Bufadienolide Compounds and Their Uses

Abstract

The present invention belongs to the field of medicinal chemistry and relates to bufadienolide compounds and their uses. The bufadienolide compounds of the present invention exhibit good anti-tumor activity and have high selectivity for EGFR T790M protein mutation.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and relates to bufadienolide compounds with anti-tumor activity or pharmaceutically acceptable salts thereof, pharmaceutical compositions containing the same, and uses thereof. Background Art

[0002] Cancer is a malignant disease with extremely high incidence and mortality rates and relatively poor treatment effects worldwide. Globally, anti-tumor drugs have become the largest therapeutic area in the drug market. Currently, commonly used anti-tumor drugs include cytotoxic drugs, hormonal drugs, molecular targeted therapy drugs, biological response modifiers, tumor differentiation inducers, tumor angiogenesis inhibitors, and drugs for adjuvant treatment of tumors, etc.

[0003] Lung cancer is known as the king of cancers due to its high incidence and mortality rates. Among them, non-small cell lung cancer (NSCLC) accounts for about 80%-90% of the total number of lung cancer patients. As one of the genes with the highest mutation frequency in non-small cell lung cancer, the development of its inhibitors has become an important strategy for the treatment of non-small cell lung cancer and has made great progress. Although the curative effect is remarkable, it is found that most patients will develop drug resistance after taking the medicine for about ten months during clinical use. Currently, the research on anti-EGFR active small molecules based on natural products has become a very promising research direction.

[0004] Venenum Bufonis is the dried secretion of the toad Bufo bufo gargarizans Cantor or B. melanostictus Schneider of the family Bufonidae, and has been widely used in the treatment of various tumors. According to literature reports and the previous research of the present inventors, it is found that bufadienolide compounds have good anti-tumor activity and have a significant inhibitory effect on lung cancer cell lines, but their clinical application is limited due to their significant toxicity. Therefore, the present inventors carried out structural modification based on the bufadienolide compounds in Venenum Bufonis as the parent nucleus to achieve the purpose of enhancing efficacy and reducing toxicity or maintaining efficacy and reducing toxicity. Summary of the Invention

[0005] The present invention uses three bufadienolide small molecules including resibufogenin, cinobufagin, and bufotalin as the cores, and reacts with malonic acid and succinic acid through esterification reactions to obtain a series of derivatives, and their structures are identified by HPLC, LC-MS / MS, and nuclear magnetic resonance. Subsequently, these derivatives are subjected to amidation reactions with L-histidine methyl ester to obtain 6 compounds, which are identified by LC-MS / MS and nuclear magnetic resonance as: 15-dehydrobufotalin-3-malonyl-L-histidine methyl ester, cinobufagin-3-malonyl-L-histidine methyl ester, bufotalin-3-malonyl-L-histidine methyl ester, resibufogenin-3-succinyl-L-histidine methyl ester, cinobufagin-3-succinyl-L-histidine methyl ester, bufotalin-3-succinyl-L-histidine methyl ester, all of which are new compounds. Through research, it is found that they have high anti-tumor activity, good selectivity, and low toxicity. These compounds or their pharmaceutically acceptable salts and their pharmaceutical compositions can be used to treat and / or prevent cancer, especially lung cancer, and in the case of EGFR L858R / T790M mutations.

[0006] The present invention provides the following bufadienolide amide compounds of Compound 1 - Compound 6 or their pharmaceutically acceptable salts:

[0007]

[0008] Preferably, they are bufadienolide amide compounds of Compound 2, Compound 3, Compound 5, or Compound 6 or their pharmaceutically acceptable salts. These compounds have good inhibitory activity against the NCI-H1975 cell line.

[0009]

[0010] More preferably, they are bufadienolide amide compounds of Compound 3 or Compound 6 or their pharmaceutically acceptable salts.

[0011]

[0012] Even more preferably, the bufadienolide amide compounds or their pharmaceutically acceptable salts have a high inhibition rate against NCI-H1975 cells, and at the same time have low toxicity to A549 cells and excellent selectivity.

[0013] The present invention also provides a preparation method for the bufadienolide amide compounds of Compound 1 - Compound 6 or their pharmaceutically acceptable salts, including the following steps (taking cinobufagin as an example):

[0014]

[0015] Cinobufagin was reacted with Meldrum's acid and succinic anhydride respectively to obtain Intermediate 1 and Intermediate 2, and Intermediate 1 and Intermediate 2 were respectively condensed with methyl histidinate to obtain Compound 2 and Compound 5. Among them, bufadienolide compounds, Meldrum's acid, succinic anhydride, and methyl histidinate can all be directly obtained commercially.

[0016] In another aspect of the present invention, the present invention provides a pharmaceutical composition comprising one or more bufadienolide amide compounds of Compound 1 - Compound 6 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0017] In still another aspect of the present invention, the present invention provides the use of bufadienolide amide compounds of Compound 1 - Compound 6 or a pharmaceutically acceptable salt thereof, and their pharmaceutical compositions in the preparation of drugs for preventing and / or treating cancer.

[0018] Preferably, the cancer is mainly lung cancer.

[0019] Preferably, the lung cancer has an EGFR L858R / T790M protein mutation, and more preferably has an EGFR T790M protein mutation.

[0020] The bufadienolide amide compounds of Compound 1 - Compound 6 or a pharmaceutically acceptable salt thereof provided by the present invention have strong inhibitory activity against tumor cells, and at the same time show obvious selectivity, with very low killing effect on wild-type EGFR cells and low toxicity. The bufadienolide amide compounds of Compound 1 - Compound 6 or a pharmaceutically acceptable salt thereof provided by the present invention have anti-tumor activity, low toxicity, strong selectivity, a mild synthesis method, simple operation, are easy to synthesize, and are suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows the inhibitory activity of the compounds of the present invention against the NCI-H1975 cell line;

[0022] Figure 2 shows the inhibitory activity of the compounds of the present invention against the NCI-H1975 and A549 cell lines;

[0023] Figure 3A is an SPR analysis chart of the time-response value of cinobufotalin binding to EGFR T790M;

[0024] Figure 3B is an SPR analysis chart of the concentration-response value of cinobufotalin binding to EGFR T790M;

[0025] Figure 4A is an SPR analysis chart of the time-response value of Compound 3 of the present invention binding to EGFR T790M;

[0026] Figure 4B SPR analysis chart of the concentration-response value of the compound 3 of the present invention binding to EGFR T790M;

[0027] Figure 5A SPR analysis chart of the time-response value of the compound 6 of the present invention binding to EGFR T790M;

[0028] Figure 5B SPR analysis chart of the concentration-response value of the compound 6 of the present invention binding to EGFR T790M;

[0029] Figure 6A SPR analysis chart of the time-response value of Olmutinib binding to EGFR T790M;

[0030] Figure 6B SPR analysis chart of the concentration-response value of Olmutinib binding to EGFR T790M. Detailed implementation manners

[0031] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the test methods without specific conditions in the following embodiments, the conventional conditions of such reactions are usually followed. The terms used in the present invention are only used for description and not to limit specific embodiments. Unless otherwise specified in the context, the meanings of all technical terms and scientific terms used in the present invention are intended to be the same as those commonly understood by those skilled in the art.

[0032] The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0033] The structures of the compounds described in the following embodiments were determined by nuclear magnetic resonance 1 1H-NMR and 13 13C-NMR. The nuclear magnetic resonance measurement instrument used was an Agilent DD2 600MH nuclear magnetic resonance spectrometer, and the measurement solvents were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), and hexadeuterated dimethyl sulfoxide (DMSO-d6), and the internal standard substance was tetramethylsilane (TMS).

[0034] The meanings represented by the abbreviations in the nuclear magnetic resonance (NMR) data in the following embodiments are as follows:

[0035] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, m: multiplet, J: coupling constant, Hz: Hertz, δ: chemical shift. The chemical shift (δ) value is given in parts per million (ppm).

[0036] Example 1: Preparation of Compound 2

[0037]

[0038] Step 1: Dissolve 30.62 mg (0.069 mmol) of cinobufagin in 2 ml of acetonitrile, add 30.04 mg (0.208 mmol) of malonic acid, reflux and stir the reaction in an oil bath for about 10 h. Evaporate the solvent under reduced pressure, recrystallize with ethyl acetate / petroleum ether, and filter to obtain 27.17 mg of white crystals (cinobufagin-3-malonate half ester), with a yield of 74.4%.

[0039] Step 2: Dissolve 19.90 mg (0.037 mmol) of cinobufagin-3-malonate half ester and 31.30 mg (0.129 mmol) of L-histidine methyl ester dihydrochloride in 2 ml of dichloromethane, stir at room temperature for 15 minutes, add 37.40 mg (0.195 mmol) of EDCI, 15.53 mg (0.114 mmol) of HOBt, and 1 drop of triethylamine. After stirring at room temperature for 30 minutes, change to reflux at 40 °C and react until the raw materials disappear. Evaporate the solvent under reduced pressure, and after purification, obtain 6.83 mg of white powder (cinobufagin-3-malonyl-L-histidine methyl ester, Compound 2), with a yield of 27.1%.

[0040] 11H NMR (600 MHz, MeOD): δ 8.02 (1H, s), 7.60 (1H, s), 7.37 (1H, s), 6.89 (1H, s), 6.24 (1H, d, J = 9.8 Hz), 5.49 (1H, d, J = 9.3 Hz), 5.10 (1H, s), 4.70 (1H, dd, J = 7.8, 5.5 Hz), 3.74 (1H, s), 3.70 (3H, s), 3.35 (1H, s), 3.12 (1H, dd, J = 14.9, 5.5 Hz), 3.03 (1H, dd, J = 14.9, 7.7 Hz), 2.92 (1H, d, J = 9.3 Hz), 1.93 (1H, s), 1.86 (3H, s), 0.99 (3H, s), 0.82 (3H, s).

[0041] 13 13C NMR (150 MHz, MeOD): δ 172.96, 171.60, 168.87, 168.38, 164.02, 150.88, 136.37, 130.85, 118.38, 114.08, 76.63, 73.39, 73.37, 60.83, 54.26, 52.80, 51.43, 46.35, 40.61, 40.28, 38.12, 36.30, 34.48, 31.35, 31.21, 30.83, 30.33, 30.21, 28.11, 26.68, 25.78, 24.02, 22.10, 21.47, 20.36, 17.49.

[0042] Example 2: Preparation of Compound 5

[0043]

[0044] Step 1: Dissolve 49.98 mg (0.112 mmol) of cinobufagin in 2 ml of dichloromethane, add 28.07 mg (0.229 mmol) of DMAP and 27.70 mg (0.276 mmol) of succinic anhydride, introduce nitrogen, reflux at 40 °C until the reaction is complete, wash with water and saturated sodium chloride 3 times respectively, 10 ml each time. After drying the organic layer with anhydrous magnesium sulfate, prepare a white powder of cinobufagin-3-succinic acid semiester by thin layer chromatography, with a yield of 79.2%.

[0045] Step 2: Dissolve 40.34 mg (0.074 mmol) of cinobufotalin-3-succinic acid semiester and 54.24 mg (0.224 mmol) of L-histidine methyl ester dihydrochloride in 4 ml of dichloromethane, stir at room temperature for 15 minutes, add 71.41 mg (0.372 mmol) of EDCI, 30.42 mg (0.225 mmol) of HOBT and 2 drops of triethylamine, stir at room temperature for 30 minutes and then reflux at 40 °C until the raw materials disappear. Evaporate the solvent under reduced pressure, and after purification, obtain 18.51 mg of white powder (cinobufotalin-3-succinyl-L-histidine methyl ester, Compound 5) with a yield of 36.1%.

[0046] 1 H NMR (600 MHz, MeOD): δ 8.02 (1H, s), 7.65 (1H, s), 7.37 (1H, s), 6.90 (1H, s), 6.24 (1H, d, J = 9.7 Hz), 5.49 (1H, d, J = 9.3 Hz), 5.06 (1H, s), 4.65 (1H, dd, J = 8.2, 5.4 Hz), 3.74 (1H, s), 3.69 (3H, s), 3.10 (1H, dd, J = 14.9, 5.5 Hz), 3.00 (1H, dd, J = 14.9, 8.1 Hz), 2.92 (1H, d, J = 9.3 Hz), 2.61–2.50 (4H, m), 1.00 (3H, s), 0.82 (3H, s).

[0047] 13 C NMR (150 MHz, MeOD): δ 174.22, 173.74, 173.32, 171.61, 164.03, 153.52, 150.89, 136.29, 118.39, 114.08, 76.63, 73.40, 72.39, 64.44, 60.83, 54.11, 52.73, 51.43, 46.35, 40.61, 40.29, 38.31, 36.32, 34.49, 31.51, 31.31, 31.20, 30.67, 30.05, 26.72, 25.87, 24.11, 22.10, 21.49, 20.36, 17.49.

[0048] The inventors of the present invention also synthesized the following compounds using a synthesis method similar to that of Example 1 and Example 2.

[0049] Table 1 Structural formulas and data analysis of the compounds of the present invention

[0050]

[0051] Example 3: In vitro activity study

[0052] The anti-proliferative activities of the compounds of the present invention against NCI-H1975 cells (EGFR L858R / T790M) and A549 cells (EGFR WT) were studied.

[0053] (1) Test method: The CCK8 method was used to study the inhibitory effect of the compounds on cell proliferation.

[0054] (2) Instruments and reagents are shown in Tables 2 and 3.

[0055] Table 2 Reagent List

[0056]

[0057] Table 3 Instrument List

[0058]

[0059] (3) Compound preparation

[0060] The compounds were dissolved in DMSO and stored at room temperature under a nitrogen atmosphere for later use. The samples in DMSO were serially diluted with the culture medium to the required concentrations.

[0061] (4) Test method

[0062] NCI-H1975 and A549 cells in the logarithmic growth phase were digested with trypsin and directly pipetted into single-cell suspensions. The NCI-H1975 cell suspension was adjusted to 1×10 5 / ml, and the A549 cell suspension was adjusted to 5×10 4 / ml. They were respectively inoculated into 96-well culture plates, 100 μL per well. After culturing and adhering in a 37°C, 5% CO2 incubator, 1 μM of each of the above compounds was added to the culture plates by changing the medium, 100 μL per well. Each sample was in triplicate, and a blank group and a control group were established simultaneously. After adding the drugs, the NCI-H1975 cells were cultured for another 48 h, and the A549 cells were cultured for another 24 h. Then, 100 μL of CCK-8 solution containing 10% was added to each well, and the culture plate was incubated in the incubator for 2 hours. The absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0063] Inhibition rate = [(Ac - As) / (Ac - Ab)] × 100% (the same hereinafter)

[0064] As: Absorbance of the experimental well (containing cells, culture medium, CCK-8 solution, and compound solution)

[0065] Ac: Absorbance of the control well (containing cells, culture medium, CCK-8 solution, without compound solution)

[0066] Ab: Absorbance of blank well (containing culture medium, CCK-8 solution, without cells and compounds)

[0067] The results are shown in Table 4 below and Figures 1-2 . Table 4 shows the inhibition rates (x±s, n = 3) of the compounds on NCI-H1975 cells (EGFR L858R / T790M) and A549 cells (EGFR WT) at 1 μM. Figure 1 is the inhibitory activity of the compound on the NCI-H1975 cell line; Figure 2 is the inhibitory activity of the compound on the NCI-H1975 and A549 cell lines.

[0068] Table 4 Inhibition rates of compounds on cells

[0069]

[0070] From Table 4 and Figures 1-2 it can be seen that bufotalin series amide derivatives (Compound 3 and Compound 6) have good inhibition rates on NCI-H1975 cells at 1 μM, and at the same time have low toxicity to A549 cells, showing excellent selectivity. In addition to the good selectivity of bufotalin series amide compounds, cinobufagin series amide compounds (Compound 2 and Compound 5) also have good selectivity.

[0071] Example 4: Surface Plasmon Resonance Technology

[0072] The in vivo antitumor activity of the compounds of the present invention against human lung cancer was studied.

[0073] (1) Instruments and reagents

[0074] Table 5 List of reagents

[0075]

[0076] Table 6 List of instruments

[0077]

[0078] (2) Pre-enrichment experiment

[0079] The recombinant TLR4 protein was immobilized on the surface of the sensor CM5 chip by the amino coupling method. Take 3 μL of the prepared EGFR T790M protein solution with a mass concentration of 200 μg / mL and dissolve it with 97 μL of sodium acetate solution at pH 4.0, respectively, and place them in EP tubes without lids and with labels. Measure 300 μL of NaOH solution and place it in another lidless EP tube for CM5 chip regeneration. During the pre-enrichment test, select channel 2 of the chip as the protein coupling channel and channel 1 as the reference channel, and set the injection program: the sample flow cell temperature is 16 °C, the detection temperature is 25 °C, inject the EGFR protein mutant solution for 30 s, wait for 10 s, regenerate with NaOH solution for 30 s, and the flow rate is 10 μL / min. Observe the RU value to determine the optimal coupling conditions.

[0080] (3) Detection of the binding ability between the compound and the EGFR protein mutant

[0081] The EGFR T790M protein was diluted to 20 μg / mL in 10 mM CH3COONa solution at pH 4.0 under the optimal ligand enrichment conditions. It was injected onto the CM5 chip at a flow rate of 10 μL / min. Then, the carboxyl groups on the sensor surface were activated by injecting a mixture of EDC and NHS (1:1), and the remaining carboxyl groups were blocked with ethanolamine to end the coupling. The target coupling amount of the EGFR T790M protein was 8000 response units (RU). After the coupling of the EGFR T790M protein was completed, a running buffer (1×PBS-P+ with 5% DMSO) and 4.5% and 5.8% solvent calibration mother liquors were prepared. The LMW kinetics method in the multi-cycle kinetic detection method was used to pass the analyte solutions of the compound (0, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 μM) diluted with 1×PBS-P+ containing 5% DMSO through the surface of the CM5 chip in turn. After setting the program, the instrument will run automatically. After each analysis cycle, it was regenerated with 50 mM NaOH. After the run, the Biacore T200 Evaluation Software was used for result analysis. The equilibrium dissociation constant KD was obtained to evaluate the affinity (the smaller the value, the stronger the affinity). Assuming the binding ratio is 1:1, the results are shown in Table 8 below and Figures 3A-6B . Among them, Figure 3A is the SPR analysis chart of the time-response value of the binding between bufotalin and EGFR T790M; Figure 3B is the SPR analysis chart of the concentration-response value of the binding between bufotalin and EGFR T790M;

[0082] Figure 4A is the SPR analysis chart of the time-response value of the binding between compound 3 and EGFR T790M;Figure 4B SPR analysis chart of the concentration-response value of compound 3 binding to EGFR T790M; Figure 5A SPR analysis chart of the time-response value of compound 6 binding to EGFR T790M; Figure 5B SPR analysis chart of the concentration-response value of compound 6 binding to EGFR T790M; Figure 6A SPR analysis chart of the time-response value of Olmutinib binding to EGFR T790M; Figure 6B SPR analysis chart of the concentration-response value of Olmutinib binding to EGFR T790M.

[0083] Table 7 5% DMSO concentration calibration curve

[0084]

[0085] Table 8 KD values of compounds for EGFR T790M protein

[0086]

[0087] Through Table 8 and Figures 3A-6B The SPR experiment further verifies the binding ability of the compounds of the present invention to EGFR T790M protein. The study found that bufotalin has extremely weak binding ability to this protein, but the binding affinity of the derivatives after structural modification (compound 3 and compound 6) to EGFR T790M protein is increased by about 100 - 1000 times compared with bufotalin, and the affinity is increased by about 5 - 60 times compared with the positive control Olmutinib.

[0088] The technical intention used in the present invention refers to the technology generally understood in the art, including changes to the technology that are obvious to those skilled in the art or substitutions of equivalent technologies.

Claims

1. Compounds 1 - 6 bufadienolide compounds or their pharmaceutically acceptable salts, 2. A pharmaceutical composition comprising one or more of the compounds 1 - 6 bufadienolide compounds or their pharmaceutically acceptable salts according to claim 1, and a pharmaceutically acceptable carrier.

3. Use of the compounds 1 - 6 bufadienolide compounds or their pharmaceutically acceptable salts according to claim 1, or the pharmaceutical composition according to claim 2, in the preparation of a medicament for preventing and / or treating cancer, wherein, The cancer is lung cancer.

4. Use according to claim 3, wherein, The lung cancer has an EGFR T790M protein mutation.

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