of MDA-MB-231 cells

[0077] The effect of HCL-23 on ferroptosis of MDA-MB-231 cells was investigated by detecting the levels of cellular lipid peroxidation and variable iron pool.

[0078] 2.6.1 Cellular lipid peroxidation assay

[0079] MDA-MB-231 cells were cultured at 2.0 × 10 5Cells were planted in 6-well plates for culture. After the cells attached to the wall, they were treated with high, medium and low concentrations of HCL-23 (20μM, 10μM and 5μM), respectively. 0.1% DMSO was used as the control group. After 4 hours of action, 1μM C11BODIPY (581 / 591) probe was added, gently shaken to mix, and incubated at 37℃ in the dark for 30 minutes, and then the cells were collected. Centrifuged at 1500rpm for 5 minutes. Resuspended in 100μL PBS and analyzed by flow cytometry. (Oxidation of the polyunsaturated butadiene part of C11 BODIPY causes the fluorescence emission peak to move from ~590nm to ~510nm, which is proportional to the production of lipid peroxidation.)

[0080] 2.6.2 Detection of variable cellular iron pools

[0081] MDA-MB-231 cells were cultured at 3.0 × 10 5 Cells were cultured in 6-well plates with 100 μM cells / well. After the cells adhered to the wall, they were treated with high, medium and low concentrations of HCL-23 (20 μM, 10 μM and 5 μM) respectively. 0.1% DMSO was used as the control group. After 4 hours of action, 0.5 μM calcein-ethoxymethyl ester (C-AM) was added, gently shaken and mixed, and incubated at 37°C in the dark for 30 minutes before collecting the cells. Centrifuged at 1500 rpm for 5 minutes, washed with PBS, and incubated at 37°C for 1 hour with or without 120 μM deferoxamine (DFO), and analyzed by flow cytometry. The level of the variable iron pool was calculated based on the difference in the average fluorescence of cells with or without deferoxamine.

[0082] like Figure 6 The results showed that after the action of HCL-23, the levels of lipid peroxidation and variable iron pool in MDA-MB-231 cells increased significantly and in a concentration-dependent manner, indicating that HCL-23 triggered ferroptosis in breast cancer MDA-MB-231 cells.

[0083] 2.7 Effect of HCL-23 on the metastatic ability of MDA-MB-231 cells

[0084] The scratch healing assay and Transwell migration and invasion assay were used to detect the effect of HCL-23 on the metastatic ability of MDA-MB-231 cells at the two-dimensional and three-dimensional levels.

[0085] 2.7.1 Scratch healing test

[0086] (1) Use a marker to draw straight lines every 1 cm on the back of the 6-well plate to facilitate observation of each area under a microscope.

[0087] (2) MDA-MB-231 cells were cultured at 6×10 5Each well was planted in a marked 6-well plate for culture. When the cells adhered to the wall and covered the bottom of the plate, a 10 μL pipette tip was used to scratch the cells perpendicular to the well plate and line.

[0088] (3) Discard the old culture medium and gently wash the cells three times with sterile preheated PBS. After removing the scratched cells, treat them with low serum culture medium (containing 2% FBS) containing high, medium and low concentrations of HCL-23 (20 μM, 10 μM and 5 μM), respectively, with 0.1% DMSO as the control group.

[0089] (4) Place the cells in a 37°C incubator and take photos at 0 h and 24 h.

[0090] (5) Image data analysis: Image Pro Plus software was used to measure the scratch distance at each time point in each group. The cell migration rate was calculated according to the following formula:

[0091] Cell migration rate (%) = (0h scratch width - scratch width after culture) / 0h scratch width × 100%

[0092] 2.7.2 Transwell migration and invasion assay

[0093] (1) MDA-MB-231 cells were cultured at 2.0×10 5 After the cells adhered to the wall, they were treated with high, medium and low concentrations of HCL-23 (20 μM, 10 μM and 5 μM), respectively, with 0.1% DMSO as the control group. After 24 h, the cells were collected and counted, and single cell suspension was prepared in serum-free medium (containing 0.05% BSA) to adjust the concentration to 1×10 5 Pieces / mL.

[0094] (2) Matrigel coating of Transwell invasion chamber: Dilute Matrigel stock solution with serum-free culture medium at a ratio of 1:5, add 100 μL / well to the Transwell invasion chamber, and place at 37°C for 4 h (Matrigel coating is not required for Transwell migration assay).

[0095] (3) Add 600 μL of culture medium containing 15% FBS to a 24-well culture plate, then gently place the Transwell chamber into the well to prevent bubbles from forming at the bottom, and then add 200 μL of blood-free monocytic suspension into the well.

[0096] (4) After culturing for 24 h, the Transwell chamber was removed, the culture medium in the chamber was discarded, the cells on the upper layer were gently wiped off with a cotton swab, the chamber was fixed with methanol for 30 min, and then stained with 0.1% crystal violet stain for 20 min.

[0097] (5) Wash the Transwell chamber three times with PBS, gently wipe off the water in the upper layer with a cotton swab, and randomly select five fields of view under an inverted microscope (200×) to take pictures and count.

[0098] like Figure 7 As shown in the scratch-healing test, the cell migration ability of the HCL-23-treated group was reduced compared with the control group, and it was concentration-dependent. The results of the Transwell migration and invasion assay showed that the number of cells in the HCL-23-treated group that passed through the lower chamber was significantly reduced, and it was concentration-dependent. This shows that HCL-23 can inhibit the migration and invasion of breast cancer cells MDA-MB-231.

[0099] 2.8 In vivo evaluation of the effect of HCL-23 on breast cancer

[0100] Take 4T1-luc mouse triple-negative breast cancer cells in the logarithmic growth phase, collect the cells, resuspend them in DMEM medium, prepare single cell suspension, and adjust the cell density to 1.25×10 7 cell·mL -1 80 μL of cell suspension was subcutaneously injected into the second nipple fat pad on the right side of each mouse. The successfully established breast cancer tumor-bearing mice were randomly divided into a negative control group and an HCL-23 group. The HCL-23 group was intraperitoneally injected with 50 mg / kg of HCL-23 daily, and the negative control group was intraperitoneally injected with an equal volume of 0.9% NaCl daily for 14 consecutive days. Starting from the first day after administration, the long diameter (A) and short diameter (B) of the tumor were measured with a vernier caliper every other day. The tumor volume = 0.5 × long diameter (A) × short diameter (B) 2 When the tumor in the negative control group grew to a certain volume, the mice in each group were anesthetized and killed, and the transplanted tumor tissues of the mice were dissected and removed, accurately weighed, and statistically analyzed.

[0101] like Figure 8 As shown, compared with the negative control group, the tumor growth rate and tumor weight in the HCL-23 group were significantly reduced, indicating that HCL-23 has the effect of inhibiting breast cancer.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A curcuminol derivative for treating tumors, characterized in that: The structural formula of the curcuminol derivative is as follows:

2. Use of the curcumol derivatives according to claim 1 in the preparation of drugs for treating tumors.

3. The use according to claim 2, characterized in that: The tumor is breast cancer.

4. A drug for treating tumors, characterized in that: The active ingredient of the medicine comprises the curcuminol derivative according to claim 1.

5. The drug according to claim 4, characterized in that The drug also includes pharmaceutically acceptable excipients.

6. The method for preparing the curcumol derivative according to claim 1, characterized in that: The steps include: The curcumin, m-chloroperbenzoic acid and a first organic solvent are mixed and subjected to epoxidation reaction to obtain an oily liquid; the oily liquid, a second organic solvent and an inorganic base are mixed and subjected to hydrolysis ring-opening and elimination reaction to obtain compound EZC-17; the compound EZC-17, p-nitrobenzoic acid, dicyclohexylcarbodiimide, a catalyst and a first organic solvent are mixed and subjected to esterification reaction to obtain compound HCL-13; the compound HCL-13, sodium hydride, chloroacetic anhydride and a first organic solvent are mixed and subjected to esterification reaction to obtain the curcumin derivative; the structural formula of the compound EZC-17 is The structural formula of the compound HCL-13 is 7. The preparation method according to claim 6, characterized in that: The first organic solvent is dichloromethane; the second organic solvent is isopropanol.

8. The preparation method according to claim 6, characterized in that: The inorganic base is one of sodium hydroxide and potassium hydroxide; and the catalyst is 4-dimethylaminopyridine.

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