Indolo delta-sultone crystal form compound and application

Abstract

The invention discloses a crystal form I of an indolo delta-sultone compound A with a structure shown in a figure as well as a preparation method and application thereof. In an X-ray powder diffraction pattern of the crystal form I, peaks exist when 2 theta is 7.1, 8.4, 12.0, 15.2, 18.3, 18.9, 20.0, 21.4, 22.0, 22.9, 22.2, 26.9, 27.3 and 29.8. The invention also provides a preparation method of the crystal form I. The successfully prepared compound A and the crystal form I thereof have certain anti-tumor activity and can be used for preparing anti-tumor drugs. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an indole delta-sultone crystalline compound and its use. Background Art

[0002] Indole rings are widely present in natural products and synthetic drugs. Related studies have shown that compounds containing indole skeleton structures have a variety of important biological activities and pharmacological effects. Sulfonates or δ-sultones are also present in many drug molecules. However, substances with both indole rings and δ-sultone structures have been less studied. In patent CN202310180410.8, indolin-3-one substances and styrene sulfonyl fluoride substances are reacted together, and the two skeleton structures are successfully combined in the same molecule to obtain a class of substances with novel structural skeletons, namely indole δ-sultone compounds. This series of compounds has certain inhibitory activity against two cancer cells, HepG2 and Caco2, and can be further developed as anticancer drugs or as lead compounds to further optimize and modify their structures. Summary of the invention

[0003] The object of the present invention is to provide a crystalline form I of an indole δ-sultone compound A and its use. The structure of compound A is shown in the following formula:

[0004]

[0005] The present invention also provides a crystalline form I of compound A, which is tested by X-ray powder diffraction, and its X-ray powder diffraction pattern has peaks at 2θ of 7.1, 8.4, 12.0, 15.2, 18.3, 18.9, 20.0, 21.4, 22.0, 22.9, 22.2, 26.9, 27.3, and 29.8 ( Figure 1 ), the data are shown in the following table:

[0006] Serial number 2-Theta d Height Height % Area Area % 1 7.1 449 18564 100.0 212741 100.0 2 8.4 495 13183 71.0 136536 64.2 3 12.0 362 1126 6.1 22020 10.4 4 15.2 5.8401 1660 8.9 24341 11.4 5 18.3 4.8437 3012 16.2 58199 27.4 6 18.9 4.7014 1160 6.2 25998 12.2 7 20.0 4.4272 18503 99.7 207019 97.3 8 21.4 4.1562 1022 5.5 17546 8.2 9 22.0 4.0331 2939 15.8 47370 22.3 10 22.9 3.8870 662 3.6 18948 8.9 11 22.2 3.6684 508 2.7 13071 6.1 12 26.9 3.3117 6678 36.0 154644 72.7 13 27.3 3.2642 1428 7.7 33797 15.9 14 29.8 2.9918 1208 6.5 31499 14.8

[0007] The present invention also provides a method for preparing the above-mentioned crystal form I, comprising the following steps:

[0008] Compound A is taken and recrystallized in an acetone-cyclohexane system, wherein the amount of acetone (ml) is 2-10 times that of compound A (g), and the amount of cyclohexane is 3-10 times that of acetone.

[0009] The present invention also provides use of compound A and its crystal form I in preparing anticancer drugs.

[0010] Furthermore, the uses of compound A and its crystalline form I in the preparation of anti-cancer drugs include: use as a preparation of anti-lung cancer drugs, use as a preparation of anti-cervical cancer drugs, use as a preparation of anti-melanoma drugs, use as a preparation of anti-colorectal cancer drugs, use as a preparation of anti-breast cancer drugs, and / or use as a preparation of anti-liver cancer drugs.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The invention obtains a new crystal compound having an indole delta-sultone skeleton structure. Experimental results show that the new compound and the crystal have anti-tumor activity and can be used for preparing anti-cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 X-ray powder diffraction pattern (XRD pattern) of Form I of Compound A

[0014] Figure 2 Compound A inhibits colony formation of human lung adenocarcinoma cell lines A549 and H1299

[0015] Figure 3 Compound A inhibits DNA replication in human lung adenocarcinoma cells A549 and H1299

[0016] Figure 4 Compound A induces apoptosis in human lung adenocarcinoma cells A549 and H1299

[0017] Figure 5 Compound A inhibits tumor growth in xenograft models DETAILED DESCRIPTION

[0018] The technical scheme of the present invention is further specifically described below through specific examples, but the present invention is not limited to these examples. The reagents used in the present invention are all commercially available, and unless otherwise specified, all are prepared using existing technologies or natural conditions at normal temperature and pressure.

[0019] Example 1. Preparation of Compound A

[0020] N-acetyl-3-indolone (175 mg, 1.0 mmol), p-methoxystyrenesulfonyl fluoride (324 mg, 1.5 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (abbreviated as DBU, 166 mg, 1.1 mmol) were added to a three-necked reaction bottle, and 5 mL of dichloromethane was added. The mixture was stirred at 10°C for 60 min and monitored by TLC until the reaction was completed. The mixture was subjected to silica gel column chromatography with an eluent of petroleum ether:ethyl acetate = 10:1-6:1. The product was collected and concentrated to dryness to obtain 278 mg of the target compound A with a yield of 75%. 1HNMR and 13 CNMR is consistent with patent CN202310180410.8.

[0021] Example 2. Preparation of Form I

[0022] Place 200 mg of compound A in a 50 ml three-necked flask, add 2 mL of acetone, heat in an oil bath, stir, and the compound is completely dissolved and clarified. While stirring, slowly drop 12 mL of cyclohexane. Remove the three-necked flask from the oil bath, cool it in an ice bath, continue stirring, and crystals slowly precipitate. Stir and grow the crystals for 1-2 hours, filter, wash the crystals with 1 mL of a mixed solvent of acetone / cyclohexane (1:6), filter, place the crystals in a vacuum oven, and dry under reduced pressure to obtain 160 mg of product. This crystalline form was tested by X-ray powder diffraction ( Figure 1 ). The X-ray powder diffraction test conditions are: 2-60 degrees, step size 0.02, scanning speed 8 degrees / min, copper target, 40KV, 15mA.

[0023] The beneficial effects of the present invention are further illustrated by test examples below.

[0024] Test Example 1: Investigation of the stability and hygroscopicity of the crystal form of the present invention

[0025] 1. Stability

[0026] The compound crystal form I of the present invention was placed in a stability test chamber for accelerated testing, and the test conditions were: temperature, 40°C ± 2°C; humidity, RH75% ± 5%, and the time was 3 months. Results: TLC and HPLC were used to determine that the crystalline compound did not undergo significant changes, indicating that the crystal form of the present invention has good stability.

[0027] 2. Hygroscopicity

[0028] The test results were as follows:

[0029] Time (days) 0 5 10 15 Compound weight gain due to moisture absorption 0％ 0.2％ 0.2％ 0.2％

[0030] The results in the above table show that the crystals of the present invention do not significantly increase in weight due to moisture absorption when placed in a humid environment for 15 days, indicating that the crystal form of the present invention can effectively prevent the compound from hygroscopic deliquescence.

[0031] Test Example 2: Antitumor Activity

[0032] Experimental method: The cell proliferation experiment is determined by the CCK-8 experimental method. The experimental principle is: during cell metabolism, the cell body is in a reducing environment compared to the cell body outside the body. When the CCK-8 detection reagent enters the cell, it can be reduced by some dehydrogenases in the mitochondria in the presence of electron coupling reagents to generate orange-yellow formazan. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color is linearly related to the number of cells. The anti-tumor activity test includes the following steps:

[0033] 1. Inoculate tumor cells into 96-well cell culture plates and treat overnight to allow cells to adhere to the plate and grow;

[0034] 2. When the cell confluence rate reaches more than 90%, add different test compounds to act on the cells for 24 hours;

[0035] 3. Then add 10 μL CCK-8 detection reagent to each well at 10% (v / v) of the culture medium volume and place at 37°C.

[0036] Incubate in a cell culture incubator for 1-4 hours;

[0037] 4. After the culture solution turns orange-yellow, use a multifunctional reader to measure its absorbance at a wavelength of 450 nm; 5. Proliferation inhibition rate (%) = 100-(OD 实验孔 -OD 空白孔 ) / (OD 对照孔 -OD 空白孔 )*100.

[0038] The results showed that when the concentration of compound A was 10 μM, the growth inhibition rate of human lung adenocarcinoma cell A549 was 81.14%, the growth inhibition rate of human cervical cancer cell HeLa was 83.58%, the growth inhibition rate of human melanoma cell A375 was 77.86%, the growth inhibition rate of human colorectal cancer cell HCT116 was 78.92%, the growth inhibition rate of human breast cancer cell MCF-7 was 68.17%, and the growth inhibition rate of human liver cancer cell SK-Hep-1 was 70.56%. It can be seen from the above experiments that compound A and its crystal form I of the present invention have good anti-tumor activity against various tumor cells.

[0039] Experimental Example 3: Tumor Cell Proliferation Inhibition Experiment

[0040] When the cells in the logarithmic growth phase grow to 80%-90% confluence, discard the old culture medium, gently wash the cells with sterile PBS buffer, discard, and add an appropriate amount of trypsin to digest the cells. When the cells can fall off by tapping the culture dish, add culture medium containing serum to terminate digestion. Transfer the cells to a centrifuge tube, centrifuge at 1000rpm for 3 minutes, discard the culture medium containing trypsin, resuspend the cells with fresh culture medium, and count them. Dilute with culture medium to an appropriate concentration and adjust the cell density to 3×10 4 / mL, inoculate into 96-well plates, add 100 μL of cell suspension to each well, and culture overnight in a 37°C, 5% CO2 incubator until the cells adhere. After compound A is prepared to the set concentration with culture medium, discard the old culture medium, add the culture medium containing compound A to the 96-well plate, add 100 μL to each well, set 3 replica wells for each concentration, and culture in the incubator for 48 hours. After the incubation is completed, remove the cells, add the reagent according to the instructions of the CCK-8 kit, and place the 96-well plate in a 37°C incubator for about 1-4 hours. Read the OD on the microplate reader 450 GraphPad 9.0 software was used to perform nonlinear fitting on the compound concentration and cell proliferation inhibition rate to calculate IC 50 .

[0041]

[0042] The test results show that compound A is more effective than the control drug cisplatin for the six tumor cell types tested.

[0043] Experimental Example 4: Plate colony formation experiment

[0044] Experimental method: The plate cloning experiment uses crystal violet dye to stain cells. Experimental principle: Crystal violet is an alkaline dye that can bind to DNA in the cell nucleus to form a purple complex, thereby staining the cell nucleus; the staining results can be used to determine the number and size of cell colonies formed, and to evaluate the ability of single cells to proliferate and form clusters and their survival. The specific steps of the plate cloning experiment are as follows:

[0045] Digest the cells in the logarithmic phase with trypsin, centrifuge at 1000rpm for 3 minutes, and then resuspend them in culture medium to form a single cell suspension. Count and dilute them with culture medium to an appropriate concentration, dilute to a cell density of about 500 / mL, and then add them to a 6-well plate with 1000 cells inoculated per well. Shake the 6-well plate slightly to ensure that the cells are evenly distributed in the 6-well plate. Culture in a 37°C, 5% CO2 incubator until the cells adhere to the wall. Add culture medium containing different concentrations of compound A to the 6-well plate, change the culture medium containing compound A every three days, and culture in an incubator for more than 7 days until the cell colony is clearly visible. After the cloned cell clusters are clearly visible in the control group, remove the 6-well plate. The culture medium was discarded, and the cells were gently washed three times with PBS buffer. 4% paraformaldehyde was added to each well to fix the cells for 30 minutes. The 4% paraformaldehyde was gently removed, and the cells were gently washed three times with PBS buffer. 0.1% crystal violet was added for staining for 20 minutes. The excess dye was gently rinsed with water, and the cells were dried in air. The cells were photographed, and the number of cell clones was counted using image J software. The results were analyzed using GraphPad 9.0 software ( Figure 2 ).

[0046] The experimental results showed that after 7 days of culture, there was no significant change in the number of cell clones in the low concentration group compared with the control group, while the number of clones of human lung adenocarcinoma cells A549 and H1299 in the medium and high concentration groups was significantly reduced, and the high concentration group had the most significant inhibitory effect on proliferation, indicating that compound A can effectively inhibit the proliferation ability of lung cancer cells.

[0047] Experimental Example 5: EdU Experiment

[0048] Experimental method: EdU experiment uses EdU dye to stain newly synthesized DNA. Experimental principle: EdU (5-ethynyl-2'-deoxyuridine) is a new thymidine analog. EdU can replace thymidine and be incorporated into newly synthesized DNA during DNA synthesis. On the other hand, the ethynyl group on EdU can covalently react with the fluorescently labeled small molecule azide probe through the catalysis of monovalent copper ions to form a stable triazole ring. The reaction is very rapid. Through this reaction, the newly synthesized DNA will be labeled with the corresponding fluorescent probe, so that the proliferating cells can be detected using fluorescence detection equipment. Therefore, the more cells proliferate, the stronger the fluorescence intensity. The specific operation steps of the EdU experiment are as follows:

[0049] Digest the cells in the logarithmic phase, centrifuge, and count. Dilute with culture medium to an appropriate concentration, inoculate into a 24-well plate, and culture overnight in a 37°C, 5% CO2 incubator until the cells adhere. After compound A is prepared with culture medium to a set concentration, discard the old culture medium, add fresh culture medium containing compound A, and continue to incubate at 37°C, 5% CO2 for 48 hours. Add the corresponding reagent according to the instructions of the EdU detection kit and incubate for the corresponding time, then stain with Hoechst33342 for 5 minutes in the dark, and observe and photograph the cells under a fluorescence microscope ( Figure 3 ).

[0050] The experimental results show that in the EdU cell proliferation detection experiment, it can be observed that the red fluorescent EdU labeling indicates cells with strong proliferation ability, and the blue fluorescent Hoechest labeling indicates tumor cell nuclei. In human lung adenocarcinoma cells A549 and H1299, the number of EdU positive cells decreased in a dose gradient, especially in the high-dose group, indicating that compound A can effectively inhibit the proliferation of lung cancer cells.

[0051] Experimental Example 6: Flow cytometry apoptosis detection

[0052] Experimental method: Flow cytometry apoptosis detection uses Annexin V / PI co-staining to detect the apoptosis rate. Experimental principle: In the early stages of apoptosis, cells will externalize phosphatidylserine to the cell surface, and Annexin V with the fluorescent label FITC can bind to the phosphatidylserine externalized to the cell surface. Propidium iodide (PI) can stain necrotic cells or cells that have lost cell membrane integrity in the late stages of apoptosis, presenting another type of fluorescence. For necrotic cells, since the integrity of the cell membrane has been lost, Annexin V-FITC can enter the cytoplasm and bind to the phosphatidylserine on the inner side of the cell membrane, thereby also making the necrotic cells present green fluorescence. Therefore, flow cytometry can be used to distinguish between apoptotic and normal cells. The specific operating steps for flow cytometry apoptosis detection are as follows:

[0053] Digest the cells in the logarithmic phase, centrifuge, and count. Dilute with culture medium to an appropriate concentration, inoculate into a 6 cm dish, and incubate overnight in a 37 ° C, 5% CO2 incubator. After compound A is prepared with culture medium to a set concentration, discard the old culture medium, add fresh culture medium containing compound A, and continue to incubate at 37 ° C, 5% CO2 for 48 hours. After the incubation, take the cells out of the incubator, aspirate the old culture medium of the 6 cm dish into a suitable centrifuge tube, wash the cells once with PBS buffer, and combine the PBS buffer and the old culture medium. Add an appropriate amount of trypsin to digest the cells, and be careful not to over-digest. Gently blow down the cells with the collected cell culture medium, transfer to a centrifuge tube, centrifuge at 1000 rpm for 3 minutes, and discard the supernatant. Gently resuspend the cells with pre-cooled PBS buffer, centrifuge at 1000 rpm for 3 minutes, and discard the supernatant. Repeat the above process of washing with PBS buffer once again to completely remove the influence of trypsin. Add the corresponding reagents according to the instructions of the flow apoptosis kit for incubation, then place on ice and detect on the machine within 1 hour. After the sample is tested by flow cytometry, the data is exported ( Figure 4 ).

[0054] The experimental results showed that compound A significantly increased the number of human lung adenocarcinoma cell A549 and H1299 Annexin V positive cells, and the cell apoptosis rate showed a concentration gradient increase. The apoptosis rates of human lung adenocarcinoma cell A549 cells were 10.497%, 20.348% and 21.24% after 48 hours of administration, and the apoptosis rates of human lung adenocarcinoma cell H1299 cells were 6.077%, 8.753% and 29.673% after 48 hours of administration. Therefore, it can be concluded from the results that compound A can induce apoptosis of lung cancer cells.

[0055] Experimental Example 7: In vivo imaging

[0056] Experimental method: The in vivo imaging detection animal model uses a mouse in vivo imaging device to detect tumor growth status in animals. Experimental principle: Using in vivo bioluminescence imaging technology, the Fluc gene is integrated into cells to express luciferase. When its substrate luciferin is given exogenously, such as intraperitoneally or intravenously, luminescence can be generated within a few minutes. This enzyme catalyzes the oxidation reaction of luciferin in the presence of ATP and oxygen to produce luminescence, so luminescence will occur in living cells. At the same depth in vivo, the detected luminescence intensity and the number of cells have a clear linear relationship. The specific operation of the in vivo imaging detection animal model is as follows:

[0057] H1299 cells with Luciferase label in logarithmic growth phase were digested, centrifuged and resuspended in PBS to 6×10 7 / ml cell suspension, and then 0.1 ml was injected into the subcutaneous axillary area of ​​nude mice to establish a subcutaneous tumor transplant model. When the volume of the implanted tumor reached about 100 cubic millimeters, the drug was started. The animals were randomly divided into 3 groups, namely, a blank group, a cisplatin group (2mg / kg) and a compound A group (10mg / kg). The drug was administered by intraperitoneal injection once every two days to observe the tumor growth. After the cell implantation, the tumor grew to a certain volume and the nude mice were killed.

[0058] The small animal in vivo imaging system detects tumor growth. The specific operation steps are as follows:

[0059] (1) 5-10 min before anesthesia, mice were intraperitoneally injected with 15 mg / ml luciferin potassium salt solution at a volume of 10 μL / g;

[0060] (2) placing the mouse in an anesthesia induction box, turning on the aerosol anesthesia system, inducing anesthesia of the mouse with isoflurane, and after the mouse is anesthetized, placing the mouse in a dark room, adjusting the position of the mouse, and selecting bioluminescence to detect the mouse;

[0061] (3) All measurements should be performed by the same person who is skilled in the operation;

[0062] (4) After all the measurements of the mouse live imaging are completed, all the data are quantitatively analyzed;

[0063] (5) Import the images of mouse live imaging into the small animal live imaging analysis system for fluorescence analysis, and export all data for quantitative analysis ( Figure 5 ).

[0064] The above description shows and describes several preferred embodiments of the invention, but as mentioned above, it should be understood that the invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention concept described herein through the above teachings or the technology or knowledge of the relevant field. Changes and modifications made by those skilled in the art should be within the scope of protection of the claims attached to the invention without departing from the spirit and scope of the invention.

Claims

1. A crystalline form I of an indole δ-sultone compound A, characterized in that: The X-ray powder diffraction pattern thereof has peaks at 2θ of 7.1, 8.4, 12.0, 15.2, 18.3, 18.9, 20.0, 21.4, 22.0, 22.9, 22.2, 26.9, 27.3, and 29.

8. The structure of the indole δ-sultone compound A is shown in the following formula:

2. The method for preparing the crystalline form I of compound A according to claim 1, characterized in that: Compound A is dissolved in acetone, and cyclohexane is added dropwise to crystallize out a solid, which is filtered and dried to obtain Form I.

3. Use of compound A according to claim 1 as a preparation of anti-tumor drugs.

4. Use of the crystalline form I according to claim 1 as a preparation of anti-tumor drugs.

5. The use according to claim 3 and claim 4, characterized in that Use as the preparation of anti-lung cancer drugs, use as the preparation of anti-cervical cancer drugs, use as the preparation of anti-melanoma drugs, use as the preparation of anti-colorectal cancer drugs, use as the preparation of anti-breast cancer drugs, and / or use as the preparation of anti-liver cancer drugs.

6. A drug, characterized in that: The invention relates to a preparation prepared by using the compound A or its crystal form I as claimed in claim 1 as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

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