A bufalin derivative, and a preparation method and application thereof

Abstract

The present application relates to a bufalin derivative and its preparation method and application, belong to the medical technical field. The present application adopts TEMPO oxidation reaction, and selectively oxidizes the primary alcohol in 19-OH bufalin to aldehyde, successfully synthesizes 19-CHO bufalin, that is, bufalin derivative. The bufalin derivative of the present application has a significant killing effect on pancreatic cancer BxPC-3 cells, liver cancer HepG2 cells and brain glioma U-87MG cells, the IC 50 of the bufalin derivative on BxPC-3 cells is 40.63 nM, the IC 50 of the bufalin derivative on HepG2 cells is 36.74 nM, and the IC 50 of the bufalin derivative on U-87MG cells is 28.73 nM, and the IC 50 of the bufalin derivative on CHO-K1 cells is greater than 30 μM, indicating that the toxicity to the heart is weak, and the bufalin derivative can be applied to the application of anti-pancreatic cancer, anti-liver cancer and anti-brain glioma drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to a bufalin derivative, a preparation method and application thereof, and belongs to the technical field of medicine. BACKGROUND

[0002] Tumor is one of the main causes of human death, and its incidence and mortality rate are generally on the rise. Malignant tumor has become the primary cause of death for urban and rural residents. Therefore, the prevention and treatment of tumors are extremely urgent. Drug treatment is one of the main means of tumor treatment. Although a large number of antitumor 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 antitumor drugs still face great challenges. For example, most of the antitumor 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 targeting tumor signaling proteins, such as imatinib and trastuzumab, have shown promising therapeutic effects and lower toxic side effects in clinical studies. However, the emergence of acquired drug resistance and the variability of tumor genome have also posed great challenges to targeted therapy.

[0003] Toad venom is the dried secretion of Bufo bufo andrewsi Schmidt or Bufomelanostitus Schneider, which is a rare traditional Chinese medicine. It is pungent and warm, and has the effects of detoxification, swelling, refreshing, opening the orifice, strengthening the heart, and pain relief. The main active ingredient of toad venom is bufadienolides, which belongs to cardiac steroid compounds. A large number of bufadienolides have been discovered and reported, such as bufalin, resibufogenin, cinobufagin, and cinobufotalin. Among them, bufalin, a cardiac glycoside, has the molecular formula C 24 H 34 O4, and the structural formula is as follows, which is the most active toxic base extracted from toad venom.

[0004]

[0005] Chinese patent document CN118845798A (application number 202410911116.4) discloses the application of 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 application of bufalin in antitumor drugs. Bufalin can inhibit the proliferation of cancer-related fibroblasts, and the IC 50 concentration is 800-1000 nM, and the cancer is colorectal cancer.

[0006] Although these natural steroidal compounds have good inotropic activity, they also have strong cardiotoxicity and cytotoxicity, which seriously affects their clinical application. Therefore, it is necessary to develop steroidal compounds with high efficiency and low toxicity. Chinese patent document CN112209985A (application number 202010644392.0) discloses an application of acetyl bufalin in the preparation of an antitumor drug. The acetyl bufalin can improve the antitumor efficacy, especially for lung cancer cells. SUMMARY

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

[0008] The technical scheme of the present application is as follows:

[0009] A bufalin derivative, the structural formula of which is shown as formula I:

[0010] Formula I.

[0011] The preparation method of the above bufalin derivative uses 19-OH bufalin as a reaction substrate, and obtains 19-CHO bufalin through an oxidation reaction, thereby obtaining the bufalin derivative.

[0012] According to the present application, the preparation method of the bufalin derivative specifically comprises the following steps:

[0013] The 19-OH bufalin, TEMPO (tetramethylpiperidine oxide), and TBACl (tetrabutylammonium chloride) are sequentially added to a solvent composed of dichloromethane and a NaHCO3-K2CO3 aqueous buffer solution, and then NCS (N-chlorosuccinimide) is added, and the reaction is stirred. After the reaction is completed, the organic layer is separated, the water phase is extracted with dichloromethane, and then the organic phase is collected, washed, dried, and purified to obtain the target compound bufalin derivative.

[0014] Further preferably, the mass-volume ratio of the 19-OH bufalin to the solvent is 1: (1-2), mg / mL.

[0015] Further preferably, the molar ratio of the TEMPO (tetramethylpiperidine oxide) to the 19-OH bufalin is (0.1-0.5): 1.

[0016] Further preferably, the molar ratio of the TBACl (tetrabutylammonium chloride) to the 19-OH bufalin is (0.1-0.5): 1.

[0017] Further preferably, the molar ratio of the NCS (N-chlorosuccinimide) to the 19-OH bufalin is (1.0-1.5): 1.

[0018] Further preferably, the volume ratio of dichloromethane to the 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.

[0019] The application of the bufalin derivative in the preparation of an antitumor drug.

[0020] According to the application, the tumor includes pancreatic cancer, liver cancer and brain glioma.

[0021] Advantages:

[0022] 1. The TEMPO oxidation reaction is adopted in the application to selectively oxidize the primary alcohol at the 19th position in 19-OH bufalin into aldehyde, and 19-CHO bufalin is successfully synthesized, i.e. the bufalin derivative.

[0023] 2. The bufalin derivative has a significant killing effect on pancreatic cancer BxPC-3 cells, liver cancer HepG2 cells and brain glioma U-87MG cells, the IC 50 of the bufalin derivative on BxPC-3 cells is 40.63nM, the IC 50 of the bufalin derivative on HepG2 cells is 36.74nM, and the IC 50 of the bufalin derivative on U-87MG cells is 28.73nM, the IC 50 of the bufalin derivative on CHO-K1 cells is greater than 30uM, which indicates that the toxicity on the heart is weak, and the bufalin derivative can be applied in the anti-pancreatic cancer, anti-liver cancer and anti-brain glioma drugs. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The H NMR spectrum of the target compound. 1

[0025] Figure 2 The C NMR spectrum of the target compound. 13

[0026] Figure 3 The MS spectrum of the target compound.

[0027] Figure 4 The HPLC spectrum of the target compound.

[0028] Figure 5 The cell toxicity activity curve of BxPC-3 cells.

[0029] Figure 6 The cell toxicity activity curve of HepG2 cells.

[0030] Figure 7 ​​The cytotoxic activity curve for U-87MG cells.

[0031] Figure 8 The Na + -K + The remaining enzyme activity curve of the α1 subunit of Na

[0032] Figure 9 The scatter plot of the hERG current inhibition rate of bufalin derivative on CHO-K1 cells. DETAILED DESCRIPTION

[0033] The technical solutions of the present application are further described below in combination with examples, but the protection scope of the present application is not limited to this. The reagents and materials used in the examples are all ordinary commercially available products if not specifically stated. The experimental operations involved in the examples are all conventional experimental methods in the art if not specifically stated.

[0034] The main reagents and materials used in the examples are as follows:

[0035] Table 1. The main reagents and materials used in the examples

[0036]

[0037] Example 1:

[0038] A bufalin derivative, the structural formula of which is shown as formula I:

[0039] Formula I,

[0040] The preparation steps thereof are as follows:

[0041]

[0042] 50 mg of 19-OH bufalin, 1.94 mg of TEMPO (tetramethylpiperidine oxide) (0.1 eq), 3.45 mg of TBACl (tetrabutylammonium chloride) (0.1 eq) were sequentially added to 25 mL of dichloromethane and 25 mL of a buffered aqueous solution of NaHCO3-K2CO3 (pH = 8.6), then 18.25 mg of NCS (N-chlorosuccinimide) (1.1 eq) was added and stirring was continued, and thin layer chromatography was used to monitor the reaction; after 24 h of reaction, the organic layer was separated, and the aqueous phase was extracted with dichloromethane (3 × 15 mL); after the extraction was completed, the organic phase was collected and washed with saturated brine (3 × 15 mL), dried over anhydrous magnesium sulfate, and dried under reduced pressure, and purified by PE:EA:MeOH = 10:10:1 thin layer chromatography (PTLC) to obtain 9 mg of the target compound, which was a white solid, with a yield of 36.2%, and a molecular formula of C 24 H 32 O5.

[0043] NMR spectra of the target compounds are shown in Figure 1 and Figure 2 The NMR shifts (δ) are given in units of 10-0 (ppm). NMR was measured with a Bruker DRX (600 MHz) type nuclear magnetic resonance apparatus, and deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3) was used as the measuring solvent, and tetramethylsilane (TMS) was used as the internal standard. The NMR results are as follows:

[0044] 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).

[0045] 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.

[0046] Mass spectra (MS) of the target compounds are shown in Figure 3MS was measured by using API 4000 mass spectrometer. The MS results are as follows:

[0047] MS (ESI): m / z 401.25 [M+H] +.m / z 423.25 [M+Na]+.m / z 823.10 [2M+Na]+.

[0048] The high performance liquid chromatography (HPLC) spectrum of the target compound is shown in Figure 4 The determination of HPLC uses Agilent 1260 DAD liquid chromatograph. The results show that the purity of the target compound is 98%.

[0049] Example 2:

[0050] Determination of the inhibitory effect of the bufalin derivative prepared in Example 1 on tumor cell proliferation by CTG method

[0051] BxPC-3 cells, HepG2 cells and U-87MG cells were inoculated in 384-well cell culture plates, respectively, with an inoculation amount of 35 μL and a cell number of 1000 cells per well, and were placed in a 37°C, 5% CO2 incubator for overnight culture. A 10 mM bufalin derivative stock solution was prepared with DMSO, and a 4-fold gradient dilution was performed with DMSO from the 10 mM bufalin derivative stock solution, with a total of 9 dilution gradients. An ECHO pipettor was used to add 35 nL of bufalin derivative solution of different concentrations to each well of the 384-well cell culture plate, with a final DMSO concentration of 0.1%, and the cell culture plate was placed in a 37°C, 5% CO2 incubator for 3 days. During the culture, on day 0, 20 μL of CellTiter-Glo detection reagent was added to each well of the D0 cell culture well, and the light was incubated at room temperature for 30 minutes. The chemiluminescence value was read by an Envision multi-label microplate detector. On day 3 of the culture, 20 μL of CellTiter-Glo detection reagent was added to each well of the D3 cell culture well, and the light was incubated at room temperature for 30 minutes. The chemiluminescence value was read by an Envision multi-label microplate detector. The tumor cell inhibition rate was calculated by software, the cytotoxic activity curve was drawn, and the IC 50 value was calculated.

[0052] The cytotoxic activity curve of BxPC-3 cells is shown in Figure 5 The IC 50 of the bufalin derivative on BxPC-3 cells is 40.63 nM.

[0053] The cytotoxic activity curve of HepG2 cells is shown in Figure 6 The IC 50 of the bufalin derivative on HepG2 cells is 36.74 nM.

[0054] The cytotoxic activity curve of U-87MG cells is shown in Figure 1, and the IC50 of the bufalin derivative against U-87MG cells is 28.73 nM. Figure 7 50

[0055] Example 3:

[0056] The ADP-Glo Max assay kit (Promaga, product number V7001) was used to detect the inhibitory effect of the bufalin derivative prepared in Example 1 on Na + -K + -ATPase α1 subunit, and the specific steps are as follows:

[0057] (1) Prepare a 10 mM bufalin derivative stock solution with DMSO, and dilute the 10 mM bufalin derivative stock solution with DMSO by 3-fold gradient, a total of 10 gradient doses; use an ECHO pipettor to add 5 nL of bufalin derivative solution of different concentrations to each well of a 384-well cell culture plate, and the final concentration of DMSO in the reaction system is 0.1%;

[0058] (2) Centrifuge the 384-well cell culture plate at 1000 rpm for 1 minute;

[0059] (3) Prepare a 0.12 mU / μL Na + / K + -ATPase α1 subunit (Sigma, product number A7510) solution with ADP-Glo buffer, and add 2.5 μL of Na + / K + -ATPase α1 subunit solution to each well of the 384-well cell culture plate, and add the same volume of blank buffer to the blank control group;

[0060] (4) Incubate at 37°C for 30 minutes;

[0061] (5) Add 2.5 μL of 2x ATP solution to each well of the 384-well cell culture plate;

[0062] (6) Incubate at 37°C for 30 minutes;

[0063] (7) Add 5 μL of ADP-Glo Reagent to each well of the 384-well cell culture plate, and incubate at 37°C for 30 minutes;

[0064] (8) Add 5 μL of ADP-Glo Max Detection Reagent to the 384-well cell culture plate, and incubate at 37°C for 60 minutes;

[0065] (9) Read the chemiluminescence value with an envision multi-label microplate detector, and calculate the Na​​+ - K + - Residual enzyme activity of ATPase α1 subunit, draw residual enzyme activity curve, calculate IC 50 value.

[0066] Na + - K + - Residual enzyme activity curve of ATPase α1 subunit as shown in Figure 8 , the bufonidine derivative has a strong inhibitory effect on Na + - K + - IC of ATPase α1 subunit 50 103.16 nM, indicating that the bufonidine derivative has a strong inhibitory effect on Na + - K + - ATPase α1 subunit, and the dose will not affect the activity of normal cells. + - K + - ATPase α1 subunit in tumor cells is higher than that in normal cells, and the dose will not affect the activity of normal cells.

[0067] Example 4:

[0068] The bufonidine derivative prepared in Example 1 was tested for cardiotoxicity using a SyncroPatch 384i system. The components of the extracellular fluid and intracellular fluid used in the experiment are as follows:

[0069] Extracellular fluid: NaCl 80 mM, NMDG (N-methyl-D-glucamine) 60 mM, KCl 4 mM, CaCl2 2 mM, MgCl2 1 mM, D-glucose 5 mM, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 10 mM, pH 7.4, sterilized through membrane and stored at 4°C for standby;

[0070] Intracellular fluid: EGTA (ethylene glycol-bis(2-aminoethylether)-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 through membrane and stored at 4°C for standby.

[0071] After culturing and digesting the CHO-K1 stable cell strain (purchased from B'SYS GmbH), a cell suspension with a cell density of 5-7.5 x 10 5 The cell suspension was transferred to a special Teflon plate for the SyncroPatch 384i system, and placed in the cell incubation tank of the automatic patch clamp system, incubated at 15°C, 200 rpm for 30 minutes, and then the experiment could begin.

[0072] The 10 mM bufadienolide derivative stock solution was prepared with DMSO, and 3-fold gradient dilution was performed with DMSO from the 10 mM bufadienolide derivative stock solution, and 4 gradient doses (10, 3.33, 1.11, 0.37 mM) were obtained. The above 4 doses of bufadienolide derivative solution were diluted 500 times with extracellular fluid, and the 10 mM bufadienolide derivative stock solution was also diluted, and 5 doses (60, 20, 6.66, 2.22, 0.74 μM) were obtained. The working solution was prepared. The working solution and the extracellular fluid were added to the corresponding plate position in the instrument at a volume ratio of 1:1, and the experiment was ready to start. The actual dosing gradient concentration should be 30, 10, 3.33, 1.11, 0.37 μM, and the final DMSO content was 0.1%-0.3%.

[0073] 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 15 μM β-Escin as a perforator, and the cell suspension was added to the chip. The cell pressure was -150 mBar, and the cells were adsorbed on the microwells at the bottom of the chip, and each microwell only contained a single cell. At this time, the cell bottom was exposed to the intracellular fluid on the other side of the chip. The pressure should be maintained at -50 mBar during the experiment to prevent the cells from falling off the microwells. The clamping voltage was set to 500 ms and -90 mV; the current sampling frequency was 500 Hz, and the filtering frequency was 3 kHz; the detection condition of the leakage current was -80 mV, and the time course was 500 ms.

[0074] The hERG current test method is as follows:

[0075] 1) A 4.8-second depolarization voltage was applied to depolarize the membrane potential from -90 mV to +30 mV, and then an instant repolarization voltage was applied to reduce the membrane potential to -50 mV, lasting for 5.2 seconds to remove channel inactivation, so that the hERG tail current could be observed, and the peak of the tail current was the size of the hERG current. The sampling interval of this stimulation mode was 15 seconds. The cells used for detecting the test compound were continuously recorded for 120 seconds before administration to evaluate the stability of the hERG current generated by the test cells. Only stable cells within the acceptance range of evaluation criteria could have their subsequent test results be trusted.

[0076] 2) Test of the inhibitory effect of the test compound on hERG current: First, the cells were perfused with extracellular solution containing 0.1% DMSO for 6 times to determine the stable hERG current as the detection baseline, and the baseline current value was the average of 5 stable sampling points. After the hERG current remained stable, the solution containing the test compound was perfused around the cells, and 10 minutes were waited for the compound to fully act on the cells and record the hERG current synchronously. After the current tends to be stable, 5 stable hERG current values are read, and their average is taken as the final current value at a specific concentration. If it does not reach a stable state within 10 minutes, the last 5 current peak values recorded are taken as the reading value. Cisapride was used as a positive drug for synchronous determination of 6 concentration points in the experiment to verify the stability of the experimental cells and the accuracy of the experimental results. After testing the compound, 450 nM dofetilide was added to all test cells to completely inhibit their current, as a complete positive control for the cells.

[0077] The tail current inhibition rate was calculated according to the following formula:

[0078] Inhibition rate (%) = (1- ) x 100%

[0079] In the formula, the tail current 化合物 is the average of 5 consecutive current values stably obtained after perfusion of the test compound;

[0080] The tail current 阳性对照 is the average of 5 consecutive current values stably obtained after perfusion of dofetilide;

[0081] The tail current 空白 is the average of 5 consecutive current values stably obtained after perfusion of blank extracellular solution.

[0082] The scatter plot of the bufonidine derivative on the hERG current inhibition rate of CHO-K1 cells is shown in Figure 9 , and the IC 50 of the bufonidine derivative on CHO-K1 cells is greater than 30 μM. Combined with the IC 50 values of the bufonidine derivative on different tumor cells in Example 2, it can be seen that at the working concentration for inhibiting tumor cells, the toxicity to the heart is weak, and the safety is high.

Claims

1. Use of bufalin derivatives in the preparation of antitumor drugs, said tumor being pancreatic cancer, liver cancer, brain glioma, said bufalin derivatives having the structural formula as shown in formula I: ###0001### Formula I.

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