An isodomesticenol compound, a preparation method and application thereof

Abstract

The application discloses an isodiallylartemisia ketone compound and a preparation method and application thereof, and has a structure shown in formula I: The isodiallylartemisia ketone compound provided by the application has a strong inhibiting effect on histone deacetylase and a strong inhibiting effect on cancer cells, and can form a new generation of anticancer leading compound; compared with the histone deacetylase inhibitor vorinostat which has been put on the market, the activity of the isodiallylartemisia ketone compound provided by the application on the histone deacetylase is significantly improved, and the effect on solid tumors such as lung cancer is better, and the problems of low activity and poor effect on solid tumors of the existing histone deacetylase inhibitors are overcome.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and in particular to an isocarboxylic acid lactone compound, its preparation method, and its application. Background Technology

[0002] Histone deacetylases (HDACs) are a class of proteases widely present in eukaryotic cells. HDACs participate in various cellular physiological processes through the acetylation of histone and non-histone substrates, and are closely related to the occurrence and development of cancer. HDAC inhibitors can inhibit cancer cell proliferation and promote apoptosis, and have become an effective means of cancer treatment. However, currently marketed histone deacetylase inhibitors (HDACis) are significantly less effective against solid tumors than against hematological malignancies.

[0003] Therefore, it is necessary to develop a new compound that has a strong inhibitory effect on histone deacetylase and can improve the therapeutic effect on solid tumors. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the first aspect of the present invention proposes an isothomonolactone compound that has a strong inhibitory effect on histone deacetylase and can improve the therapeutic efficacy against solid tumors.

[0005] A second aspect of the present invention also provides a method for preparing isocarboxylic acid lactone compounds.

[0006] A third aspect of the present invention also provides a histone deacetylase inhibitor.

[0007] A fourth aspect of the present invention also provides a pharmaceutical composition.

[0008] The present invention also provides an application.

[0009] According to a first aspect of the present invention, an isocarboxylic acid lactone compound has the structure shown in Formula I:

[0010]

[0011] The isocarboxylic acid lactone compounds according to embodiments of the present invention have at least the following beneficial effects:

[0012] The isotretinoin lactone compounds provided by this invention have a strong inhibitory effect on histone deacetylase and a strong inhibitory effect on cancer cells, and can form a new generation of anticancer lead compounds. Compared with the already marketed histone deacetylase inhibitor vorinostat, the isotretinoin lactone compounds provided by this invention significantly improve the activity of histone deacetylase and have a better effect on solid tumors such as lung cancer, overcoming the problems of low activity and poor efficacy of existing histone deacetylase inhibitors against solid tumors.

[0013] The method for preparing isocarboxylic acid lactone compounds according to a second aspect embodiment of the present invention includes the following steps:

[0014] Compound 1a, trifluoroacetic acid, triethylsilane and organic solvent were mixed and reacted, and then post-treated to obtain the isocarboxylic acid lactone compound shown in Formula I;

[0015] The structural formula of compound 1a is as follows:

[0016]

[0017] According to some embodiments of the present invention, the organic solvent is selected from at least one of dichloromethane and chloroform.

[0018] According to some embodiments of the present invention, the volume molar ratio of the organic solvent to compound 1a is 1 mL: (0.05–0.2) mmol.

[0019] According to some embodiments of the present invention, the volume ratio of the organic solvent, trifluoroacetic acid and triethylsilane is 1:(0.01-0.03):(0.01-0.03).

[0020] According to some embodiments of the present invention, the post-processing includes the steps of purification, filtration and drying.

[0021] A histone deacetylase inhibitor provided according to a third aspect of the present invention comprises the above-described isothomyl lactone compounds.

[0022] A pharmaceutical composition according to a fourth aspect of the present invention comprises the above-described isothomyl lactone compound or the above-described histone deacetylase inhibitor; and pharmaceutically acceptable excipients.

[0023] According to some embodiments of the present invention, the excipients include at least one of a slow-release agent, a filler, a disintegrant, an adsorbent carrier, an absorbent, a surfactant, or a lubricant.

[0024] According to some embodiments of the present invention, the dosage form of the pharmaceutical composition is a solution, emulsion, pill, tablet, capsule or powder.

[0025] The use of the above-described isocarboxylide compounds, or the above-described histone deacetylase inhibitors, or the above-described pharmaceutical compositions in the preparation of antitumor drugs, according to the fifth aspect of the present invention.

[0026] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Detailed Implementation

[0027] The following are specific embodiments of the present invention, and the technical solutions of the present invention will be further described in conjunction with the embodiments, but the present invention is not limited to these embodiments.

[0028] Unless otherwise specified, the reagents, methods and equipment used in this invention are all conventional reagents, methods and equipment in this technical field.

[0029] The reaction equation and preparation steps for compound 1a are as follows:

[0030]

[0031] Under argon protection, in a 100 mL reaction flask, 1 h (100 mg, 0.40 mmol, 1 eq), BOC-D-alanine (76 mg, 0.40 mmol, 1 eq), N,N'-diisopropylcarbodiimide (61 mg, 0.48 mmol, 1.2 eq), and 4-dimethylaminopyridine (5 mg, 0.04 mmol, 0.1 eq) were added, followed by 10 mL of dichloromethane. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed by TLC monitoring, the reaction was stopped, and 15 mL of saturated NaHCO3 solution was added. The mixture was extracted three times with dichloromethane, and the organic phase was washed with saturated brine. The mixture was dried overnight with anhydrous magnesium sulfate. After drying, the anhydrous magnesium sulfate was filtered off, and the solvent was removed by vacuum distillation. The crude product was separated by silica gel column chromatography to obtain 1 g of intermediate (131 mg), with a yield of 78%. 1H NMR(500MHz,Chloroform-d)δ6.15(s,1H),5.61(s,1H),5.38(s,1H),5.15(s,1H),5.04(d, J=8.1Hz,1H),4.73(s,1H),4.56-4.50(m,1H),4.30(t,J=7.4Hz,1H),3.06-2.98(m,1H),2. 27-2.20(m,2H),2.18(d,J=2.5Hz,1H),1.89-1.76(m,2H),1.72(ddd,J=13.9,7.1,2.6Hz,1 H),1.59-1.55(m,2H),1.44(s,9H),1.36(d,J=7.2Hz,3H),1.34-1.24(m,2H),0.84(s,3H).

[0032] Under argon protection, 1 g (142 mg, 0.34 mmol, 1 eq) of intermediate was added to a 50 mL reaction flask, dissolved in 3 mL of dichloromethane, and then trifluoroacetic acid (1163 mg, 10.2 mmol, 30 eq) was added. The reaction was carried out at room temperature for 1 h, concentrated under reduced pressure, and the solution was adjusted to neutral by adding saturated sodium bicarbonate. The solution was extracted with dichloromethane to obtain 103 mg of yellow solid 1f, with a yield of 95%. No purification was required before it could be added to the next step reaction.

[0033] Under argon protection, 1e (0.82 g, 5 mmol, 1 eq), malonic acid (1.56 g, 15 mmol, 3 eq), pyridine (0.4 mL), and 20 mL of DMF were added to a 100 mL reaction flask. The mixture was dissolved and reacted at 90 °C for 10 h. After the reaction was complete, the mixture was allowed to return to room temperature, and 60 mL of distilled water was added to precipitate the product. The product was filtered under reduced pressure and dried under vacuum to give 979 mg of intermediate product 1d, a white solid, with a yield of 90%. ¹H NMR (500 MHz, DMSO-d⁶) δ 12.58 (s, 1H), 7.97 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 16.1 Hz, 1H), 6.66 (d, J = 16.1 Hz, 1H), 3.86 (s, 3H).

[0034] Under argon protection, compound 1d (634 mg, 3.08 mmol, 1 eq), O-triphenylmethylhydroxylamine (848 mg, 3.08 mmol, 1 eq), HATU (1.4 g, 3.70 mmol, 1.2 eq), and N,N-diisopropylethylamine (1.5 mL, 9.24 mmol, 3 eq) were added to a 100 mL reaction flask and dissolved in 10 mL of DMF. The reaction was carried out at room temperature for 8 h. 50 mL of distilled water was added, and the mixture was extracted three times with ethyl acetate. The organic phase was extracted once with saturated sodium bicarbonate solution and once with 10% dilute hydrochloric acid. The mixture was washed with saturated brine and dried over anhydrous sodium sulfate overnight. After drying, the anhydrous sodium sulfate was filtered off, and the solvent was removed by vacuum distillation. The crude product was separated by silica gel column chromatography to obtain 913 mg of white solid 1c, with a yield of 64%. 1H NMR (500MHz, Chloroform-d) δ7.95 (d, J = 8.1Hz, 2H), 7.53-7.24 (m, 17H), 6.15 (d, J = 15.9Hz, 1H), 3.90 (s, 3H).

[0035] Under argon protection, 1 c (648 mg, 1.40 mmol, 1 eq) was added to a 100 mL reaction flask, dissolved in 2 mL of THF, and then 5.2 mL of distilled water was added. At 0 °C, 2.8 mL of a 2M aqueous solution of lithium hydroxide (134 mg, 5.6 mmol, 4 eq) was added. After reacting at 0 °C for 10 minutes, the ice bath was removed, and the reaction was allowed to proceed at room temperature for 8 hours. After the reaction was complete, the solution was adjusted to acidity with 10% dilute hydrochloric acid, extracted with ethyl acetate, and the solvent was removed by vacuum distillation to obtain 584 mg of white solid 1b, white solid, yield 93%: ¹H NMR (500 MHz, DMSO-d6) δ 13.01 (s, 1H), 10.51 (s, 1H), 7.91 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 7.41–7.22 (m, 15H), 6.61 (d, J = 15.9 Hz, 1H).

[0036] Under argon protection, compounds 1b (485 mg, 1.08 mmol, 1 eq), 1f (329 mg, 1.08 mmol, 1 eq), HATU (409 mg, 1.30 mmol, 1.2 eq), and N,N-diisopropylethylamine (0.5 mL, 3.08 mmol, 3 eq) were added to a 100 mL reaction flask and dissolved in 10 mL of DMF. The reaction was carried out at room temperature for 8 h. 50 mL of distilled water was added, and the mixture was extracted three times with ethyl acetate. The organic phase was extracted once with saturated sodium bicarbonate solution and once with 10% dilute hydrochloric acid. The mixture was washed with saturated brine and dried overnight with anhydrous sodium sulfate. After drying, the anhydrous sodium sulfate was filtered off, and the solvent was removed by vacuum distillation. The crude product was separated by silica gel column chromatography to obtain 518 mg of white solid 1a, with a yield of 64%. 1H NMR (500MHz, Chloroform-d) δ7.95 (d, J = 8.1Hz, 2H), 7.53-7.24 (m, 17H), 6.15 (d, J = 15.9Hz, 1H), 3.90 (s, 3H).

[0037] Example 1

[0038] Example 1 provides an isocarboxylic acid lactone compound, the reaction equation of which is as follows:

[0039]

[0040] Its preparation method is as follows:

[0041] Under argon protection, compound 1a (75.0 mg, 0.1 mmol) was added to a 100 mL reaction flask, dissolved in 1 mL of dichloromethane, followed by the addition of 0.02 mL of trifluoroacetic acid and 0.02 mL of triethylsilane. The reaction was allowed to proceed for 15 min. Then, 17 mL of a mixed solution of n-hexane:petroleum ether:diethyl ether (10:10:1) was added. The mixture was collected by filtration as a 28 mg yellow solid, yielding 56%.

[0042] Its 1H and 1C NMR spectra are as follows: 1H NMR (500MHz, DMSO-d6) δ10.83(s,1H),9.12(s,1H),8.84(d,J=7.2Hz,1H),7.95(d,J=8.0Hz,2H),7.69(d,J=8.0Hz,2H),7.5 0(d,J=15.8Hz,1H),6.55(d,J=15.8Hz,1H),5.96(s,1H),5.74(s,1H),5.25(s,1H),5.07(s,1H),4.66(s,1H),4.46(t,J=7.2 Hz,1H),4.43-4.37(m,1H),2.92-2.83(m,1H),2.09(d,J=12.5Hz,1H),1.94(d,J=15.0Hz,1H),1.78-1.64(m,2H),1.57(ddd ,J=13.9,7.1,2.6Hz,1H),1.52-1.42(m,1H),1.41(d,J=7.3Hz,3H),1.31-1.20(m,2H),1.09(q,J=12.8Hz,1H),0.68(s,3H). 13 C NMR (125MHz, DMSO) δ171.41,169.89,165.80,162.41,145.57,142.05,137.84,137.32,134.15,128.10,127.39 ,120.99,120.45,112.09,76.17,75.26,48.77,40.46,35.57,33.37,26.61,26.35,16.75,16.54.HRMS(ESI)m / z calculated for C 28 H 33 N₂O₇[M+H] + 509.2282, found 509.2279.

[0043] Comparative Example 1

[0044] Comparative Example 1 provides an isocarboxylidene, CAS No. 470-17-7; commercially available.

[0045] Example 2

[0046] Activity study of the isothomyl lactone compounds prepared in Example 1 against human histone deacetylase 6 (HDAC6):

[0047] (1) Experimental materials

[0048] Example 1 shows the preparation of isothomyl lactone compounds and positive controls vorinostat (SAHA), human histone deacetylase 6 (Abnova, ab42632), Trypsin (Sigama-Aldrich), microplate reader (Biotek NEO2), and black 96-well plates.

[0049] (2) Experimental methods

[0050] The black 96-well plate contained three systems: a blank group, a negative group, and a sample group, with three replicates for each system. The blank group (Bw) consisted of 40 μL buffer, 10 μL HDAC6 substrate, and 50 μL trypsin stop solution; the negative group (F0) consisted of 39 μL buffer, 1 μL HDAC6 enzyme, 10 μL substrate, and 50 μL trypsin stop solution; and the sample group (F) consisted of 39 μL buffer, 1 μL HDAC6 enzyme, 10 μL of different concentrations of compounds, and 10 μL HDAC6 substrate. The plates were incubated at 37°C for 10 min in a microplate shaker. Then, 50 μL of developer was added to each well, and the plates were incubated for another 35 min. Fluorescence intensity was measured using a Biotek NEO2 microplate reader at an excitation wavelength of 350 nm and an emission wavelength of 450 nm. The inhibition rate of different concentrations of compounds against HDAC6 enzyme was calculated. Nonlinear curve fitting analysis was performed on the inhibition rate at each concentration to obtain the half-maximum inhibitory concentration (IC50). 50 Value. Half-maximal inhibitory concentration (IC50) 50 The lower the value, the stronger the inhibitory effect of the compound on the HDAC6 enzyme.

[0051] As shown in Table 1, the half-maximal inhibitory concentration (IC50) of the positive control SAHA against human histone deacetylase 6 (HDAC6) was 24.55 nM, while the half-maximal inhibitory concentration (IC50) of the isothomoleactone compounds prepared in Example 1 was 7.69 nM, which was better than the positive control. This shows that the target compounds have strong inhibitory activity against HDAC6, while isothomoleactone has no activity against HDAC6 at 1000 μM.

[0052] Table 1

[0053]

[0054] Example 3

[0055] Study on the effect of isocarboxylic acid lactone compounds prepared in Example 1 on cancer cell proliferation

[0056] (1) Experimental materials

[0057] Example 1 includes the preparation of isothomyl lactone compounds and positive control vorinostat (SAHA), trypsin, washing solution PBS, fetal bovine serum, human lung cancer cells A549, double antibodies (penicillin and streptomycin), CCK-8 kit (Beyotime), microplate reader (Biotek NEO2) and 96-well plate.

[0058] (2) Experimental methods

[0059] Human lung cancer cells A549 were obtained from the Shanghai Institute of Biochemistry and Cell Biology. Cells were cultured in a 37°C incubator containing 5% CO2 in DMEM medium containing 10% serum and 1% penicillin and streptomycin.

[0060] For the SRB assay, cells in the logarithmic growth phase were first digested and counted. Cells were seeded into 96-well plates and incubated overnight. After cell attachment, different concentrations of the compound were added, with 7 concentrations for each compound and 3-5 replicates for each concentration. Cells and drugs were cultured for another 72 hours. Cells were then removed, and 50 μL of 50% (m / v) trichloroacetic acid was added to each well to fix the cells. The cell plate was first incubated at room temperature for 5 minutes, then at 40°C for 1 hour. The fixative was discarded, and the cells were washed 5 times with distilled water and air-dried. Next, 100 μL of 0.4% SRB solution was added to each well, and the cells were incubated at room temperature for 30 minutes, then washed at least 10 times with 1% acetic acid solution and air-dried. Finally, 150 μL of Tris solution was added, and absorbance was measured using a Biotec NEO2 microplate reader at 570 nm. The inhibition rate at each concentration was analyzed using a nonlinear curve fitting method to obtain the half-maximal inhibitory concentration (IC50). 50 value.

[0061] As shown in Table 2, the half-maximal inhibitory concentration (IC50) of the positive control SAHA against human lung cancer cells A549 was 2.47 μM, and the half-maximal inhibitory concentration (IC50) of the isocarboxylic acid lactone compound prepared in Example 1 was 0.12 μM, which was superior to the positive control and Comparative Example 1, showing that the target compound has a strong inhibitory activity against cancer cells.

[0062] Table 2

[0063]

[0064] Example 4

[0065] Evaluation of the in vivo antitumor activity of isocarboxylic acid lactone compounds prepared in Example 1

[0066] (1) Experimental materials

[0067] The experimental animals were 6-8 week old female BALB / c nu / nu mice (weighing about 20g), purchased from Shanghai Slack Laboratory Animal Co., Ltd., and the cell line was human non-small cell lung cancer cell line A549.

[0068] (2) Experimental steps

[0069] A lung cancer A549 nude mouse xenograft model was used, with 5 mice in each group. The mice were administered compound 1 (5 mg / kg) and SAHA (10 mg / kg) via tail vein injection every three days. The control group received only saline. Tumor size and body weight were measured and recorded every 3 days. Mice were sacrificed after 18 days, tumor tissue was weighed, and internal organ damage was examined. Data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 19.0 software. The t-test or ANOVA was used to evaluate whether the differences in means were statistically significant. A p-value < 0.05 was indicated by *; a p-value < 0.01 was indicated by **; and a p-value < 0.001 was indicated by ***. All experimental data are expressed as mean ± standard deviation (SD). Tumor inhibition rate (%) = (1 - tumor volume in the drug-treated group / tumor volume in the control group) * 100.

[0070] (3) Experimental Results

[0071] The in vivo antitumor activity of the isothomyl lactones prepared in Example 1 was further evaluated using the A549 nude mouse xenograft model. The tumor inhibition rate of the SAHA 10 mg / kg group was 39.0%, and the isothomyl lactones showed significant antitumor activity. When treated with 5 mg / kg of isothomyl lactones, the tumor inhibition rate was 52.7% (p < 0.05), indicating that the isothomyl lactones can significantly inhibit tumor growth.

[0072] The above embodiments demonstrate that compound 1 provided by the present invention has a strong inhibitory effect on histone deacetylase and cancer cells. Compared with the marketed histone deacetylase inhibitor SAHA, the isothomyl lactone histone deacetylase inhibitor provided by the present invention has significantly improved activity and can be developed into a new generation of anticancer drugs.

[0073] The present invention has been described in detail above with reference to the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A compound of isocarboxylic acid lactone, characterized in that, It has the structure shown in Equation I:

2. The method for preparing isocarboxylic acid lactone compounds according to claim 1, characterized in that, Includes the following steps: Compound 1a, trifluoroacetic acid, triethylsilane, and an organic solvent were mixed and reacted to obtain the isocarboxylic acid lactone compound shown in Formula I; the volume molar ratio of the organic solvent to compound 1a was 1 mL:(0.05–0.2) mmol; the volume ratio of the organic solvent, trifluoroacetic acid, and triethylsilane was 1:(0.01–0.03):(0.01–0.03). The structural formula of compound 1a is as follows:

3. The preparation method according to claim 2, characterized in that, The organic solvent is selected from at least one of dichloromethane and chloroform.

4. A histone deacetylase inhibitor, characterized in that, Including the isocarboxylic acid lactone compounds as described in claim 1.

5. A pharmaceutical composition, characterized in that, It includes the isothomyl lactone compound of claim 1 or the histone deacetylase inhibitor of claim 4; and pharmaceutically acceptable excipients.

6. The pharmaceutical composition according to claim 5, characterized in that, The excipients include at least one of the following: a slow-release agent, a filler, a disintegrant, an adsorbent carrier, an absorbent, a surfactant, or a lubricant.

7. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is a solution, emulsion, pill, tablet, capsule, or powder.

8. The use of the isothomyl lactone compound of claim 1; or the histone deacetylase inhibitor of claim 4; or the pharmaceutical composition of any one of claims 5 to 7 in the preparation of an antitumor drug.

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