CN116621812B - Tetrahydroindazole compound, preparation method and application thereof in treating esophageal squamous carcinoma - Google Patents

Tetrahydroindazole compound, preparation method and application thereof in treating esophageal squamous carcinoma Download PDF

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CN116621812B
CN116621812B CN202310529793.5A CN202310529793A CN116621812B CN 116621812 B CN116621812 B CN 116621812B CN 202310529793 A CN202310529793 A CN 202310529793A CN 116621812 B CN116621812 B CN 116621812B
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CN116621812A (en
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刘凯胜
苏苑
张卓
蓝妮
任哲
王一飞
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Shenzhen Peoples Hospital
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Abstract

The invention relates to the technical field of medicine synthesis, in particular to a tetrahydroindazole compound, a preparation method and application thereof in treating esophageal squamous carcinoma, wherein the structural formula of the compound is as followsThe 4- ((2-carbamoyl-5- (3, 6-trimethyl-4-oxo-4, 5,6, 7-tetrahydro-1H-indazol-1-yl) phenyl) amino) piperidine-1-yl benzoate prepared by the research of the invention has stronger activity of inhibiting the growth of tumor cells, has a generally lower IC50 value, can particularly obviously cause apoptosis of esophageal squamous carcinoma cells at low concentration, reduces the cloning capacity and migration capacity of the esophageal squamous carcinoma cells, inhibits the proliferation of the cells by blocking the cells in the G2/M phase, and has great significance for developing novel antitumor drugs.

Description

Tetrahydroindazole compound, preparation method and application thereof in treating esophageal squamous carcinoma
Technical Field
The invention relates to the technical field of medicine synthesis, in particular to a tetrahydroindazole compound, a preparation method and application thereof in treating esophageal squamous cell carcinoma.
Background
In the last decades, cancer has become one of the important causes of life health hazards for humans due to various causes of environmental pollution, unhealthy lifestyles, and the like. The present antitumor drugs can be classified into alkylating agents, antimetabolites, antitumor antibiotics, hormonal drugs, antitumor plant drugs, other antitumor drugs and adjuvant therapeutic drugs according to different structures, sources of drugs and different mechanisms of action on tumor cells. Although in recent years, anti-tumor drug research has made great progress, the mortality rate of various cancers including node esophageal squamous cell carcinoma has remained high and has not been significantly reduced. Tetrahydroindazole compounds have been recognized as drugs with great antitumor potential due to the indazole group. However, the existing tetrahydroindazoles generally have the defects of poor antitumor activity, large toxic and side effects, poor targeting property and the like, so that the existing tetrahydroindazole compounds are further improved, and the research and development of the tumor medicament with lower use concentration and lower toxic and side effects of the body are of great significance to medical research.
Disclosure of Invention
In a first aspect, the present invention provides tetrahydroindazole compounds having the structural formula I:
in a second aspect, the present invention provides a process for preparing a tetrahydroindazole compound, said process comprising: reacting a compound of formula II with an aminopiperidine compound;
further, the preparation method comprises the following steps: the compound of formula II is reacted with 1-Boc-4-aminopiperidine.
Further, the reaction of the compound II with 1-Boc-4-aminopiperidine comprises the following steps:
s1, taking 5a, 1-Boc-4-aminopiperidine and DMSO in a round bottom flask, carrying out reflux reaction overnight, cooling to room temperature (25 ℃), pouring the reaction solution into ice water, carrying out suction filtration on the solid, drying, and recrystallizing with methanol to obtain a white solid product which is marked as 5b.
S2, adding the intermediate 5b, KOH, DMSO into a single-neck round-bottom flask, ice-bathing, and dropwise adding H 2 O 2 Reacting at room temperature (25 ℃), pouring into ice water, stirring, filtering to obtain solid, and drying. Methanol recrystallisation gives the product 5c as a white solid.
S3, taking the intermediate 5c and DCM in a round-bottomed flask, slowly dripping TFA in an ice bath, gradually heating to room temperature (25 ℃) for reaction overnight, concentrating the solvent, adding the DCM and repeatedly concentrating to obtain yellow oily substance 5e.
S4, taking the intermediate 5e, and adding TEA and DMF into a round bottom flask containing the Boc-removed product under ice bath condition. Stirring and adding Na 2 HPO 4 、(PhCO 2 ) 2 The reaction was carried out overnight at room temperature (25 ℃). Pouring the reaction solution into ice water, filtering the solid, and drying. Recrystallization from methanol gives the product as a white solid, designated compound i (i.e., 4- ((2-carbamoyl-5- (3, 6-trimethyl-4-oxo-4, 5,6, 7-tetrahydro-1H-indazol-1-yl) phenyl) amino) piperidin-1-ylbenzoate, designated JD-14).
Further, the reaction of the compound II with 1-Boc-4-aminopiperidine comprises the following steps:
s1, 5a (1 g,3.36 mmol), 1-Boc-4-aminopiperidine (1.35 g,6.74 mmol) and DMSO (20 mL) are taken and reacted in a 125mL round bottom flask at 110 ℃ under reflux overnight, cooled to room temperature (25 ℃), the reaction solution is poured into ice water, the solid is filtered off with suction, dried and recrystallized from methanol to give a white solid product, which is recorded as 5b.
S2, adding the intermediate 5b (1 g,2.10 mmol), KOH (0.06 g,1.07 mmol) and DMSO (10 mL) into a 125mL single-neck round-bottom flask, ice-bathing, and dropwise adding 30% H 2 O 2 (0.07 g,2.06 mmol), reacted at room temperature (25 ℃ C.) for 2 hours, poured into ice water, stirred, suction filtered and dried. Methanol recrystallisation gives the product 5c as a white solid.
S3, taking the intermediate 5c (0.5 g,1.01 mmol) and DCM (10 mL) in a 100mL round bottom flask, ice-bathing, slowly dripping 3mL of LTFA, gradually heating to room temperature (25 ℃) for reaction overnight, concentrating the solvent, adding DCM and repeatedly concentrating to obtain yellow oily substance 5e.
S4, taking the intermediate 5e, adding TEA (0.26 g,2.56 mmol) and DMF (10 mL) into a round bottom flask containing the Boc-removed product under ice bath. Stirring for 30 min, adding Na 2 HPO 4 (0.29g，2.04mmol)、(PhCO 2 ) 2 (0.27 g,1.11 mmol) was reacted overnight at room temperature (25 ℃ C.). Pouring the reaction solution into ice water, filtering the solid, and drying. Recrystallisation from methanol gives the product as a white solid, designated asCompound I (i.e., 4- ((2-carbamoyl-5- (3, 6-trimethyl-4-oxo-4, 5,6, 7-tetrahydro-1H-indazol-1-yl) phenyl) amino) piperidin-1-ylbenzoate, designated JD-14).
The preparation reaction process of the invention is as follows:
in a third aspect, the invention provides a pharmaceutical composition for combating esophageal squamous carcinoma, the active ingredient of said pharmaceutical composition comprising a tetrahydroindazole compound or a pharmaceutically acceptable salt thereof.
In some embodiments, the pharmaceutical composition is an oral, external, tablet, capsule, powder, pill, granule, gel, injection, or emulsion.
In a fourth aspect, the invention provides the use of a tetrahydroindazole compound or pharmaceutical composition in the manufacture of a medicament for treating esophageal squamous carcinoma.
In a fifth aspect, the invention provides the use of a tetrahydroindazole compound or pharmaceutical composition in the manufacture of a medicament for inhibiting the expression of genes associated with esophageal squamous carcinoma proliferation.
In a sixth aspect, the invention provides methods of using tetrahydroindazole compounds or pharmaceutical compositions that inhibit the growth of esophageal squamous carcinoma cells at a concentration of 0.01-100 μm.
In some embodiments, the esophageal squamous carcinoma cells include one or more of Kyse140, eca109, SW620, A549, and MDA-MB-231.
Compared with the prior art, the invention has the following beneficial effects:
the 4- ((2-carbamoyl-5- (3, 6-trimethyl-4-oxo-4, 5,6, 7-tetrahydro-1H-indazol-1-yl) phenyl) amino) piperidine-1-yl benzoate prepared by the invention is researched, and a plurality of groups of experiments prove that the 4- (4-hydrazone-3, 6-trimethyl-1-tetrahydroindazole) -2- (4-hydroxycyclohexylamino) benzamide compound has stronger tumor cell growth inhibition activity, has a generally lower IC50 value, can particularly obviously cause apoptosis of esophageal squamous carcinoma cells under low concentration, reduces the cloning capacity and migration capacity of the esophageal squamous carcinoma cells, inhibits cell proliferation in G2/M phase by blocking the esophageal squamous carcinoma cells, and has great significance for developing novel antitumor drugs.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the cell viability of Compound I (JD-14) prepared in example 1 as measured in example 2.
FIG. 2 is a graph showing the results of cloning experiments on Kyse140 cells and Eca109 cells corresponding to Compound I (JD-14) measured in example 3.
FIG. 3 is a graph showing the results of apoptosis experiments on Kyse140 cells and Eca109 cells corresponding to Compound I (JD-14) measured in example 4.
FIG. 4 is a graph showing the results of a scratch test on Kyse140 cells and Eca109 cells corresponding to Compound I (JD-14) measured in example 5.
FIG. 5 is a graph showing the results of a cell cycle arrest assay for Kyse140 cells and Eca109 cells corresponding to Compound I (JD-14) as determined in example 6.
FIG. 6 is a nuclear magnetic resonance carbon spectrum of intermediate 5b obtained in example 1.
FIG. 7 is a nuclear magnetic resonance hydrogen spectrum of intermediate 5b obtained in example 1.
FIG. 8 is a nuclear magnetic resonance carbon spectrum of intermediate 5c obtained in example 1.
FIG. 9 is a nuclear magnetic resonance hydrogen spectrum of intermediate 5c obtained in example 1.
FIG. 10 is a nuclear magnetic resonance carbon spectrum of the compound I (JD-14) produced in example 1.
FIG. 11 is a nuclear magnetic resonance hydrogen spectrum of the compound I (JD-14) produced in example 1.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
This example provides a process for preparing a tetrahydroindazole compound, comprising the steps of:
s1, 5a (1 g,3.36 mmol), 1-Boc-4-aminopiperidine (1.35 g,6.74 mmol) and DMSO (20 mL) were taken and reacted in a 125mL round bottom flask at 110℃under reflux overnight, cooled to room temperature (25 ℃), the reaction solution was poured into ice water, the solid was suction filtered, dried and recrystallized from methanol to give 1.49g of a white solid product, recorded as 5b, yield 92.5%.1HNMR (400 MHz, CDCl 3) delta 7.49 (d, J=7.9 Hz, 1H), 6.92 (s, 1H), 6.70 (d, J=7.8 Hz, 1H), 4.65 (s, 1H), 4.08 (s, 2H), 3.61 (s, 1H), 2.97 (s, 2H), 2.81 (s, 2H), 2.54 (s, 3H), 2.41 (s, 2H), 2.06 (d, J=12.2 Hz, 2H), 1.75 (s, 2H), 1.48 (s, 9H), 1.12 (s, 6H), 13CNMR (101 MHz, CDCl 3) delta 193.30,154.65,150.54,149.84,149.14,143.55,133.97,117.71,117.07,110.77,105.80,95.17,79.96,77.26,52.29,49.99,37.80,35.91,31.90,28.42 (5C), 13.42.
S2, adding the intermediate 5b (1 g,2.10 mmol), KOH (0.06 g,1.07 mmol) and DMSO (10 mL) into a 125mL single-neck round-bottom flask, ice-bathing, and dropwise adding 30% H 2 O 2 (0.07 g,2.06 mmol), reacted at room temperature (25 ℃ C.) for 2 hours, poured into ice water, stirred, suction filtered and dried. Methanol recrystallisation gives 0.95g of the product as a white solid, recorded as 5c, in 91.35% yield. 1HNMR (400 MHz, CDCl 3) δ8.24 (d, J=6.8 Hz, 1H), 7.51 (d, J=8.0 Hz, 1H), 6.82 (s, 1H), 6.60 (d, J=8.3 Hz, 1H), 3.94 (d, J=11.1 Hz, 2H), 3.57 (d, J=3.1 Hz, 1H), 3.09 (t, J=10.8 Hz, 2H), 2.81 (s, 2H), 2.54 (s, 3H), 2.40 (s, 2H), 2.06-
1.94(m,4H),1.53(d,J＝11.1Hz,2H),1.47(s,9H),1.11(s,6H).13CNMR(101MHz,CDCl3)δ193.51，171.41，154.80，150.08，150.00，149.08，142.70，129.76，117.26，112.27，108.95，106.62，79.75，52.33，50.70，48.79，37.66，35.84，31.58，28.44(3C)，28.41(2C)，13.42.
S3, taking an intermediate 5c (0.5 g,1.01 mmol) and DCM (10 mL) in a 100mL round-bottom flask, carrying out ice bath, slowly dripping 3mL of LTFA, gradually heating to room temperature (25 ℃) for reaction overnight, concentrating a solvent, adding DCM, repeatedly concentrating to obtain yellow oily matter 5e, and directly carrying out the next reaction without purification; TEA (0.26 g,2.56 mmol), DMF (10 mL) was added to a round bottom flask containing the debonded Boc product under ice-bath. Stirring for 30 min, adding Na 2 HPO 4 (0.29g，2.04mmol)、(PhCO 2 ) 2 (0.27 g,1.11 mmol) was reacted overnight at room temperature (25 ℃ C.). Pouring the reaction solution into ice water, filtering the solid, and drying. Recrystallization from methanol gave 0.32g of the product as a white solid, designated as compound i (i.e., 4- ((2-carbamoyl-5- (3, 6-trimethyl-4-oxo-4, 5,6, 7-tetrahydro-1H-indazol-1-yl) phenyl) amino) piperidin-1-ylbenzoate, designated JD-14) in 61.5% yield. 1HNMR (400 MHz, CDCl 3) delta 8.33 (d, J=6.7 Hz, 1H), 7.49 (d, J=8.5 Hz, 1H), 7.41 (s, 5H), 7.26 (s, 1H), 6.86 (s, 1H), 6.62 (d, J=8.2 Hz, 1H), 5.74 (s, 2H), 4.39 (s, 1H), 3.73 (s, 2H), 3.30 (s, 2H), 2.81 (s, 2H), 2.55 (s, 3H), 2.40 (s, 2H), 2.09 (d, J=47.3 Hz, 2H), 1.32 (d, J=47.1 Hz, 1H), 1.11 (s, 6H), 13CNMR (101 MHz, CDCl 3) delta 193.38,171.20,170.49,150.17,149.99,149.03,142.88,135.94,129.72,129.65,128.54 (2C), 126.86 (2C), 117.36,112.20,109.07,106.69,77.24,52.36,48.65,37.71,35.87,28.44 (2C), 13.44.
Example 2
This example demonstrates the growth curve and IC50 value determination of Compound I (JD-14) for tumor cell inhibition:
(1) Kyse140 cells, eca109 cells, SW620 cells, A549 cells and MDA-MB-231 cells are selected as tumor cells to be tested, the culture condition of the MDA-MB-231 cell strain is DMEM culture medium added with 10% FBS, and the culture condition of Kyse140 cell strain, eca109 cell strain, SW620 cell strain and A549 cell strain is RPMI1640 culture medium added with 10% FBS. All cell lines were in the presence of 5% CO 2 Culturing in a 37℃incubator, and measuring cell viability with a 96-well plateForce and growth.
(2) Primary screening: eca109 cells, A549 cells and MDA-MB-231 cells are taken as primary screening experimental cells, a series of tetrahydroindazole compounds synthesized in the laboratory are respectively added into each hole to be taken as experimental groups after the cells are grown in an adherence manner, no medicine is added as control groups, and the cells respectively contain 5% CO 2 After culturing in an incubator at 37℃for 48 hours and then adding 10. Mu.LCCK 8 to each well for 2 hours, absorbance was measured at 450nm with a microplate reader, and survival was calculated:
survival = [ (absorbance of experimental group-absorbance of blank well)/(absorbance of control group-absorbance of blank well) ]x100%. A fitted curve of drug concentration versus cell viability was plotted with GraphPadPrism 8.
(3) Kyse140 cells, eca109 cells, SW620 cells, A549 cells and MDA-MB-231 cells are taken as tumor cells to be tested, after the cells are attached to the wall and grow, the compound I (JD-14) and JD-02 are respectively added into each hole to be taken as experimental groups, and no drug is added to be taken as control groups, and the experimental groups respectively contain 5% CO 2 After culturing in an incubator at 37℃for 48 hours and then adding 10. Mu.LCCK 8 to each well for 2 hours, absorbance was measured at 450nm with a microplate reader, and survival was calculated:
survival = [ (absorbance of experimental group-absorbance of blank well)/(absorbance of control group-absorbance of blank well) ]x100%. A fitted curve of drug concentration versus cell viability was plotted with GraphPadPrism 8.
In order to verify the antitumor effect of the JD-14 of the present invention, a series of tetrahydroindazole compounds (JD-01, JD-03, JD-04, JD-05, JD-06, JD-07, JD-08, JD-09, JD-11, JD-12, JD-15, JD-16) were prepared simultaneously, and compared with the anti-tumor effect of the JD-14, the IC50 values of the inhibition of Eca109 cells, A549 cells and MDA-MB-231 cells were shown in Table 1. As can be seen from Table 1, in Eca109 cells, A549 cells and MDA-MB-231 cells, compound I (JD-14) was more potent than other tetrahydroindazole compounds, so Compound I (JD-14) was selected for further study and comparison with JD-02. The cell viability curves corresponding to the compound I (JD-14) are shown in FIG. 1, and the IC50 value measurement results of the tumor cell activity inhibition by the compound I (JD-14) and JD-02 are shown in Table 2, which illustrate that the compound I (JD-14) of the present invention has concentration-dependent and time-dependent inhibition effects on cell viability of Kyse140 cells, eca109 cells, SW620 cells, A549 cells and MDA-MB-231 cells. In particular to the inhibition effect on the activity of esophageal squamous carcinoma Kyse140 and Eca109 cell lines.
TABLE 1
TABLE 2
The JD-02 is a novel tetrahydroindazole antitumor compound disclosed in Chinese patent No. CN202210158435.3, and is totally called 4- (4-hydrazone-3, 6-trimethyl-1-tetrahydroindazole) -2- (4-hydroxycyclohexylamino) benzamide, and the structural formula is as followsAlso has effect in inhibiting tumor cell growth activity.
The preparation methods of the JD-01, the JD-03, the JD-04, the JD-05, the JD-06, the JD-07, the JD-08, the JD-09, the JD-11, the JD-12, the JD-15 and the JD-16 are as follows:
(1) Synthesis of key intermediates
5, 5-dimethyl-1, 3-cyclohexanedione (10.00 g,71.34 mmol) was dissolved in 25mL of ethyl acetate, then Et3N (21.66 g,214.05 mmol), DMAP (4.36 g,35.69 mmol) and acetyl chloride (8.40 g,107.00 mmol) were added and the reaction was allowed to proceed at room temperature for 12 hours. The precipitate was removed by filtration, and the organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was extracted under reduced pressure to give 12.58g of a yellow oil (intermediate 2) in 96.78% yield.
2, 4-difluorobenzonitrile (10.00 g,71.89 mmol) was added to 20mL of ethanol at room temperature followed by 80% hydrazine hydrate (4.10 g,81.96 mmol). Reflux reaction at 80℃for 8 hours. Thereafter, the reaction solution was poured into ice water, and a white solid was precipitated. Ethanol is used for: recrystallisation from water (8:1) gives the product as a white solid (intermediate 4) 5.99g, 55.14% yield.
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 250mL round bottom flask and reacted at 80℃for 10 hours. And separating out solid after the reaction is finished, filtering the solid, cooling the solid to room temperature, and soaking, cleaning and filtering the solid by petroleum ether to obtain red crystalline solid. The mother liquor was concentrated and recrystallized twice from ethanol to give a reddish brown solid product which was 9.15g of solid product (5 a) in total with a yield of 92.99%.
Intermediate 5a (5.00 g,16.82 mmol) and p-aminocyclohexanol (4.26 g,37.00 mmol) were dissolved in 25mL DMSO and reacted at 110℃for 12 hours. After TLC monitoring substrate disappearance the reaction solution was cooled to room temperature, poured into ice water, filtered to remove solids, ethanol recrystallized to give 5.20g of pale white solid (intermediate 7) in 78.79% yield.
Intermediate 7 (5.00 g,12.74 mmol), KOH (0.07 g,1.27 mmol) and DMSO (25 mL) were added to a 125mL single neck round bottom flask, ice-bath, 30% H2O2 (0.43 g,12.74 mmol) was added dropwise, reacted at room temperature for 2H, poured into ice water, stirred, suction filtered, and dried. Methanol was recrystallized to give 4.34g of a white solid product (AT 533), yield 83%.
(2) Synthesis of target Compound JD-01
AT533 (2.0 g,4.87 mmol), hydroxylamine hydrochloride (0.68 g,9.79 mmol), sodium acetate (0.80 g,9.75 mmol) and EtOH (10 mL) were taken and reacted in a 125mL round bottom flask AT 80℃under reflux overnight, cooled to room temperature to precipitate a solid, which was filtered off with suction to give 1.46g of a pale yellow solid, the mother liquor was concentrated and recrystallized from ethyl acetate to give 0.35g of a pale yellow solid product, which gave 1.81g of the total product in 87.4% yield.
(3) Synthesis of target Compound JD-03
AT533 (0.2 g,0.49 mmol) and EtOH (10 mL) were taken in a 100mL round bottom flask, ice-cooled, naBH4 (0.06 g,1.59 mmol) was added, reacted overnight AT room temperature, the solvent was concentrated, extracted with saturated brine and ethyl acetate, the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and recrystallized from ethanol to give 0.16g of a white solid product in 80% yield.
(4) Synthesis of target Compounds JD-04, JD-05
Raw material AT533 (2.0 g,4.87 mmol) and 20mL of ethylene glycol were taken in a 125mL single neck round bottom flask, and then 4eq 6N HCl was added dropwise and reacted AT 100deg.C under reflux overnight. After 12h of reaction, cooling to room temperature, concentrating the solvent to give a yellow oil, recrystallizing with ethyl acetate to remove a small amount of the starting material, purifying by column chromatography, and recrystallizing with methanol to give 0.63g of a white solid product (JD-04) in 55.3% yield.
Operating according to the described synthetic route for the preparation of JD-04, 0.34g of the product was obtained as a white solid in 37.8% yield.
(5) Synthesis of target Compound JD-06
Intermediate 7 (1.0 g,2.55 mmol), hydroxylamine hydrochloride (0.71 g,10.21 mmol), naOAc (0.84 g,10.24 mmol), etOH (20 mL) was taken in a 125mL round bottom flask and reacted overnight at 70℃under reflux. The solvent was dried by spinning, extracted three times with saturated brine and ethyl acetate, and the organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and the solid was purified by filtration through a silica gel column to give 0.35g of a white solid with a yield of 31.3%.
(6) Synthesis of target Compound JD-07
JD-05 (0.2 g,0.45 mmol) was taken in a 100ml round bottom flask and DMSO was added: H2O (4:
1) CuCl2 (0.15 g,0.88 mmol) was added as a solvent, stirred overnight at room temperature, the reaction solution was poured into ice water, the solid was suction filtered, extracted with ethyl acetate, washed with saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate and concentrated, and purified by silica gel column filtration to give 0.12g of a white solid JD-06 in 63.2% yield.
(7) Synthesis of target Compound JD-08
5a (0.5 g,1.68 mmol), N-aminomorpholine (0.38 g,3.70 mmol) and DMSO (10 mL) were taken and reacted in a 100mL round bottom flask at 100℃overnight under reflux, cooled to room temperature, the reaction solution was poured into ice water, the solid was suction filtered and dried, and recrystallized from methanol to give 0.38g of the white solid product (8 a) in 59.37% yield.
Intermediate 8a (0.2 g,0.53 mmol), KOH (0.03 g,0.53 mmol), DMSO (10 mL) were added to a 125mL single neck round bottom flask, ice-bath, 30% H2O2 (0.02 g,0.58 mmol) was added dropwise, reacted at room temperature for 2H, poured into ice water, stirred, suction filtered, and dried. Methanol was recrystallized to yield 0.18g of product with 85.7% yield.
(8) Synthesis of target Compound JD-09
5a (0.55 g,1.84 mmol), 1-amino-4-methylpiperazine (0.46 g,3.99 mmol) and DMSO (10 mL) were taken and reacted in a 100mL round bottom flask at 100deg.C under reflux overnight, cooled to room temperature, the reaction solution was poured into ice water, the solid was suction filtered, dried and recrystallized from methanol to give 0.35g of yellow crystalline product (9 a) in 48.6% yield.
Intermediate 9a (0.2 g,0.51 mmol), KOH (0.03 g,0.53 mmol), DMSO (10 mL) were added to a 125mL single neck round bottom flask, ice-bath, and 30% H2O2 (0.03 g,1.01 mmol) was added dropwise and reacted at room temperature for 3H. Pouring the reaction mixture into ice water, stirring, filtering the solid by suction, and drying. Methanol was recrystallized to give 0.17g of the product in 81.0% yield.
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(9) Synthesis of target Compound JD-11
Trans-1, 4 cyclohexanediol (0.78 g,6.711 mmol), anhydrous DMSO (20 mL) was taken in a 125mL single neck round bottom flask, naH (0.16 g,6.67 mmol) was added under ice bath conditions and gradually warmed to room temperature and stirred for 1h. While stirring, 5a (0.5 g,1.68 mmol) was added, the reaction mixture was stirred at room temperature for 1h, gradually warmed to 110 ℃ and refluxed overnight, cooled to room temperature, the reaction solution was poured into ice water, the solid was suction filtered, dried, and recrystallized from methanol to give 0.56g of a white solid product (11 a) in 84.8% yield.
Intermediate 11a (0.3 g,0.76 mmol), KOH (0.04 g,0.71 mmol) and DMSO (10 mL) were added to a 125mL single neck round bottom flask, ice-bath, 30% H2O2 (0.03 g,0.76 mmol) was added dropwise, reacted at room temperature for 2H, poured into ice water, stirred, suction filtered, and dried. Methanol was recrystallized to give 0.17g of yellow crystalline product (JD-11) in 81.0% yield.
(10) Synthesis of target Compound JD-12
5a (0.5 g,1.68 mmol), trans-cyclohexanediamine (0.58 g,5.08 mmol) and DMSO (10 mL) were taken and reacted in a 100mL round bottom flask at 110℃overnight under reflux, cooled to room temperature, the reaction solution was poured into ice water, the solid was suction filtered and dried, and recrystallized from methanol to give 0.58g of the yellow solid product (12 a) in 87.9% yield.
Intermediate 12a (0.3 g,0.77 mmol), KOH (0.04 g,0.77 mmol), DMSO (10 mL) were added to a 125mL single neck round bottom flask, ice-bath, 30% H2O2 (0.03 g,0.88 mmol) was added dropwise, reacted at room temperature for 3H, poured into ice water, stirred, suction filtered, and dried. Methanol was recrystallized to give 0.25g of a white solid product (JD-12) in 79.7% yield.
(11) Synthesis of target compounds JD-14 and JD-15
5a (1 g,3.36 mmol), 1-Boc-4-aminopiperidine (1.35 g,6.74 mmol) and DMSO (20 mL) were taken and reacted in a 125mL round bottom flask at 110℃under reflux overnight, cooled to room temperature, the reaction solution was poured into ice water, the solid was suction filtered, dried and recrystallized from methanol to give 1.49g of the product as a white solid in 92.5% yield.
Intermediate 5b (1 g,2.10 mmol), KOH (0.06 g,1.07 mmol) and DMSO (10 mL) were added to a 125mL single port round bottom flask, ice-bath, 30% H2O2 (0.07 g,2.06 mmol) was added dropwise, reacted at room temperature for 2H, poured into ice water, stirred, suction filtered, and dried. Methanol was recrystallized to give 0.95g of white solid product in 91.35% yield.
Taking intermediate 5c (0.5 g,1.01 mmol) and DCM (10 mL) in a 100mL round bottom flask, ice-bathing, slowly dripping 3mL of LTFA, gradually heating to room temperature for reaction overnight, concentrating the solvent, adding DCM, repeatedly concentrating to obtain yellow oily substance 5e, and directly carrying out the next reaction without purification; TEA (0.26 g,2.56 mmol), DMF (10 mL) was added to a round bottom flask containing the debonded Boc product under ice-bath. After stirring for 30 minutes Na2HPO4 (0.29 g,2.04 mmol), (PhCO 2) 2 (0.27 g,1.11 mmol) was added and reacted overnight at room temperature. Pouring the reaction solution into ice water, filtering the solid, and drying. Recrystallisation from methanol gives 0.32g of the product as a white solid (JD-14) in 61.5% yield.
Intermediate JD-14 (0.2 g,0.39 mmol), KOH (0.07 g,1.25 mmol) and EtOH (20 mL) were taken in a 100mL round bottom flask and reacted at 110℃under reflux for 12h, the solvent was concentrated, the solid was extracted with saturated brine and ethyl acetate, the organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated to give 0.08g of a white solid (JD-15) in 50.12% yield.
(12) Synthesis of target Compound JD-16
5a (2 g,6.73 mmol), tert-butyl 4-aminopiperidine-1-carboxylate (2.98 g,14.80 mmol) and DMSO (25 mL) were taken and reacted in a 125mL round bottom flask at 110℃under reflux overnight, cooled to room temperature, the reaction solution was poured into ice water, the solid was suction filtered, dried and recrystallized from methanol to give 2.51g of the product (6 b) as a white solid in 77.91% yield.
Intermediate 6b (1 g,2.09 mmol), KOH (0.01 g,1.07 mmol), DMSO (10 mL) were added to a 125mL single port round bottom flask, ice-bath, 30% H2O2 (0.07 g,2.09 mmol) was added dropwise, reacted at room temperature for 2H, poured into ice water, stirred, suction filtered, and dried. Methanol recrystallisation gives the product (6 c) as a white solid (0.56 g) in 53.97% yield.
Taking intermediate 6c (0.5 g,1.01 mmol) and DCM (10 mL) in a 100mL round bottom flask, ice-bathing, slowly dripping 3mL of LTFA, gradually cooling to room temperature for reaction overnight, concentrating the solvent, adding DCM, repeatedly concentrating to obtain a white solid product 6e, and directly carrying out the next reaction without purification; TEA (0.26 g,2.56 mmol) and DMF (10 mL) were added to a round bottom flask containing 6e under ice-bath. After stirring for 30 minutes Na2HPO4 (0.29 g,2.04 mmol), (PhCO 2) 2 (0.27 g,1.11 mmol) was added and reacted overnight at room temperature. Pouring the reaction solution into ice water, filtering the solid, and drying. Recrystallisation from methanol gives 0.31g of the product as a white solid in 59.62% yield.
Example 3
On the basis of example 2, this example further demonstrates the effect of compound I (JD-14) on the clonogenic potential of esophageal squamous carcinoma cells, as studied using a clonogenic assay.
(1) Taking Eca109 cells and Kyse140 cells in logarithmic growth phase and good growth state, digesting to prepare uniform single cell suspension, inoculating 500 cells per well into 6-well plate, and placing into a culture medium containing 5% CO 2 Is cultured in an incubator at 37 ℃. After the cells were attached, different concentrations of Compound I (JD-14) were added as the experimental group, and no Compound I was added and only medium was added as the control group. The culture was continued by changing fresh, drug-free medium containing 10% fbs for 24h, with fresh medium changing every 48h.
(2) The incubation was continued for 14 days, the old medium was discarded, washed with PBS, and fixed with paraformaldehyde fixative solution for 30 minutes. After the fixation was completed, the mixture was washed with PBS, and stained with 0.2% crystal violet solution for 20 minutes. After the dyeing is finished, residual crystal violet is washed off by using clean water, the pore plate is dried in the air, and the record is photographed.
As shown in FIG. 2, the compound I (JD-14) can significantly reduce the clone numbers and single clone areas of esophageal squamous carcinoma Kyse140 and Eca109 cells relative to the control group, which indicates that the compound I (JD-14) can inhibit the cell clone formation of esophageal squamous carcinoma Kyse140 and Eca109 cells.
Example 4
On the basis of example 2, this example further demonstrates the effect of compound I (JD-14) on the growth and survival of esophageal squamous carcinoma cells, whose effect on apoptosis was examined using DAPI staining.
DAPI staining principle: DAPI (4, 6-diamidino-2-phenylindole) is a commonly used nucleic acid dye which can penetrate cell membranes and combine with double-stranded DNA in cell nuclei to play a role of labeling, and generates fluorescence which is 20 times stronger than DAPI itself, and has high staining sensitivity on double-stranded DNA. Commonly used for apoptosis detection, common nuclear staining, and in some specific cases double stranded DNA staining.
The experimental steps are as follows:
(1) Taking logarithmic growth phaseAnd the Eca109 cells and Kyse140 cells with good growth state are digested to prepare uniform single cell suspension, 15w cells per hole are inoculated in a 6-hole plate and put in the culture medium containing 5% CO 2 Is cultured in an incubator at 37 ℃. After the cells are attached, compound I (JD-14) with different concentrations is added as a dosing group, and a blank control group without adding compound I and with only culture medium is used for 48 hours.
(2) After the drug action, the 6-well plate was removed from the incubator, the medium was removed by pipetting, washed with PBS, and 1 mL/well paraformaldehyde was added for fixation at room temperature (25 ℃) for 30 minutes.
(3) After the fixation was completed, the mixture was washed once with PBS, 1 mL/well DAPI working solution was added, and the mixture was stained at room temperature (25 ℃) for 30 minutes in a dark place. After the staining was completed, the staining was washed with PBS and then examined under a fluorescence microscope.
The results are shown in FIG. 3, and the blank group has clear cell edges and uniform dyeing and abundant cytoplasm. The cells of the drug-added group become small cytoplasm to concentrate, the nucleus chromosome is highly condensed and marginalized, the nucleus is broken into fragments, typical crescent apoptosis bodies appear, the cell number is continuously reduced along with the increase of the drug concentration, the cell proportion of the apoptosis bodies is increased, and the compound I (JD-14) can promote the apoptosis of the esophageal squamous carcinoma Kyse140 and Eca109 cells and inhibit the proliferation of the esophageal squamous carcinoma Kyse140 and Eca109 cells.
Example 5
On the basis of example 2, the influence of the compound I (JD-14) on the migration ability of esophageal squamous carcinoma cells is further verified, and the influence on the migration ability of the esophageal squamous carcinoma cells is detected by using a scratch experiment.
(1) Pre-drawing lines: the marker pen is used to uniformly mark the transverse lines behind the 6-hole plate, and every 0.5-1 cm of the transverse lines cross the through holes, and each hole passes through at least 5 lines.
(2) Taking Eca109 cells and Kyse140 cells in a logarithmic growth phase and in a good growth state, digesting the Eca109 cells and the Kyse140 cells to prepare uniform single cell suspension, inoculating 50w cells per hole into a 6-hole plate, and placing the mixture into a chamber containing 5% CO 2 Is cultured in an incubator at 37 ℃. And (5) when the cell wall is full.
(3) The 10 mu L gun head is used for drawing parallel lines in the direction perpendicular to the marking lines compared with a straight ruler, and the gun head is required to hang downStraight, cannot tilt. Washing with PBS buffer at least twice, and washing off the scraped floating cells as much as possible. Compound I (JD-14) was added at various concentrations as a dosing group and medium alone was used as a control group without compound I. Adding 5% CO 2 Is cultured in an incubator at 37 ℃ and photographed under an inverted microscope at 0 and 24 hours, respectively, to observe the migration of cells.
As shown in FIG. 4, the wound healing distance between the scratches of the cells of the drug-added group is obviously higher than that of the control group, and the horizontal migration and the vertical migration capacity are obviously reduced, which indicates that the compound I (JD-14) can inhibit the cell migration of esophageal squamous cell carcinoma Kyse140 and Eca 109.
Example 6
On the basis of example 2, this example further demonstrates the effect of compound I (JD-14) on esophageal squamous carcinoma cell cycle, as measured by PI single-staining and flow cytometry.
PI single-dye detection principle: propidium iodide (PI for short) is a fluorescent dye of double-stranded DNA. Propidium iodide can generate fluorescence after being combined with double-stranded DNA, and the fluorescence intensity is proportional to the content of double-stranded DNA. After the DNA in the cells is stained by propidium iodide, the DNA content of the cells can be measured by a flow cytometer, and then the cell cycle analysis can be performed according to the distribution of the DNA content.
The experimental steps are as follows:
(1) Taking Eca109 cells and Kyse140 cells in a logarithmic growth phase and in a good growth state, digesting to prepare uniform single cell suspension, inoculating 15w cells per hole into a 6-hole plate, and placing the mixture into a chamber containing 5% CO 2 Is cultured in an incubator at 37 ℃. After the cells are attached, compound I (JD-14) with different concentrations is added for action.
(2) Sample collection and fixation are carried out at 0h, 12h, 24h, 36h and 48h respectively: old medium was aspirated, washed with PBS, and digested with 500. Mu.L of pancreatin. After complete digestion, pancreatin was aspirated, cells were blown down with PBS, 1000g, centrifuged at 25℃for 5min, and the supernatant removed. The mixture was washed with PBS, centrifuged, and the supernatant was removed, and 1mL of 75% pre-chilled ethanol was added for resuspension and fixed overnight at 4 ℃.
(3) After the fixation was completed, 1000g was centrifuged at 25℃for 5min, ethanol was removed by pipetting, washing the supernatant with PBS, adding 500. Mu.L of PI dye containing 10% RNaseA for resuspension, incubating for 1h in the dark, and detection on a flow cytometer.
Cell cycle results are shown in FIG. 5, and the cell cycle arrest of esophageal squamous cell carcinoma Kyse140 and Eca109 in the G2/M phase can be obviously caused after the compound I (JD-14) acts for 24h, 36h and 48h.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

Claims (5)

1. The tetrahydroindazole compound is characterized in that the structural formula of the compound is shown as formula I:
2. the method for preparing a tetrahydroindazole compound according to claim 1, characterized in that said method comprises the steps of:
3. a pharmaceutical composition for combating esophageal squamous carcinoma, characterized in that the active ingredient of said pharmaceutical composition comprises a tetrahydroindazole compound according to claim 1 or a pharmaceutically acceptable salt thereof.
4. A pharmaceutical composition according to claim 3, wherein the pharmaceutical composition is in the form of a tablet, capsule, powder, pill, granule, gel, injection or emulsion.
5. Use of a tetrahydroindazole compound according to claim 1 or a pharmaceutical composition according to claim 3, for the preparation of an anti-esophageal squamous carcinoma drug.
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Citations (5)

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CN102020640A (en) * 2009-09-18 2011-04-20 上海恒瑞医药有限公司 Tetrahydroindolone and tetrahydroindolone derivative as well as preparation method and medical application thereof
CN107428740A (en) * 2014-09-11 2017-12-01 埃萨内克斯股份有限公司 Indazolyl and indoles yl-benzamide derivatives
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