Preparation method and application of tetrahydroindazole antitumor compounds

Abstract

The present application relates to the technical field of medicine (IPC classification A61P31 / 22), and particularly relates to a preparation method and application of a tetrahydroindazole antitumor compound, the preparation method comprising: reacting a compound of formula I with adamantanol to obtain a 4-(4-hydrazone-3,6,6-trimethyl-1-tetrahydroindazole)-2-(4-hydroxycyclohexylamino) benzamide compound, compared with a 4-(4-hydrazone-3,6,6-trimethyl-1-tetrahydroindazole)-2-(4-hydroxycyclohexylamino) benzamide compound in the prior art, the compound has stronger inhibition of tumor cell growth activity, has generally lower IC50 values, and in particular can significantly reduce the clonogenicity of non-small cell lung cancer at a low concentration, and causes apoptosis of non-small cell lung cancer cells, and has great significance for development of a novel antitumor drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine (IPC classification A61P31 / 22), and in particular to a preparation method of tetrahydroindazole antitumor compounds and application thereof. BACKGROUND

[0002] Antitumor drugs are drugs that can inhibit the growth of tumor cells and fight and treat malignant tumors. In the past, pharmacology divided antitumor drugs into six categories according to their properties and sources: alkylating agents, antimetabolites, antibiotics, plant drugs, hormones, and miscellaneous. After years of development, the field of antitumor drugs has made remarkable achievements. The research and development of new drugs, especially new compounds, have laid a solid foundation for the development of individualized treatment of antitumor drugs. Drugs with high selectivity, high efficiency, and low toxicity are the direction of future new drug research and development. With the continuous improvement of people's requirements for medical level, it is required that the drug has a strong inhibitory activity on tumor cell growth while having a lower use concentration and a lower body toxicity, which brings challenges to drug research and development, especially compound research and development. SUMMARY

[0003] A first aspect of the present application provides a preparation method of tetrahydroindazole antitumor compounds, which comprises: reacting a compound of formula I with adamantanol to obtain a compound of formula II,

[0004] Formula I.

[0005] In some embodiments, the adamantanol comprises at least one of trans-4-amino-1-adamantanol, cis-4-amino-1-adamantanol, 2-propyl-2-adamantanol, 2-butyl-2-adamantanol, 2-ethyl-2-adamantanol, 3-ethyl-1-adamantanol, (5R,7S)-2-amino-1-adamantanol, 2-phenyl-2-adamantanol, 2-isopropyl-2-adamantanol, 2-ethyl-1,3-dimethyl-2-adamantanol, 1-phenyl-2-adamantanol, 1-(4-methoxyphenyl)-2-adamantanol, 2-adamantanol, 3-methyl-1-adamantanol, 3-amino-1-adamantanol, 2-methyl-2-adamantanol, 3,5-dimethyl-1-adamantanol, 1-adamantanol, and 1-acetylamino-3-adamantanol.

[0006] In some embodiments, the molar ratio of the compound of formula I to adamantanol is (1-2):(4-6).

[0007] Further, the compound of formula I is prepared by the following reaction:

[0008]

[0009] Further, the preparation method of the compound of formula I comprises the following steps:

[0010] S1. 5,5-dimethyl-1,3-cyclohexanedione is dissolved in ethyl acetate, then Et3N, DMAP and acetyl chloride are added for reaction, the organic layer is washed with saturated brine, filtered with a sand core funnel, and the solvent is extracted under reduced pressure to obtain yellow oil, which is recorded as intermediate 2.

[0011] S2. 2,4-difluorobenzonitrile is added to ethanol at room temperature, then hydrazine hydrate is added for reaction, the reaction solution is poured into ice water, and white solid is precipitated, which is recrystallized to obtain white solid product, which is recorded as intermediate 4.

[0012] S3. Intermediate 2 and intermediate 4, glacial acetic acid and ethanol are added to a round-bottom flask for reaction, and after the reaction is completed, a solid is precipitated, which is filtered, cooled, washed and filtered to obtain red crystalline solid. The mother liquor is concentrated, and the red-brown solid product is obtained after recrystallization, which is recorded as 5a, i.e. formula I.

[0013] Further, the preparation method of the compound of formula I comprises the following steps:

[0014] S1. 5,5-dimethyl-1,3-cyclohexanedione (10.00 g, 71.34 mmol) is dissolved in 25 mL of ethyl acetate, then Et3N (21.66 g, 214.05 mmol), DMAP (4.36 g, 35.69 mmol) and acetyl chloride are added, and the reaction is allowed to proceed at room temperature (25°C) for 12 hours, then the organic layer is washed with saturated brine, filtered with a sand core funnel, and the solvent is extracted under reduced pressure to obtain 12.58 g of yellow oil, with a yield of 96.78%, which is recorded as intermediate 2.

[0015] S2. 2,4-difluorobenzonitrile (10.00 g, 71.89 mmol) is added to 20 mL of ethanol at room temperature (25°C), then 80% hydrazine hydrate (4.10 g, 81.96 mmol) is added. The reaction is refluxed at 90°C for 5 hours. Then, the reaction solution is poured into ice water, and white solid is precipitated. Recrystallization is performed with ethanol: water (mass ratio 8:1) to obtain 5.99 g of white solid product, with a yield of 55.14%, which is recorded as intermediate 4.

[0016] S3. Intermediate 2 (6.03 g, 33.08 mmol) and intermediate 4 (5.00 g, 33.08 mmol), glacial acetic acid (2.98 g, 49.62 mmol) and ethanol (20 mL) were added into a 250 mL round-bottom flask and reacted at 80 °C for 12 hours. After the reaction was completed, a solid was precipitated, which was filtered and cooled to room temperature (25 °C), and then washed by soaking in petroleum ether to obtain a red crystalline solid. The mother liquor was concentrated and recrystallized twice with ethanol to obtain a red-brown solid product, a total of 9.15 g, a yield of 92.99%, recorded as 5a, i.e. Formula I.

[0017] Further, the reaction of the compound of Formula I with adamantanol includes the following steps:

[0018] S1. 5a, adamantanol, DMA, DMSO were taken in a round-bottom flask to react, and after cooling, the reaction solution was poured into ice water, the solid was filtered and dried, and recrystallized to obtain a white solid product, recorded as 10a.

[0019] S2. Intermediate 10a, KOH, DMSO were added into a round-bottom flask, ice-bathed, and H2O2 was added dropwise to react, the reaction solution was poured into ice water, stirred, the solid was filtered and dried, and recrystallized to obtain a white solid product, recorded as JD-10.

[0020] Further, the reaction of the compound of Formula I with adamantanol includes the following steps:

[0021] S1. 5a (0.5 g, 1.68 mmol), trans-4-amino-1-adamantanol (0.84 g, 5.02 mmol), DMAP (0.41 g, 3.36 mmol), DMSO (10 mL) were taken in a 100 mL round-bottom flask, and refluxed at 100 °C overnight, cooled to room temperature (25 °C), and the reaction solution was poured into ice water, the solid was filtered and dried, and recrystallized with methanol to obtain a white solid product 0.74 g, a yield of 92.5%, recorded as 10a.

[0022] S2. Intermediate 10a (0.5 g, 1.05 mmol), KOH (0.06 g, 1.07 mmol), DMSO (10 mL) were added into a 125 mL round-bottom flask, ice-bathed, and 30% H2O2 (0.11 g, 3.23 mmol) was added dropwise, and reacted at room temperature (25 °C) for 3 h, poured into ice water, stirred, the solid was filtered and dried, and recrystallized with methanol to obtain a white solid product 0.46 g, a yield of 93.8%, to obtain a product 2-(((1S,5S)-5-hydroxyadamantan-2-yl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide, recorded as JD-10, with a structural formula of .

[0023] The preparation reaction process of the present application is as follows:

[0024]

[0025] The second aspect of the present application provides an anti-tumor pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the prepared tetrahydroindazole anti-tumor compound or a pharmaceutically acceptable salt thereof.

[0026] In some embodiments, the pharmaceutical composition is an oral agent, a topical preparation, a tablet, a capsule, a powder, a pill, a granule, a gel, an injection or an emulsion.

[0027] The third aspect of the present application provides the use of the prepared tetrahydroindazole anti-tumor compound or the pharmaceutical composition in the preparation of an anti-tumor drug.

[0028] The fourth aspect of the present application provides the use of the prepared tetrahydroindazole anti-tumor compound or the pharmaceutical composition in the preparation of a drug for inhibiting the expression of a tumor proliferation-related gene.

[0029] The seventh aspect of the present application provides a method for using the prepared tetrahydroindazole anti-tumor compound or the pharmaceutical composition, wherein the concentration of the tetrahydroindazole anti-tumor compound for inhibiting tumor cell growth is 0.01-1 μM.

[0030] Further, the tumor cells include one or more of HCT116, Eca109, A549, MDA-MB-231 and NCI-H358.

[0031] Compared with the prior art, the present application has the following beneficial effects:

[0032] The present application studies the prepared 2-(((1S,5S)-5-hydroxyadamantan-2-yl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide, and a large number of experiments have confirmed that, compared with the prior art 4-(4-hydrazone-3,6,6-trimethyl-1-tetrahydroindazole)-2-(4-hydroxycyclohexylamino) benzamide compound, the prepared 2-(((1S,5S)-5-hydroxyadamantan-2-yl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide has stronger inhibitory activity on tumor cell growth, has a generally lower IC50 value, and can significantly reduce the clonogenicity of non-small cell lung cancer at a low concentration. It can cause inhibition of cell growth by weakening the population dependence and proliferation ability of non-small cell lung cancer cells, and can also cause apoptosis of non-small cell lung cancer cells and inhibit cell survival. The above-mentioned pathways play an anti-cancer role, and have great significance for the development of new anti-tumor drugs. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the specific embodiments of the present application or the prior art, the accompanying drawings required by the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and all other drawings obtained by those of ordinary skill in the art without creative effort based on these drawings also belong to the protection scope of the present application.

[0034] Figure 1 Cell survival rate curve corresponding to the tetrahydroindazole antitumor compound (JD-10) prepared in Example 1 determined in Example 2.

[0035] Figure 2 A549 cell and NCI-H358 cell apoptosis test results corresponding to the tetrahydroindazole antitumor compound (JD-10) determined in Example 3.

[0036] Figure 3 A549 cell and NCI-H358 cell apoptosis test results corresponding to the tetrahydroindazole antitumor compound (JD-10) determined in Example 4.

[0037] Figure 4 NMR carbon spectrum of intermediate 10a obtained in Example 1.

[0038] Figure 5 NMR hydrogen spectrum of intermediate 10a obtained in Example 1.

[0039] Figure 6 NMR carbon spectrum of tetrahydroindazole antitumor compound (JD-10) prepared in Example 1.

[0040] Figure 7 NMR hydrogen spectrum of tetrahydroindazole antitumor compound (JD-10) prepared in Example 1. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0042] Example 1

[0043] The present embodiment provides a preparation method of tetrahydroindazole antitumor compound (JD-10), comprising the following steps:

[0044] S1. 5,5-dimethyl-1,3-cyclohexanedione (10.00 g, 71.34 mmol) was dissolved in 25 mL of ethyl acetate, then Et3N (21.66 g, 214.05 mmol), DMAP (4.36 g, 35.69 mmol) and acetyl chloride were added. After the reaction was carried out at room temperature (25 °C) for 12 hours, the organic layer was washed with saturated brine, filtered with a sand core funnel, and the solvent was extracted under reduced pressure to obtain 12.58 g of yellow oil, with a yield of 96.78%, which was recorded as intermediate 2.

[0045] S2. 2,4-difluorobenzonitrile (10.00 g, 71.89 mmol) was added to 20 mL of ethanol at room temperature (25 °C), then 80% hydrazine hydrate (4.10 g, 81.96 mmol) was added. The reaction was refluxed at 90 °C for 5 hours. After that, the reaction solution was poured into ice water to precipitate white solids. Recrystallization was performed with ethanol: water (mass ratio 8:1) to obtain 5.99 g of white solid product, with a yield of 55.14%, which was recorded as intermediate 4.

[0046] S3. Intermediate 2 (6.03 g, 33.08 mmol) and intermediate 4 (5.00 g, 33.08 mmol), glacial acetic acid (2.98 g, 49.62 mmol) and ethanol (20 mL) were added to a 250 mL round-bottom flask and reacted at 80 °C for 12 hours. After the reaction was completed, solids were precipitated, filtered and cooled to room temperature (25 °C), then washed and filtered with petroleum ether to obtain red crystalline solids. The mother liquor was concentrated and recrystallized twice with ethanol to obtain red-brown solid product, with a total yield of 9.15 g, with a yield of 92.99%, which was recorded as 5a.

[0047] S4. Take 5a (0.5 g, 1.68 mmol), trans 4-amino-1-adamantanol (0.84 g, 5.02 mmol), DMAP (0.41 g, 3.36 mmol), DMSO (10 mL) in a 100 mL round bottom flask, reflux at 100 °C overnight, cool to room temperature (25 °C), pour the reaction into ice water, filter the solid, dry, recrystallize with methanol to get the product as white solid 0.74 g, yield 92.5%, record as 10a.1H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 8.2 Hz, 1H), 6.84 - 6.68 (m, 2H), 5.00 (d, J = 6.5 Hz, 1H), 3.69 (s, 1H), 2.81 (s, 2H), 2.54 (s, 3H), 2.41 (s, 2H), 2.27 (s, 2H), 2.21 (s, 1H), 1.89 (d, J = 13.4 Hz, 6H), 1.82 (d, J = 11.6 Hz, 3H), 1.69 (s, 2H), 1.56 (d, J = 12.8 Hz, 3H), 1.12 (s, 6H).13C NMR (101 MHz, CDCl3) δ 193.32, 150.52, 150.02, 149.07, 143.59, 133.81, 117.68, 117.22, 110.72, 105.72, 95.06, 77.25, 67.46, 55.50, 52.29, 45.23, 44.23 (2C), 37.82, 35.89, 33.22 (2C), 30.14 (2C), 29.46, 28.43 (2C), 13.43.

[0048] S5. Intermediate 10a (0.5 g, 1.05 mmol), KOH (0.06 g, 1.07 mmol), DMSO (10 mL) were added into a 125 mL round bottom flask, ice bath, 30% H2O2 (0.11 g, 3.23 mmol) was added dropwise, and the reaction was allowed to proceed at room temperature (25 °C) for 3 h. The reaction mixture was poured into ice water, stirred, and the solid was filtered and dried. The product was recrystallized from methanol to obtain a white solid, 0.46 g, yield 93.8%, and the product 2-(((1S,5S)-5-hydroxyadamantan-2-yl)amino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indazol-1-yl)benzamide, noted as JD-10 (tetrahydroindazole antitumor compound).1H NMR (400 MHz, CDCl3) δ 8.68 (d, J = 7.2 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 6.76 – 6.58 (m, 2H), 5.74 (s, 2H), 3.64 (d, J = 4.7 Hz, 1H), 2.81 (s, 2H), 2.55 (s, 3H), 2.40 (s, 2H), 2.26 (s, 2H), 2.18 (s, 1H), 1.97 (d, J = 13.2 Hz, 2H), 1.87 (dd, J = 26.4, 11.6 Hz, 4H), 1.79 (s, 2H), 1.68 (s, 5H), 1.50 (d, J = 12.0 Hz, 3H), 1.11 (s, 6H), 0.84 (s, 1H).13C NMR (101 MHz, CDCl3) δ 193.45, 171.41, 150.27, 150.11, 148.96, 142.91, 129.73, 117.31, 111.64, 108.69, 106.51, 77.24, 67.73, 55.17, 52.35, 45.43, 44.40 (2C), 37.77, 35.83, 33.37 (2C), 30.38 (2C), 29.68, 28.44 (2C), 13.45.

[0049] Example 2

[0050] Growth curve and IC50 value determination of tetrahydroindazole antitumor compound on tumor cells (JD-10):

[0051] (1) Select human colorectal cancer cells HCT116, human esophageal squamous cell carcinoma cells Eca109, human non-small cell lung cancer cells A549 and NCI-H358, human breast cancer cells MDA-MB-231 as tumor cells to be tested, the culture conditions of MDA-MB-231 cell strain are DMEM culture medium added with 10% FBS, the culture conditions of Eca109, A549 and NCI-H358 cell strains are RPMI1640 culture medium added with 10% FBS, and the culture conditions of HCT116 cell strain are McCoy's 5A culture medium added with 10% FBS. All cell strains are cultured in a 37℃ incubator containing 5% CO2, and cell viability and growth are determined by using a 96-well plate.

[0052] (2) After the cells are adherently grown, different concentrations of JD-10 and JD-02 are added to each well as experimental groups, and no drug is added as a control group, and then cultured in a 37℃ incubator containing 5% CO2 for 48h, and then 10μL CCK8 is added to each well and cultured for 2h, and then the absorbance value is detected at 450nm by using an enzyme-labeled instrument, and the survival rate is calculated according to the following formula:

[0053] Survival rate = [(absorbance of experimental group-absorbance of blank well) / (absorbance of control group-absorbance of blank well)]*100%.

[0054] The fitting curve of drug concentration corresponding to cell survival rate is drawn by using GraphPad Prism8, and the cell survival rate curve corresponding to compound JD-10 is shown in Figure 1 , which shows that JD-10 of the present application has a concentration-dependent and time-dependent inhibitory effect on the viability of HCT116, Eca109, A549, MDA-MB-231 and NCI-H358 cells.

[0055] The JD-02 is a tetrahydroindazole antitumor compound disclosed in Chinese invention patent CN202210158435.3, and the full name is 4-(4-hydrazone-3, 6, 6-trimethyl-1-tetrahydroindazole)-2-(4-hydroxycyclohexylamino) benzamide, and the structural formula is , which also has the effect of inhibiting tumor cell growth.

[0056] (3) The IC50 value determination results of JD-10 on the viability of the above tumor cells after 48h are shown in Table 1.

[0057] Table 1

[0058]

[0059] Example 3

[0060] On the basis of Example 2, in order to further verify the effect of tetrahydroindazole antitumor compound (JD-10) on the colony formation ability of non-small cell lung cancer, a colony formation experiment was performed.

[0061] (1) After trypsin digestion of A549 and NCI-H358 cells, a uniform single cell suspension was prepared, 400 cells were inoculated per well into a 6-well plate, and placed in a 37°C incubator containing 5% CO2. After 24h, the cells adhered, and JD-10 was prepared as a drug working solution after the culture medium containing 10% FBS was prepared, and was given as an experimental group. The control group was replaced with fresh culture medium without drugs, and after 48h of treatment, fresh, drug-free culture medium containing 10% FBS was replaced, and the culture was continued for 2 weeks, during which fresh culture medium was replaced every 48h.

[0062] (2) After 2 weeks, the original culture medium was aspirated, washed with PBS, and 4wt% paraformaldehyde fixing solution was added for 20min, then the fixing solution was washed clean, and Giemsa staining solution was added for 15min, then the staining solution was washed clean with water, the plate was dried, and photographed. The results, as shown in Figure 2 , JD-10 can cause a decrease in the number of A549 cell and NCI-H358 cell clones and the area of individual clones.

[0063] Example 4

[0064] On the basis of Example 3, in order to further verify the effect of tetrahydroindazole antitumor compound (JD-10) on the growth and survival of non-small cell lung cancer cells, DAPI staining was used to detect the effect on cell apoptosis.

[0065] Principle: DAPI (4,6-diamidino-2-phenylindole) is a fluorescent dye that can strongly bind to DNA, and can bind to AT base pairs in the minor groove of double-stranded DNA. The fluorescence intensity of DAPI molecules bound to double-stranded DNA increases by about 20 times, and the fluorescence intensity can be observed by fluorescence microscopy, and is commonly used to observe cell nucleus morphology. During apoptosis, the cell nucleus will deform, condense, and fragment, so the morphology of the cell nucleus can be detected by DAPI staining to determine whether the cell is apoptotic.

[0066] Experimental procedure: the digested NCI-H358 cells or A549 cells were prepared into a uniform single cell suspension, and the cell density was adjusted to 16w per hole; 6-hole plates were taken, 2mL of cell suspension was inoculated in each hole, and was placed in a 37℃ incubator containing 5% CO2 for culture. After the cells adhered, JD10 containing 10% FBS was prepared as a drug working solution for drug treatment for 48h. After the 6-hole plate was taken out of the incubator, it was observed under a microscope, then the culture medium was discarded, and the cells were washed once with PBS, 1mL of polyethylene glycol was added to each hole, and the cells were fixed at room temperature for 30min. During the fixing, DAPI working solution was prepared according to the proportion of DAPI: PBS = 1: 1. After the fixing was completed, the cells were washed once with PBS, 1mL of DAPI working solution was added to each hole, and the cells were stained at room temperature for 20-30min. After the staining was completed, the cells were washed once with PBS, and then observed and photographed under a fluorescence microscope.

[0067] The results are shown in Figure 3 As shown in the results, after the A549 cells and NCI-H358 cells were treated with JD-10 drug for 48h, and then stained with DAPI and photographed under a fluorescence microscope, it was observed that the cells in the blank control group had clear edges, uniform staining and abundant cytoplasm; the cells treated with JD-10 drug at low, medium and high doses all showed fluorescence, and with the increase of the dose, the number of cells decreased, the number of cells with strong fluorescence increased significantly, most of the cells shrank, the cytoplasm condensed, the nucleus and chromosomes highly condensed, the edges of the nucleus and chromosomes, the nucleus and chromosomes fragmented into pieces, and typical crescent-shaped apoptotic bodies appeared. It is shown that JD-10 drug can inhibit the proliferation of A549 cells and NCI-H358 cells and promote their apoptosis.

[0068] The above is the preferred embodiment of the present application, and it should be pointed out that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A process for the preparation of tetrahydroindazole antitumor compounds, characterized in that, The preparation method comprises the following steps: reacting a compound of formula I with adamantanol to prepare a compound of formula II, Formula I; The tetrahydroindazole antitumor compound has a structural formula 。 2. The method for preparing the tetrahydroindazole antitumor compound according to claim 1, characterized in that, The adamantanol is trans-4-amino-1-adamantanol.

3. Use of the tetrahydroindazole antitumor compound prepared according to claim 1 in the preparation of an antitumor drug for inhibiting the growth of tumor cells, wherein the tumor cells are selected from one or more of HCT116, Eca109, A549, MDA-MB-231 and NCI-H358.

4. Use of the tetrahydroindazole antitumor compound prepared according to claim 1 in the preparation of a drug for inhibiting the expression of a tumor cell proliferation-related gene, wherein the tumor cells are selected from one or more of HCT116, Eca109, A549, MDA-MB-231 and NCI-H358.

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