A dehydrocostus lactone derivative and its preparation method and use

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a dehydrocostus lactone derivative, a preparation method and use thereof. The structure of the dehydrocostus lactone derivative is shown in Formula I. The compound exhibits a significantly better proliferation inhibition effect than dehydrocostus lactone on human lung cancer cells, human liver cancer cells, human colon cancer cells, human esophageal cancer cells, human renal clear cell adenocarcinoma cells, human bladder cancer cells, human prostate cancer cells, human breast cancer cells and human brain tumor cells. The compound has a significant anti-colon cancer effect, can induce concentration-dependent stagnation of HCT116 cells in the G0 / G1 phase, and at the same time concentration-dependently causes early apoptosis and late apoptosis of HCT116 cells. The dehydrocostus lactone derivative has broad application prospects in the preparation of anti-tumor drugs. .

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a dehydrocostus lactone derivative and a preparation method and application thereof. Background Art

[0002] Cancer, also known as malignant tumor, is a type of disease characterized by abnormal cell proliferation. It is a pathological phenomenon in which human cells lose normal growth and division regulation after genetic material (DNA) is damaged. Normal cells follow certain growth and apoptosis rules under the regulation of the body. However, when DNA mutations or other mechanisms cause the failure of cell growth control, these abnormal cells may form tumors, destroy normal tissues through diffusion and invasion, leading to organ failure; and invade surrounding tissues or transfer to other parts of the body through the blood and lymphatic systems, causing metastatic damage. At the same time, cancer cells consume a lot of nutrients, and patients may develop cachexia, which manifests as extreme weight loss and weakness; it may also cause complications such as infection, bleeding, and decreased immune function. Ultimately, it aggravates systemic damage and directly threatens life.

[0003] Natural products have always been an important source of candidate compounds for new drug development. Currently, about 40% of anti-tumor drugs in the world are derived from natural products and their derivatives or analogs. Developing anti-tumor drugs with better activity and higher safety based on the structure of natural products has always been a hot topic in this field.

[0004] Dehydrocostus lactone is a guaiac-type sesquiterpenoid compound distributed in Asteraceae plants and is also a traditional Chinese medicine Yunmuxiang ( Aucklandiacostus Falc.) has been reported in many literatures to have good anti-tumor effects. Its structural formula is as follows:

[0005]

[0006] In order to meet the needs of clinical applications, it is necessary to further improve the anti-tumor effect of dehydrocostus lactone. Therefore, it is of great significance to modify the structure based on the dehydrocostus lactone skeleton and find anti-tumor compounds with better activity. However, in the process of structural modification of dehydrocostus lactone, it was found that derivatives with different substituents showed great differences in anti-tumor activity. Therefore, how to design the molecular structure to obtain dehydrocostus lactone derivatives with excellent anti-tumor activity is an important topic in this field. Summary of the invention

[0007] In order to solve the above problems existing in the prior art, the object of the present invention is to provide a dehydrocostus lactone derivative and a preparation method and use thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention provides a dehydrocostus lactone derivative, or a stereoisomer thereof, or a solvate thereof, or a metabolite thereof, or a deuterated product thereof, or a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a cocrystal thereof. The structure of the dehydrocostus lactone derivative is shown in Formula I:

[0010] .

[0011] The present invention also provides a method for preparing the above-mentioned dehydrocostus lactone derivative, which specifically comprises the following steps:

[0012] ;

[0013] Step 1: Dehydrocostus lactone reacts with an oxidizing agent to obtain intermediate A;

[0014] Step 2: The intermediate A reacts with the raw material B to obtain the compound of formula I.

[0015] Furthermore, in step 1, the oxidizing reagent is selenium dioxide and tert-butyl hydroperoxide; the solvent of the reaction is dichloromethane; the temperature of the reaction is -4~45ºC; the reaction time is 3~30 h; in step 2, the reaction is carried out under the action of a condensing agent and a catalyst; the solvent of the reaction is dichloromethane; the temperature of the reaction is 15~45ºC; the reaction time is 3~30 h.

[0016] Furthermore, in step 1, the molar ratio of selenium dioxide, tert-butyl hydroperoxide, and dehydrocostus lactone is 1:4~5:7~8; the reaction temperature is 0~30ºC; and the reaction time is 24 h; in step 2, the condensation agent is N,N'-dicyclohexylcarbodiimide; the catalyst is 4-dimethylaminopyridine; the molar ratio of 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide, raw material B, and intermediate A is 1:5~7:7~8:23~27; the reaction temperature is 30ºC; and the reaction time is 24 h.

[0017] Furthermore, in step 1, the molar ratio of selenium dioxide, tert-butyl hydroperoxide, and dehydrocostus lactone is 1:4.4:7.8; in step 2, the molar ratio of 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide, raw material B, and intermediate A is 1:6:7.5:25.

[0018] The present invention also provides a pharmaceutical composition, which is prepared by using the above-mentioned compound, or its stereoisomer, or its solvate, or its metabolite, or its deuterated product, or its prodrug, or its pharmaceutically acceptable salt, or its cocrystal as an active ingredient and adding a pharmaceutically acceptable excipient.

[0019] The present invention also provides use of the above-mentioned pharmaceutical composition in preparing anti-tumor drugs.

[0020] Furthermore, the tumor is lung cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer or brain tumor, the kidney cancer is preferably renal clear cell adenocarcinoma, and the brain tumor is preferably glioma.

[0021] The present invention also provides the use of the above-mentioned compound, or its stereoisomer, or its solvate, or its metabolite, or its deuterated product, or its prodrug, or its pharmaceutically acceptable salt, or its cocrystal in the preparation of anti-tumor drugs.

[0022] Furthermore, the tumor is lung cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer or brain tumor, the kidney cancer is preferably renal clear cell adenocarcinoma, and the brain tumor is preferably glioma.

[0023] The present invention has achieved the following beneficial effects:

[0024] The present invention optimizes the structure of dehydrocostus lactone and provides a new guaiacyl sesquiterpene lactone-6-nicotinate compound DLDA. The compound exhibits a significantly better proliferation inhibition effect than dehydrocostus lactone on human lung cancer cells (A549), human liver cancer cells (HepG2), human colon cancer cells (HCT116), human esophageal cancer cells (TE-1), human renal clear cell adenocarcinoma cells (786-O), human bladder cancer cells (5637), human prostate cancer cells (DU145), human breast cancer cells (MCF-7) and human brain tumor cells (SF126). The half inhibitory concentration (IC 50 ) were 0.86, 1.24, 0.24, 2.23, 0.97, 1.35, 1.64, 0.78 and 1.85 μM, respectively. Among them, the anti-proliferation effect on human colon cancer cell HCT116 was the strongest. Preliminary in vitro mechanism studies found that the compound could induce concentration-dependent arrest of HCT116 cells in the G0 / G1 phase, and concentration-dependently cause early and late apoptosis of HCT116 cells. This compound is a new compound that has not been reported yet, and its mechanism of action can be further explored, and it can be used to develop drugs or lead compounds for the treatment of the above cancers.

[0025] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0026] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The dehydrocostus lactone derivative DLDA of the present invention 1 H NMR spectrum.

[0028] Figure 2 The dehydrocostus lactone derivative DLDA of the present invention 13 C NMR spectrum.

[0029] Figure 3 The figure is the high resolution mass spectrum (HRESIMS) of the dehydrocostus lactone derivative DLDA of the present invention.

[0030] Figure 4 It is a test of different concentrations of DLDA to block the HCT116 cell cycle.

[0031] Figure 5 This is a test of different concentrations of DLDA inducing apoptosis in HCT116 cells. DETAILED DESCRIPTION

[0032] The reagents and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0033] Normal temperature is 25±5℃.

[0034] Example 1 Compound DLDA and its preparation method

[0035] The preparation method of compound DLDA is as follows:

[0036]

[0037] Add 200 mg (1.8 mmol) of selenium dioxide and 721 mg (8 mmol) of tert-butyl hydroperoxide to an eggplant-shaped bottle, then add 20 mL of dichloromethane and activate at 0°C for 30 min. Then dissolve 3.22 g (14 mmol) of dehydrocostus lactone in 5 mL of dichloromethane, add dropwise to the activated mixed solution of selenium dioxide and tert-butyl hydroperoxide at 0°C, and then transfer it to room temperature for reaction for 24 h. Monitor by TLC with sulfuric acid ethanol as the display agent. After the reaction is complete, add saturated Na2S2O3 solution to quench the reaction. Place the reaction solution in a separatory funnel and let it stand for stratification. Collect the lower layer of the reaction solution, and neutralize the residual Na2S2O3 in the reaction solution with saturated NaHCO3 solution. The solvent in the reaction solution is dried by vortexing and the sample is separated and purified by Flash fast column to obtain white solid intermediate A with a yield of about 42%.

[0038] Then, 98 mg (0.4 mmol) of intermediate A, 230 mg (1.2 mmol) of raw material B and 20 mg (0.16 mmol) of 4-dimethylaminopyridine were added to a 25 mL eggplant-shaped bottle, 10 mL of dichloromethane was added as solvent, and then 198 mg (0.96 mmol) of N,N'-dicyclohexylcarbodiimide was added. The reaction was stirred for one day at room temperature. TLC monitoring was performed. After the reaction was completed, the solvent was dried and the sample was separated and purified using a Flash fast column to obtain a white solid product. The yield was 45% and the purity was 99.6%.

[0039] The H NMR spectrum of the product DLDA is as follows: Figure 1 The carbon spectrum results are shown in Figure 2 The specific data are as follows:

[0040] (3a S ,6a R ,8 R ,9a R ,9b S )-3,6,9-trimethylene-2-oxododecahydroazuleno[4,5-b]furan-8-yl4,6-dichloronicotinate (DLDA). White solid, 45% yield.C 21 H 19 Cl2NO4. HRESIMS: m / z 442.0588 [M+Na] + (calcd. for C 21 H 19 Cl2NO4Na, 442.0589). 1 H NMR (400 MHz, CDCl3) δ 8.84 (s, 1H), 7.47 (s, 1H), 6.25 (d, J = 3.5 Hz, 1H),5.95 (t, J = 5.9 Hz, 1H), 5.65 (s-like, 1H), 5.55 (s-like, 1H), 5.53 (d, J = 3.1Hz, 1H), 4.99 (s, 1H), 4.85 (s, 1H), 3.97 (q, J = 9.2 Hz, 1H), 3.14–3.20 (m,2H),2.91 (m, 1H), 2.55 (dt, J = 13.1, 4.7 Hz, 1H), 2.41 (ddd, J = 14.2, 6.9, 4.5Hz, 1H), 2.28 (m, 1H), 2.09–2.19 (m, 2H), 1.44 (m, 1H). 13 C NMR (100 MHz, CDCl3) δ 170.16, 162.97, 154.88, 152.38, 148.36, 147.94, 146.09, 139.28, 126.18,125.06,120.93, 117.13, 113.84, 84.92, 78.50, 49.89, 45.50, 45.06, 37.36,36.60, 31.12.

[0041] The high-resolution mass spectrometry results of the product DLDA are as follows Figure 3 The specific data are shown in Table 1:

[0042] Table 1 High-resolution mass spectrometry results of the product DLDA

[0043]

[0044] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0045] Experimental Example 1 Antitumor Activity Experiment of Compound DLDA

[0046] 1. Experimental Methods

[0047] (1) Test cell lines and their culture

[0048] The following 9 test cell lines were selected: human lung cancer cells (A549), human liver cancer cells (HepG2), human colon cancer cells (HCT116), human esophageal cancer cells (TE-1), human renal clear cell adenocarcinoma cells (786-O), human bladder cancer cells (5637), human prostate cancer cells (DU145), human breast cancer cells (MCF-7) and human brain tumor cells (SF126) for anti-tumor activity testing; the cell lines used were obtained through regular channels. All cells were cultured in a culture medium containing 10% fetal bovine serum. All experiments used the same batch of cell lines between the 2nd and 10th generations.

[0049] (2) CCK-8 assay to determine cell proliferation activity

[0050] The compound DLDA prepared according to the method of Example 1 was prepared into a 10 mM DLDA stock solution using cell-grade dimethyl sulfoxide (DMSO) as solvent for subsequent cell experiments. Cell counts were performed when the cells grew to the logarithmic phase. The cells were inoculated in a 96-well plate at 20,000 cells / well (90 μL / well) and pre-cultured in an incubator at 5% CO2 and 37°C for 24 hours. The DLDA stock solution was diluted and prepared into 8 concentrations of sample solutions (the concentrations were 0.5, 1.5, 4.5, 13.5, 40.5, 121.5, 364.5 and 1093.5 μM, respectively), the concentration of dehydrocostus lactone was set as above, and the concentration of the positive control cisplatin was set as above. Then, 10 μL of sample solution containing different concentrations was added to each well of the experimental group, and 3 replicates were set for each concentration; 10 μL of dehydrocostus lactone solution containing different concentrations was added to each well of the dehydrocostus lactone group, and 3 replicates were set for each concentration; 10 μL of cisplatin solution containing different concentrations was added to each well of the positive group, and 3 replicates were set for each concentration; 10 μL of culture medium containing 10% fetal bovine serum was added to each well of the blank control group, and the solvent control group was added with an equal volume (10 μL) of culture medium containing 10% fetal bovine serum containing DMSO equal to the highest experimental concentration of the drug, and 3 parallel wells were set for each group; the cells were placed in an incubator at 5% CO2 and 37°C for 72 h. After the drugs acted for 72 h, the cell proliferation activity was determined by the CCK-8 method, and 10 μL of CCK-8 working solution was added to each well, and the cells were cultured for 1-4 h in the incubator at 5% CO2 and 37°C, and the culture was terminated. The absorbance at a wavelength of 450 nm was detected on an ELISA reader. The inhibition rate was calculated according to the absorbance values ​​at different concentrations, and the IC 50 value.

[0051] Inhibition rate = [(solvent control OD value - experimental group OD value) / (solvent control OD value - blank control group OD value)] × 100%, calculate the inhibition rate of each experimental group.

[0052] After calculating the inhibition rate of each concentration of the compound on cell proliferation activity, the compound treatment concentration and its corresponding inhibition rate were fitted with the dose-response curve using Graphpad prism software, and the IC 50 The experimental results are expressed as “mean ± SD”.

[0053] IC 50 Value: The concentration of sample solution corresponding to an inhibition rate (%) of 50%.

[0054] The experimental results are shown in Table 2.

[0055] (3) Cell cycle detection

[0056] Flow cytometry is an experimental method used to study the distribution and proportion of cells in different life cycle stages. HCT116 cells growing in the logarithmic phase were collected in a centrifuge tube, centrifuged and discarded the supernatant, and RPMI-1640 complete medium was added to the cell pellet to resuspend the cells to prepare a cell suspension. Count the cells and calculate the number of cells at 1×10 6 Cells were plated at a density of 100 μM / well in 6 wells, and 1.9 mL of cell suspension was added to each well. The cells were cultured in an incubator at 5% CO2 and 37 °C for 8 h. The DLDA stock solution was diluted to prepare sample solutions with concentrations of 10, 20, 40 and 100 μM, and then 100 μL of sample solutions of different concentrations were added to each well of the experimental group to treat the cells for 24 h. An equal volume (100 μL) of RPMI-1640 complete medium without drugs was added to the blank control wells. After 24 h of drug treatment, the 6-well plate was removed, the cell suspension was collected in a centrifuge tube, washed twice with pre-cooled PBS, and the supernatant was discarded by centrifugation. 500 μL of pre-cooled 75% ethanol was slowly added to the cell pellet, and the cells were dispersed by gently mixing with a pipette tip. The sample was fixed at 4 ºC overnight. The fixed cells were centrifuged at 1500 rpm for 3 min and the supernatant was removed. Wash twice with PBS, then resuspend the cells in the pre-prepared PI / RNAse solution (50 µg / mL). After incubation at room temperature in the dark for 10-15 min, the cell cycle of the sample was detected by flow cytometry.

[0057] (4) Cell apoptosis detection

[0058] Flow cytometry detection of cell apoptosis is an experimental method used to study cell apoptosis. It is mainly divided into four subpopulations by using Annexin V and PI double staining, namely surviving cells (Annexin V- / PI-), early apoptotic cells (AnnexinV+ / PI-), late apoptotic cells (Annexin V+ / PI+) and necrotic cells (AnnexinV- / PI+). First, collect HCT116 cells growing in the logarithmic phase in a centrifuge tube, centrifuge and discard the supernatant, add RPMI-1640 complete medium to the cell pellet and resuspend the cells to prepare a cell suspension. Count and count according to 1×10 6 Cells were plated at a density of 100 μM / well in 6 wells, and 1.9 mL of cell suspension was added to each well. The cells were cultured in an incubator at 5% CO2 and 37 °C for 8 h. The DLDA stock solution was diluted to prepare sample solutions with concentrations of 10, 20, 40 and 100 μM, and then 100 μL of sample solutions of different concentrations were added to each well of the experimental group to treat the cells for 48 h. An equal volume (100 μL) of RPMI-1640 complete medium without drugs was added to the blank control wells. After 48 h of drug treatment, the 6-well plate was removed, the cell suspension was collected in a centrifuge tube, washed once with pre-cooled PBS, and then washed once with binding buffer, and the supernatant was discarded by centrifugation. According to the instructions of the kit, the cells were resuspended with 500 μL binding buffer, and the cells were transferred to the flow tube. 5 μL of Annexin V staining solution was added to each flow tube, and after flicking evenly, the cells were stained in the dark at room temperature for 10 min. After washing once with binding buffer, resuspend the cells with 500 μL binding buffer, add 5 μL PI dye and mix gently, and stain again for 10 min. Use flow cytometry to detect apoptosis of cell samples, and complete flow cytometry within 1 h.

[0059] 2. Experimental Results

[0060] (1) In vitro antitumor activity of DLDA

[0061] Table 2. In vitro antitumor activity (IC 50 , μM)

[0062]

[0063] The results of in vitro antitumor experiments are shown in Table 2. The IC values ​​of compound DLDA against human lung cancer cells (A549), human liver cancer cells (HepG2), human colon cancer cells (HCT116), human esophageal cancer cells (TE-1), human renal clear cell adenocarcinoma cells (786-O), human bladder cancer cells (5637), human prostate cancer cells (DU145), human breast cancer cells (MCF-7) and human brain tumor cells (SF126) are shown in Table 2. 50 The inhibitory effects of DLDA on human colon cancer cells HCT116 were 0.86, 1.24, 0.24, 2.23, 0.97, 1.35, 1.64, 0.78 and 1.85 μM, respectively, showing good proliferation inhibition, and their inhibitory effects were better than those of dehydrocostus lactone and the positive control cisplatin. Among them, the compound DLDA had the strongest antiproliferative effect on human colon cancer cells HCT116, with IC 50 is 0.24 μM.

[0064] (2) DLDA induces HCT116 cell cycle arrest

[0065] The above experiments have proved that the compound DLDA has a good anti-proliferative effect on a variety of tumor cells, among which the anti-proliferative effect on human colon cancer cell HCT116 is the strongest. Therefore, HCT116 cells were selected to further investigate the effect of the compound DLDA on the cell cycle. HCT116 cells were treated with different concentrations of DLDA (0, 0.5, 1, 2 and 5 μM) for 24 hours and then stained with PI. The experimental results are as follows Figure 4 As shown, flow cytometry analysis revealed that the compound DLDA could induce G0 / G1 arrest of HCT116 cells in a concentration-dependent manner, accompanied by a decrease in the total number of cells in the S phase and G2 / M phase.

[0066] (3) DLDA induces apoptosis of HCT116 cells

[0067] The ability of the compound DLDA to induce cell apoptosis was further investigated. HCT116 cells were treated with DLDA at different concentrations (0, 0.5, 1, 2, and 5 μM) for 48 h, and the apoptosis of cells was detected by flow cytometry using Annexin V and PI double staining: live cells (Annexin V- / PI-), early apoptotic cells (Annexin V+ / PI-), late apoptotic cells (Annexin-V+ / PI+), and necrotic cells (Annexin-V- / PI+). The results are shown in Figure 2. Figure 5 As shown, the compound DLDA can induce early and late apoptosis of HCT116 cells in a concentration-dependent manner.

[0068] In summary, the present invention provides a new compound DLDA, which has a significantly better proliferation inhibitory effect on a variety of tumor cells than dehydrocostus lactone, especially the strongest anti-proliferation effect on human colon cancer cells HCT116. It can significantly induce G0 / G1 phase arrest and apoptosis of HCT116 cells, providing a new reference for broadening the screening and development of anti-tumor drugs.

Claims

1. A dehydrocostus lactone derivative or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the dehydrocostus lactone derivative is shown in Formula I:

2. The method for preparing the dehydrocostus lactone derivative according to claim 1, characterized in that: The specific steps include: Step 1: Dehydrocostus lactone reacts with an oxidizing agent to obtain intermediate A; Step 2: The intermediate A reacts with the raw material B to obtain the dehydrocostus lactone derivative shown in formula I.

3. The preparation method according to claim 2, characterized in that: In step 1, the oxidizing agent is selenium dioxide and tert-butyl hydroperoxide; the solvent of the reaction is dichloromethane; the reaction temperature is -4 to 45°C; the reaction time is 3 to 30 hours; In step 2, the reaction is carried out under the action of a condensing agent and a catalyst; the solvent of the reaction is dichloromethane; the reaction temperature is 15 to 45° C.; and the reaction time is 3 to 30 hours.

4. The preparation method according to claim 3, characterized in that: In step 1, the molar ratio of selenium dioxide, tert-butyl hydroperoxide, and dehydrocostus lactone is 1:4-5:7-8; the reaction temperature is 0-30° C.; and the reaction time is 24 hours; In step 2, the condensation agent is N,N'-dicyclohexylcarbodiimide; the catalyst is 4-dimethylaminopyridine; the molar ratio of 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide, raw material B, and intermediate A is: 1:5-7:7-8:23-27; the reaction temperature is 30°C; and the reaction time is 24h.

5. The preparation method according to claim 4, characterized in that: In step 1, the molar ratio of selenium dioxide, tert-butyl hydroperoxide and dehydrocostus lactone is 1:4.4:7.8; In step 2, the molar ratio of 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide, raw material B and intermediate A is 1:6:7.5:

25.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition is prepared by using the dehydrocostus lactone derivative or a pharmaceutically acceptable salt thereof as claimed in claim 1 as an active ingredient and adding pharmaceutically acceptable excipients.

7. Use of the pharmaceutical composition according to claim 6 in the preparation of anti-tumor drugs.

8. The use according to claim 7, characterized in that: The tumor is lung cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer or brain tumor.

9. Use of the dehydrocostus lactone derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor is lung cancer, liver cancer, colon cancer, rectal cancer, esophageal cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer or brain tumor.

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