Bufalin derivative as well as preparation method and application thereof

Abstract

The invention relates to a bufalin derivative as well as a preparation method and application thereof, and belongs to the technical field of medicines. According to the invention, TEMPO oxidation reaction is adopted to selectively oxidize 19-site primary alcohol in 19-OH bufalin into aldehyde, so that 19-CHO bufalin is successfully synthesized, namely the bufalin derivative is obtained. The bufalin derivative has a remarkable killing effect on pancreatic cancer BxPC-3 cells, liver cancer HepG2 cells and brain glioma U-87MG cells, the IC50 of the bufalin derivative to the BxPC-3 cells is 40.63 nM, the IC50 of the bufalin derivative to the HepG2 cells is 36.74 nM, the IC50 of the bufalin derivative to the U-87MG cells is 28.73 nM, and the IC50 of the bufalin derivative to CHO-K1 cells is larger than 30 [mu] M, so that the bufalin derivative is relatively weak in toxicity to the heart, and the bufalin derivative has a remarkable killing effect on the liver cancer BxPC-3 cells, the liver cancer HepG2 cells and the brain glioma U-87MG cells. The compound can be applied to medicines for resisting pancreatic cancer, liver cancer and brain glioma.

Description

Technical Field

[0001] The invention relates to a bufalin derivative and a preparation method and application thereof, belonging to the technical field of medicine. Background Art

[0002] Tumors are one of the main causes of human death, and their morbidity and mortality rates are generally on the rise. Malignant tumors have become the leading cause of death among urban and rural residents. It can be seen that the prevention and treatment of tumors are very urgent. Drug therapy is one of the main means of tumor treatment. Although many anti-tumor drugs have been developed, which can effectively prolong the life of patients or improve the quality of life of patients, the research and development of tumor drugs still faces huge challenges. For example, most anti-tumor drugs are cytotoxic drugs, which have obvious side effects, limiting the clinical application of these drugs. In recent years, targeted therapy for tumors has developed rapidly. Drugs such as imatinib and trastuzumab that target tumor signaling proteins have shown promising therapeutic effects and low toxic side effects in clinical studies. However, the emergence of acquired drug resistance and the variability of tumor genomes have also made targeted therapy face huge challenges.

[0003] Toad venom is the dried secretion of the Chinese giant toad (Bofo bufo andrewsi Schmidt) or the black-rimmed toad (Bufomelanostitus Schneider) of the Toad family. It is a precious Chinese medicine, pungent, warm, and toxic. It has the effects of detoxification, swelling, refreshing, opening the mind, strengthening the heart, and relieving pain. The main cardiotonic active ingredient in toad venom is bufotoxin compounds, which belong to the cardiotonic steroid compounds. At present, a large number of bufotoxin compounds have been discovered and reported, such as bufotoxin, resibufogenin, cinobufolin, and cinobufolin. Among them, bufotoxin belongs to the cardiac glycosides, and its molecular formula is C 24 H 34 O4, with the structural formula shown below, is the most active toxic ligand extracted from the traditional Chinese medicine toad venom.

[0004]

[0005] Chinese patent document CN118845798A (application number 202410911116.4) discloses the use of the compound bufalin or its pharmaceutically acceptable salt in the preparation of a drug for preventing and treating acute pancreatitis. Chinese patent document CN118078840A (application number 202410315551.0) discloses the use of bufalin in anti-tumor drugs. Bufalin can inhibit the proliferation of cancer-related fibroblasts, IC 50 The concentration is 800-1000 nM, and the cancer is colorectal cancer.

[0006] Although these natural steroid compounds have good cardiotonic activity, most of them also have strong cardiotoxicity and cytotoxicity, which seriously affects their clinical application. Therefore, it is necessary to develop high-efficiency and low-toxic steroid compounds. Chinese patent document CN112209985A (application number 202010644392.0) discloses the use of acetobuflin in the preparation of anti-tumor drugs, which can improve the anti-tumor efficacy, especially has a good inhibitory effect on lung cancer cells. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides a bufalin derivative and a preparation method and application thereof.

[0008] The technical solution of the present invention is as follows: A bufalin derivative, the structural formula of which is shown in Formula I: Formula Ⅰ.

[0009] The preparation method of the bufalin derivatives is to use 19-OH bufalin as a reaction substrate, and obtain 19-CHO bufalin through an oxidation reaction, thereby obtaining the bufalin derivatives.

[0010] Preferably, according to the present invention, the method for preparing the bufalin derivative comprises the following steps: 19-OH bufalin, TEMPO (tetramethylpiperidinoxide), and TBACl (tetrabutylammonium chloride) are added in sequence to a solvent consisting of dichloromethane and NaHCO3-K2CO3 buffered aqueous solution, and then NCS (N-chlorosuccinimide) is added and stirred for reaction. After the reaction is completed, the organic layer is separated, the aqueous phase is extracted with dichloromethane, and the organic phase is collected. The target compound bufalin derivative is obtained after washing, drying, and purification.

[0011] Further preferably, the mass volume ratio of 19-OH bufalotoxin to the solvent is 1:(1-2), mg / mL.

[0012] More preferably, the molar ratio of TEMPO (tetramethylpiperidinyl oxide) to 19-OH bufalin is (0.1-0.5):1.

[0013] Further preferably, the molar ratio of the TBACl (tetrabutylammonium chloride) to 19-OH bufalin is (0.1-0.5):1.

[0014] Further preferably, the molar ratio of NCS (N-chlorosuccinimide) to 19-OH bufalin is (1.0-1.5):1.

[0015] Further preferably, the volume ratio of dichloromethane and NaHCO3-K2CO3 buffer aqueous solution in the solvent is 1:(0.7~1.2), and the pH of the NaHCO3-K2CO3 buffer aqueous solution is 8.5~9.0.

[0016] Application of the above-mentioned bufalin derivatives in the preparation of anti-tumor drugs.

[0017] Preferably according to the present invention, the tumor includes: pancreatic cancer, liver cancer, and brain glioma.

[0018] Beneficial effects: 1. The present invention adopts TEMPO oxidation reaction to selectively oxidize the primary alcohol at position 19 in 19-OH bufalin to aldehyde, and successfully synthesizes 19-CHO bufalin to obtain bufalin derivatives.

[0019] 2. The bufalin derivatives of the present invention have significant killing effects on pancreatic cancer BxPC-3 cells, liver cancer HepG2 cells, and brain glioma U-87MG cells. The IC 50 The IC value for HepG2 cells is 40.63 nM. 50 The IC value for U-87MG cells is 36.74 nM. 50 The IC of bufalin derivatives against CHO-K1 cells was 28.73 nM. 50 >30μM, indicating that the toxicity to the heart is weak and can be used in anti-pancreatic cancer, anti-liver cancer, and anti-glioma drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 For the target compound 1 H NMR spectrum.

[0021] Figure 2 For the target compound 13 C NMR spectrum.

[0022] Figure 3 is the MS spectrum of the target compound.

[0023] Figure 4 HPLC spectrum of the target compound.

[0024] Figure 5 This is the cytotoxic activity curve of BxPC-3 cells.

[0025] Figure 6 This is the cytotoxic activity curve of HepG2 cells.

[0026] Figure 7 This is the cytotoxic activity curve of U-87MG cells.

[0027] Figure 8 for Na + -K + -Residual enzyme activity curve of ATPase α1 subunit.

[0028] Fig. 9 It is a scatter plot of the inhibition rate of bufalin derivatives on hERG current of CHO-K1 cells. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and materials used in the embodiments are all common commercially available products unless otherwise specified. The experimental operations involved in the embodiments are all conventional experimental methods in the art unless otherwise specified.

[0030] The main sources of reagents and materials in the examples are: Table 1. Sources of main reagents and materials in the examples

[0031] Embodiment 1: A bufalin derivative, the structural formula of which is shown in Formula I: Formula I, The preparation steps are as follows:

[0032] 50 mg 19-OH bufalin, 1.94 mg TEMPO (tetramethylpiperidin oxide) (0.1 eq), and 3.45 mg TBACl (tetrabutylammonium chloride) (0.1 eq) were added to 25 mL dichloromethane and 25 mL NaHCO3-K2CO3 buffer solution (pH = 8.6) in sequence, and then 18.25 mg NCS (N-chlorosuccinimide) (1.1 eq) was added and continued to stir. The reaction was monitored by thin layer chromatography. After 24 h of reaction, the organic layer was separated and the aqueous phase was extracted with dichloromethane (3 × 15 mL). After the extraction, the organic phase was collected and washed with saturated brine (3 × 15 mL), dried over anhydrous magnesium sulfate, dried under reduced pressure, and purified by thin layer chromatography (PTLC) with PE:EA:MeOH = 10:10 to obtain 9 mg of the target compound as a white solid with a yield of 36.2%. The molecular formula was C 24 H 32 O5.

[0033] The nuclear magnetic resonance (NMR) spectrum of the target compound is as follows Figure 1 and Figure 2As shown. NMR shift (δ) is given in units of 10-0 (ppm). NMR was measured using a Bruker DRX (600 MHz) nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3) as the solvent and tetramethylsilane (TMS) as the internal standard. The NMR results are as follows: 1 H NMR (600 MHz, CDCl3) δ 9.50 (d, J = 1.7 Hz, 1H), 7.85 (dd, J = 9.7, 2.6Hz, 1H), 7.25 (dd, J = 2.7, 1.2 Hz, 1H), 6.35 – 6.24 (m, 1H), 4.27 – 4.16 (m,1H), 2.48 (d, J = 3.0 Hz, 1H), 2.35 – 2.29 (m, 1H), 2.20 (dt, J = 13.1, 9.6 Hz,1H), 2.03 (dt, J = 13.2, 10.1 Hz, 1H), 1.93 – 1.85 (m, 1H), 1.84 – 1.66 (m,7H), 1.63 – 1.51 (m, 5H), 1.46 (ddd, J = 17.3, 10.5, 4.7 Hz, 2H), 1.40 – 1.23(m, 3H), 0.78 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 206.08, 162.91, 148.77, 147.11, 122.95,115.48, 85.28, 65.86, 51.20, 51.18, 48.60, 42.61, 40.97, 35.03, 32.37, 31.98,28.91, 28.71, 28.17, 26.42, 21.49, 21.20, 21.10, 16.56. The mass spectrometry (MS) spectrum of the target compound is as follows Figure 3 As shown. MS was measured using an API 4000 mass spectrometer. The MS results are as follows: MS (ESI): m / z 401.25 [M+H] + .m / z 423.25 [M+Na]+ .m / z 823.10 [2M+Na]+ . The high performance liquid chromatography (HPLC) spectrum of the target compound is as follows Figure 4 As shown. HPLC determination was performed using an Agilent 1260DAD liquid chromatograph. The results showed that the purity of the target compound was 98%.

[0034] Embodiment 2: The inhibitory effect of the bufalin derivative prepared in Example 1 on tumor cell proliferation was determined by the CTG method BxPC-3 cells, HepG2 cells and U-87MG cells were inoculated in 384-well cell culture plates, with an inoculation volume of 35 μL per well and 1000 cells per well, and cultured overnight in a 37°C, 5% CO2 incubator. A 10 mM bufalin derivative stock solution was prepared with DMSO, and a 4-fold gradient dilution was performed from the 10 mM bufalin derivative stock solution with DMSO, for a total of 9 dilution gradients. Use an ECHO pipette to add 35nL of different concentrations of toad venom derivative solution to each well of a 384-well cell culture plate, with a final DMSO concentration of 0.1%. Place the cell culture plate in a 37°C, 5% CO2 incubator for 3 days. On the 0th day of culture, add 20μL of CellTiter-Glo detection reagent to each well of the D0 cell culture well, incubate at room temperature for 30 minutes in the dark, and read the chemiluminescence value with an envision multi-label microplate reader; on the 3rd day of culture, add 20μL of CellTiter-Glo detection reagent to each well of the D3 cell culture well, incubate at room temperature for 30 minutes in the dark, and read the chemiluminescence value with an envision multi-label microplate reader. Use software to calculate the tumor cell inhibition rate, draw the cytotoxic activity curve, and calculate the IC 50 value.

[0035] Cytotoxicity curve of BxPC-3 cells Figure 5 As shown, the IC values ​​of bufalin derivatives on BxPC-3 cells 50 It is 40.63nM.

[0036] Cytotoxicity curve of HepG2 cells Figure 6 As shown, the IC values ​​of bufalin derivatives on HepG2 cells 50 It is 36.74nM.

[0037] The cytotoxic activity curve of U-87MG cells is shown in Figure 7 As shown, the IC values ​​of bufalin derivatives on U-87MG cells 50 It is 28.73nM.

[0038] Embodiment 3: The ADP-Glo ​​Max detection kit (Promaga, product number V7001) was used to detect the activity of the bufalin derivative prepared in Example 1 on Na + -K + -Inhibition of ATPase α1 subunit, the specific steps are as follows: (1) Prepare a 10 mM stock solution of bufalin derivatives with DMSO, and dilute the 10 mM stock solution of bufalin derivatives 3-fold with DMSO, for a total of 10 gradient doses; use an ECHO pipette to add 5 nL of bufalin derivative solutions of different concentrations to each well of a 384-well cell culture plate, and the final DMSO concentration in the reaction system is 0.1%; (2) Centrifuge the 384-well cell culture plate at 1000 rpm for 1 minute; (3) Prepare 0.12 mU / μL Na + / K + -ATPase α1 subunit (Sigma, product number A7510) solution, add 2.5 μL Na + / K + -ATPase α1 subunit solution to a 384-well cell culture plate, and an equal volume of blank buffer was added to the blank control group; (4) Incubate at 37°C for 30 min. (5) Add 2.5 μL of 2× ATP solution to each well of a 384-well cell culture plate; (6) Incubate at 37°C for 30 min. (7) Add 5 μL ADP-Glo ​​Reagent to each well of a 384-well cell culture plate and incubate at 37°C for 30 min. (8) Add 5 μL ADP-Glo ​​Max Detection Reagent to a 384-well cell culture plate and incubate at 37°C for 60 min. (9) Use the Envision multi-label microplate reader to read the chemiluminescence value and use the software to calculate the Na + -K + - Residual enzyme activity of ATPase α1 subunit, draw residual enzyme activity curve, calculate IC 50 value.

[0039] Na + -K + -ATPase α1 subunit residual enzyme activity curve Figure 8 As shown, the bufalin derivatives have an + -K + -IC of ATPase α1 subunit 50 is 103.16nM, indicating that the bufalin derivatives have a strong affinity for Na + -K+ -ATPase α1 subunit has a strong inhibitory effect, Na + -K + -The expression of ATPase α1 subunit in tumor cells is higher than that in normal cells, and this dose will not affect the activity of normal cells.

[0040] Embodiment 4: The SyncroPatch 384i system was used to detect the cardiotoxicity of the bufalin derivative prepared in Example 1. The components of the extracellular fluid and intracellular fluid used in the experiment were as follows: Extracellular fluid: NaCl 80mM, NMDG (N-methyl-D-glucamine) 60mM, KCl 4mM, CaCl22mM, MgCl21mM, D-glucose 5mM, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 10mM, pH 7.4, sterilized by membrane and stored at 4°C for future use; Intracellular solution: EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) 10 mM, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 10 mM, KCl 10 mM, NaCl 10 mM, KF 110 mM, pH 7.2, sterilized by membrane and stored at 4°C until use.

[0041] CHO-K1 stable cell line (purchased from B'SYS GmbH) was cultured and digested to prepare a cell density of 5-7.5×10 5 Transfer the cell suspension to the SyncroPatch 384i system-specific Teflon plate, place it in the cell incubation tank of the automatic patch clamp system, and incubate it at 15°C and 200 rpm for 30 minutes to start the experiment.

[0042] Prepare 10 mM bufalin derivative stock solution with DMSO, dilute the 10 mM bufalin derivative stock solution 3 times with DMSO, and obtain 4 gradient doses (10, 3.33, 1.11, 0.37 mM), dilute the above 4 doses of bufalin derivative solution 500 times with extracellular fluid again, and dilute the 10 mM bufalin derivative stock solution at the same time, and obtain 5 doses (60, 20, 6.66, 2.22, 0.74 μM), and the working solution is ready. Add the working solution and the extracellular solution to the corresponding plate position in the instrument at a volume ratio of 1:1, and prepare to start the experiment. Therefore, the actual drug administration gradient concentration should be 30, 10, 3.33, 1.11, 0.37 μM, and the final DMSO content is 0.1%-0.3%.

[0043] The SyncroPatch 384i system was used to record electrophysiological signals. The bottom of the NPC-384 chip dedicated to the SyncroPatch 384i system was filled with intracellular fluid containing 15μM β-Escin as a perforator, and the cell suspension was added to the chip. The cell grabbing pressure was -150mBar, so that the cells were adsorbed on the micropores at the bottom of the chip, and each micropore only accommodated a single cell. At this time, the bottom of the cell was exposed to the intracellular fluid on the other side of the chip. During the experiment, the pressure should be maintained at -50mBar to prevent the cells from falling off the micropores. Set the clamping voltage to 500ms and -90mV; the current sampling frequency is 500Hz, and the filtering frequency is 3kHz; the detection conditions of the leakage current are -80mV and the time course is 500ms.

[0044] The hERG current test method is as follows: 1) Apply a depolarizing voltage for 4.8 seconds to depolarize the membrane potential from -90mV to +30mV, then instantly apply a repolarizing voltage to reduce the membrane potential to -50mV for 5.2 seconds to remove channel inactivation, so that the hERG tail current can be observed. The peak value of the tail current is the magnitude of the hERG current. The sampling interval of this stimulation mode is 15 seconds. The cells used to detect the test compounds were recorded continuously for 120 seconds before administration to evaluate the stability of the hERG current generated by the test cells. Only stable cells within the acceptable range of the evaluation criteria can be trusted in subsequent test results.

[0045] 2) Test of the inhibitory effect of the test compound on hERG current: First, wash the cells 6 times with extracellular solution containing 0.1% DMSO, and measure the stable hERG current as the detection baseline. The baseline current value is the average of 5 stable sampling points. After the hERG current remains stable, the solution containing the test compound is perfused around the cells, and wait for 10 minutes to allow the compound to fully act on the cells and record the hERG current synchronously. After the current tends to stabilize, read 5 stable hERG current values, and take the average value as the final current value at a specific concentration. If the stable state is not reached within 10 minutes, the last 5 current peaks recorded are used as the readings. In the experiment, cisapride was used as a positive drug for synchronous determination of 6 concentration points to verify the stability of the experimental cells and the accuracy of the experimental results. After testing the compound, 450nM dofelide was added to all test cells to completely inhibit their current as a complete positive control for the cell.

[0046] The tail current suppression rate was calculated according to the following formula: Inhibition rate (%) = (1- )×100% In the formula, the tail current 化合物It is the average of 5 consecutive current values ​​obtained stably after perfusion of the test compound; Tail current 阳性对照 It is the average value of 5 consecutive current values ​​stably obtained after perfusion of Dofelide; Tail current 空白 It is the average of 5 consecutive current values ​​obtained stably after perfusing blank extracellular solution.

[0047] The scatter plot of the inhibition rate of bufalin derivatives on hERG current of CHO-K1 cells is shown in Fig. 9 As shown, the IC values ​​of bufalin derivatives on CHO-K1 cells 50 >30μM, combined with the IC values ​​of the bufalin derivatives on different tumor cells in Example 2 50 It can be seen from the values ​​that at the working concentration for inhibiting tumor cells, the toxicity to the heart is weak and the safety is high.

Claims

1. A bufalin derivative, characterized in that: Its structural formula is shown in Formula I: Formula Ⅰ.

2. The method for preparing the bufalin derivative according to claim 1, characterized in that: Using 19-OH bufalin as a reaction substrate, 19-CHO bufalin is obtained through oxidation reaction, thus obtaining a bufalin derivative.

3. The preparation method according to claim 2, characterized in that: The preparation method of the bufalin derivative specifically comprises the following steps: 19-OH bufalin, tetramethylpiperidinoxide and tetrabutylammonium chloride are added in sequence to a solvent consisting of dichloromethane and NaHCO3-K2CO3 buffered aqueous solution, and then N-chlorosuccinimide is added and stirred for reaction. After the reaction is completed, the organic layer is separated, the aqueous phase is extracted with dichloromethane, and the organic phase is collected. The target compound bufalin derivative is obtained after washing, drying and purification.

4. The preparation method according to claim 3, characterized in that: The mass volume ratio of the 19-OH bufalin to the solvent is 1:(1-2), mg / mL.

5. The preparation method according to claim 3, characterized in that: The molar ratio of the tetramethyl piperidine oxide to 19-OH bufalin is (0.1-0.5):

1.

6. The preparation method according to claim 3, characterized in that: The molar ratio of the tetrabutylammonium chloride to 19-OH bufalin is (0.1-0.5):

1.

7. The preparation method according to claim 3, characterized in that: The molar ratio of the N-chlorosuccinimide to 19-OH bufalin is (1.0-1.5):

1.

8. The preparation method according to claim 3, characterized in that: The volume ratio of dichloromethane to NaHCO3-K2CO3 buffer solution in the solvent is 1:(0.7-1.2), and the pH of the NaHCO3-K2CO3 buffer solution is 8.5-9.

0.

9. Use of the bufalin derivative according to claim 1 in the preparation of anti-tumor drugs.

10. The use according to claim 9, characterized in that The tumors include: pancreatic cancer, liver cancer, and brain glioma.

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